George J Banwart Basic Food Microbiology 1979

BASIC FOOD MICROBIOLOGY
Micrograph of bacteria growing on alfalfa sprouts.

BASIC FOOD
MICROBIOLOGY
Second Edition
George J. Banwart
Professor Emeritus
Department of Microbiology
The Ohio State University
CHAPMAN & HALL
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Librnry of Congress Cataloging-in-PubUcation Data
Banwart, George J.
Basic tood microbiology, 2/e .
. An A VI book."
Includes index.
ISBN13: 9781-4684-64559 o-ISBN13: 9781-468464535
DOl: 10.1007/978-1468464535
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Contents
Preface vii
1 General Aspects of Food 1
2 Estimating the Number of Microorganisms 11
3 Microorganisms Associated with Food 49
4 Factors That Affect Microbial Growth in Food 101
5 Sources of Microorganisms 165
6 Foodborne Agents Causing Illness 195
7 Indicator Organisms 371
8 Food Spoilage 393
9 Useful Microorganisms 433
10 Control of Microorganisms 505
11 Control of Microorganisms by Retarding Growth 545
12 Control of Microorganisms by Destruction 651
13 Regulations and Standards 725
Index 751
Preface
The second edition of Basic Food Microbiology follows the same general
outline as the highly successful first edition. The text has been revised
and updated to include as much as possible of the large body of infor-
mation published since the first edition appeared. Hence, foodborne ill-
ness now includes listeriosis as well as expanded information about
Campylobacter jejuni.
Among the suggestions for altering the text was to include flow sheets
for food processes. The production of dairy products and beer is now
depicted with flow diagrams.
In 1954, Herrington made the following statement regarding a review
article about lipase that he published in thejournal of Dairy Science: "Some
may feel that too much has been omitted; an equal number may feel that
too much has been included. So be it."
The author is grateful to his family for allowing him to spend the
time required for composing this text. He is especially indebted to his
partner, Sally, who gave assistance in typing, editing, and proofreading
the manuscript.
The author also thanks all of those people who allowed the use of
their information in the text, tables, and figures. Without this aid, the
book would not have been possible.
1
General Aspects of Food
BASIC NEEDS
Our basic needs include air that contains an adequate amount of oxy
gen, water that is potable, edible food, and shelter. Food provides us with
a source of energy needed for work and for various chemical reactions.
Food also supplies chemicals for growth, for repair of injured or worn
out cells, and for reproduction. Food consumption can be a pleasurable
experience, and a time for meeting with family and friends. Food is so
necessary for our existence that the search for food has been the main
occupation of human beings throughout history.
FOOD NEEDS
In the United States, supermarket shelves are so well stocked that an
uninformed observer might assume that our search for an adequate food
supply has been successful. However, it is believed that 12.5 percent of
the earth's people receive considerably less food than they need; as many
as 50 percent might be receiving a marginal level of food (Kahn 1981).
The reasons for this widespread hunger problem include unequal distri
bution of food and money, as well as cultural, religious, and superstitious
beliefs.
The main problem facing the world today is its increasing population
(Fig. 1.1). In recent years the birth rate has declined, but so has the death
rate. The present world population is over 5 billion, and the annual in
crease is estimated to be 60 to 80 million people. Predictions for the
future food supply range from very pessimistic, with famines expected
to start in the near future, to the very optimistic viewpoint that there will
be plenty of food for a population of almost limitless numbers. The most
widely held opinion seems to be that, unless the rate of population in
crease can be substantially reduced, the demand for food will eventually
outrun the supply, regardless of the efficiency of food production.
Although world agricultural production has been increasing, the va
garies of nature (floods, droughts, freezing, and other adverse climatic
conditions) could cause a severe setback. With the expected increase in
G. J. Banwart, Basic Food Microbiology
Van Nostrand Reinhold 1989
2 BASIC FOOD MICROBIOLOGY
z
4
2

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Figure 1.1. Estimated world population. Will we be able to feed everyone in the fu-
ture?
population, the search for food will continue to be the primary endeavor
of many food scientists, including food microbiologists.
SOURCES OF FOODS
Our supply of food depends upon the photosynthetic reaction be-
tween solar energy and plants that contain chlorophyll. Through photo
synthesis, carbon dioxide and water are converted to glucose. Further
cellular reactions produce the various organic compounds-carbohy.
drates, fats, proteins, and vitamins.
Both the amount of food and its dietary quality can be increased. To
do this, we can improve the utilization of our land and fishery resources,
upgrade the quality of plant proteins, convert waste materials into edible
foods, and prevent losses or deterioration of our food supplies.
Land Resources
Increasing the productivity of land resources will require utilization
of more land and higher yields of foods. However, there is a finite
GENERAL ASPECTS OF FOOD 3
amount of land, and not all of the land that is available can be used
for agriculture. Even less can be used for crop production. Experts have
estimated that the amount of land used for agriculture can be doubled.
This will not necessarily double production. These added lands are of
marginal quality and will require increased water, fertilizer, technology,
and energy to make them productive.
One problem in American food production is the loss of good agri
cultural land for the construction of highways, airports, shopping cen
tel's, housing, and factories. With an increasing population, there will be
an even greater demand for land for these purposes. The United States is
now losing about 3 million acres of arable land per year to development.
Fishery Resources
Oceans and seas cover more than 70 percent of the earth's surface,
yet in the United States less than 10 percent of our protein comes from
fishery resources. It is likely that this resource could be utilized more
effectively as a source of food for the future. As on land, the principal
foodproducing organisms in the sea are plants (phytoplankton). These
plants use the photosynthetic process to produce food for herbivorous
animals in the sea. These, in turn, furnish food for small carnivorous
animals, larger fish, and ultimately human beings.
Microorganisms as Food
The use of microorganisms in food products is not a new idea. The
action of yeast in the fermentations producing wine and beer and the
leavening of doughs has been known for at least 4,000 or 5,000 years.
The nature of the action in these fermentations did not become estab
lished until the latter part of the nineteenth century when the relation
of living yeast cells to fermentation was discovered. Microorganisms are
used in the fermentation of various foods and are consumed as part of
these foods. This is especially evident in cheese. Penicillium roquejorti, the
blue mold of Roquefort cheese, and Penicillium camemberti, the white mold
of Camembert cheese, are consumed with the cheese. Therefore, the con
cept of using microorganisms as part of the food supply should not be
completely objectionable.
Although we can synthesize amino acids and polypeptides commer
cially, microorganisms can produce not only these substances, but pro
teins, antibiotics, vitamins, steroids, and many other products. The devel
opment of microorganisms as a food source will involve many
disciplines, but food microbiologists will play an especially important
role.
Bacteria, yeasts, or molds cannot create foods, but they can grow on
cellulosic compounds that would otherwise be wasted. Algae can utilize
solar energy to produce food. The production of microbial protein is
discussed in Chapter 9.
Wastes as Food
For every kilogram of food produced, between 5 and 10 kg of waste
materials are left in the field or at the processing plant. These substances
are considered wastes because their economic value is such that it is not
profitable to utilize them. Surprisingly, not long ago, fat was removed
from milk, fish, and oilseeds for human use, while the more valuable
protein was wasted or fed to animals. Hence, the "waste" products of
today may have food value in the future.
Some wastes are not readily usable because they are seasonal, diluted
with water, or require transportation to amass a large quantity for pro-
cessing. With waste disposal becoming an ever-increasing problem, and
food shortages becoming more critical, it is difficult to believe that the
problems involved in utilizing acceptable wastes cannot be resolved.
Processes such as reverse osmosis and ultrafiltration can be used to
concentrate dilute wastes (Moon 1980). Alternative systems for concen-
tration of wastes in the meat industry have been discussed (Hansen 1983).
Progress is being made in the recovery of wastes and in the utilization of
these materials in human foods and in animal feeds (Cherry, Young, and
Shewfelt 1975; Cooper 1976; Kamm et al. 1977; Knorr 1983; Toyama
1976).
Legal Aspects
U.S. food laws and regulations must be considered before any new
or novel food can be sold. The U.S. Department of Agriculture (USDA)
regulates red meat and poultry processing operations. In all other cases,
the U.S. Food and Drug Administration (FDA) decides what is an accept-
able food or food ingredient. Besides the dictates of these federal agen-
cies, the food laws of states and local jurisdictions must be followed.
If an ingredient is to be used in a food, it cannot create a health
hazard. The Food, Drug, and Cosmetic Act provides that a food shall be
deemed to be adulterated if it consists in whole or in part of any filthy
substances. What will be the FDA's reaction to single-cell protein ob-
tained from microorganisms grown on unconventional substrates? If a
food shortage does develop, we may need to reevaluate the aesthetic as-
pects of our foods. The health criteria for processed wastes have been
discussed (Taylor et al. 1974).
Preventing Losses
The data on losses of plant and animal products are neither adequate
nor reliable enough to allow us to conduct investigations to fully deter
mine the causes of the losses. We do know that deterioration, waste, and
loss occur in almost every step from production to consumption. Since
we already have a shortage of food and will need more food in the future,
we must make an increased effort to protect our food supply from fur
ther losses. If losses could be reduced or prevented, our food supply
would increase with no additional utilization of land or sea resources.
The main losses of foods result from the action of microorganisms,
insects, rodents, birds, nematodes, and the enzymes inherent in the food.
One study (Ennis, Dowler, and Klassen 1975) estimated that 30 percent
of crops worldwide are lost to pests. Albrecht (1975) suggested that pests
destroy 25 percent of all crops in the United States. He believed that
government regulations prevent adequate pest controL Schweigert
(1975) estimated that food losses from production to consumption range
from 20 to 50 percent. The monetary loss from producer to consumer is
reported to be more than $30 billion in the United States.
Reasons for Food Preservation
Preservation can be defined as a process by which foods are treated
to retard decay or spoilage. There are many reasons for preserving foods.
Several plant foods are harvested only once each year. To have a supply
of these foods throughout the year, rather than only at harvest time, pres
ervation is necessary. In case of a crop failure caused by a natural disaster
such as drought, wind, hail, flood, fire, freezing, or insect and disease
infestations, or by human disasters, such as war, the preservation of previ
ously produced excess food becomes paramount.
With preservation, one can obtain a more varied diet because a crop
can then be used throughout the year and because crops native to only
a small area can be transported and used anywhere in the world. One of
the reasons developing countries have food shortages is that they do not
have facilities for preservation and transportation of foods. Thus, certain
areas have a temporary surplus of food while other areas have a shortage.
Flesh foods deteriorate rapidly if held at ambient temperatures. In
some countries, although fish is plentiful in the coastal areas, there is a
protein shortage inland because refrigeration and rapid transit are lack
ing to transport the fish protein without spoilage.
Preservation allows the holding of foods so that they can be used as
ingredients for mixed foods. Many of our convenience foods are combi
nations of various foods. Some systems used to preserve food also destroy
many of the organisms and toxic factors that are hazards in food prod-
ucts.
METHODS OF FOOD PRESERVATION. The chief methods of food
preservation can be listed in four basic categories: asepsis (preventing
entry of microorganisms into foods); removing microorganisms; inhibit-
ing growth by controlling the environment; and destroying microorgan-
isms. Various systems have evolved from these basic procedures (Table
1.1).
Most methods of preserving food are merely modifications of systems
used in ancient times. The addition of salt as a chemical preservative,
fermentations, smoking, and cold storage have been practiced for over
2,000 years. Comparatively, canning might be considered a modern
method, although the process of preserving food by putting it into a
closed container and heat-treating it was patented by Appert in 1810. A
much newer system for preserving food involves the use of radiation; at
this time, however, irradiation has been approved by the FDA only for
specific purposes (see Chapter 12).
FOOD HAZARDS
From the beginning of life until death, a person is subjected to poten-
tially harmful environments. During one's lifetime, some hazards disap-
pear and others take their place, so that the problems of safety are not
static. The ingestion of food is no exception. Food may serve as a carrier
of chemical and biological substances, either added or acquired as con-
taminants from soil, water, air, food handlers, equipment, and other
sources. The possible subtle relationships between these substances in
foods and physical vigor, mental alertness, longevity, resistance to infec-
tion, and the onset of degenerative diseases are not fully understood.
Since we do not have all the answers regarding the safety of all possible
substances, there are many controversies concerning the overall safety of
TABLE 1.1. FOOD-PRESERVATION METHODS
Asepsis
Cen trifugation
Filtration
Refrigeration
Freezing
Drying
Freeze-drying
Chemicals
Smoking
Gas or vacuum packing
Acidification
Fermentation
Fumigation
Pasteurization
Cooking
Canning
Radiation
food. Although absolute safety of food is an ideal goal, for all practical
purposes, it is unattainable. Since a human being is a biological system,
and since biological systems vary, a food that causes no ill effects in one
person may cause problems in another person.
Wodicka (1977) listed six principal categories of food hazards: micro
biological hazards, malnutrition, environmental contaminants, naturally
occurring toxins, pesticides, and conscious food additives. Drug residues
or filth in foods might be hazardous when ingested. In addition, muta-
gens and carcinogens may be formed when certain foods are heated
(Grose et al. 1986; Jigerstad et al. 1983). Many chemicals that can be in-
gested relatively safely at low levels may be hazardous when ingested at
high levels. Even a high-fiber diet may reduce the absorption of essential
vitamins and minerals from the digestive tract.
Naturally Occurring Toxins
Several books and articles have been written concerning toxic agents
naturally present in foods (Coon 1975; Elton 1981; Gori 1979; Hatfield
and Brady 1975; Hironi 1981,Jadhav, Sharma, and Salunkhe 1981; Lewis
and Endean 1983; McMichael 1984; Munro 1976; Onoue et al. 1983; Op-
penheimer 1985; Panasiuk and Bills 1984; Shupe and James 1983; Swain,
Truswell, and Loblay 1984; Taylor 1982, 1985; van der Hoeven et al. 1983;
Wilson, McGann, and Bushway 1983). These naturally occurring toxins
include estrogens, tumorigens, carcinogens, cyanogens, as well as seafood
toxins, fungal toxins (mycotoxins), nutritional inhibitors, and antigens
that produce allergies. The quantities of these substances in foods are
usually low and, during processing, some of these substances are altered
to reduce their potency.
Problems resulting from the natural toxicants are often due to people's
eating raw foods or too much of only one type offood, or mistaking toxic
plants for similar, edible plants. Potatoes contain the alkaloid solanine,
a potent cholinesterase inhibitor that interferes with the transmission of
nerve impulses. The amount of potatoes an average person eats each
year contains enough solanine to be fatal if consumed in one dose.
At high levels, even polyunsaturated fats reportedly increase the inci-
dence of tumors and gallstones, increase the body's requirement of vita-
min E, and cause premature aging in laboratory animals. When exces-
sively heated, these fats are reported to contain toxic substances. One
product, malonaldehyde, is carcinogenic. Potentially carcinogenic lipid
peroxides are easily formed from polyunsaturated fats by autoxidation.
Many substances not generally considered toxic may present a poten-
tial hazard to some people. For example, some consumers of milk de-
velop severe distress of the digestive system because of an enzyme defi-
ciency that results in an intolerance to lactose.
Microorganisms
Data compiled by the Centers for Disease Control (CDC 1981a, 1981b,
1983) show that microbiological hazards are by far the most common
type of food hazard (Table 1.2). Since not all foodborne illnesses are reo
ported, the CDC data are not exact, but they are the most complete data
presently available. Each year, more than 60 percent of foodborne out
breaks are the result of bacterial etiologies, while less than 30 percent
are due to chemicals from various sources. The CDC has discontinued
listing foodborne outbreaks caused by unknown etiologies. In the past,
such outbreaks accounted for 25 to 30 percent of the total number.
Although not listed as causing any foodborne outbreaks, mycotoxins,
the toxins produced by molds, have received much attention in the past
ten to fifteen years. They are considered to be naturally occurring toxins
in foods.
Besides microorganisms, there are other biological entities that pre
sent a potential health hazard. Trichinella spiralis (the agent of trichinosis),
Taenia solium (pork tapeworm), Taenia saginata (beef tapeworm), Ascarias
(a roundworm) and Entamoeba histolytica (which causes amoebic dysen
tery) are a few of the agents that have been found in foods.
ROLE OF THE MICROBIOLOGIST
The food microbiologist is concerned with the biochemical reactions
of microorganisms in and on foods. These reactions result in spoilage,
public health hazards, and fermentation products. Determining the num
bers and types of microorganisms associated with food, and knowing
the sources of microorganisms, factors affecting their multiplication, and
systems that can be used for their control are important to food micro
biologists. However, we cannot look only at these aspects, but must also
examine other facets of foods, such as their chemical and physical charac
TABLE 1.2. CONFIRMED FOODBORNE OUTBREAKS, 1978-1980
1978 1979 1980
Cause No. %
No. %
No.
%
Bacterial 105 68.2 119 69.2 136 61.5
Viral 5 3.2 6 3.5 12 5.4
SL:IITOTAL 110 71.4 125 72.7 148 66.9
Parasitic 7 4.5 11 6.4 7 3.2
Chemical 37 24.0 6 20.9 66 29.9
TcrrAL 154 142 221
SOURCE: Data from CDC (l981a, 1981b, 1983)
teristics and the various attributes that are referred to as quality. We can
sterilize a food to destroy all microorganisms, but if the process makes
the food inedible or depletes its nutritional value, then the sterilization
process is not satisfactory.
With an understanding of food science, a food microbiologist can
better relate his or her role to the very important endeavor of providing
all people with an adequate supply of safe, wholesome foods. The role
of the food microbiologist in the food industry was discussed by Bauman
(1982) and Winslow (1982).
REFERENCES
Albrecht,].]. 1975. The cost of government regulations to the food industry. Food Technol.
29(10): 61, 64-65.
Bauman, H. E. 1982. The food microbiologist's role in the decisionmaking process. Food
Technol. 36(12): 58-59.
CDC. 1981a. Foodborne Disease Surveillance, Annual Summary, 1978. Atlanta, Ga.: Cen
ters for Disease Control.
--. 1981 b. Foodborne Disease Surveillance, Annual Summary, 1979. Atlanta, Ga.: Cen
--. 1983. Foodborne Disease Surveillance, Annual Summary, 1980. Atlanta, Ga.: Cen
Cherry,]. P.; Young, C. T.; and Shewfelt, A. L. 1975. Characterization of protein isolates
from keratinous material of poultry feathers.]. Food Sci. 40: 331-335.
Coon,]. M. 1975. Natural toxicants in foods.]. Amer. Diet. Assoc. 67: 213-218.
Cooper,]. L. 1976. The potential of food processing solid wastes as a source of cellulose
for enzymatic conversion. Proceedings of Biotechnol. Bioeng. Symp. 6: 251-271.
Elton, G. A. H. 1981. Additives and contaminants in the food supply. Food Technol. Aust.
33(4): 184-188.
Ennis, W. B.,Jr.; Dowler, W. M.; and Klassen, W. 1975. Crop protection to increase food
supplies. Science 188: 593-598.
Gori, G. B. 1979. Food as a factor in the etiology of certain human cancers. Food Technol.
33(12): 48-56.
Grose, K. R; Grant,]. L.; Bjeldanes, L. F.; Andresen, B. D.; Healy, S. K.; Lewis, P. R.; Felton,
]. S.; anrl Hatch, F. T. 1986. Isolation of the carcinogen IQ from fried egg patties.].
Agr. Food Ghern. 34: 201-202.
Hansen, C. 1983. Methods for animal waste recovery and energy conservation. Food Tech
nolo 37(2): 77-80, 84.
Hatfield, G. M., and Brady, L. R. 1975. Toxins of higher fungi. Lloydia 38: 36-55.
Hironi, 1. 1981. Natural carcinogenic products of plant origin. Grit. Rev. Toxieol. 8(3):
235-277.
Jadhav, S.].; Sharma, R P.; and Salunkhe, D. K. 1981. Naturally occurring alkaloids in
foods. Grit. Rev. Toxieol. 8(3): 21-104.
Jiigerstad,.M.; Reutersward, A. L.; Oste, R; Dahlqvist, A.; Grivas, S.; Olsson, K.; and Nyham
mar, T. 1983. Creatinine and Maillard reaction products as precursors of muta
genic compounds formed in fried beef. In The Maillard Reaction in Foods and Nutrition
(G. R Waller and M. S. Feather, editors), pp. 507-519. ACS Symposium Series, No.
215.
Kahn, S. G. 1981. World hunger: An overview. Food Technol. 35(9): 93-98.
Kamm, R.; Meacham, K.; Harrow, L. S; and Monroe, F. 1977. Evaluating new business
opportunities from food wastes. Food Technol. 31(6): 36,38-40.
Knorr, D. 1983. Recovery of functional proteins from food processing wastes. Food Tech
nol. 37(2): 71-76.
Lewis, R.].; and Endean, R. 1983. Occurrence of a ciguatoxinlike substance in the Span
ish mackerel (Scomberomoruscommersoni). Toxicon 21: 19-24.
McMichael, A.]. 1984. Dietary influences upon human carcinogenesis. Food Technol. Aust.
36(10): 460-463, 465.
Moon, N. J. 1980. Maximizing efficiences in the food system: A review of alternatives for
waste abatement.]. Food Prot. 43: 231-238.
Munro,1. C. 1976. Naturally occurring toxicants in foods and their significance. Clin.
Toxicol. 9: 647-663.
Onoue, Y.; Noguchi, T.; Maruyama,].; Hashimoto, K.; and Seto, H. 1983. Properties of
two toxins newly isolated from oysters.]. Agr. Food Chem. 31: 420-423.
Oppenheimer, S. B. 1985. Humanmade carcinogens vs. natural food carcinogens:
Which pose the greatest cancer risk? Amer. Clin. Prod. Rev. 4(2): 16,18-19.
Panasiuk, 0., and Bills, D. D. 1984. Cyanide content of sorghum sprouts.]. Food Sci. 49:
791-793.
Schweigert, B. S. 1975. Food processing and nutrition-Priorities and needed outputs.
Food Technol. 29(9): 36, 38.
Shupe,]. L., and James, L. F. 1983. Teratogenic plants. Vet. Human Toxicol. 25: 415-421.
Swain, A.; Truswell, A. S.; and Loblay, R. H. 1984. Adverse reactions to food. Food Tech
nolo A ust. 36: 467-468, 471.
Taylor,]. C.; Gable, D. A.; Graber, G.; and Lucas, E. W. 1974. Health criteria for processed
wastes. Fed. Proc. 33: 1945-1946.
Taylor, S. L. 1982. An overview of interactions between foodborne toxicants and nutri
ents. Food Technol. 36(10): 91-95.
1985. Food allergies. Food Technol. 39(2): 98-105.
Toyama, N. 1976. Feasibility of sugar production from agricultural and urban cellulosic
wastes with Trichoderma viride cellulase. Proceedings of Biotechnol. Bioeng. Symp. 6: 207-
219.
van der Hoeven,]. C.; Laqerweij, W.].; Bruggeman, 1. M.; Voragen, F. G.; and Koeman,
]. H. 1983. Mutagenicity of extracts of some vegetables commonly consumed in the
Netherlands.]. Agr. Food Chem. 31: 1020-1026.
Wilson, A. M.; McGann, D. F.; and Bushway, R. ]. 1983. Effect of growth location and
length of storage on glycoalkaloid content of roadside stand potatoes as stored by
consumers.]. Food Prot. 46: 119-121, 125.
Winslow, R. L. 1982. The food microbiologist's role in the professional execution of in
dustry's goals for a safe, wholesome food supply. Food Technol. 36(12): 60-62.
Wodicka, V. O. 1977. Food safety-rationalizing the ground rules for safety evaluation.
Food Technol. 31(9): 75-77, 79.
Estimating the Number
of Microorganisms
2
An important aspect of food microbiology is the examination of food or
other materials for microorganisms.
NUMBERS OF MICROORGANISMS IN FOOD
The number of microorganisms in a food as determined by the aero
bic plate count (APC) is variable because of the original contamination,
increase or decrease of microorganisms during processing, recontamina
tion of processed product, and growth or death during storage, retailing,
and handling. The microbial flora are changing constantly. In foods such
as refrigerated fresh meat, the microbial numbers increase during stor
age, whereas in dried or frozen foods, the viable organisms tend to de
crease in number. The APes for a food may vary from less than 10 to
over 100,000,000 microorganisms per gram, depending upon the prod
uct, how long it was stored, and the temperature of storage. The loga
rithms of the range of APes reported for various foods are listed in Table
2.1. The microbial contents of various foods were reported by Pizzo,
Purvis, and Waters (1982).
The usual range of organisms in most animal products is 1,000 to
10,000 per gram. Ground meat is more contaminated than whole cuts of
meat because of the type of meat that is used in the product, the extra
handling during grinding, and the release of meat juices that allow bacte
ria to multiply. Foods that receive a heat treatment generally have lower
microbial numbers than foods not heated. Even then, poorquality ingre
dients, poor sanitation, unsatisfactory heating, recontamination, or poor
handling and storage, cause some heated products to have high numbers
of microorganisms.
An estimate of the number of microorganisms in or on foods is
needed in order to determine if a product meets the microbial levels
expressed in specifications, guidelines, or standards. Spoilage of some
foods is imminent when the APe reaches very high numbers (10
7
-10
8
/g).
Hence, the microbial count can be used to help predict the shelf life of
11
TABLE 2.1. AEROBIC PLATE COUNTS OF VARIOUS FOODS
Food
Animal Products
Beef (steaks, roasts)
Beef (ground)
Pork sausage
Ham
Bacon
Dry sausage
Chicken carcasses (cm
2
)
Fish (fresh)
Fish (smoked)
Fish sticks or crab cakes
Shrimp (raw)
Shrimp (raw, breaded)
Milk (raw, grade A)
Milk (pasteurized)
Milk (dry)
Butter
Plant Products
Raw
Almonds
Beans or peas
Broccoli or kale
Carrots, potatoes, or spinach
Corn or cucumbers
Tomatoes
Frozen
Asparagus, beans, or peas
Corn
Squash
Dried
Carrots
Garlic
Parsley
Spices
Cinnamon
Cloves
Ginger
Nutmeg
Oregano
Pepper
Sage
Mixed dried
Soup (meat type)
Soup (vegetabletype)
Salads
Chicken or ham
Green
Macaroni
Shrimp
Tuna
a Reported in logarithms of bacteria per gram.
12
Overall Range"
2-6
3-8
4-6
1-8
3-7
3-7
2-7
2-8
1-7
2-6
2-7
2-8
2-5
2-4
1-6
3-5
0-4
3-7
6-7
4-7
5-7
3-7
2-5
2-7
2-4
2-4
4-6
2-5
1-5
2-3
2-7
2-4
2-6
6-7
3
3-5
2-5
1-7
3-8
3-6
3-7
2-6
Usual Range"
4
5-7
5
4
4
4-5
3-4
4-5
2-4
3-4
4-5
4-6
3
2
2-3
4
3
4-5
2-3
3
3-4
7
4
3-4
3-5
5-6
4-5
6
3-4
ESTIMATING THE NUMBER OF MICROORGANISMS 13
certain foods. To a limited extent, the microbial numbers might be used
to evaluate the potential safety of foods. The count also might indicate
if the product was produced under sanitary conditions, or if the product
was mishandled during harvesting, processing, or storage. In general, as
the microbial count increases, the quality of the food is reduced. This
generalization does not apply to fermented foods, since microorganisms
are used in their production. There are cases in which the number of
microorganisms in a food has little or no relationship to potential shelf
life, spoilage, or a health hazard. Other factors to be considered include
the type of food, the type of microorganisms present, and the storage
conditions.
To ensure production of food with a low number of microorganisms,
the producer must assay not only the final food product but also such
things as ingredients, processing equipment, packaging, and environ
mental samples. These determinations aid in the evaluation of general
sanitary practices prevailing during processing and handling of food,
and the potential sources of contamination. The determination of micro-
bial numbers is needed to evaluate the effectiveness of methods of pres
ervation.
The presence of particular types of microorganisms, especially poten
tial pathogens or toxin producers, is more important than the estimate
of the total number of microorganisms. In general, the main difference
in these analyses is that specific types of microorganisms are determined
with selective or differential media rather than with noninhibitory me
dia. Thus, for purposes of simplicity, this discussion will be limited to
total number estimations. Some of the special procedures are discussed
with specific organisms in later chapters of this text.
Although the term total count has been used, no single method or me
dium is capable of detecting all of the microorganisms in a food. Thus,
the counts that are obtained are merely estimates of the actual microbial
population. Errors of 90 percent in counts are not unusual when the
level is 10,000 to 100,000 per gram (Collins and Lyne 1976). Besides the
errors, many assumptions are involved in microbial estimations. Also,
there are factors that affect the growth of microorganisms and influence
the results when the viability of the cells is involved in the enumeration
technique. With all of these considerations, it is essential that the techni
cian doing the testing does not further influence the results by using
poor technique.
For microbial analysis, a sample and a system for estimating the num
ber of microorganisms in the sample are needed. After the data from the
evaluation are obtained, the information must be reported and, when
necessary, followup checks should be made. If the report is for manage
ment, an interpretation of the results might be included. What do they
mean? Are the levels of microorganisms acceptable, or are they too high?
THE SAMPLE
If the samples are not delivered to the laboratory, it might be neces
sary to establish a sampling procedure. The samples of food might be
obtained from the processing line, from warehouse storage, or from reo
tail shelves. Food is processed as liquid, solid, mixed solid and liquid, or
semisolid, and in many shapes and sizes. Since there are many variables
in the food and many places of sampling, several sampling plans will be
needed. Sampling suggestions have been made for various factors in
food products (AOAC 1985; APHA 1984; Barrow 1983; FDA 1978; Jones
1979; Kilsby and Pugh 1981; Montagna 1982; Rao and Koehler 1979; Rob
erts, MacFie, and Hudson 1980; Schutz 1984).
The sampling plan should reflect the ultimate use of the analysis, the
potential health hazard of the food, or potential for spoilage. If the reo
suits are needed to satisfy the requirements of a microbiological stan
dard, the sampling plan as outlined in the standard should be followed.
If the results are for the producer's information, a less restrictive sam
piing plan can be used. Sampling plans have been suggested for micro
biological standards (Biltcliffe et al. 1983; ICMSF 1974; Martin 1979) and
for salmonellae (health hazard) testing (Olson 1975). Further discussion
of these sampling plans is presented in appropriate chapters of this text.
A sample will yield significant and meaningful information only if it
represents the mass of material being examined, is collected in a manner
that protects it against microbial contamination, and is protected from
changes in the population that might occur between collection and anal
ysis.
Representative Samples
The need for a representative sample cannot be overemphasized. The
results of the analysis can be no more reliable than the sample on which
they were based. Usually microorganisms are not distributed homoge
neously, so thorough mixing of the product prior to sampling is impor.
tanto Thorough mixing is not as easy for nonliquid foods as for liquid
foods.
The size of the particles being sampled may influence the sampling
procedure, since many particles of a product such as powdered milk can
be obtained; but if the product were sides of beef, a different procedure
would be necessary.
Sampling material in motion, such as on a production line, tends to
minimize variables and gives a more representative sample than sam-
pling material at rest, such as in stacks in a warehouse or on retail shelves.
With on-line sampling, automatic sampling devices might be considered.
These devices usually give a more random and reliable sample and at
less cost than manual sampling of the product.
If cases or containers are stacked as a lot, the person collecting the
samples must randomly select containers throughout the entire pile. If
only containers around the edges or in front of the stack are selected, he
or she is introducing a bias into the results of the analysis.
The laboratory analysis is usually more expensive than obtaining the
sample, so cutting corners in sampling is not the way to save money.
Number of Samples
The number of samples needed, or the frequency of sampling, de-
pends upon many factors. The uniformity or homogeneity of the prod-
uct, the size of the many particles, previous knowledge of the material,
and experience will help dictate the amount of sampling needed. Either
too few samples or too many samples waste product, laboratory material,
and labor.
For microbiological standards, the number of samples to be obtained
and analyzed is included in the standard. One of the prime consider-
ations that influence the number of samples to be analyzed is the poten-
tial health hazard of the foods. Statistical sampling schemes will help en-
sure that the samples give an acceptable assessment of the microbial
conditions of the food, ingredient, or other substance being analyzed.
Aseptic Collection of Samples
Aseptic technique is needed when samples are collected. To prevent
possible contamination, if the samples are in individual containers, such
as cans, bottles, or boxes of food, they should be taken directly to the
laboratory for analysis. On the other hand, if the product is in bulk or
in containers of impractical size to submit directly to the laboratory, rep-
resentative portions must be transferred to sterile containers using asep-
tic technique.
Since there is little interest in bacteria associated with sampling de
vices or sample containers, the instruments must be sterile. If possible,
the instruments should be sterilized in the laboratory rather than at the
place of sampling. After the sampling equipment is cleaned, the pre-
ferred methods of sterilization are (1) steam at 121.5C in an autoclave
for 15 to 30 min (the time for exposure depends on how bulky the mate-
rial is and how closely the material is packed in the chamber), or (2) a
hot air oven. The suggested conditions for hot air sterilization vary from
1 to 3 hr at 160 to 180C. If protected from recontamination, the steri
lized instruments may be stored. Alternative systems for sterilizing are
needed when neither an autoclave nor a hot air oven is available. These
include (1) expose to steam (l00C) for 1 hr and use the same day, (2)
immerse in water at 100C for 5 min and use immediately, (3) immerse
in 70 percent alcohol and flame to burn off alcohol immediately before
use, or (4) flame with hydrocarbon (propane or butane) torch so that all
working surfaces contact the flame before use. Using the alternative sys-
tems has been questioned. According to the FDA (1978), alcohol flaming
is unsatisfactory because the instrument does not get hot enough to be
effectively sterilized, and the flaming alcohol creates a fire hazard. The
FDA recommends using a propane torch. Tansey (1973) suggested using
a heavy-duty butane lighter rather than an unwieldy torch.
When obtained, the sample should be placed in a sterile container. A
wide-mouth screw-capped jar is recommended (APHA 1984; FDA 1978);
however, plastic bags or other acceptable containers can be used.
The methods of sampling and the types of instruments needed are
determined by the substance to be sampled.
LIQUIDS AND SMALL PARTICLES. These foods can be mixed and
sampled with sampling tubes, dippers, teaspoons, tablespoons, spatulas,
or similar instruments.
LARGE MATERIALS. If these substances can be cut, they may be sam-
pled with a knife or cheese trier. For many materials, such as animal
carcasses or processing equipment, the surface is sampled.
SURFACES. Since microorganisms are on the surfaces of equipment
as well as on foods such as animal carcasses, the sampling and analysis
of surfaces are important. The system to use for sampling depends upon
the type of surface, the amount of contamination, and the use of the data
that are obtained. Some of the surface sampling systems that have been
used are listed in Table 2.2. Each system has certain advantages and dis-
advantages. No single method is the best for all of the diverse surfaces of
foods and equipment. Hence, several have been proposed and compared
(Dewhurst, Rawson, and Steele 1986; Dickens, Cox, and Bailey 1986;
Goulet et al. 1983; Lee and Fung 1986; Scott, Bloomfield, and Barlow
1984; Speers, Lewis, and Gilmour 1984). Microorganisms become at-
tached to surfaces, which makes them difficult to recover for analysis.
Excision and maceration of tissue yield higher numbers of microorgan-
isms than do systems such as swabbing, rinsing, or contact agar or tape
TABLE 2.2. SURFACE SAMPLING METHODS
Swab
Cotton
Alginate
Glass sampler
Cylinder template
Scrape
Excise tissue
Washrinse
Vacuum probe
Contact systems
Agarsyringe
Agar-sausage
Agar plate (RODAC)
Tape
Membrane filter pad
Agar spray
Drip or exuded juice
Abrasive discs
(Anderson et al. 1987; Lillard and Thompson 1983; Morgan, Krautil, and
Craven 1985).
AIR. The two general methods for air sampling are solid and liquid
impingement. The systems for solid impingement include the settling
plate, slit samples, the sieve or Anderson sampler, the centrifugal sam-
pler, and the membrane sampler. Except for the settling plate, specific
volumes of air are sampled. Systems of air sampling have been evaluated
and compared (Lundholm 1982; Macher and First 1983; Placencia et al.
1982).
Holding of Sample
For best results, the sample should be analyzed immediately. When
this is not possible, the sample should be refrigerated to prevent growth
of any microorganisms. Alternatively, the sample can be packed in ice. If
shipment to another city is necessary, or if the sample is a frozen product,
dry ice should be placed in the package. Refrigeration is preferred to
freezing, because freezing may cause death or damage to some cells,
which may then give erroneous results when the sample is analyzed.
Preparation of Sample
Many methods of analysis require some preparation of the sample.
The main consideration is to get the bacteria into a homogeneous sus-
pension so they can be pipetted. If a food is a liquid such as milk, an
aliquot can be mixed and pipetted, but if the food is a solid, such as
hamburger, it is necessary to blend the food with a diluent to obtain a
suspension. The rinse or wash samples from surfaces are treated as liquid
samples, while swabs are placed in sterile diluent and shaken to suspend
the bacteria.
SOLID FOOD. Solid food is generally mixed with a sterile diluent in a
sterile mechanical blender or other system to obtain a 1:10 dilution of
the food (Fig. 2.1). This 1:10 dilution also is referred to as Yio or 10-
1
dilution. A 1:10 dilution means that in 10 g of the mixture, there is 1 g
of food, or in 1 g of the mixture, there is 0.1 g of food, with associated
organisms. Thus, if 1 g of the 1:10 dilution is analyzed, the microbial
count is that of 0.1 g of food. To report the count as the number per
gram, it is multiplied by 10. The first dilution of the food may be 1:2 or
1:4, such as for shellfish (APHA 1970; Cook and Pabst 1984).
As an alternative to blending, a sterile plastic bag containing the sam-
ple and diluent is placed in a device called a stomacher (Fig. 2.2). In the
stomacher, the compression and shearing forces of the pounding result
in a homogeneous suspension of sample and microorganisms (Deibel
and Banwart 1982; Purvis et al. 1987; Sharpe and Jackson 1972; Thomas
and McMeekin 1980; Thrasher and Richardson 1980).
Various systems might be used to make further dilutions of the
blended food sample. One is the loop-tile method (Hudson, Roberts, and
Whelehan 1983)_
DILUENTS. Several diluents have been suggested and used_ Although
AOAC (1985) recommends the use of Butterfield's buffered phosphate,
0.1 percent peptone water is also accepted. Peptone water is easy to pre-
pare, and it protects the organisms during dilution and plating. One dis-
advantage of this preparation is that, if the prepared dilution is allowed
to remain at room temperature for extended periods, the organisms will
multiply. Not more than 20 min should elapse between the first dilution
in phosphate buffer until the last plate is poured in the series (APHA
Too,
'd450 ml DILUENT
10-
1
MIN, LOW SPEED
l" ,,_.
IIml IIml IIml IIml
99ml
DILUTION 10-
2
10-
3
10-
4
10-
5
10-
6
Figure 2.1_ Suspension and dilu-
tion of food sample for microbial
analysis_
Iml Alml
00
Figure 2.2. Systems for blending food samples with diluents. From left to right:
Waring blender, stomacher, Osterizer.
1984). An increase in count up to 10 percent can be expected in this 20
min period.
According to Harrewijn (1975), some pertinent aspects to be consid-
ered are the composition, temperature, and pH of the diluent; anaerobic
or aerobic condition; carryover of inhibitors with the food; and any treat
ments needed to allow the recovery of cells injured during food process
ing or preparation of the sample. The recovery of injured waterborne
coliforms is aided by diluents containing peptone or milk (McFeters,
Cameron, and Le Chevallier 1982).
The soaking of mustard seeds in a sterile diluent for 10 min prior to
analysis resulted in an increased aerobic plate count (Cowlen and Marshall
1982). Perhaps other such samples should be soaked before analysis.
DILUTIONS NEEDED. For the plate count, only plates with less than
250 or 300 colonies are considered to be countable (FDA 1978; Tomasie
wicz et al. 1980). Hence, for moderately to highly contaminated foods,
dilutions are needed beyond the 1:10 ratio of the original suspension.
A 1:10 dilution of a 1:10 dilution is a 1:100 dilution. This 1:100 dilu
tion is prepared by aseptically transferring 10 ml of the 1:10 dilution
to a screw-capped bottle containing 90 ml of sterile diluent (or 11 ml
transferred to 99 ml)_ The bottle is shaken (twenty-five times through a
30-cm arc in 7 sec) to distribute the organisms homogeneously_ Further
dilutions can be 'made in this manner as far as needed.
The dilutions needed to estimate the number of microorganisms in
a food can be determined by experience or by the requirements of stan-
dards, guidelines, or specifications. If 50,000 organisms per gram are al-
lowed in a specification, a 1:1000 dilution can be used for the plate count.
If fewer than fifty organisms are observed on the incubated plate, the
food is within the limit, but if more than fifty colonies are observed, it
does not meet the requirement. Usually two or three dilutions are ana-
lyzed to increase the chances of obtaining an acceptable plate to count;
for the most probable number (MPN), at least three dilutions are needed.
ANALYSIS
Several procedures can be used to estimate a microbial population
(Table 2.3). Not all of these procedures are readily adaptable to all foods,
however. The ideal test should be accurate, rapid, inexpensive, and use-
ful for most types of samples.
TABLE 2.3. SYSTEMS TO ESTIMATE THE MICROBIAL LOAD OF FOOD
Direct microscopic count (DMC)
Breed clump count
Electronic particle count
Pour plate (APC, SPC)
Spread plate
Spiral plate
Drop plate
Plate loop
Roll tube
Oval tube
Burri strip/slant
Little plate
Tube dilution
Most probable numbers (MPN)
Membrane filter
Hydrophobic grid (HGMF)
Direct epifluorescent filter technique
(DEFT)
Microtiter-Spot plate
Dry rehydratable film
Petrifilm
Electrical
Conductance
Impedance
Capacitance
Voltage drop
Spectrophotometric (optical density)
Adenosine triphosphate (ATP)
Reductase tests
Easicult-TTC
Respiration rates
Limulus amoebocyte lysate
Chemical indicators (decomposition prod-
ucts)
pH
Agar droplets
Millipore sampler
Bactoscan
Microcalorimetry
Flow cytometry
NOTF.: For the systems not discussed in text, see the following references: Ackland, Manvell,
and Bean 1984; Bailey and May 1979; Ginn, Packard, and Fox 1984; King and Mabbitt
1984; Kramer 1977; O'Toole 1974; Schoon et al. 1970; Sharpe and Kilsby 1971; Swientek
1981. 1983.
Total Cell Counts
Some systems make no differentiation of living or dead cells. All mi
crobial cells are counted. Two of these are the direct microscopic count
and the electronic particle count.
DIRECT MICROSCOPIC COUNT (DMC). With this method, the reo
suIts are obtained sooner than with most other procedures because no
incubation period is needed for the cells to metabolize and multiply.
Liquid foods may be determined directly, but solid foods must be put
into a suspension (1:10 dilution) before analysis. A counting chamber
can be used, but for food, usually a portion (0.01 ml) of the material
(measured with a standard loop or micropipet) is spread uniformly over
a prescribed area on a glass slide (usually 1 cm
2
).
For products such as eggs or cream, xylene or another suitable solvent
is added prior to staining to remove the fat from the material. After dry
ing, the slide is then fixed by dipping in ethyl alcohol for 1 to 2 min
before staining.
Several stains have been suggested for and used in the DMC. The
stained films are examined with a microscope, using the oil immersion
objective. The number of fields to be examined and counted is inverse
to the number of cells and clumps observed in each field.
To calculate the organisms per gram of food, the diameter of the field
that is examined must be known. The diameter (d) is measured with a
stage micrometer to the nearest 0.001 mm. Since the field is a circle, the
area can be calculated (A = 7r r2).
The average number of cells or clumps per field is calculated and
divided by the area of the field to obtain the number per mm
2
To deter
mine the number of cells or clumps per cm
2
, the number per mm
2
must
be multiplied by 100 (since there are 100 mm
2
per cm
2
). The resultant
number is then multiplied by the dilution factor, which, in the case of
liquid food (milk) is 100 (0.01 ml was used), or 1,000 for solid food (0.01
ml of a 1:10 dilution).
Values of the DMG. The DMC is a rapid method because an estimate of the
bacterial load can be obtained in a short time. This is of value when on-
the-spot alterations or adjustments are needed in the processing opera-
tion to remedy any problems.
Other values of the DMC that have been suggested include the follow-
ing: (1) little work is required; (2) the test is not too difficult; (3) very
little apparatus or equipment is needed except for a microscope; (4) the
prepared slide can be stored and maintained as a permanent record; (5)
some idea as to the type of organism (cocci or rods) is obtained; (6) counts
represent organisms in the original product (if it has been treated, such
as by heat); (7) preservatives can be added to the sample for holding prior
to analysis, for shipment, or to hold for futher study, so that organisms
do not multiply; (8) only a small amount of sample is needed, which is
of value if the product is expensive.
Since both living and dead cells are counted, there is some question
as to the value of the microscopic count. However, high numbers of cells,
whether living or dead, in pasteurized products indicate poor quality of
product before processing, survival or multiplication of bacteria during
processing, or recontamination or growth, or both, after processing.
The value of the DMC is limited to samples with high cell loads. How-
ever, with increased technology and efforts in sanitation, the bacterial
load in many foods has been reduced. The DMC has little or no value
for foods with low microbial loads.
Besides being used to evaluate the microbial content of a food, the
DMC can be used to evaluate the number of body cells (leucocytes or
lymphocytes) in milk. This is especially valuable in indicating mastitis in
cows.
The value of the DMC depends upon the type of food and the type
of organisms associated with the food. For products that have received a
treatment such as heat to control the microorganisms, it is doubtful that
the DMC could predict shelf life of the product. It is also doubtful tha.
the DMC would have any value in determining the public health hazard
of the product.
Assumptions and Errors. In any microbiological method of analysis, many
assumptions and errors are made. There are errors inherent in the ana-
lytical procedure, as well as those introduced by the technician doing the
test.
Assumptions are also made regarding the sample. It is assumed that
(1) the sample is representative of the entire lot of product; (2) the sub-
sample used for analysis is representative of the sample; (3) cells are dis-
tributed homogeneously in the sample as well as the subsample and, if
not originally homogeneous, that operations such as mixing, blending,
or shaking have produced a homogeneous mixture; (4) the weighing or
measuring of the subsample, diluents, and aliquots is accurate; and (5)
the sample or subsample has been handled so that there is no contamina-
tion or multiplication of cells during sampling or analysis.
Errors in the DMC can occur during the preparation and staining of
the cells, counting the cells or clumps, or in the calculations involved in
converting the raw data into the count per gram of product.
It is easier to spread the sample uniformly in circles than in squares.
If the material is not spread uniformly, the cells will not be distributed
homogeneously. It is assumed that the smear on the slide will dry into
flat layers of uniform density. However, the smears have been found to
vary in thickness from one area to another. For some foods, such as liquid
egg, the film on the slide may be of such thickness that it obscures many
bacterial cells, which cannot then be counted. Organisms that are lightly
stained are difficult to discern, and with an unevenly stained back
ground, it is difficult to distinguish dirt or other particles from bacterial
cells. Improper illumination with resulting eye strain and fatigue is
another cause of errors in cell counting. The staining process itself can
also wash some cells from the slide or can result in the counting of pre
cipitated stain as cells.
For other errors, see APHA (1978).
ELECTRONIC PARTICLE COUNT. The electronic counter is based
on the principle that cells are poor electrical conductors as compared to
an electrolyte solution. A dilute suspension of cells in saline or other
suitable electrolyte is drawn through a minute aperture conducting an
electric current between two electrodes-usually platinum. Each cell
passing through the aperture displaces an equal volume of the electrolyte
solution and causes a momentary increased impedance to the flow of
electric current. The resulting voltage pulse is proportional to the size
or volume of the particle passing through the aperture. These pulses are
amplified and counted. They simultaneously appear on the screen of an
oscilloscope.
Since background particles, such as those that occur in foods, also
would produce pulses as they pass the aperture, or could clog the aper
ture, the particles must be removed. Although electronic particle
counters are used successfully to count somatic cells in milk (Dijkman et
al. 1979), blood cells, and mammalian cells, much work needs to be done
before they are useful in determining microorganisms in foods. However,
Kogure and Koike (1987) reported satisfactory results when a particle
counter was used to determine the bacterial biomass of seawater.
Viable Counts
Several methods exist to estimate the number of viable microorgan
isms in foods. Most of the systems are based on the plate count or tube
dilution methods.
PLATE COUNT (POUR). The standard plate count (SPC) has been the
usual technique for estimating the living microorganisms in foods. The
procedure is relatively simple. Appropriate dilutions are plated immedi
ately by transferring a measured aliquot to a sterile petri plate and add
ing sterile melted and cooled (42 to 45C) agar. The type of agar used
for the SPC is non inhibitory and nutritious, unless specific microbial
types are to be determined. For the aerobic plate count the media usually
used are plate count agar or tryptone glucose extract agar, but various
other agars have been employed. The medium and inoculum should be
mixed thoroughly to distribute the cells uniformly. After solidification of
the agar, the prepared plates are inverted (turned upside down) to pre
vent condensation of moisture on the agar surfaces, and then incubated.
The temperature and time of incubation will vary, depending upon the
type of cells that are being determined (psychrotrophs, mesophiles, or
thermophiles). A temperature of 32C for three days is used for eggs and
egg products, while 35C for 48 2 hr is listed for frozen, chilled, or
prepared foods (AOAC 1985). Huhtanen (1968) found the highest bacte-
rial counts in raw milk when the plates were incubated at 27C. However,
these counts were not significantly different from those obtained at a
range from 10 to 30C.
During the incubation period, growth and multiplication of cells will
occur until a visible colony is formed. These colonies are then counted
on the plates that contain from 30 to 300 colonies (AOAC 1985; FDA
1978). Cowell and Morisetti (1969) furnished evidence that greater preci
sion is obtained from plates containing from 80 to 320 colonies. A range
of 25 to 250 colonies was suggested in a later study (Tomasiewicz et ai.
1980). The number of colonies is multiplied by the dilution factor and
reported as the number of colony-forming units (CFU) per gram of food.
Desirable Characteristics. The pour plate procedure is simple, can cover a
large concentration range, and, at present, is probably the most precise
method for determining those bacteria that will grow in an agar medium
(Gilchrist et al. 1973). Besides these virtues, the organisms can be recov
ered for further study. The results should reflect the level of viable micro-
organisms in the food at the time of sampling.
The data obtained from the pour plate should reveal information
such as the source of microorganisms, potential shelf life, or possible
public health hazards of the product. With the present aerobic plate sys-
tem, the source of microorganisms generally is not determined (Blanken-
agel 1976).
In most foods, microbial growth causes undesirable changes. Hence,
the plate count might be used as an indicator of potential shelf life or of
incipient spoilage. No relationship was found to exist between the bacte-
rial count and potential shelf life of iced shrimp (Cobb et al. 1973), or
pasteurized milk (Watrous, Barnard, and Coleman 1971). The usual plate
count system does not differentiate types of organisms that cause
spoilage.
It is generally agreed that any potential health hazard is not deter-
mined by the aerobic plate count. Some people believe that a high micro-
bial count indicates improper handling with possible pathogens being
present. Quite often the reverse is true, and low-count products contain
potential pathogens. Microbial toxins can be present after the bacteria
are destroyed by processing.
Undesirable Characteristics. There are many facets of the pour plate system
that are undesirable. Of most concern are time, expense, technical re-
quirements, information obtained, and accuracy.
The prepared plates must be incubated so that the organisms can
produce a visible colony prior to counting. This incubation period may
range from two to ten days. For highly perishable products, or for deter-
mining production or processing conditions, it is desirable to obtain the
results as soon as possible. If a ten-day incubation period is needed, the
potential shelf.life of a food can be determined more easily by incubating
the food directly.
Since the pour plate system is so common in the United States, we
might not realize that it is rather expensive compared to other methods.
In some countries, other, less expensive methods are used in preference
to the plate count.
The pour plate method seems simple to do, but a trained technician
is needed to perform the test. The accuracy of the pour plate depends
upon the ability of the technician as well as on assumptions and errors
inherent in the technique.
Assumptions and Errors. The same assumptions and errors in sampling as
discussed for the DMC apply equally to the pour plate. The technical
ability and concern of the technician during cleaning of glassware, prep-
aration of dilutions and media, sampling, plating, counting, and calculat
ing can influence the reported CFU.
Two major assumptions of the pour plate system are that (1) microor-
ganisms are in suspension as dissociated single-cell units so that each
colony on the plate arises from an individual cell; and (2) all cells planted
in the medium will multiply and produce a visible colony. Neither as-
sumption is accurate.
Quite often bacteria grow in chains or clusters. Mixing, shaking, and
other procedures do not always separate these chains or clumps into in
dividual cells. Hence, when plated, a colony may arise from not only one,
but several bacterial cells.
The environment in which the organisms are placed (medium, tem-
perature, oxygen) as well as previous treatments of the cells (sublethal
heating, freezing, radiation) and even the presence of other types of mi-
croorganisms influence the ability of the cell to multiply and produce a
visible colony. No one environmental condition will support the growth
of all of the types of microbial cells that might be present in a food prod-
uct. Hence the bacterial count should be referred to as CFU per gram
of food.
The main value of a plate count is to be able to compare the results
of various samples taken at different times from the different laborato-
ries. This is possible only when the results are reproducible. It is impor-
tant that standardized procedures be followed so that results can be com-
pared.
PLATE COUNT (SURFACE). In this system, the sterile melted and
cooled agar is poured in sterile petri plates. After solidification, the
plates are preincubated overnight. The incubation dries the surface of
the agar so that, when planted, the organisms do not coalesce. Before
using, the dried agar surface should be observed for any possible contam-
ination.
Aliquots of dilutions are added to the dry surface and uniformly
spread over the agar by means of a sterile glass rod, bent into the shape
of a hockey stick. Various amounts of aliquots have been suggested. Inas-
much as we usually work with dilutions in the order of 10, it is much
easier to calculate the results per gram of product if O.l-ml aliquots are
used.
For simplification, calibrated loops can be used in place of pipets for
preparing dilutions as well as for inoculating the pour plate or the sur-
face of the spread plate. In the plate loop system, a calibrated loop is
fitted into the barrel of a repeating syringe. The loopful of sample is
flushed onto the agar surface in a petri plate with sterile diluent in the
syringe. According to O'Connor (1984), the precision and accuracy of the
plate loop system are within acceptable limits.
With the drop plate method, 0.02 ml of inoculum is allowed to drop
onto the surface so that it spreads over an area 1.5 to 2.0 cm in diameter.
Six to eight drops are placed on an agar surface in a petri plate, with no
further manual spreading. After inoculation, the plates are inverted and
incubated, and the resultant colonies counted as with the pour plate
method.
Automated devices for distributing the samples over the agar surface
have been described and evaluated (Gilchrist et aL 1973; Gilchrist et aL
1977; Jarvis, Lach, and Wood 1977; Kramer, Kendall, and Gilbert 1979;
Tilton and Ryan 1978; Trotman and Byrne 1975). One type of automatic
plating system is the spiral plater (Fig. 2.3) (Gilchrist et aL 1973; Gilchrist
et aL 1977)_ This system was adopted as an official method by the AOAC
(1981). With this mechanical device, a stylus dispenses the sample, or di-
lution, in a spiral, in varying amounts, on an agar surface from the center
Figure 2.3. The spiral plater.
of the dish to the outer edge. By varying the amount of inoculum, the
equivalent of three dilutions can be plated on one agar surface. After
incubation of the inoculated plates, a laser colony counter, developed for
the spiral plater, follows the spiral from the outer edge toward the center,
counts the colonies, and determines the CFU for the inoculum. Also, the
colonies can be counted manually with the use of a spiral grid system.
Surface VS. Pour Plates. The desirable aspects listed for the pour plate are
equally applicable to the surface plate.
It is well recognized that higher counts are obtained by surface spread
plates than by pour plates. The possibility of heat-sensitive organisms
being damaged by hot agar during the preparation of pour plates is over-
come by using the spread plate technique. Obligate aerobic organisms
will grow faster on the surface than in the depth of agar in pour plates.
Surface colonies are always detectable sooner and are much larger and
easier to count than are colonies in a pour plate.
The main advantage of surface plates as compared to pour plates is
that surface plating can be automated. With automated analyses such as
the spiral plate system, both work and materials are saved. One problem
with the spiral plate system is that the stylus is easily clogged if food
particles are present in the sample. Hence, filtration of the sample may
be needed to remove these particles prior to plating. Hoben and Soma-
segaran (1982) found the drop plate method to be more economical than
either the spread or pour plate systems.
The undesirable characteristics of the spread plate are similar to
those discussed for the pour plate. With the spread plate system, some
of the organisms might cling to the glass rod used for spreading. Treating
of the glass rod with silicone helps to overcome the problem. Reportedly,
precision with the pour plate is better than with the spread plate.
Dry, Rehydratable Film. As an alternative to the petri plates used in the
aerobic plate count systems, plastic films with a dry, rehydratable me-
dium coated upon them (Petrifilm SM) have been developed. The dry
medium contains nutrients, a cold water-soluble gel, and 2,3,5-triphenyl-
tetrazolium chloride that is reduced by microbial growth from white to
red. The prepared samples or dilutions are added at the rate of 1.0 ml
per plate. The sample is spread over an area of about 20 cm
2
by applying
pressure with a plastic spreader on the overlay film. The liquid in the
sample rehydrates the medium, then the gel is allowed to solidify before
the prepared films are incubated for bacterial growth. Reportedly, the
Petrifilm SM system was a satisfactory alternative to the aerobic plate
count for poultry (Bailey and Cox 1987), pasteurized fluid milk (Senyk
et aL 1987), and ground beef (Smith, Fox, and Busta 1985). Petrifilm
methods were adopted as official first action by the AOAC (Brickey et aL
1986).
ROLL TUBE. The basic idea of the roll tube is the same as for the pour
plate method, except that screw-capped test tubes or bottles are used in
place of petri plates. Test tubes are sterilized with 2 to 4 ml of plate count
agar (with 2 percent agar). When the melted agar is cooled to 42 to
45C, 0.1 ml of the appropriate dilution of the sample is added and the
tube rolled in cool water in a horizontal position until the agar is solidi-
fied in a thin layer on the inner wall of the tube.
The roll tubes are incubated upside down so that any water that con-
denses collects below the inoculated agar and does not smear the colo-
nies. After incubation, the colonies that develop are counted with the aid
of a low-power magnifier. Multiplying the colony count by the dilution
factor yields the number of organisms per gram of food.
Although the basic idea of the roll tube is similar to the plate count,
there are obvious differences. Since test tubes are used rather than petri
plates, the cost of the procedure may be lower or higher, depending upon
the relative cost of these items. Less plate count agar is used in the roll
tube method.
Hartman (1968) stated that the roll tube requires less space, materials,
and time, with less risk of contamination and less desiccation of the me-
dia in the tubes than in plates during long incubation periods_ He also
reported that there is no waiting for agar to solidify to invert and incu-
bate such as in the pour plate system_ There are machines for rolling the
tubes_
It would seem that the colonies would be more difficult to discern
and count in the roll tube than in the pour or spread plate techniques_
In his review, Hartman (1968) did not find counting of the colonies to be
a problem in the roll tube_ Devices are available to assist in the counting
of colonies in roll tubes_ The roll tube technique can be used to deter-
mine anaerobic types of microorganisms in foods (Gray and Johnson
1976)_
BURRI STRIP OR SLANT. This method involves the spreading of a
sample over an agar slant with a calibrated loop. Test tubes can be used,
but the oval tube gives a larger surface for the growth of colonies. The
agar surface must be dry to prevent colonies from coalescing_ After incu-
bation (32 or 37C for 24 hr) in a horizontal position, the surface is
examined for microbial growth. Colonies may be counted or compari-
sons can be made as to the extent of growth that occurs so that high- and
low-count products can be distinguished.
The Burri slant method is a simple test for the evaluation of plant
sanitation.
LITTLE PLATES. Since Frost introduced the little plate system in
1916, many modifications to the system have been proposed. The origi-
nal procedure was to mix 0.1 ml of milk with about 2 ml of nutrient agar,
and this was spread uniformly over a 4 cm
2
area on a glass slide. After
incubation for 3 to 8 hr in a moist chamber, the slides were air-dried,
flame-fixed, and stained for counting. The colonies were observed and
counted with a microscope.
Modifications have been suggested in the procedure, such as the types
of slide used, the method of inoculation and incubation, as well as type
of stains. A similar procedure was described by Postgate (1969) to distin-
guish viable cells from dead cells, since to observe colonies on the slide,
the cells must be viable.
This system is a more rapid method than the plate count, since only
3 to 8 hr of incubation are used. Besides being rapid, an estimate of the
viable number of cells is obtained, which is not the case with DMC. The
little plate, slide plate, and microplate methods give results comparable
those for the plate count.
MEMBRANE FILTERS. When fluids are filtered through a membrane
filter (MF), all particles, bacteria, or cells larger than the pores are re-
tained on the filter surface.
The procedure has been useful for analyzing processed water, various
beverages, or air when the microbial count is relatively low. Such low
contamination is difficult to evaluate with the APC. More recently, MF
systems have been used to analyze foods with relatively high numbers of
bacteria. Prefilters are used to remove food particles that might clog the
MF. In some cases, surfactants and enzymes, such as proteases, are used
to degrade the food so that it can be filtered (Bourgeois et al. 1984; Entis,
Brodsky, and Sharpe 1982; QALL 1981).
The retained microorganisms can be cultured by aseptically transfer-
ring the filter onto a nutrient agar or one that is selective, differential,
or both. After incubation for 6 to 8 hr, the microcolonies can be counted
with a microscope similarly to that used in the little plate or microplate
method. After incubation for 24 to 48 hr, the colonies can be counted
similarly to the APC.
The bacterial cells can be stained with 0.1 percent toluidine blue
(O'Toole 1983a, 1984). After destaining the filter and making it transpar-
ent, the dye retained by the cells is determined with a spectrophotometer.
Reportedly, this reading is related to the number of cells on the filter.
A membrane filter with hydrophobic material in a grid pattern is
called a hydrophobic grid membrane filter (HGMF). The grids are com-
partments of equal and known size, and the hydrophobic material deters
the spreading of colonies. After the organisms are grown on the filter,
the number of squares containing colonies is enumerated and converted
to a most probable number- The results can be determined manually or
with an automated counting system (sample analyzer). A disposable filter
unit has been developed for the HGMF (Tsuji and Bussey 1986). The
HGMF system was given official status by the AOAC (AOAC 1983; Entis
1986).
In one system, the microorganisms on the filter are subjected to a
fluorescent dye, acridine orange, which stains viable cells, and then ob-
served with an epifluorescent microscope. This direct epifluorescent fil-
ter technique (DEFT) was reviewed by Pettipher (1986). The DEFT is a
rapid method and is especially useful for samples of food containing
high numbers of organisms (Pettipher 1987; Qvist and Jakobsen 1985;
Shaw et al. 1987). The method was not suitable for heated samples
(Hunter and McCorquodale 1983; Rodrigues and Kroll 1986). However,
a double staining system using acridine orange and janus green B al-
lowed the differentiation of viable and heat-killed cells (Rodrigues and
Kroll 1986). A modified Gram-staining procedure using acridine orange
as the counterstain allows the differentiation of Gram-positive and Gram-
negative cells (Rodrigues and Kroll 1985). Microorganisms on DEFT
slides can be counted automatically (Pettipher 1986).
TUBE DILUTION. The tube dilution method is essentially the aseptic
inoculation of a series of tubes of sterile nutrient broth with a series of
dilutions of the food. After incubating the inoculated tubes, the broth is
observed for turbidity, which indicates growth of organisms. If no turbid
ity is evident, it is assumed that no microorganisms were present or were
able to multiply. With broth that appears turbid due to inoculated food,
growth can be detected by streaking on an agar surface and observing
growth after a few hours of incubation, or by spreading some turbid
broth on a slide and looking for microorganisms with the aid of a micro
scope.
If the tube with the 1:100 dilution showed growth and the tube with
1:1000 had no growth, there were between 100 and 1,000 organisms in
the food. Sometimes this rough estimate is all that is needed. It gives only
an estimate of the range of bacteria that are present.
MOST PROBABLE NUMBERS (MPN). By using several tubes at each
dilution and recording the positive (showing growth) tubes and negative
(no growth) tubes, you get a more accurate estimate of the number of
organisms present. In the tube dilution example, if you inoculated 10
tubes with 1 ml of the 1:1,000 dilution, there would be as much total
inoculum as in the 1:100 tube which showed growth. Theoretically, one
or more of the 10 tubes with the 1:1,000 dilution also should be turbid.
The relationship of positive and negative tubes has been determined
mathematically and MPN tables have been derived (Tables 2.4 and 2.5).
To use the MPN system, at least three dilutions are needed. Ideally, the
least dilute tubes should all be positive and the most dilute tubes (of the
three dilutions) should all be negative. This is not always the case, so the
rule that has been established is to select the highest dilution in which
all portions tested are positive (no lower dilution giving negative results),
and the two succeeding dilutions are then chosen. The more tubes that
are used in each dilution, the more accurate is the estimate, but for rea
sons of convenience, threetube or five tube series are adopted. After se
lecting the three series of dilutions, consult the appropriate MPN table,
obtain a most probable number that satisfies the number of positive
tubes, and multiply this by the dilution factor to obtain the MPN per
gram of product.
Assumptions and Errors (MPN). The assumptions and errors due to sam
pIing and diluting apply to the MPN technique. It is assumed that a single
viable cell inoculated into a tube of broth will multiply so that a change
TABLE 2.4. MOST PROBABLE NUMBER (MPN) PER GRAM OF SAMPLE
Twosided 95% One
Number of Positives Program Values Conf. Limits sided
Upper 95%
1.0 0.1 O.oI MPN St. Error Lower Upper Limit
0 0 0
<
0.03
1 0 0 0.36 0.36 0.05 2.54 1.85
1 1 0 0.74 0.52 0.18 2.94 2.36
1 1 1 1.12 0.64 0.36 3.47 2.89
2 0 0 0.92 0.65 0.23 3.67 2.94
2 1 0 1.47 0.85 0.47 4.55 3.80
2 1 1 2.05 1.02 0.77 5.46 4.66
2 2 0 2.11 1.05 0.79 5.61 4.79
2 2 1 2.76 1.24 1.15 6.64 5.76
2 2 2 3.48 1.42 1.56 7.74 6.80
3 0 0 2.31 1.33 0.74 7.17 5.98
3 1 0 4.27 2.14 1.60 11.38 9.72
3 1 I 7.49 3.35 3.12 17.99 15.63
3 2 0 9.33 4.17 3.88 22.41 19.47
3 2 1 14.94 6.10 6.71 33.25 29.23
3 2 2 21.46 8.11 10.23 45.02 39.97
3 3 0 23.98 17.41 5.78 99.49 79.15
3 3 1 46.22 17.47 22.03 96.96 86.07
3 3 2 109.89 38.87 54.94 219.82 196.65
3 3 3 > 110.00
SOURCE: Data Courtesy of Robert J. Parnow (personal communication).
NOTE: Standard error, upper and lower 95% confidence limits, and onesided upper 95%
confidence limits when three dilutions are used with three tubes in each dilution at levels
of 1.0, 0.1. and 0.01 g per tube.
such as turbidity or production of acid or gas can be observed. Because
dilution to extinction is necessary, good aseptic technique is needed,
since any contamination during inoculation of the tubes of broth could
result in growth. The MPN is less precise than the agar plating methods
(Pike et al. 1972).
Some people become confused when 1 g of sample is added to a tube
with 9 ml of broth for the MPN series. They feel that since this is a 1:10
dilution, somehow it has to be considered when the dilution factor for
the MPN is determined. It does not make any difference if there are 8,
9, 10, or 11 ml of nutrient media per tube. The only consideration is the
amount of original sample that is added to the tube (0.01, 0.001, 0.0001
g, or whatever).
Advantages of the MPN. The MPN is, in some ways, easier or simpler to do
than the plate count. Broth can be dispensed into tubes with an auto
matic pipetter. Selective or differential media can be used so that certain
types of organisms can be determined. The MPN is particularly useful
TABLE 2.5. FREQUENTLY OCCURRING MOST PROBABLE NUMBER (MPN) PER
GRAM OF SAMPLE
Twosided 95%
Onesided
Number of Positives Program Values Conf. Limits
Upper 95%
1.0 0.1 0.01 MPN St. Error Lower Upper Limit
0 0 0 <0.18
1 0 0 0.19 0.19 0.03 1.34 0.98
1 1 0 0.40 0.28 0.10 1.60 1.28
1 1 1 0.60 0.35 0.19 1.86 1.55
2 0 0 0.44 0.31 0.11 1.76 1.41
2 1 0 0.68 0.39 0.22 2.11 1.76
2 1 1 0.92 0.46 0.34 2.45 2.09
2 2 0 0.93 0.46 0.35 2.48 2.12
2 2 1 1.17 0.52 0.49 2.81 2.44
2 2 2 1.42 0.58 0.64 3.16 2.78
3 0 0 0.77 0.44 0.25 2.39 1.99
3 1 0 1.07 0.54 0.40 2.85 2.44
3 1 1 1.36 0.61 0.57 3.27 2.84
3 2 0 1.38 0.62 0.57 3.32 2.88
3 2 1 1.69 0.69 0.76 3.76 3.31
3 2 2 2.02 0.76 0.96 4.24 3.76
3 3 0 1.72 0.70 0.77 3.83 3.37
3 3 1 2.05 0.77 0.98 4.30 3.82
4 0 0 1.27 0.64 0.48 3.38 2.89
4 1 0 1.68 0.75 0.70 4.04 3.50
4 1 1 2.11 0.86 0.95 4.70 4.13
4 2 0 2.16 0.88 0.97 4.81 4.23
4 2 1 2.64 1.00 1.26 5.54 4.92
4 2 2 3.17 U2 1.58 6.34 5.67
4 3 0 2.70 1.02 1.29 5.66 5.03
4 3 1 3.25 U5 1.62 6.50 5.81
4 3 2 3.86 1.29 2.01 7.42 6.68
4 4 0 3.35 U8 1.68 6.70 5.99
4 4 1 3.98 1.33 2.07 7.65 6.89
5 0 0 2.31 1.03 0.96 5.55 4.82
5 1 0 3.29 1.34 1.48 7.32 6.44
5 1 1 4.56 1.72 2.17 9.56 8.49
5 2 0 4.93 1.86 2.35 10.34 9.18
5 2 1 6.99 2.47 3.50 13.98 12.50
5 2 2 9.43 3.14 4.91 18.12 16.32
5 3 0 7.92 2.80 3.96 15.84 14.17
5 3 1 10.86 3.62 5.65 20.87 18.79
5 3 2 14.05 4.44 7.56 26.11 23.64
5 3 3 17.49 5.27 9.68 31.58 28.72
5 4 0 12.99 4.33 6.76 24.97 22.48
5 4 1 17.23 5.45 9.27 32.02 28.99
5 4 2 22.11 6.67 12.24 39.92 36.31
5 4 3 27.80 8.02 15.79 48.95 44.70
5 4 4 34.54 9.58 20.06 59.49 54.51
5 5 0 23.97 7.58 12.90 44.55 40.33
5 5 1 34.76 10.48 19.25 62.77 57.08
5 5 2 54.22 15.65 30.79 95.48 87.18
5 5 3 91.78 25.46 53.28 158.09 144.86
5 5 4 160.94 43.06 95.26 271.90 249.92
5 5 5 > 1,600
SOURCE: Data Courtesy of Robert]. Parnow (personal communication).
NOTE: Standard error, upper and lower 95% confidence limits, and onesided upper 95%
confidence limits when three dilutions are used with five tubes in each dilution at levels
of 1.0, 0.1 and 0.01 g.
33
for samples with only a few organisms and can be used to detect orga-
nisms in samples larger than 1 g_
Estimations Based on Metabolism
Microbial metabolism is used in general microbiology to determine
fermentation of sugars, starch hydrolysis, production of hydrogen sul-
fide, indole, or reduction of nitrate_ The metabolic products that are pro-
duced can be determined and used to estimate microbial populations or
the quality of the food_
REDUCTASE TESTS. Organisms obtain energy from chemical reac-
tions involving either organic or inorganic compounds. This involves an
oxidation-reduction reaction; the energy source becomes oxidized, while
another compound is reduced. Oxygen mayor may not be involved be-
cause oxidation-reduction reactions concern electron transfers. When a
compound loses an electron it becomes oxidized, and another com-
pound which accepts this electron is reduced.
Compounds vary in their oxidation-reduction potential, which is the
tendency for a compound to give up electrons. Since these reactions con-
sist of electron transfers, they can be measured electrically with a potenti-
ometer and are expressed by the electrical unit, the volt. The oxidation-
reduction potential also is called the redox potential.
Besides being determined potentiometrically, the redox potential can
be determined with indicators or dyes. Many compounds undergo color
changes when oxidized or reduced. If such a compound is added to a
substrate containing metabolizing bacteria, electrons may be transferred
to the indicator, and its color will be altered.
Since the color change of the indicator depends on the metabolic rate
of a microbial culture, the larger the number of cells, the sooner the
indicator will show a color change. The reduction time is inversely pro-
portional to the number of cells present (Fig. 2.4). Although several oxi-
dation-reduction indicators could be used, methylene blue, resazurin,
and the tetrazoliums are the ones used most often in food analysis. The
reductase tests usually are called dye reduction tests, apparently because
the dye methylene blue is used. However, resazurin and the tetrazoliums
are not dyes, but indicators (Conn 1961).
During reduction, methylene blue becomes colorless. This dye has
been used to determine the bacterial quality of milk and dairy products
such as ice cream (Anderson and Whitehead, 1974). Also, it has been
suggested as a means to predict the sterility of heated food (Hall 1971)
and to estimate the number of bacteria in ground beef (Emswiler et al.
1976).
9
8
""0;
z=
5 ~ 7
(.)'0
J ~
<t" _D
a:E
1oJ'"
.... c 6
(.).,.
<t.3
I D ~
!5
4
2
6
DYE REDUCTION TIME (HOURS)
Figure 2.4. Relationship between reduction time and microbial
load. The slope of the line depends upon the types of microorgan-
isms that are present.
Two color changes occur during the reduction of resazurin. The blue
color goes through various shades of purple and mauve to pink. This
color change is not due to an electron transfer, but to the loss of a loosely
bound oxygen atom. This color change to pink is not reversible by atmo
spheric oxygen. The second color change results in the indicator becom
ing colorless and is reversible by atmospheric oxygen. The resazurin test
is a simple, relatively rapid, inexpensive system to determine the quality
of fresh scallop meat (Webb et al. 1972) and frozen shrimps (Kiimmerlin
1982).
The tetrazolium salt most often used for testing food is 2,3,5-triphen
yltetrazolium chloride (TTC), since it is less toxic to bacteria than are
other tetrazolium salts. The TTC is colorless in the oxidized state but
forms intensely colored pink to red pigments (formazans) when reduced.
The red color that develops when TTC is sprayed onto a surface indicates
sites of bacterial activity. This indicator can be used to distinguish bacte
rial colonies from food particles in an SPC.
Comparison of Reductase Tests to Viable Count Tests. The reductase test gener
ally gives an estimate of the bacterial contamination in a shorter time
than the SPC. The information obtained from reductase tests can, at best,
be used to obtain a rough estimate of the number of microorganisms
present in or on a food.
Not all organisms cause a lowering of the redox potential at the same
rate. If a clump or chain of bacteria is plated in agar, a single colony will
develop, but the metabolic activity in the reductase test will be the sum
of the total number of cells in the clump or chain. This will result in a
more rapid color change in the indicator than the plate count would
suggest.
For a cell to be counted in the SPC, it must multiply and form a visible
colony. Cells may be metabolizing, but not reproducing. These cells
could cause a color change in the redox indicator and not be included
in the SPC.
Methylene blue and tetrazolium are inhibitory to certain microorgan
isms. Sometimes tetrazolium is added after the organisms have grown,
such as by flooding an agar surface, due to its potential for inhibiting the
cells. It has been suggested that reducing enzymes naturally present in
foods can cause color changes of these indicators. In this case, the reduc
tase test would indicate more contamination than is present.
CHEMICAL INDICATORS OF DECOMPOSITION. Everyone makes
organoleptic evaluations of food by sight, smell, taste, or touch. The food
industry relies on these organoleptic tests to determine certain quality
attributes of foods. This type of analysis is very subjective, and many ar
guments can develop between seller and buyer. Chemical indicators can
be used to evaluate the quality of food in a more objective manner. Food
is composed of various chemical compounds that are subject to biochem
ical changes. These changes may be desirable or undesirable, depending
upon the food, the microorganisms that are present, and the end prod-
ucts of the reaction. Decomposition of a food with resulting quality dete-
rioration is an undesirable change.
The main reactions occurring in foods are catalyzed by enzymes.
These enzymes may be tissue enzymes naturally present in the food, they
may be produced by microorganisms associated with the food, or they
may be added to catalyze a desirable reaction (see Chapter 9). Some
chemical reactions, such as oxidation, occur in foods without specific en-
zymes to catalyze them. The extent of change occurring in the food may
or may not be related to the number of microorganisms present.
The type and amount of metabolic products formed depends upon
the kind of food (protein, carbohydrate or fat), the type of microorgan-
ism (proteolytic, saccharolytic, or lipolytic), the availability of oxygen
(aerobic-oxidation, decay, or oxidative rancidity; anaerobic-fermentation,
putrefaction, or hydrolytic rancidity), the temperature (psychrotrophic,
mesophilic, or thermophilic organisms) and the types of inhibitors that
might be present.
Criteria for Chemical Indicators. For a chemical to be a useful indicator, it
must meet the following criteria: (1) it must be absent or at very low levels
in sound food; (2) it should be produced by the predominant spoilage
flora and not used as a nutrient; (3) it should not be detected quantita-
tively with simple and rapid tests and the tests should never yield false
positive results; (4) it should not have a useful function in the food; and
(5) it should preferably be able to distinguish poor quality from poor
processing operations.
Possible Chemical Indicators_ Fields, Richmond, and Baldwin (1968) pre-
sented a comprehensive review of chemical indicators. Some potential
chemicals for estimating the microbial or other quality of food are listed
in Table 2.6.
Because of variations in a food and its microbial flora, none of these
chemical indicators is entirely satisfactory_ However, the presence of cer-
tain indicators in some foods does correlate with the microbial count or
organoleptic evaluation. In general, a group of compounds such as vola
tile reducing substances, total volatile acids, or bases gives a better indica-
tion of quality than a single indicator such as ammonia, indole, or alco-
hol. One problem with many of these indicators is that by the time there
is a significant change in the amount that is present, deterioration of the
food is very evident. Perhaps incubation of the food for a few hours be-
fore analysis could be used to develop the presence of indicators more
rapidly. However, this higher temperature could alter the dominant
spoilage flora so that the metabolic products would differ from those
expected to develop under normal storage conditions.
TABLE 2.6. POTENTIAL CHEMICAL INDICATORS OF FOOD QUALITY
Ammonia
Trimethylamine
Dimethylamine
Total volatile bases
Total volatile acids
Free fatty acids
Water insoluble acids (oleic, palmitic)
Organic acids (acetic, lactic, pyruvic, suc-
cinic)
Indole
Ethanol
Furfural
Hydrogen sulfide
Total reducing substances
Volatile reducing substances
Histamine
Hypoxanthine
Diacetyl
Acetylmethylcarbinol
PHYSICAL TESTS. Microbial growth causes alterations in foods, such
as acid content or pH. As the pH is varied, the water-binding capacity of
protein changes. This difference can be determined by the extract re-
lease volume test.
The fluorescence of liquid egg is related to mustiness and growth of
certain bacteria, while pyoverdine, a fluorescent pigment produced by
pseudomonads, has been determined in frozen whole egg and on poultry
carcasses_
pH. Since basic compounds such as ammonia and amines are formed
during deterioration of protein foods, the pH will tend to rise. If carbo-
hydrates are present and fermented, the pH will tend to decline. Shelef
and Jay (1970) recommended blending 10 g of meat with water and titrat-
ing to pH 5.0 with 0.02N HCl. If more than 2.0 ml of acid is required,
the meat is in some form of incipient spoilage.
Extract Release Volume (ERV). As meat deteriorates, there is an increase in
the amount of water retained and a decrease in ERY. Shelef (1974) re-
ported the relationship between pH and ERY. Regardless of the micro-
bial quality of the meat, the maximum ERV occurred at pH 5.5.
ADENOSINE TRIPHOSPHATE (ATP). During metabolism, cells form
high-energy phosphate bonds stored in ATP. Not only microbial cells, but
also other living cells contain ATP.
When an animal dies and the muscle glycogen is utilized by anaerobic
glycolysis, the amount of ATP decreases. It has been established that
when bacterial cells are killed, the ATP disappears. In starved cells, the
ATP falls to low levels before the loss of viability is evident. Since all
microbial cells contain ATP, it should be possible to estimate the number
of cells by quantitating the ATP in a system.
The method for determining ATP is based on the firefly reaction as
shown in Figure 2.5. A purified extract from the firefly, containing luci-
ferin and luciferase, when reacted with ATP in the presence of magne-
sium ions, causes a light emission. Crude extracts from the firefly, when
reacted with adenosine diphosphate (ADP) have caused light emission.
When the purified firefly extract is added to bacterial ATP, the light emis-
sion can be measured with a photometer. For the assay, it is necessary to
eliminate the nonbacterial ATP and then to release the bacterial ATP to
react with the luciferin-luciferase system.
One method used to eliminate nonbacterial ATP is to rupture the
somatic cells and hydrolyze the released ATP (Anon. 1974; Thore et al.
1975). Another method is the centrifuging and/or filtration of a food to
separate the bacterial cells from food cells (Jarvis 1982; Picciolo et al.
1981; Stannard and Wood 1983).
After elimination of food ATP, the microbial ATP can be released
+
E
Luciferin Luciferase
E LH
2
AMP
+
+
ATP
Adenosine
triphosphate
PP
Mg++
:::;::::!=
Luciferyl adenylate Pyrophosphate
complex
E LH2 AMP + O
2
-- E + LO + CO
2
+ AMP + Light
Oxyluciferin Adenosine
monophosphate
Figure 2.5. Reactions involved in adenosine triphosphate determinations.
from the cells and assayed with the luciferinluciferase system, and an
estimate of the number of cells is calculated.
The ATP system is relatively simple and rapid. The system is report
edly reliable and acceptable for food and water analysis (J arvis 1982; Pic
ciolo et al. 1981). Firefly bioluminescence was reviewed by McElroy and
DeLuca (1983).
MEASUREMENT OF GAS PRODUCTION. When organisms metabo
lize compounds, carbon dioxide is produced as a metabolic product and
oxygen is consumed. One method for determining CO2 production uses
14Clabeled glucose and measures the reactivity of the released 14C0
2
The
detection time of 14C0
2
is proportional to the logarithm of the original
inoculum (Waters 1972). This procedure has been used for determining
various bacteria in water and food (Hatcher et al. 1977; Lampi et al. 1974;
Mafart et al. 1978; Rowley, Previte, and Srinivasa 1978).
Headspace gas of bottled fruit juice was analyzed for CO
2
by an infra
red system (Threlkeld 1982). Infrared analysis detected 89 percent of the
samples that were positive by conventional methods. Most of the false
negative samples lacked the presence of fermentative organisms.
IMPEDANCE. The metabolic activity of cells changes the chemical
composition of a medium, a process that results in altering the imped
ance (FirstenbergEden and Zindulis 1984). The impedance is the opposi
tion to the flow of alternating electrical current and can be measured by
a sensitive meter. This system has been used to estimate microorganisms
in cooked food (Rowley et al. 1979), cereal grain (Sorrells 1981), milk
(FirstenbergEden 1984; Firstenberg-Eden and Tricarico 1983; Gnan and
Luedecke 1982; Martins et al. 1982), meat (Firstenberg-Eden 1983; Mar-
tins and Selby 1980), frozen vegetables (Hardy et al. 1977), and orange
juice (Weihe, Seibt, and Hatcher 1984).
OTHER INSTRUMENTATION OR METHODS. Besides those systems
described, other systems have been used by microbiologists to develop
rapid methods for microbial detection. A pyrolysisgas chromatography-
mass spectrometry experiment was used to analyze Martian soil for or-
ganic compounds that would indicate life on Mars (Klein 1976; Sim
monds 1970). Sanders and Parkes (1970) attempted to correlate infrared
analysis of swabbings from broiler skin with bacterial numbers.
Gas chromatography or gasliquid chromatography has been used to
detect metabolic products of microorganisms which can be used to differ
entiate bacterial species or estimate the count or quality (Carlsson 1973;
Staruszkiewicz and Bond 1978).
Limulus amoebocyte lysate forms a gel in the presence of small
amounts of endotoxin from Gram-negative bacteria. This assay has been
suggested as a system to estimate Gramnegative bacteria in various sam-
ples such as meat Gay 1981; Seiter and Jay 1980), milk (Hansen, Mik-
kelsen, and M<t>ller-Madsen 1982), fish (Sullivan et al. 1983), cooked
turkey (Dodds, Holley, and Kempton 1983), and drinking water (Sykora
et al. 1980).
It is not possible to discuss all of the procedures that might be used
to determine microorganisms in foods. Some other procedures were de-
scribed by Bordner (1981), Feldman (1982), Goldschmidt and Fung (1978,
1979), Jarvis (1982), Newsom (1978), and Southern (1979).
Although agar is the usual solidifying agent in solid media, various
substitutes have been suggested (Cranston 1983; Kang et al. 1982; Lin
and Cas ida 1984; Shungu et al. 1983).
Comparison of Methods
There are many procedures that can be used to estimate the total
microbial population of a food product. The test that is selected depends
upon the use of the information that is obtained. If the results are to be
used to satisfy a microbiological standard, then the specified method,
usually the standard plate count, must be used. If the results are for inter-
nal quality control, some other method that is simpler, faster, or less ex
pensive might be used. The procedure selected will depend on the accu
racy, reliability, or precision that is needed. The laboratory equipment
and personnel, both now and in the future, will probably dictate the type
of analysis that can be conducted. The cost of supplies, materials, instru-
ments, and labor must be compared with other factors to determine
which method or methods give the desired information with ease, sim-
plicity, speed, and low cost.
In selecting the test, not only precision but also accuracy must be
considered. Precision is an index of the random error in a group of de-
terminations and is not related to accuracy. The term accuracy considers
the relationship of the determined value and the actual value. Since there
is no way to know the actual or true number of microorganisms in a
sample, it is difficult to assess the accuracy of a method. Quite often, the
results of a method are compared to those obtained with the APC. The
results obtained with the APC may not be accurate, but the method yields
acceptable reproducibility or precision.
Rapid tests such as those based on metabolic products and instrumen
tation are valuable for control purposes. Time is important in analyzing
highly perishable foods. Also, it is desirable to keep the inventory of
processed food at a reasonable level. If unacceptable product is shipped
from storage, it might need to be recalled. A recall of a product and the
bad publicity that often results is not desirable for a processor.
If microbial inhibitors are present in the food, these will be carried
over into the growth medium. In these cases, as the sample is diluted,
higher counts might be obtained due to dilution of the inhibitors. If a
food containing glucose is added to a medium designed to determine
lactose fermentation, erroneous results may be obtained due to fermen-
tation of the added glucose.
In processed foods, there may be cells that are sublethally injured.
These are of special importance when the types of organisms are deter
mined with selective media.
Comparisons of various methods of estimating microbial counts have
been published (Greenwood et al. 1984; O'Toole 1983b). Perhaps food
microbiologists have emphasized the socalled total count too much,
since spoilage organisms and potential pathogens are more important in
a food than are other types of organisms. However, at the present time,
the total count is usually the first microbial characteristic determined for
almost any food.
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Microorganisms Associated
with Food
3
The significance of microorganisms in foods depends upon several con-
ditions: (1) the numbers found; (2) the types of microorganisms; (3) the
type of food; (4) the treatments to which the food has been exposed; (5)
the processing or storage treatments the food will receive; (6) whether
the food is to be eaten as is or heated; and (7) the individuals who might
consume the food. In this text, it is necessary to limit the discussion of
microorganisms in foods to bacteria, fungi, and viruses.
Microorganisms may have at least one of four functions in a food.
They may have a useful function, cause spoilage, be a health hazard, or
be inert. The inert microorganisms do not find an environment favor-
able for growth. In most cases of foodborne illness, spoilage, or useful
activity, the microorganisms grow and multiply. Organisms that cause
foodborne illness are of more concern than are other types of microorga-
nisms.
Spoilage is the result of undesirable changes in the odor, color, flavor,
texture, or appearance of food. Some organisms that do not directly
cause changes in a food may alter the flora so that spoilage organisms
can grow. An example is the bacteriophages that attack useful organisms,
allowing undesirable organisms to grow and cause spoilage.
Useful organisms are those which produce desirable changes in food,
such as converting milk to cheese, sugar to alcohol, and cabbage to sauer-
kraut. These changes are referred to as fermentations. The microorganism
does not always have to be present to have a useful function, since en-
zymes can be separated from the organism, and the enzymes used to pro-
duce the desired reaction. Another function of useful organisms is the
production of single-cell protein which can be used as food. Spoilage
organisms and useful organisms are similar in that they both produce
changes in the food. The difference between them is that one is desirable
and the other is undesirable.
Although it is easy to establish categories for microorganisms, it is
difficult to place an organism in only one category, since it may have
different functions in different foods. An organism may spoil one food
but be inert in another, because of the different characteristics of foods.
49 G. J. Banwart, Basic Food Microbiology
However, we do tend to associate certain organisms with particular func-
tions in food, and these will be considered as much as possible_
DETERMINING TYPES OF MICROORGANISMS
The basic systems of microbial analysis discussed in Chapter 2 can be
used to determine the specific types of microorganisms that are present
in or on a food_ However, differential or selective media are substituted
for the noninhibitory, nonselective media_ Quite often, prior to deter-
mining whether certain types of organisms are present, an enrichment
procedure is used to increase the probability of detection_ After these
enrichment processes, the organisms are detected on differential or
selective agar.
Various tests are used to differentiate organisms isolated from selec-
tive or differential agars_ The usual staining reactions and morphological
characteristics are determined_ The required biochemical tests depend
upon the microorganisms being considered_ Various commercial kits and
systems for determining biochemical reactions have been evaluated (Costi-
gan and Hollick 1984; Cox and Mercuri 1979; Cox, Bailey, and Thomson
1983; Cox et aL 1984; Ferraro, Edelblut, and Kunz 1981; Fung, Gold-
schmidt, and Cox 1984; Griffiths and Phillips 1982; Izard et aL 1984; Kelly
and Latimer 1980; Odlaug et aL 1982)_ Serological reactions can utilize
cellular, flagellar, or other antigens_ The development of monoclonal an-
tibodies (L0vborg 1984) and immunoassays such as radioimmunoassay
(RIA), enzyme immunoassay (EIA) (Milby and Zare 1984), and enzyme-
linked immunosorbent assay (ELISA) are used to detect specific microor-
ganisms as well as toxins and other antigenic agents_ The production of
pigment, antimicrobial resistance pattern, phage typing, and bacteriocin
typing can be useful for the differentiation of strains of some organisms
(Anderson and Engley 1978; Kaneko and Hashimoto 1982)_
In the future, it is likely that genetic relatedness will become the sys-
tem to identify microorganisms (Krieg and Holt 1984)_ This includes de-
oxyribonucleic acid (DNA) and ribonucleic acid (RNA) homology (Paller-
oni 1983), as well as DNA colony hybridization and gene-specific DNA
probes (Claus, Moseley, and Falkow 1983)_
Besides the genetic information in the chromosome, many bacteria
contain extrachromosomal elements called plasmids_ The genetic mate-
rial in the plasmids is not absolutely essential for the cells to survive, but
it may give the cell a selective advantage in certain environments_
When a bacterium that contains plasmids divides, the plasmids are
replicated so there is at least one copy of the plasm ids in each of the
MICROORGANISMS ASSOCIATED WITH FOOD 51
daughter cells. Under certain conditions, the plasmids from one cell can
be transferred to another cell by conjugation.
The plasmids have been separated into several classes according to
the characteristics they confer upon the cells. Class F plasmids, the fertil
ity plasmids, have the ability to transfer genetic material by conjugation.
Other plasmids are involved with antibiotic, chemical, or radiation resist
ance; enzyme coding, so that metabolic reactions different from the usual
ones occur; virulence and production of toxins, such as enterotoxins; and
various other traits (Helinski 1976; Mach and Grimes 1982; Murray et al.
1983; Taylor, Levine, and Kouvelos 1982; Veltkamp 1979).
MICROBIAL TYPES IN FOOD
The microbial analysis of food products yields many diverse types of
microorganisms. However, we are concerned with the predominant types
and those which may cause spoilage or be a health hazard.
Various genera of bacteria have been isolated from red meat shortly
after slaughter of the animal. Lee, Fung, and Kastner (1982) reported the
prevalent genera on hot boned beef as Staphylococcus, Micrococcus, Brevi
bacterium, and Bacillus. Others that have been isolated from red meat in
clude Lactobacillus, Corynebacterium, Flavobacterium, Pseudomonas, Alcaligenes,
Streptococcus, Pedicoccus, Acinetobacter, Microbacterium, and Aerococcus. Quite
often coliforms, salmonellae, and Clostridium perfringens are found on red
meats. The temperature of holding and the packaging materials influ
ence the types of organisms that become dominant on fresh meat. Spe
cies of Micrococcus and Lactobacillus are important on cured meat. Also,
Bacillus, Vibrio, coliforms, fecal streptococci, and various yeasts can be
isolated from cured meats.
The same genera found on red meat tend to be present on poultry
and fishery products. Although the potential pathogens, such as Salmo
nella and Campylobacter, have been isolated from poultry, generally less
than 25 percent of the carcasses are contaminated (Cunningham 1982;
Kraft et al. 1982; McKinley and Avens 1981; Norberg 1981). Fishery prod
ucts, especially those from the oceans, contain Vibrio species (Sobsey et
al. 1980; Sochard et al. 1979).
Animal products such as milk and eggs tend to have bacteria similar
to those of meat and poultry (Coghill 1982; Cousin 1982; Johnston and
Bruce 1982; Sashihara et al. 1979; Schwab et al. 1982).
Many types of microorganisms are associated with plant products. The
most common bacteria in vegetables are Lactobacillus and Leuconostoc. The
genus Erwinia is important in vegetable spoilage. Other genera include Aer-
omonas, Alcaligenes, Bacillus, Corynebacterium, Enterobacter, Flavobacterium,
Klebsiella, Pseudomonas, Serratia, and Xanthomonas. Molds on vegetables in
clude Alternaria, Aspergillus, Aureobasidium, Botrytis, Chaetomium, Cladospor
ium, Epicoccum, Fusarium, Mucor, Penicillium, Phoma, and Rhizopus (Andrews
et al. 1982; Geeson 1979; Webb and Mundt 1978). During processing, the
initial microflora are distributed throughout the product and are supple
mented with other microorganisms.
The important organisms on fruits are molds (Penicillium, Alternaria,
Aspergillus). Lactobacillus species cause spoilage in fruit juices.
Molds such as Aspergillus, Fusarium, and Penicillium are found on dry
products such as grain and peanuts.
Several genera of yeasts are important in the spoilage of foods, espe
cially fruits and foods containing sugar. Yeasts are also useful in fermen
tation and the production of singlecell protein.
BACTERIA
Compared to the total number of species of bacteria, relatively few
have any importance in food, and most of these are the common types
that are discussed in general microbiology. Individual species of bacteria
are discussed with their functions in later chapters, so we will be con
cerned mainly with the genera that are important.
In Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons
1974), the bacteria are grouped according to the Gram reaction, mor
phology, and relation to growth with or without oxygen. In essence, that
system is used by Krieg and Holt (1984) as well as in this text for bacteria
found in food. Various morphological types of bacteria are shown in Fig
ures 3.1 and 3.2.
Aerobic/Microaerophilic, Motile, Helical/Vibroid
Gram-Negative Bacteria
In this group only one genus, Campylobacter, is important in food.
CAMPYLOBACTER. These Gramnegative, oxidase-positive organisms
have a singular polar flagellum at one or both ends and are motile with
a typical corkscrew motion. Unlike most bacteria, campylobacters use
amino acids or tricarboxylic acid intermediates rather than carbohy-
drates as a source of energy. The species of concern is C. jejuni. This or-
ganism is found on and in animal products and is a cause of gastroenter-
itis in human beings. (See Chapter 6.)
o 8fj m
tP flJ b
Cocci , I'-i", llllllIods 01
multipl ication. G, SftptOCOCC .. ;
b, Micr_; e ...,;, SaIaro
Hic;lher bclc:,.,;a
Figure 3.1. Morphological forms of bacteria.
Courtesy of Weiser, Mountney, and Gould (1971).
Gtanul" in _'eria

QI()

Figure 3.2. Structures of Bacteria,
Courtesy of Weiser, Mountney, and Gould (1971).
Gram-Negative, Aerobic Rods, and Cocci
000 CD Q? 00
a:m(J
c::l c:::> CO a Cl
Bac1eroal Cel l dl. ision
BacterIa wi'" flGQe lla
Included in this group are the family Pseudomonadaceae with the
genera Pseudomonas and Xanthomonas; the family Halobacteriaceae with
the genera Halobacterium and Halococcus; the family Acetobacteriaceae
with the genera Acetobacter and Gluconobacter; the family Neisseriaceae
with the genus Acinetobacter and four other genera (Alcaligenes, Alteromonas,
Brucella, and Flavobacterium) of uncertain status (Krieg and Holt 1984).
PSEUDOMONAS. This genus is divided into four sections on the basis
of RNA homology and a fifth section which contains species whose rela-
tionships to the others are generally unknown (Krieg and Holt 1984).
Oyaizu and Komagata (1983) divided the pseudomonads into nine groups,
primarily on the basis of cellular fatty acid composition.
The pseudomonads are straight to curved, motile (polar flagella) rods.
They are widely distributed in nature and are found on both animal and
plant products. Some pseudomonads are pathogenic for plants (Wong
and Preece 1980). Others, such as P aeruginosa, produce toxic substances
and are opportunistic pathogens for human beings (DeBell 1979;
Homma 1978). Some Pseudomonas species have been suggested as caus-
ative agents of foodborne illness, but there is incomplete proof that this
is so (Bryan 1973).
The pseudomonads are noted for their biochemical activity, being
able to attack a wide variety of organic compounds, including aromatic
types, and some synthetically produced chemicals. Some species (P aerugi-
nasa and P cepacia) have been found to grow in distilled water. The
pseudomonads produce catalase and most produce oxidase. They can
produce acids oxidatively from glucose, maltose, or both.
Some species of pseudomonads produce pyoverdine or fluorescein,
which are water-soluble fluorescent pigments (Leisinger and Margraff
1979). These pigments can be observed in spoiled foods by using an ultra-
violet light. They are usually yellow-green but may appear blue or orange,
depending on the species and environmental factors.
The pseudo monads produce enzymes that catalyze proteolytic (Mc-
Kellar 1982; Patel, Bartlett, and Hamid 1983) and lipolytic (Roy 1980)
reactions that contribute to spoilage of refrigerated fresh animal prod
ucts (Shaw and Latty 1982). Some pseudomonads produce pectolytic en-
zymes that can cause soft rot of fleshy vegetables (Cuppels and Kelman
1980; Palleroni and Holmes 1981).
The psychrotrophic pseudomonads are found in almost all types of
refrigerated and frozen foods. Since they are not very heat resistant, they
are not found in heat-processed foods unless the food is recontaminated
after heating. They are not very resistant to drying or gamma irradiation.
P aeruginosa is notoriously resistant to quaternary ammonium com-
pounds.
Several recent reports of selective media for Pseudomonas have been
published (Gould et al. 1985; Katoh and Itoh 1983; Wu and Thompson
1984).
XANTHOMONAS. The organisms in this genus are plant pathogens.
They are straight, motile (polar flagellum) rods and are catalase positive.
A medium to isolate a strain of X. campestris from walnuts was described
by Mulrean and Schroth (1981).
Being plant pathogens, these organisms have been involved with var
ious types of rots of these products. X. campestris produces xanthan gum,
which is used in the food industry.
HALOBACTERIACEAE. The two genera Halobacterium and Halococcus
are extreme halophiles requiring about 15 percent salt for growth. They
are found in solar salt (produced by evaporation of seawater).
Their halophilic character prevents them from growing in most
foods. However, in foods preserved by salting, these organisms can pro
duce a red pigment, bacteriorubein.
The high salt concentration is necessary for activity of the enzymes,
stability of membranes and ribosomes, and synthesis of protein. A low
salt concentration results in Halobacterium changing from rods to spheri
cal forms.
ACETOBACTER. These cells are ellipsoidal to straight or slightly
curved rods. Young cultures are Gram negative, while older cultures are
Gram variable. They are motile (peritrichous flagella) or nonmotile.
The Acetobacter are noted for the oxidation of ethanol to acetic acid.
They oxidize acetate and lactate to CO
2
and H
2
0. They are found on
fruits and vegetables and are involved in the souring of fruit juices and
alcoholic beverages (beer and wine).
A selective medium for differentiating Acetobacter and Gluconobacter
was described by Cirigliano (1982).
GLUCONOBACTER. G. oxydans represents this genus. This organism is
ellipsoidal to rodshaped, Gram negative to weakly Gram positive in
older cultures. The cells occur singly, in pairs, or in chains. They are
strictly aerobic and oxidize ethanol to acetic acid.
G. oxydans is found in various food products such as vegetables, fruits,
baker's yeast, beer, wine, cider, and vinegar. It is involved in spoilage,
causing the souring of fruits.
ACINETOBACTER. These organisms are very short, plump rods or
coccobacilli, predominantly in pairs or short chains. They are strictly aero
obic, mesophilic saprophytes.
They are found in soil and water and also in animals and human
beings. Organisms identified as Acinetobacter have been isolated from var
ious types of raw and prepared foods, including beef and poultry car
casses (Lahellec et aL 1975). They are potential spoilage organisms. Meth
ods for enumeration of Acinetobacter were described by LaCroix and
Cabelli (1982) and Spino and Geldreich (1981).
ALCALIGENES. The four species in this genus are motile (four to
eight peritrichous flagella) rods, coccal rods, or cocci. They are strict aero
obes and oxidase positive.
They are widespread in nature, being found in soil, water, decaying
matter, and the intestinal tract of animals. These organism are involved
with the spoilage of protein foods (eggs and dairy products).
ALTEROMONAS. This genus comprises Gramnegative, motile (polar
flagellate), nonfermentative bacteria with a guanine + cytosine (GC) ra
tio between 43.2 and 48 mol percent (Baumann et al. 1972). This ratio is
listed as 38:50 mol percent by Krieg and Holt (1984).
These organisms are common in marine environments and on fish,
causing fish spoilage (Chan et al. 1978; Gillespie 1981; Gray and Stewart
1980). Alteromonas was isolated from ground beef (Parker and Levin
1983).
BRUCELLA. These coccobacilli or short rods are nonmotile. Brucella
melitensis (pathogenic for goats and sheep), B. abortus (pathogenic for cat
tle), and B. suis (pathogenic for pigs) can affect people, causing bru
cellosis (undulant fever). The sources of infection are raw milk or dairy
products, uncooked meat or sausage products, discharges from infected
animals, and human' carriers.
Since these organisms are susceptible to the heat treatment used for
pasteurization of milk and cooking of meat, they should be of limited
importance in food.
There are between 100 and 200 cases of brucellosis in the United
States annually. The majority of cases involve meat packing plant work
ers, and most of these are involved with hogslaughter operations. Most
of the other cases involve livestock producers, veterinarians, and govern
ment inspectors.
FLAVOBACTERIUM. This genus contains diverse bacteria. The spe
cies includes nonmotile rods that produce yellow, orange, or greenish
yellow pigment (Hayes 1977). Environmental factors (substrate and
temperature) affect the synthesis of pigment and resultant hue. These
organisms prefer temperatures below 30C, although some strains can
grow at 37C.
Flavobacterium species have been isolated from water, soil, animals, hu
mans, and various food products. They can produce discoloration on
some foods. The organisms have been found on thawing frozen vegeta
bles, fresh vegetables, refrigerated fish and shellfish, meat, meat prod-
ucts, poultry, and in cannery environments.
Gram-Negative, Facultative Anaerobic Rods
In this group, the family Enterobacteriaceae contains several genera,
including Escherichia, Shigella, Salmonella, Citrobacter, Klebsiella, Enterobacter,
Erwinia, Serratia, Edwardsiella, Proteus, and Yersinia. The family Vibrionaceae
includes Vibrio and Aeromonas. There are other genera in these families
and in this section, but they are not very important in food microbiology.
ESCHERICHIA. The principal species is E. coli. The organisms are
members of the coliform group, which are indicators of fecal contamina-
tion (see Chapter 7). E. coli may cause spoilage of food, and certain strains
are enteropathogenic or enterotoxigenic.
E. coli is found in soil and water, on plants, in the intestinal tract
of animals, and in various foods, especially animal products and foods
handled by people. Since the cells are heat sensitive, their presence in
heat-pasteurized or cooked products indicates recontamination after the
treatment.
SHIGELLA. The four species in this genus are nonmotile, nonspore-
forming rods. The normal habitat is the intestinal tract of human beings
and other primates. Rarely are these organisms isolated from other ani-
mals.
The shigellae are the causative agent of shigellosis, a foodborne gas-
troenteritis. The number of foodborne illnesses due to these organisms
is not great, but the illness can be severe. Additional information about
Shigella and shigellosis is in Chapter 6 and in Dodd and Jones (1982).
SALMONELLA. The genus Salmonella is divided into five subgenera
(I-V). These subgenera are differentiated on the basis of minor biochemi-
cal differences. Subgenus I includes the majority of serotypes of this ge-
nus, especially the more important serotypes, and subgenus III includes
the organisms previously called the Arizona group. Most of the different
types were differentiated on the basis of serological reactions, so they are
called serotypes or serovars. The number of known serovars is approach-
ing 1900.
The salmonella organisms are catalase positive and oxidase negative,
with both respiratory and fermentative metabolism. Most of the sero-
types are motile (peritrichous flagella), do not ferment lactose, and grow
well in simple media containing glucose, inorganic nitrogen, and mineral
salts. Although salmonellae do not ferment sucrose, strains of S. arizonae
contain a plasmid that gives them the ability to ferment this sugar (Bart
lett and Trust 1980).
The salmonellae are said to be ubiquitous, being worldwide and
found in or on soil, water, sewage, animals, humans, processing equip
ment, feed, and various food products. The natural habitat is the intesti
nal tract of humans and animals. It is logical that humans, animals, and
their environments are the primary sources of salmonellae. Some sero
types seem to be localized in a region or a country, but with national and
international travel and trade, the organisms are easily disseminated.
The serotypes of Salmonella are an important cause of foodborne ill
ness. Further information about these organisms is found in Chapter 6.
CITROBACTER. The species in this genus are motile (peritrichous
flagella) rods that utilize citrate as the sole carbon source. Most ferment
lactose. They are members of the coliform group of indicator organisms.
They are normal intestinal inhabitants and are found on various foods,
especially animal products. These organisms are listed as causing enteri
tis in humans, but proof of transmission by foods is inconclusive (Bryan
1973). They have been associated with various human infections. Citro
bacter can also cause spoilage of foods.
Crossreactions with antisera of other Enterobacteriaceae indicate
that there are close relationships between Citrobacter, Salmonella, and Esch
erichia.
KLEBSIELLA. The klebsiellae are nonmotile, encapsulated rods. They
are found on grain and fresh produce and in frozen foods. They are
members of the coliform group, can cause food spoilage, and are poten
tial health hazards, causing gastroenteritis. According to Bryan (1973),
proof that transmission of these organisms is by means of food is incon
clusive.
ENTEROBACTER. Organisms in the Enterobacter genus are similar to
the klebsiellae, except they are motile (peritrichous flagella).
They can be found in soil, water, sewage, the intestinal tract of people
and animals, and in various food products. The organisms are important
in food as a potential health hazard and as indicators of spoilage.
ERWINIA. Organisms in this genus are small, mostly motile (peritrich
ous flagella), predominantly single, straight rods. They are oxidase nega
tive and catalase positive.
The organisms cause various plant diseases and soft rots. E. carotovara
is the main spoilage organism of stored vegetables (Chapter 8). At least
one species is associated with humans and animals.
SERRATIA. The cells of Serratia are motile (peritrichous flagella) rods.
They are important as potential foodspoilage organisms. S. marcescens is
noted for the production of red pigment and is used as a biological
marker (Yu 1979).
EDWARDSIELLA. These organisms have peritrichous flagella. They
produce indole from tryptophan and produce H
2
S on triple sugar iron
agar.
Although these organisms cause enteritis in humans, proof that trans-
mission is by means of food is not conclusive (Bryan 1973). E. tarda has
a wide geographical distribution and infects numerous animal species.
PROTEUS. Organisms in this genus are highly motile (peritrichous
flagella) rods that occur singly, in pairs, or in short chains. On moist agar
surfaces, some Proteus species tend to swarm, which makes it difficult to
pick isolated colonies for further study. Swarming is more evident at
20C than at 37C.
The organisms are widely distributed. They are common inhabitants
of the gastrointestinal tract, and are found in sewage, soil, in decompos-
ing animal protein, and in various foods.
The importance of Proteus in food includes potential health hazards
and spoilage. Although strains of Proteus can cause enteric infection in
humans, according to Bryan (1973), proof of transmission by food is in-
conclusive.
YERSINIA. The cells in this genus are ovoid to rod shaped. Included
in this genus is Y. enterocolitica, which is associated with gastroenteritis,
meseteric lymphadenitis, terminal ileitis, and pseudoappendicitis (Berco-
vier et al. 1980)_ This organism has been isolated from red meat, chicken,
shellfish, milk, ice cream, and vegetables. These organisms may be more
prevalent in food than reported.
VIBRIO. This genus is in the family Vibrionaceae. The cells are short,
motile, oxidase positive, curved, or straight rods. Some strains fail to grow
without the presence ofNaCI, the optimum concentration being 3.0 per-
cent.
Strains of V. costicola can tolerate NaCI concentrations of 23 percent.
This species has been found in cured meats and curing brines (Gardner
1980-1981). A medium for isolating these salt-tolerant organisms was de-
scribed by Gardner (1973). Methods for enumeration, phage typing, and
characterizing V. cholerae were discussed by various researchers (Bocke-
muhl and Meinicke 1976; Morris et al. 1976; Salles and Momen 1981).
V. cholerae and V. parahaemolyticus are important pathogens, causing
gastroenteritis in humans. V. cholerae is found in the intestinal tract of
people and animals, in water, and occasionally in food. V. parahaemolyticus
is found in the ocean, in seafoods, and in the intestinal contents of in
fected humans. Various other vibrios have been incriminated in out-
breaks of foodborne illness (Roberts and Gilbert 1979).
AEROMONAS. The cells are rods with rounded ends to coccoid. They
are motile (polar flagella), and are oxidase and catalase positive.
These organisms are frequently mistaken for members of the family
Enterobacteriaceae because of their similarity in growth and biochemical
characteristics. A positive oxidase test and nitrate reduction help differ-
entiate the Aeromonas from Enterobacteriaceae.
The main habitat of Aeromonas is water. Some strains cause disease
(hemorrhagic septicemia) in fish, eels, and frogs. Some strains cause en-
teritis in humans. One source of this infection may be fish or other sea-
food. Aeromonas may playa role in spoilage of fish and other animal prod-
ucts.
Gram-Negative, Anaerobic Straight, Curved, and
Helical Rods
In this category, the family Bacteroidaceae contains twelve genera,
including Bacteroides. These organisms are nonmotile, straight rods. Be-
ing nonsporeforming obligate anaerobes, they probably cause no
changes in foods. However, they are found in fecal material in very high
numbers. Hence, they might be useful as indicators of fecal contamina
tion of food as well as water (Fiksdal et al 1985).
Gram-Positive Cocci
The Gram-positive cocci include aerobic or facultative anaerobic bac
teria in the family Micrococcaceae with the genera Micrococcus and Staphy-
lococcus. Other Gram-positive cocci include the genera Streptococcus, Leuco-
nostoc, Pediococcus, and Aerococcus.
MICROCOCCUS. These spherical cells are strict aerobes, catalase posi-
tive, occur singly or in pairs, and characteristically divide in more than
one plane to form irregular clusters, tetrads, or cubical packets. They can
grow in the presence of 5 percent salt.
The micrococci are found in soil, water, and 'dust, and on the skin
of people and other animals. They are found in several types of foods,
especially milk and dairy products, on animal carcasses, and in meat
products. They are important as potential spoilage organisms.
STAPHYLOCOCCUS. These nonmotile cells occur singly, in pairs, or
in irregular clusters. They are facultative anaerobes with respiratory and
fermentative metabolism. Most strains can grow in 7.5 percent to 15 per-
cent salt. The organisms usually are sensitive to chlorine, chloramine,
iodine, and iodophors. Although usually sensitive to heat, they are mod-
erately resistant to radiation.
Both S. aureus and S. epidermidis are commonly found on the skin and
mucous membranes of humans and warm-blooded animals. They are po
tential pathogens, being either the primary pathogen or secondary in-
vader.
The staphylococci are found in many types of food products. In gen-
eral, they are not able to compete very well with other organisms. S.
aureus produces enterotoxins, which are one of the main causes of food
borne illness. S. aureus is found in pimples, boils, acne, wound infections,
and in the nose. It is easily transferred to foods by careless food handlers.
This species is differentiated by its ability to produce coagulase (which
clots blood plasma) and a heatstable nuclease. S. aureus is described fur-
ther in Chapter 6.
Other species have been found in food (Devriese et al. 1983; Schleifer
and Fischer 1982). S. hyicus reportedly produces an enterotoxin (Hoover,
Tatini, and Maltais 1983).
STREPTOCOCCUS. The bacterial species in this genus have various
characteristics and functions. These organisms have been divided into
several groups (Bridge and Sneath 1983). The groups of primary impor-
tance in food are the lactic streptococci and enterococci. Researchers
have suggested that organisms in the latter group be put into the genus
Enterococcus (Collins et al. 1984; Schleifer and KilpperBalz 1984).
These spherical cells occur in pairs or chains. With the exception of
some strains, they are not motile. They are facultative anaerobes with
a fermentative metabolism, fermenting glucose primarily by the hexose
diphosphate pathway and producing mainly lactic acid. Thus, they are
called homofermentative.
The fermentation of carbohydrates to lactic acid is desirable in prod-
ucts such as cultured milk, cheese, and sauerkraut, but it is undesirable
in products such as fresh milk. They can utilize sugar in an alcohol fer-
mentation, resulting in lower alcohol and higher acid than is wanted.
Some strains utilize citric acid and form acetoin and diacetyl. The ability
to produce diacetyl in milk is desirable in the manufacture of cultured
sour cream, buttermilk, and butter.
Various functions such as lactose metabolism, proteolytic activity, and
hemolysin and bacteriocin production are mediated by plasmids (Ander-
son and McKay 1977; Gasson 1983; McKay 1983; Oliver, Brown, and
Clewell 1977).
The streptococci are widely distributed, being found in air, water, sew-
age, soil, on plants, in the intestinal tract of people and animals, and in
various food products_
Some species and strains are somewhat heat resistant, surviving 60C
for 30 min_ The enterococci can survive freezing and frozen storage in
food products_ This makes these organisms acceptable as potential indi-
cator organisms in frozen foods_
Although some pathogenic types may be transmitted to foods, there
is more interest in the streptococci as possible indicators of fecal contam-
ination, as useful fermentative organisms, and as potential spoilage bac-
teria_
LEUCONOSTOC. These spherical to lenticular cells occur in pairs or
chains. They often have complex nutrient requirements such as vitamins,
amino acids, and a fermentable carbohydrate. They ferment glucose to
lactic acid, ethanol, and CO
2
_
The organisms are important in fermentation and spoilage of foods.
They are not pathogenic. Although they have physiological differences,
Garvie (1983) has suggested that L. cremoris, L. dextranicum, and L mesentero-
ides belong to a single deoxyribonucleic acid (DNA) homology group and
should be considered subspecies of L. mesenteroides.
PEDIOCOCCUS. These homofermentative cocci occur as single cells,
pairs, or tetrads. They have rather complex nutritional requirements,
such as vitamins and amino acids, which makes them useful to assay for
these nutrients. These organisms are found in pickles, sauerkraut, beer,
wine, and other fermenting materials_
AEROCOCCUS. This genus has only one recognized species, A. viri-
dans, which used to be a Pediococcus (P. homari) (Buchanan and Gibbons
1974). The genus is very similar to Pediococcus. On blood agar, the colonies
are surrounded by a green zone, hence the name viridans (green).
The aerococci have been found in air and dust, human infections,
meat-curing brines, and on raw and processed vegetables.
Endospore-forming Rods and Cocci
This group includes the genera Bacillus, Clostridium, Sporolactobacillus,
and Desulfotomaculum. Although Desulfotomaculum is listed with dissimila-
tory sulfate or sulfur-reducing bacteria (Krieg and Holt 1984), the discus-
sion of this genus is included with endospore-forming bacteria. The
spores that these organisms produce are different from the vegetative
cells (Fig. 3.3) so there is another entity to consider. The spores are an
inactive or dormant state of the organisms.
Figure 3.3. Spores formed in the
center of the rod: Bacillus subtilis
at left, Clostridium sporogenes at
right.
Courtesy of Weiser, Mountney, and
Gould (1971).
The organisms go through certain stages to change from a vegetative
cell to a spore and back to a vegetative cell. The stages we can consider
are cell sporulation, germination, and outgrowth.
SPORULATION. The mechanisms that trigger spore formation are not
fully known. Most strains of clostridia produce spores when incubated
in a good medium under anaerobic conditions, 3 to 8C below their
optimum growth temperature.
Muhammed, Morrison, and Boyd (1975) stated that demonstrating
the complete sporulation requirements of an organism is impossible be-
cause some nutrients essential for spore formation also may be required
for growth.
For all species, not all cells sporulate, regardless of the conditions.
There is no information as to why some cells in a culture sporulate and
others do not. However, there is agreement that spore formation begins
after the exponential growth phase during the stationary phase. Spore
formation can be arbitrarily divided into seven stages: (1) development
of axial chromatin filament; (2) spore septation; (3) engulfment of the
spore protoplast; (4) cortex formation; (5) coat formation; (6) maturation;
and (7) the free spore stage. The spore consists of a core surrounded by
several layers of mucopeptide and proteinaceous outer coats (Aronson
and Fitz:James 1976).
An organism can initiate sporulation without completing the process
and reverting to a vegetative cell. However, there is a stage at which the
cell becomes irreversibly engaged in sporulation and is committed to
completing the process. The composition of the substrate and the tem-
perature of sporulation can affect the resistance characteristics of the
resultant spores (Bayliss, Waites, and King 1981).
The main characteristic that is important to food microbiologists is
the resistance of spores to heat, radiation, chemicals, desiccation, and
freezing (Gould 1977). Quite commonly, the heat resistance of spores is
about 10
5
times, and radiation resistance is about 10 times more than
that of the corresponding vegetative cells. Spores can be stored for long
periods and retain their ability to germinate and produce vegetative cells.
GERMINATION. Since spores are dormant, they must be converted to
vegetative cells to be important in food spoilage or toxin production.
The conversion of the heat-resistant spore to vegetative cells would allow
less severe thermal processes for food preservation.
Spores may need a conditioning treatment prior to germination. This
process, called activation, may be induced by aging, heating, radiation,
altering the pH, or with chemicals. Low numbers of viable spores do not
always germinate immediately. Thus, canned foods that apparently pass
short storage tests to determine potential spoilage, can show spoilage
after extensive storage.
Activation is a reversible process and, if conditions do not allow
germination, the spore reverts to its dormant state. Although some
spores may germinate without heat shock, low levels of heat treatment,
such as 65 to 85C for 10 to 30 min, will activate most spores and induce
germination. The heat treatment used depends upon the species and
strain. Spores of species such as C. botulinum type E strains are heat sensi-
tive and should not be heated above 70C. Higher temperatures (10 to
115C) for 3 to 10 min can be used to activate spores of the thermophilic
B. stearothermophilus.
During germination, the bright, refractile spores become dark; dipi-
colinic acid is released and the cortex disintegrates. Although respiration
in the spore is undetectable, there is an abrupt onset of respiration dur-
ing germination. There is activity of a variety of enzymes, typical of the
vegetative form. The spores lose their resistance to heat, desiccation,
chemical agents, electric shock, and hydrostatic pressure. The germinat-
ing spores show a temporary rise in resistance to ultraviolet light and
ionizing radiation followed by a rapid fall in resistance.
Although pasteurized milk supported germination of B. cereus, raw
milk supported little or none (Wilkinson and Davies 1973). This helps to
explain the spoilage defect due to B. cereus in pasteurized milk but not
in raw milk. This inability to germinate in raw milk might be due to
natural inhibitory agents found in raw milk that are inactivated by pas-
teurization.
A review of bacterial spore germination by Smoot and Pierson (1982)
is suggested for further information on this subject.
OUTGROWTH. The development of a vegetative cell through the first
cell division is called outgrowth. Outgrowth proceeds by the swelling of
the spore, emergence from the spore coat, elongation, and cell division.
Germination and outgrowth can be followed by determining the synthe-
sis of RNA, proteins, and DNA, in that order.
Outgrowth occurs when germination takes place in a substrate capa-
ble of supporting vegetative growth. If the germinating medium is not
sufficient to support growth, either development is stopped or the out-
growing cell may form a second spore with no intervening cell division.
This cycle of spore-cell-spore, is called a microcycle. Microcycle sporu
lation can be induced by dilution of an acceptable medium or by sus
pending germinated spores in a glucosefree medium.
The effects of various factors on the germination and outgrowth is
important in food microbiology. It is the ability to determine viable from
nonviable spores that allows us to establish thermal processes that will
destroy spores in food. Also, if we could cause all of the spores to germi
nate prior to heat processing, the thermal treatment could be reduced.
BACILLUS. These organisms are usually Grampositive rods, but older
cultures may appear as Gram negative. The majority are motile, produce
catalase, and produce acid but not gas from glucose.
The cells in this genus vary from strict aerobes to facultative anaer
obes. The nutrient requirements vary from simple to complex. There are
psychrotrophs, mesophiles, and thermophiles. Thus, for growth, the min
imum temperature varies from - 5C to about 45C. The maximum for
some species is 25C and for others up to 75C. The minimum pH for
growth varies from pH 2.0 for B. acidocaldarius to pH 7.5 to 8.0 for B.
alcalophilus. The salt tolerance is 2 percent or less for some species. Oth
ers can grow in 25 percent salt. Due to the diversity of the species, there
have been suggestions that the genus should be divided. Five genera have
been proposed, but this differentiation has not been adopted.
Bacillus can be found in soil, water, fecal material, decaying materials,
and in various foods. Ingredients can serve as a source of Bacillus. Spices,
flour, starch, and sugar have been incriminated as sources of spores for
contamination of fermented sausages, bread, and canned food. The reo
sistance of the spores of Bacillus to various agents makes these organisms
important in food preservation. Some species, such as B. subtilis, decom
pose pectin and polysaccharides of plant tissue, causing spoilage of fresh
plant products. Lowacid canned foods are spoiled by B. stearothermophilus,
and B. coagulans causes spoilage of tomato products. B. cereus is involved
in foodborne gastroenteritis and B. anthracis causes anthrax of both ani
mals and man.
Besides potential spoilage and health hazards, some species of Bacillus
can be useful in foods. The bacilli are a source of proteolytic enzymes
that might be used to clot milk for cheese production. Some bacilli have
been suggested for use in the production of singlecell protein. Some
species are insect pathogens, which makes them useful in food produc
tion.
CLOSTRIDIUM. The species in this genus are divided into four groups
on the basis of spore position and gelatin liquefaction (Buchanan and
Gibbons 1974). The cells are usually Gram positive, at least in the early
stages of growth. They are catalase negative and, except for a few aerotol
erant species, are strictly anaerobic.
The critical tolerance of N aCI is 2.5 to 6.5 percent. Sodium nitrate at
0.5 to 1.0 percent inhibits these organisms. The lethal chlorine concentra
tion is 2.5 p,g/ml.
The principal metabolic products of a species can be determined by
chromatography (column, thinlayer, or gas). In conjunction with other
tests, gas chromatography is a valuable aid in differentiating the clos-
tridia. The analysis of soluble cellular proteins with polyacrylamide gel
electrophoresis was used to differentiate species of clostridia (Cato et al.
1982). Anaerobic methods of analysis were reviewed by Anderson and
Fung (1983).
The primary source of clostridia is soil. They are found in the intesti-
nal tract of people and animals, as well as in various foods.
The species in this genus include two involved with foodborne illness,
several that cause food spoilage, and many of no concern to food micro-
biologists. Some are free-living nitrogen-fixing organisms, some cause se-
rious illness (tetanus, gas gangrene), and others are used to p r o d u ~ com-
mercial chemicals such as butyric acid, butanol, acetone, and enzymes.
The spores of some species are very heat resistant and may survive
the heat treatment of canned foods. If the surviving spores can germinate
and the vegetative cells grow, spoilage will occur. If C. botulinum spores
survive the heat treatment, germination, and outgrowth of the spores
may result in the production of potent toxins.
DESULFOTOMACULUM. The species in this genus are similar to the
clostridia. However, they are Gram negative and have a higher DNA base
composition (G+C is 41-46 mol percent as compared to 23-43 mol per-
cent). The clostridia do not reduce sulfate, but these organisms reduce
sulfur compounds (sulfates, sulfites, and other reducible sulfur com-
pounds) to H
2
S.
D. nigrificans is a thermophilic spore former that causes sulfide spoil-
age of canned foods.
Regular, Nonsporing, Gram-Positive Rods
In this group, the genera Lactobacillus is the most important to food
microbiologists. Other genera in food are Brochothrix, Kurthia, and Lis-
teria.
LACTOBACILLUS. These organisms are straight to curved rods occur-
ring singly or in chains. The rods vary from long and slender to short
coccobacilli. (Figs. 3.4 and 3.5.) Generally they are nonmotile. Although
considered to be Gram positive, as the culture ages, the cells may become
Figure 3.4. Lactobacillus bulgari-
cus, the high-acid-producing long
rod of sour milk preparations such
as yogurt (magnification = x
2,250).
Courtesy of Pederson (1979).
Gram-negative. Growth is enhanced by 5 to 10 percent CO
2
. These orga-
nisms generally have complex nutrient requirements. Both homofermen-
tative and heterofermentative types are in this genus.
Lactobacilli are found in plant and animal material, in various places
in the body of human beings and other warm-blooded animals (including
the intestinal tract) and in various foods.
In food microbiology, the lactobacilli are useful but also cause spoil-
age. They are useful in fermentations in which lactic acid production is
desirable, whether in plant or animal products (sauerkraut, pickles, ol-
ives, fermented sausages). Some lactobacilli form slime and spoil sugar
cane. Others cause green discolorations of sausage products. Vacuum-
packaged meat becomes sour due to lactobacilli. Even in fermented vege-
tables, they may be a problem, causing pink sauerkraut or bloater for-
mation in fermented cucumbers. The lactobacilli can cause spoilage of
vinegar-preserved products, such as catsup and mayonnaise.
Figure 3.5. Lactobacillus brevis,
the common heterofermentative
species of the genus (magnifica-
tion = x 2,250).
Courtesy of Pederson (1979).
Due to their complex nutrient requirements, lactobacilli are used in
assays of food for vitamins, amino acids, and other nutrients. The lysine
excreting mutants of lactobacilli have been suggested for use in food and
feed enrichment (Sands and Hankin 1974). Vandercook and Smolensky
(1976) suggested using L. plantarum to detect adulteration of orange juice
with imitation orange beverages. The imitation juice does not support
the growth of this organism as well as does real orange juice.
BROCHOTHRIX. The species B. thermosphacta was previuusly named Mi
crobacterium thermosphacta. It is a Grampositive, facultatively anaerobic
psychrotroph. It is associated with red meat and meat products, and may,
in certain cases, cause spoilage (a sweet off.odor) of these foods.
KURTHIA. These Grampositive cells are regular, unbranched rods in
young cultures but become coccoid in older cultures by fragmentation
of the rods. The cells are strict aerobes.
These organisms are found in intestinal contents, stagnant water,
fresh and spoiling meat and meat products, meat processing plants, and
milk. According to Gardner (1969), K. zopfii is not known to cause spoil
age of refrigerated meat, but its presence indicates that the meat was
exposed to temperatures higher than refrigerator temperatures during
processing, distribution, or retailing. In meat held at 2C, Kurthia is over
grown by Pseudomonas species and other organisms. Although present in
food, their importance might be only as an indicator of mishandling.
Irregular, Nonsporing, Gram-Positive Rods
This group of bacteria includes the genera Corynebacterium, Arthro
bacter, Brevibacterium, and Propionibacterium.
CORYNEBACTERIUM. This genus is divided into three sections: (1)
human and animal parasites and pathogens; (2) plant pathogens; and (3)
nonpathogenic.
The coryneform bacteria are characterized by their pleomorphism.
The Corynebacterium cells are straight to slightly curved rods but have a
tendency to form club and pointed shapes. Generally they are not motile
and are Gram positive. The best growth is aerobic, but they can grow in
anaerobic conditions.
These organisms are widely distributed in nature. They are found in
water, soil, plants, and animals. These organisms can be found in food
products derived from both animals and plants. They have been associ
ated with spoiling food, but it is doubtful they are primary spoilage orga
nisms.
ARTHROBACTER. Organisms in this genus show considerable pleo-
morphism_ The coccal form may appear as spheres, ovoid, or slightly
elongated_ When large cocci are transferred to a fresh medium, from
one to three (seldom four) germination tubes arise from the celIs- These
develop into rods which vary in size and shape_ Upon aging, the rods
change almost completely into cocci- The cocci are Gram positive and the
rods have Gram-positive granules surrounded by Gram-negative cellular
materials.
The organisms are found in soil, in and on meat and poultry prod-
ucts, in milk products, dairy waste, activated sludge, and brewery and fish
slime.
According to Buchanan and Gibbons (1974), the organism referred
to as Brevibacterium linens, which is important in cheese, is related to and
should perhaps be placed in the genus Arthrobacter.
BREVIBACTERIUM. Although this genus is listed in Bergey's Manual
(Buchanan and Gibbons 1974), no species are recognized. However, Su-
zuki and Komagata (1983) listed several species of Brevibacterium, includ-
ing B. linens. Brevibacterium linens is important in flavor production in
cheese, especially limburger cheese.
PROPIONIBACTERIUM. Generally these organisms are Gram-positive
rods, but they may be diphtheroid, club-shaped, coccoid, elongate, bifid,
or branched. They are anaerobic to aero tolerant.
Propionibacters are found on people and in the intestinal tract of
people and animals. During fermentation, propionbacters produce pro-
pionic acid and acetic acid, with lesser amounts of other organic acids.
The species of most interest in food microbiology is P. freudenreichii subsp.
shermani. This is used in the manufacture of Swiss cheese. Due to the
production of propionic acid and CO
2
, the organism is responsible for
the characteristic flavor and the eyes in this cheese. These bacteria syn-
thesize large quantities of vitamin B12 and produce propionic acid and
can be used in the commercial production of these compounds.
Mycobacteria
Mycobacterium tuberculosis causes tuberculosis. So that this disease
could be controlled, pasteurization of milk was inaugurated. Today, tu-
berculosis is primarily an airborne disease, rather than foodborne. Myco-
bacterium strains are found in raw milk, oysters, pork, and vegetables
sprayed with sewage effluent (Hosty and McDurmont 1975; Thoen, Jar-
nagin, and Richards 1975; Van Donsel and Larkin 1977).
Streptomycetes and Their Allies
Some Streptomyces may be useful. Streptomyces produce extracellular
exgalactosidase (Lyons, Pridham, and Hesseltine 1969). According to
these workers, this enzyme may be useful during beetsugar processing
(aids in crystallization of sucrose) and be used for determining raffinose
and fot eliminating the agent in beans that induces flatulence. Streptomyces
was suggested as a possible source of singlecell protein, protease en
zymes, and glucose isomerase.
The Rickettsias and Chlamydias
In this grouping, the family Rickettsiaceae includes the genus Coxiella.
COXIELLA. These cells are short rods, occasionally appearing as diplo
bacilli or spheres. There are no flagella or capsules. The cells are Gram
negative, but under certain conditions they may appear to be Gram posi
tive. They are resistant to chemicals and elevated temperatures that nor
mally inactivate Rickettsia species. These organisms will not grow on agar
media but require host cells.
Since these organisms do not grow outside a host cell, they are not
important in food spoilage. However, food can serve as a carrier of the
organisms so that they may infect humans. Coxiella burnetii is the causative
agent of Q fever. Infected cows, sheep, and goats shed the organism in
their milk, which is the food source for human infection if raw milk is
consumed. This organism is more heat resistant than the vegetative cells
of most pathogens. Therefore, the time and temperature for pasteuriza
tion of milk are determined primarily to destroy these organisms. Heat
ing at 62.SoC for 30 min or 71.7C for 15 sec is adequate to eliminate C.
burnetii from milk.
MOLDS
Molds are part of a larger group of microorganisms called fungi. The
exact number of fungi is not known. Some fungi have not been isolated
and identified, while others have been given more than one name. The
fungi are ubiquitous, but soil, air, water, and decaying organic matter are
prime sources.
Fungi are heterotrophic organisms that lack the definite root, stem,
or leaves of higher plants. They possess a thallus and are called thallo
phytes. They are differentiated from the algae and higher plants by their
lack of chlorophyll. Hence, they are saprophytic or parasitic. They differ
from bacteria by their more complex structure and greater size. The
fungi may be multicellular or unicellular.
The fundamental structural units of molds are filaments or tubes
called hyphae. By formation of crosswalls or septa, some hyphae form
chains of cells that are septate. Others may not form septa and the hy.
phae are nonseptate or coenocytic. The septa have pores that allow the
movement of cytoplasm from one cell to another. As the hyphae elon
gate, they intertwine. A mass of these intertwined branched hyphae is
called a mycelium. Part of the mycelium grows into the substrate and
absorbs food. This is known as the vegetative mycelium. The mycelium
that remains in the air above the substrate and bears spores is called the
aerial or reproductive mycelium. When the spores find a proper sub
strate, the cycle is repeated.
Molds can reproduce sexually, asexually, or by both systems. A fungus
that has a sexual phase is known as a perfect fungus, while one that has
no sexual phase is an imperfect fungus.
There are several types of spores formed by the fungi. The asexual
fungi produce spores directly from or by the mycelium. These are called
thallospores, conidiospores, or sporangiospores. There are three types
of thallospores: blastospores, chlamydospores, and arthrospores. Sexual
spores include oospores, zygospores, ascospores, and basidiospores.
Enumeration
With bacteria, one cell is usually responsible for the growth of a col
ony. However, a mold colony may result from a single cell, a spore, a
piece of mycelium, or a number of cells. Thus there is a poor onetoone
relationship for molds. The problems involved with estimating growth
by evaluation of the mycelium were discussed by Calam (1969) and Sut
ton and Starzyk (1972).
In the past, media for enumerating molds and yeasts were acidified to
pH 4 or 5. The low pH inhibited bacteria and allowed the fungi to grow.
However, fungi that have experienced a sublethal treatment might not
grow on an acidified medium. Hence, media incorporating antibiotics
rather than acidification to inhibit bacteria have been developed and
tested for fungal enumeration (Baggerman 1981; Beuchat 1979; Henson
et al. 1982; Mossel, Vega, and Put 1975; Nelson 1972). Dichloran has been
added to media to inhibit the spreading of mold colonies (Henson 1981).
The addition of sodium thiosulfate and sodium tetrathionate to media
reduced the effect of heavy metal toxicity on the growth of fungi (Wain
wright and Grayston 1983). Higher fungal counts were obtained using
the spiral plater than the pour plate method (Zipkes, Gilchrist, and
Peeler 1981). Microscopic methods are used to enumerate mold filaments
in tomato products as well as in other canned fruits and vegetables
(AOAC 1985; Bandler and Cichowicz 1981). Determining the fungal
chitin by conversion to glucosamine is useful in estimating fungal my
celia in tomato products (Bishop et al. 1982). These and other methods
used for fungal analysis were reviewed by Jarvis et al. (1983).
Fungi in Food
Several types of fungi have been isolated from food. The extent of
contamination is influenced by the prevalence in the environment. Peni
cillium and Aspergillus enjoy favorable conditions throughout the year,
while some other fungi are limited to warm temperatures. Cladosporium
and Alternaria are prevalent during the summer and early autumn.
Although important in all types of foods, molds are more apt to cause
spoilage or be a health hazard in foods such as grain, flour, nuts, or fruit,
which do not support the growth of bacteria due to low water activity or
low pH.
Throughout the world, fungi rank second only to insects in causing
the loss of stored products (Christensen and Kaufman 1974). In countries
that have implemented rodent and insect controls, fungi destroy more
stored products than does any other agent.
Importance of Molds
We usually consider that the appearance of mold on a food is an
indication of spoilage. However, even before growth is evident to the
naked eye, these organisms can be working to cause degradation of
foods.
Although molds associated with food are not considered to be patho
genic, some produce mycotoxins. These toxic substances pose a potential
health hazard to humans.
Molds can be useful in the processing of many foods such as cheese.
Their enzyme systems can be isolated and used in many food processes.
Some fungi are used to convert wastes into usable food (protein) for ani
mal food or human use. Also, they are a source of vitamins.
The production of antibiotics by molds is well known. Antibiotics
have been of great value in medicine, and they have been investigated
for use as food preservatives.
Obviously many inert molds are associated with foods. They simply
cannot grow due to an unsatisfactory environment or the overgrowth by
bacteria that cause spoilage before the molds get a chance to multiply.
Zygomycetes
These fungi produce asexual spores (sporangiospores, arthrospores,
and conidiospores) or sexual spores (zygospores). The hyphae contain
no septa, but older hyphae may have septa.
The organisms of interest in food microbiology are in the order Mu
corales. The family Mucoraceae include the genera Mucor and Rhizopus.
The family Thamnidiaceae includes the genus Thamnidium.
MUCOR. Like other dimorphic fungi, species of Mucor can develop as
either individual spherical cells that multiply by budding (as yeasts), or
they form typical mycelia of coenocytic hyphae (Fig. 3.6). In normal aero
bic conditions, growth is usually filamentous. Stoloniferous growth does
not occur in mucor, which differentiates this genus from Rhizopus.
Organisms in this genus occur in soil and manure, and in fruits, vege
tables, stored grain, and other foods. Mucor species are used in the Orient
in food fermentations. M. pusillus produces an extracellular protease that
has milk clotting activity. Mucor is a spoilage organism of berries.
Figure 3.6. Hyphae and sporangia of Mucor.
Courtesy of Continental Can Co.
RHIZOPUS. These organisms have coenocytic mycelia and spread by
stolons. Usually they reproduce asexually with sporangiospores or ar-
throspores_
These are common spoilage organisms of various stored foods. Rhizo-
pus stolonifer is the common bread mold. Pectinolytic enzymes are se-
creted by Rhizopus species. The degradation of pectin results in the soft
rot of various plant products. Due to the heat stability of the pectinolytic
enzyme, the infection of fruit before heat processing can result in post-
process softening.
Rhizopus produces high yields of fumaric acid from fermentable
sugars. Species are used in the production of fermented foods such as
tempeh.
THAMNIDIUM. Organisms in this genus have coenocytic mycelia.
They are found on meat as well as on soil and animal excrement. They
grow on refrigerated meat, causing a defect referred to as whiskers. A
process using T. elegans to tenderize meat was patented by Williams
(1957). However, Kotula, Campano, and Kinsman (1982) found that the
lipolytic and proteolytic enzymes of this organism had little effect on
meat at 4C, the typical temperature at which beef is held in coolers.
Ascomycetes
Both molds and yeasts are present in this class of fungi. Ascomycetes
develop sexual ascospores in a saclike structure called an ascus. Besides
this sexual phase, they may grow an extension of the hyphal tip or have
an imperfect state in which asexual spores are produced. Those fungi for
which no perfect state has been observed are listed in Deuteromycetes
(Fungi Imperfecti). When a sexual phase is found for a fungus in Fungi
Imperfecti, it is given a name according to the structure and form of the
perfect state. This name takes precedence over the imperfect, asexual
state.
There are between 1,900 and 2,000 genera in Ascomycetes. Only a
few are of importance in foods.
BYSSOCHLAMYS. Besides asexual reproduction, the two species of
importance (B. fulva and B. nivea) sexually produce heat-resistant as co-
spores. These organisms can grow at relatively low pH levels and in re-
duced oxygen tension. They produce strong pectolytic enzymes. With
this combination of properties, they have been implicated in the spoilage
of canned fruit and fruit juice. Spoilage mayor may not be accompanied
by gas. If gas is formed, the can may bulge slightly.
Besides spoilage, B. fulva produces a mycotoxin that is toxic to brine
shrimp, chicken embryos, and rats (Kramer, Davis, and Diener 1976).
Chu, Nei, and Leung (1973) studied a renninlike enzyme (byssochlamyo
peptidase A) that might be useful in milk clotting for cheese manufac
ture.
CLAVICEPS. The species Claviceps purpurea is of interest to food micro
biologists because it produces toxic alkaloids on cereals. These contain a
tetracyclic ring called lysergic acid. When ingested, these alkaloids cause
numerous symptoms, especially hallucinations. With improved grain
handling, the illness is rare in humans. The last major outbreak was in
1951.
NEUROSPORA. The name Neurospora is derived from the characteristi
cally ribbed ascospores. The ascospores of N. crassa remain viable for
many years. Heat shock for 20 min at 60C aids in the germination of
the spores.
In asexual reproduction, branched chains of pink conidia develop
from upright stalks. Budding of the terminal conidia of the chain pro
duces further conidia. When the terminal conidia form two buds, the
chain branches.
Neurospora crassa and N. sitophila are two wellknown species of this
genus. Wild type strains of Neurospora have simple nutrient requirements.
They have been used as tools in genetics and biochemical research. Neuro
spora can be found in warm, humid environments. N. sitophila causes
problems in bakeries and is the red or pink bread mold. N. sitophila is
used in the fermentation of red or orange ontjom, a fermented peanut
press cake used in the Orient.
Deuteromycetes
This heterogeneous group of fungi has branching, septate hyphae
and reproduces asexually by conidia or sclerotia. There are various sys
terns used to classify the Fungi Imperfecti. Rather than attempt to segre
gate them, those of importance in food microbiology are discussed in
alphabetical order.
ALTERNARIA. These organisms are characterized by muriform, dark
colored spores. The aerial mycelia are described as wooly, gray, brown,
and olivegreen (Fig. 3.7).
Alternaria is one of the most prevalent of the molds that cause spoilage
of tomatoes in the field, attacking injured or weakened tissue. Due to
darkening of the tissues, the defect is called black rot. Late-harvested to
matoes are particularly susceptible. These organisms cause defects ofvar-
Figure 3.7. Hyphae and spores of Alternaria, a cause of rot of tomatoes.
ious plant products, including internal mold of peppers (HalfonMeiri
and Rylski 1983). Also they are involved in the development of rancid
flavors in dairy products.
A. alternata produces the enzyme (3'D-galactosidase which, according
to Macris (1982), is useful in reducing the lactose level in dairy products.
The Alternaria may be a health hazard because it produces mutagenic
substances (Scott and Stoltz 1980).
ASPERGILLUS. There are more than 100 species in this genus. These
organisms, along with penicillia, are known as storage fungi of grain.
They discolor infected grain and reduce or destroy germination of the
seed. These organisms can cause spoilage of a wide range of food prod-
ucts.
Certain aspergilli are very useful in food microbiology. Species such
as A. oryzae are used to break down rice starch to glucose in the manufac-
ture of sake and similar alcoholic beverages. Some strains are used in the
production of shoyu (soy sauce) and miso (bean paste).
Strains of Aspergillus are used in the commercial production of citric,
gluconic, and gallic acids. Almost all of the commercial citric acid is pro
duced by A. niger growing in sucrose solutions. The organisms are a
source of amylase and pectinolytic enzymes. A proteolytic enzyme of
Aspergillus is able to clot milk and might be a substitute for rennet in
cheesemaking.
These fungi may be useful as a source of protein. The mycelium of
A. niger grown on brewery wastes contained 29 percent crude protein
and might be used as a feed supplement (Hang, Splittstoesser, and Wood-
ams 1975). Reade and Gregory (1975) described the use of A. fumigatus to
convert the starchy root cassava to microbial protein for food or feed.
Several species of Aspergillus produce substances that are toxic to
other biological systems Strains of A. jlavus and A. parasiticus produce
aflatoxins. There is considerable information that implicates aflatoxins
with human illness.
The aspergilli are common contaminants of organic materials and
soils. They are found on fruits, vegetables, stored grain, peanuts, and
other food products. Aspergillus is shown in Figure 3.8.
BOTRYTIS. The asexual form of Botrytis reproduces by conidia. The
conidiophores develop from a sclerotium and are irregularly branched.
The conidia occur on short sterigmata. The positions and numbers of
sterigmata cause the conidia to appear in grapelike clusters (Fig. 3.9).
B. cinerea is the common species of Botrytis. It is the gray mold of var-
ious plants and plant products, especially lettuce, tomato, strawberry,
raspberry, and grape. It can be classed as a field mold, since it is a com-
mon soil contaminant and attacks fruits and vegetables in the field. It
enters fruits through cracks and injured areas.
CLADOSPORIUM. These organisms are common in the soil. They can
grow on connective tissue or the fat covering of meat when it is refriger
ated for several days. This results in black spots on the meat. C. carpophi-
tum causes peach scab, numerous dark circular lesions on the fruit. The
organisms are associated with stored grains and dairy products.
COLLETOTRICHUM. Molds in this genus are involved with spoilage
of foods. C. circinans causes onion smudge. Colored onions are resistant.
C. phomoides causes anthracnose rot of tomatoes. Invasion by this or-
ganism is not dependent upon an injury to the fruit.
FUSARIUM. The conidia produced by these organisms have various
shapes. The colonies may be fluffy and spreading, with colors varying
Figure 3.8. Aspergillus, showing mycelia and conidial heads.
from white, through shades of pink, red, brown, yellow, orange, and blue,
to purple.
The fusaria are widespread in nature, being found in soil, decaying
material, and foods. Some fusaria are associated with plant diseases. A
disease in rice due to F. moniliforme led to the discovery of gibberellic acid,
a plant growth stimulant.
Fusaria cause tomato rot, entering the fruit through insect or other
damage of the skin. F. solani causes a decay of potatoes called powdery
rot, dry rot, or white rot.
The fusaria produce mycotoxins that affect various animals and possi-
bly human beings. Animals refuse to eat feed that is highly infected with
fusaria.
GEOTRICHUM. This yeastlike fungus grows rapidly at room temper-
ature, forming white to cream-colored colonies. The septate, branching
Figure 3.9. Mycelia and spores of Botrytis.
hyphae fragment into chains of rectangular, barrelshaped, or spherical
arthrospores that readily break apart. These arthrospores are a means of
reproduction. Geotrichum is depicted in Figure 3.10.
Geotrichum is a spoilage organism. It has been called dairy mold, be-
cause it is found growing on dairy products. It also causes watery rot, a
common spoilage of tomatoes.
G. candidum is called machinery mold because it will grow on
equipment on which are attached food particles or juices . .As the food
product is processed, it becomes contaminated with the mold. Hence, the
mold is found in many types of processed foods. The mold is killed by
heat used in thermal processing of food, but the hyphae can be deter-
Figure 3.10. Geotrichum-machinery mold.
courtesy of Continental Can Co.
mined by microscopic examination. The presence of these mold fila
ments in processed foods has been considered to be an adulterant and
an indication of inadequate sanitation in the processing plant. However,
Splittstoesser et al. (1980) found little correlation between the aerobic
plate count and the incidence of the mold in frozen blanched vegetables.
PENICILLIUM. There are many species of Penicillium. They are closely
related to the aspergilli. The penicillia are characterized by branching
of the conidiophore to form a brushlike conidial head (Fig 3.11). The
organisms in this genus can be divided into three groups on the basis of
the type of conidiophore branching. The conidia may be white, green,
graygreen, bluegreen, or yellowgreen.
These organisms are widely distributed in nature and are found on
many foods. They are important spoilage organisms of various fruits.
This is one of the storage fungi of grain. The species most often en
countered are P. cyclopium and P. viridicatum. This is probably because they
are able to grow at relatively low levels of moisture and temperature.
Figure 3.11. Mycelia and conidial heads of Penicillium.
P. martensii and P. viridicatum are associated with blue-eyes, a blue-green
discoloration of corn germs. Species of Penicillium are found growing on
the fatty layer or connective tissue of meat that is stored in a refrigerator
for several days, and on moldy bread.
Species of Penicillium are useful in various ways. Antibiotics produced
by penicillia include penicillin_ Some species are used in cheese manu-
facture. P. camemberti and P. caseicolum are important in Camembert, Brie,
and similar cheeses, while P. roqueforti is used in Roquefort, Gorgonzola,
and blue-veined cheeses. Schwimmer and Kurtzman (1972) suggested us-
ing P. crustosum to remove caffein from coffee. The penicillia produce
enzymes such as glucose oxidase as well as proteins that can be used by
the food industry.
Certain species of penicillia can be a health hazard_ Some species
have been associated with pulmonary and urinary tract infections. Peni-
cillia produce mycotoxins_ P. islandicum, P. citrinum, and P. citreoviride were
involved in yellow rice disease which caused the deaths of several people.
Although P. roqueforti is used in cheese processing, toxin-producing
strains of these species have been isolated (Scott et al. 1977), and roque-
fortine, a neurotoxin, has been isolated from blue cheese (Scott and
Kennedy 1976).
SCOPULARIOPSIS. This genus produces conidia in brushlike clusters
similar to Penicillium. The colonies appear cottony and may be cream
colored, yellow, brownish, chocolate brown, or almost black. They are
never green like the Penicillium. One characteristic of S. brevicaulis is that
it produces a poisonous gas (diethylarsine) with a garliclike odor when
grown in the presence of arsenical compounds.
Scopulariopsis is found on all types of decaying matter and grows well
on highprotein substrates.
Being proteolytic, Scopulariopsis is involved with the development of
offflavors in dairy products (especially Camembert cheese), and spoilage
of meat. It has been detected on ham (both on the surface and in deeper
tissues), stored eggs, and peanuts.
SPOROTRICHUM. The colonies are usually white but may be yellow,
gray, pink, red, or green. It is a common soil inhabitant. S. carnis grows
at low temperatures (- 5 to - SoC) and can cause a defect, called white
spot, of refrigerated meat.
S. thermophile has an optimum growth temperature near 40C. It pro
duces cellulase and has been suggested for use in converting cellulose to
simple carbohydrates (Coutts and Smith 1976). The use of S. pulverulen-
tum as a potential food source was studied by Eriksson and Larsson
(1975).
TRICHODERMA. This genus has irregularly branched conidiophores.
The conidia are produced in slime balls.
Species are common in soil and on organic matter. The nitrogen con-
tent of the cells is about 5 percent, of which 60 to 70 percent is protein.
Since the species T. viride is cellulolytic and has a high protein content,
it has been considered for converting cellulosic wastes into foods.
YEASTS
The yeasts have been defined as fungi in which the usual dominant,
or conspicuous, form is unicellular. The unicellular form gives yeasts an
advantage over the mycelial form of molds. There is a greater surface-to-
volume ratio that allows a higher metabolic activity. Also, the unicellular
form is more readily distributed than is the mycelial form. Some yeasts
produce a true mycelium by fission of cells that remain attached. A
pseudomycelium, formed by budding, has constrictions between the
cells.
There are some 350 species recognized and classified into thirtynine
genera (Kregervan Rij 1969; Lodder 1970). The classification of yeasts is
based on morphological, cultural, sexual, and physiological characteris
tics.
On the basis of method of reproduction, the yeasts can be separated
into four groups. Only two of these groups contain yeasts involved with
foods. One group produces sexual ascospores in asci and is in the class
Ascomycetes. These are the so called true yeasts. The yeasts in the other
group form no sexual spores and have no sexual life cycle. These are the
false yeasts, Fungi Imperfecti, or Deuteromyces. All yeasts can reproduce
asexually, and this is the only method for about 50 percent of them. The
usual asexual (vegetative) reproduction is by budding (Fig. 3.12). Some
yeasts reproduce by fission or by an intermediate system called bud fis
sion. If the new cell or bud appears at the short end of the mother cell,
it is called polar budding. If a bud forms at both ends, it is bipolar bud
ding. The appearance of buds any place on the mother cell is multilateral
budding.
Nearly all of the yeasts that produce sexual ascospores and are associ
ated with food are in the class Ascomycetes, order Endomycetales, and
family Saccharomycetaceae. For most yeasts, the maximum number of
spores per ascus is four, but a few yeasts can produce eight. Strains of
Kluyveromyces may produce a large number of spores (up to 100) per as
cus. These ascospores have many shapes, including spheroidal, ovoid,

Figure 3.12. Cells of Saccharomyces cerevisiae, showing budding.
Courtesy of Amerine, Berg, and Cruess (1979).
reniform, elliptical, bean-shaped, cylindrical, and needle-shaped. Some
spherical or oval spores have a ledge in the middle or to one side, which
makes them appear saturn-shaped or helmet-shaped.
The production of ascospores may playa role in survival or in adapta-
tion to altered environmental conditions. However, the main purpose is
the rearrangement of heritable qualities.
The vegetative cells may have shapes similar to those of ascospores.
Young yeast cells may be small and spherical and, as they grow older and
larger, they may assume other shapes. The older cells become misshapen
and scarred due to budding. Aged cells tend to become shriveled.
Some molds are dimorphic and have a yeast-like phase. Some yeasts
produce a pseudomycelium or true mycelium. Hence, these organisms
may be confused when a culture is examined, especially for borderline
genera. The mold Geotrichum resembles the yeast Trichosporon. They both
form a true mycelium and arthrospores. However, Trichosporon can repro-
duce by budding.
Most yeast cultures are cream, tan, or gray. However, some are yellow,
pink, red, green, or brown.
The physiology of yeasts was discussed by Kreger-van Rij (1969) and
MacMillan and Phaff (1973). Information about fermentation, assimila-
tion, and nutritive requirements aids in identification of yeast species.
This information was compiled by Lodder (1970) and is too extensive to
repeat. The heat resistance of various yeasts has also been documented
(Put et al. 1976; Put and De Jong 1982)_
Yeasts are associated with nearly all types of food products. Foods
such as fresh vegetables, meat, poultry, and cheese often contain yeasts,
but in these foods, bacteria usually outgrow the yeasts. When bacterial
inhibitors are added, yeasts can dominate. Osmophilic yeasts such as Sac-
charomyces rouxii can tolerate high sugar concentrations. These yeasts are
found in foods such as honey, molasses, sugar, and fruit. Salt-tolerant
yeasts grow as films on brined food and on salted food and ham.
Certain strains of yeasts are very important in the baking industry, in
chemicals, in single-cell protein, in sewage disposal, and in the fermenta-
tion of alcoholic beverages.
Methodology
Sublethally stressed yeast is recovered at maximum levels with a me-
dium at pH 8.0 or over (Nelson 1972). Therefore, the use of potato dex-
trose agar (PDA) acidified to pH 3.5 is not satisfactory for enumeration
of yeasts in many food products. To inhibit bacteria and to allow yeasts
to grow, a combination of antibiotics is used in place of acidification.
Fluorescent microscopy can be used to detect yeasts in certain products
(Andrews 1982; Aries 1976; Cranston and Carver 1974).
Classification
In this text, the genera and species of yeasts are those listed in Lodder
(1970). As more information accumulates regarding the relationships of
yeast such as DNA base composition and serological reactions, there may
be some revisions in the classification (Price, Fuson, and Phaff 1978).
The yeasts are divided into Ascomycetes (the ascosporogenous yeasts)
and Deuteromycetes (the asporogenous yeasts).
ASCOMYCETES. These yeasts produce sexual ascospores as well as reo
produce asexually. Unless otherwise noted, asexual reproduction is by
budding. Certain genera such as Citeromyces, Dekkera, and Endomycopsis
have been isolated from food and may be involved with spoilage or fer
mentation. Due to space limitations, these are not discussed in this text.
Debaryomyces. The vegetative cells are often spherical to globose and reo
produce by multilateral budding. The sexual ascospores, produced by
conjugation of the mother cell and bud, are spherical or oval. There are
usually one or two spores per ascus. Fermentation is weak, slow or absent.
Nitrate is not assimilated.
D. hansenii has a high salt tolerance, being able to grow in media with
18 to 20 percent salt. It has been isolated from brined food and salted
meat products and can use nitrate as the sole source of nitrogen. Debaryo-
myces species are perhaps the most widely distributed film-forming yeasts
associated with food brines.
Species of Debaryomyces have been associated with spoiled mushrooms,
cheeses, cider, white wine, tomato puree, sausage, and salted beans.
Hanseniaspora. These yeast cells are diploid, lemon-shaped, ovoid, or long
ovoid. They are the ascospore stage of the genus Kloeckera. They have a
vigorous fermentation, but have a low tolerance to alcohol (about 4 to 6
percent). All species require inositol and pantothenate for growth. Due
to this need, they can be used to assay for these compounds.
These yeasts are found in the soil of orchards and vineyards and in
all insects. They have been isolated from grape berries and grapes and
have been associated with fermenting spoiled fruit.
Hansenula. This genus reproduces by multilateral budding and asco-
spores. A pseudomycelium or a true mycelium may be formed. These
yeasts assimilate nitrate.
Species of Hansenula have been isolated from foods such as grain,
fruit, shrimp, and brines of fermenting cucumbers and olives.
Some species and strains are useful. According to Batra and Millner
(1974), certain species are used in India to produce kanji (a beerlike bev
erage) and murcha (rice wine). A strain of Hansenula grows on methanol
to produce singlecell protein (Levine and Cooney 1973).
Kluyveromyces. These organisms reproduce by multilateral budding. The
cells may be spherical, ellipsoidal, cylindrical, or elongate. Some species
produce a red pigment, but the colonies are grayishwhite, brownish
cream, yellowish gray, sometimes tinged with pink. The organisms show
a vigorous fermentation. They grow at temperatures of from 5 to 46C.
This genus has a widespread distribution. They are found in various
foods such as fruits, preserves, corn meal, grape must, milk, and other
dairy products. The species that ferment lactose, such as K. lac tis, are
found in dairy products. Some species are osmophilic. Organisms of this
genus cause spoilage of foods, especially figs and dairy products. Certain
strains have been suggested as a source of the enzyme polygalacturonase.
Pichia. The cells have many shapes. Asexual reproduction is by multilat
eral budding, with most species forming a pseudomycelium. There is lim
ited formation of a true mycelium.
The colonies are slimy or pasty and colored yellowishwhite, yellowish
brown, tan, white, cream, grayishwhite, or red.
These are common yeasts found in various sources. Their association
with foods includes both fermentations and spoilage. Foods in which
these yeasts are found and can cause spoilage include fruit, beer, dairy
products, sorghum, and wine. They can occur as films on brined foods.
Saccharomyces. This genus is considered to be a rather heterogeneous
group of organisms. The cells are spheroidal, ellipsoidal, cylindrical, or
elongate. Vegetative reproduction is by multilateral budding. Pseudo
mycelia may be formed, but true mycelia are absent. On agar, the colon
ies are generally white or cream colored, with a typical yeasty odor. Co
wan and Bryant (1981a, 1981b) developed a serological classification of
brewing yeasts using a microimmunoelectrophoresis technique.
The name Saccharomyces means sugar fungus. All species have a vigor
ous fermentation. Some species are among the most useful yeasts, due to
the production of alcohol (brewing) and carbon dioxide (baking). Strains
of S. uvarum and S. cerevisiae are most often used in these fermentations.
Besides these important uses, species and strains have been used to reo
move glucose from egg white prior to drying, to remove the mucilage
layer from coffee beans during processing, to assay for vitamins (biotin,
pyridoxine, pantothenic acid, thiamin), as a source of enzyme (invertase),
as autolyzed yeast extract for flavoring in place of meat extracts, as a
source of ergosterol, and as a source of singlecell protein.
Sinai et aL (1974) fed brewer's yeast (S. cerevisiae) to Rhesus monkeys
and found that the yeast significantly enhanced the monkeys' resistance
to respiratory and enteric infections. They believed the resistance was
due to the stimulation of phagocytes.
The organisms in this genus have a widespread distribution. They are
associated with a variety of foods and can cause spoilage in fruit and fruit
products, sugar, syrup, honey, vinegar, mayonnaise, salad dressing, dairy
products (buttermilk, cheese), and fermenting foods such as cucumbers
for pickles.
S. aceti, found in wine, can convert alcohol to acetic acid. S. bailii var
osmophilus is found in substances with a high concentration of sugar, alco
hoI, sulfur dioxide or acetic acid. S. bailii var bailii has been associated
with spoiled wine, vinegar, salad dressing, and mayonnaise. Two sugar
tolerant (osmophilic) species, S. rouxii and S. bisporus, are the main spoil
age organisms of dates and can cause spoilage of honey, syrup, and other
foods with up to 60 percent sugar (Restaino et aL 1983).
Occasionally, species of Saccharomyces exhibit pathogenic behavior
(Eschete and West 1980).
Schizosaccharomyces. The cells are spheroidal to cylindrical. Vegetative reo
production is by fission. The absence of budding distinguishes this genus
from other yeasts. The cells may form a rudimentary true mycelium that
can break up into arthrospores. There are four to eight spores per ascus.
The name Schizosaccharomyces indicates the close relationship to Sac
charomyces (fermentation of glucose to alcohol), but also a difference (fis
sion as compared to budding).
As with other yeasts, these species are widespread. They have been
associated with spoilage of prunes, figs, raisins, and wine.
Yang (1973) suggested using S. pombe in the fermentation to produce
wine from grapes with a low pH. The yeast converts malic acid to lactic
acid, resulting in a less acid wine than when Saccharomyces cerevisiae is
used. S. malidevorans is noted for its ability to convert malic acid to lactic
acid.
DEUTEROMYCETES. These yeasts do not produce sexual spores. Un
less otherwise stated, these asporogenous yeasts reproduce by budding.
Certain genera, such as Brettanomyces and Kloeckera, are found in foods
and may be involved in spoilage or fermentation.
Candida. This is a rather large genus of yeasts. All species form pseudomy
celia; by fission, some form true mycelia.
Organisms in this genus are widespread. Besides being found in soil,
water, and air, they are found in plants, insects, higher animals, humans,
sewage, on processing equipment, and in food products.
These organisms have been involved with spoilage of various foods
such as frankfurters, fresh fruits, vegetables, dairy products, brines, and
alcoholic beverages. C. vini (formerly Mycoderma vini) forms "flowers" on
wine and causes spoilage. C. mycoderma is a film forming yeast on ferment
ing olive and cucumber brines. The psychrophilic strains tend to become
dominant in refrigerated fruit juices.
This genus contains strains of yeast that are very useful in food indus
tries and in associated industries. They are used as food yeasts, a source
of lipid, vitamins, invertase, lactose, and lysine. C. utilis is used as a fodder
yeast for animals and is a potential food yeast for humans.
There are potentially pathogenic species of Candida for both people
and animals. However, there is no information that food is the source of
these infective agents. Candida species are found in the intestinal tract,
and it has been suggested that they may cause diarrhea in malnutrition
(Gracey et al. 1974).
Rhodotorula. The cells (spheroidal, ovoidal, elongate) reproduce by multi
lateral budding. There may be a rudimentary pseudomycelium. The orga
nisms form red or yellow carotenoid pigments. These organisms are
widespread and are involved in the spoilage of a wide range of foods.
Species of this genus may be useful as a source of lipids, cystine, and
methionine and for degrading wastes in the production of singlecell pro-
teins.
Torulopsis_ Torulopsis has been defined as a heterogeneous group of imper-
fect yeasts that cannot be placed in the homogeneous genera. The cells
are spherical or oval to elongate and reproduce by multipolar budding.
Torulopsis can be differentiated from Candida by not being able to form
a pseudomycelium, from Cryptococcus by not producing starch, and from
Rhodotorula by not producing carotenoid pigments.
The colonies are usually white to cream and are usually glossy or
shiny but may be dull. The tolerance to sodium chloride varies from 2
percent to 21 percent (W/v) for different species_ At 30C, T. halonitrato-
philia is an obligate halophile. There are osmophilic species in this genus.
Species of Torulopsis are found in various types of foods. They are
credited with causing surface slime on cottage cheese, spoilage of refrig-
erated beef, cream, butter, sweetened condensed milk, and various food
brines_
Torulopsis magnoliae has been suggested for use in the production of
glycerol. T. ernobii is a source of lipase. T. utilis is a source of single-cell
protein. Torulopsis strains have been found in sourdough, apparently aid-
ing in the leavening of this product.
Trichosporon. The cells of Trichosporon species are of various shapes.
Pseudomycelia and true mycelia may be formed. The presence of ar
throspores differentiates this genus from Candida.
These organisms are found on various foods such as fresh shrimp,
crab, butter, cheese, fruit, fruit juice, and rice. They are part of the flora
used in the production of idli (Batra and Millner 1974), and may be a
source of protein.
VIRUSES
Viruses are obligate intracellular parasites. They generally have a lim
ited host range. However, nearly all forms of life are susceptible to some
type of virus. To survive and replicate, a virus must contact and invade an
acceptable host cell. If a host cell is not available, the virus may become
inactivated. This inactivation is influenced by temperature, humidity,
pH, and substrate composition as well as other factors.
There is some doubt that viruses are alive. Cliver (1971) stated that
they are not alive, but borrow life from the host cell and direct it to
produce more viruses.
Viruses have a genetic system with either RNA or DNA. This is sur
rounded by a protein coat, called the capsid. The capsid protects the
genetic material from nucleases, serves as the vehicle of transmission
from one host cell to another, and forms a bond with the host cell during
the attachment stage of infection.
For a virus to infect a host cell, there must be an attachment site on
the host cell. The attached virus injects its nuclear material into the host.
This can result in the viral genetic material causing the cell to manufac
ture new viruses, or the genetic material may attach to the cellular chro
mosome and the cell becomes lysogenic. If new viral particles are pro
duced, usually the cell is lysed upon release of the new viruses. Then
these viruses must contact another acceptable host.
If the virus material becomes attached to the chromosome of the cell,
the genetic material of the virus is reproduced along with that of the
cell (Campbell 1976). This lysogenic state may continue for an indefinite
period. Eventually some disturbance triggers the viral genetic material to
become activated, and the cell produces new viruses, which are subse
quently released.
Although the viruses are inactive as far as the reaction on food is
concerned, they are important. Since they are obligate parasites, and
some cause disease in human beings, the presence of viruses can be con
sidered potentially harmful. They can be put into the category of an envi
ronmental condition that allows spoilage, since they, as bacteriophages,
can attack useful fermentative organisms, resulting in an unsatisfactory
product being developed. Since they are involved with genetic transfer
(transduction), the viruses might be considered helpful in the develop
ment of better strains of useful microorganisms. However, transduction
may be undesirable when it results in unacceptable mutant strains that
are less sensitive to inhibitors, are more virulent, or lose the characteris
tics that made them useful. Transduction is known to occur in only a few
groups of bacteria.
Intestinal Viruses
The primary viruses that can infect people and are foodborne are
the intestinal or enteric viruses. Cliver (1971) included the enteroviruses,
adenoviruses, reoviruses, and the agents that cause infectious hepatitis
and epidemic viral diarrheas as intestinal viruses.
The enteroviruses are spherical and about 20 to 30 nm. They have
singlestranded RNA. These viruses are stable at pH 3.0. This tolerance
to low pH allows them to survive passage through the stomach. They
replicate in the intestinal tract, are shed in the feces and, if they contami
nate food that is subsequently ingested, the process is repeated. Wilner
(1973) included poliovirus, coxsackievirus A, coxsackievirus B, and echo
virus as enteroviruses.
The adenoviruses are hexagonal, about 70 to 90 nm in diameter. They
contain doublestranded DNA. They are acid stable. There are thirty
three serotypes of adenoviruses that infect human beings (Wilner 1973).
The reoviruses are hexagonal, and about 70 to 80 nm in diameter.
They contain double stranded RNA. The reoviruses, like the other intesti
nal viruses, are stable at pH 3 to 5.
The agents that cause infectious hepatitis have been described (Prov
ost et al. 1975). They are spherical, about 27 nm in diameter. Viral diar
rheal agents have been isolated or characterized. However, these agents
pass through bacterial filters and can be transmitted through several
people and still cause diarrhea. If the agent were merely a chemical toxin,
this transmission would dilute it beyond the ability to cause illness.
Since these viruses are intestinal, they are found in feces of infected
persons and resultant sewage. Present sewage treatment does not inacti
vate all of the viruses from the effluent or the sludge (Goddard, Bates,
and Butler 1982; Irving and Smith 1981; Larkin, Tierney, and Sullivan
1976). When sewage wastes are used to irrigate or fertilize crops, the vege
tation is contaminated. Larkin, Tierney, and Sullivan (1976) found that
poliovirus persisted on radishes and lettuce for up to thirtysix days. Such
foods are eaten raw, so any contaminating viruses that are not removed
by washing are ingested. When sewage contaminated with viruses is
dumped into coastal water, the shellfish growing in these waters tend to
bioaccumulate these viruses (Metcalf et al. 1980). Shellfish have been the
source of enteric viruses in several outbreaks of gastroenteritis (Eyles,
Davey, and Huntley 1981).
The procedure for the detection of viruses in or on foods consists of
several parts. First, the viruses are separated from a food suspension by
differential filtration or centrifugation. Then, the viruses are concen
trated by centrifugation or by filtration. Bacterial cells are destroyed in
the viral concentrate by adding antibiotics. Aliquots of the treated viral
suspension are planted on a monolayer of susceptible host cells. After
incubation, the monolayer of cells is observed for plaques (holes) pro
duced by lysis of cells due to the viruses. The number of plaque forming
units (PFU) is determined by counting the plaques and multiplying by
the calculated dilution factor. Several modified and improved methods
for concentrating viruses from water have been suggested (GuttmanBass
and Armon 1983; Keswick et al. 1984; Lipson and Stotzky 1983; Shields,
Berenfeld, and Farrah 1985; Wait and Sobsey 1983). Absorption, concen
tration, and detection of viruses in fecal samples were described by Pon
tefract and Bergeron (1985). The methods used to concentrate and detect
viruses were reviewed by Cliver, Ellender, and Sobsey (1983a, 1983b) and
by Richman et al. (1984). Enzymelinked immunosorbent assays have
been described for the detection of viruses (DeBokx and Maat 1979; Lo
camini et al. 1979; Marco and Cohen 1979). Fortunately, the viral contam
ination of food is very low to nonexistent (Kostenbader and Cliver 1977;
Larkin 1981).
Bacterial Viruses (Bacteriophages)
The taxonomy of bacteriophages was discussed by Ackermann and
Eisenstark (1974). The phages can have at least four relationships with
their host cells. These are as follows: (1) Virulent phages cannot combine
with the host's chromosomes to establish a lysogenic relationship; (2)
pseudolysogenic phages can establish a carrier state in the host and may
act as transducing agents; (3) defective temperature phages form parti
cles with morphological attributes of a normal phage, but cannot repli
cate; and (4) true temperate phages are able to replicate and lysogenize
their host cells.
The genetic material of temperate phages can become attached to the
chromosome of the host cell. The properties of the phage are modified
and it becomes a prophage. The prophage acts as a bacterial gene, divid
ing the bacterial chromosome and being transmitted to daughter cells. A
bacterium that carries prophage is called lysogenic. Agents such as UV
light, Xrays, organic peroxides, nitrogen mustard, and mitomycin C can
cause the release of phage material from the chromosome, with resultant
production of phage and lysis of the cell when the phage is released.
Bacteriophages can be stored by freezing with liquid nitrogen, using or
not using glycerol (19 percent) as a protective additive, and holding
at -196C. They can be held for about two years in a broth lysate stored
at 4C, with only slight loss of titer. Freeze-drying or aerosolization re-
sults in a loss of titer of phage (Cox, Harris, and Lee 1974). The loss
occurs during rehydration of the phage_
Bacteriophages can be enumerated by determining the clearance of
susceptible bacterial cultures in broth or by the formation of plaques on
lawns of the bacteria_ In some cases, the phages must be concentrated
prior to enumeration (Seeley and Primrose 1982)_
Perhaps the most important function of phages in foods is their role
in destroying cultures of lactic acid bacteria during the fermentation of
dairy products_
There is a possibility that phages could be used to control unwanted
bacteria in various food products. The main problems appear to be the
host specificity of the phages and the development of lysogenic bacte-
ria. The lysogenic bacteria can continue metabolizing and cause spoil-
age of foods.
Phages can be useful in epidemiological work. Since phages are
rather host specific, a set of phages can be devised and used for typing
bacterial species or strains. Phage typing can aid in tracing the origin
of contamination or infection of foodborne outbreaks of gastroenteritis.
One reason that phages can lyse an entire bacterial culture is that
they have a high rate of replication. If a bacterium has a generation time
of 20 min, in 1 hour there will be eight cells. A phage may require from
40 to 80 min for replication, but the burst size (number of phages re-
leased on lysis of cell) may be from 2 to over 100. It is obvious that
with a large burst size, in only a short time the phages will outnumber
susceptible bacterial cells_
Fungal Viruses
Lemke and Nash (1974) reviewed the fungal viruses. According to
them, viruses have been reported for more than sixty species of fungi_
They believe the metabolism and genetics of fungi may be influenced by
these viruses. All of the fungal viruses have double-stranded RNA, and
most have a polyhedral shape. In a review of viruslike particles of yeast,
Bruenn (1980) questioned referring to these agents as viruses. According
to him, no extracellular particles are present and they lack the criterion
of infectivity normally associated with viruses.
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Factors That Affect Microbial
Growth in Food
4
Food microbiologists need a knowledge of the factors that influence mi
crobial growth. Desirable growth conditions are needed for enumera
tion, fermentations, or the production of singlecell protein. Undesirable
conditions are used for food preservation.
Discussions concerning environmental conditions for growth attempt
to relate the macroenvironment of the system to the microenvironment
in which a microorganism exists. This has been necessary because little or
no information exists about the microenvironments in foods and other
products. There are many microenvironments with different conditions
in a food product. The microenvironments in a liquid food are less het
erogeneous than are those in a solid food. Some microorganisms that
could not grow in the measured macroenvironment might be able to
grow in a microenvironment of the food.
The microenvironments in food products are changing constantly.
The reactions catalyzed by enzyme systems naturally inherent in some
foods result in heat being generated, atmospheric oxygen used, and car
bon dioxide and other gases given off. These changes in the food affect
the processes of microbial systems. The environment determines the con
ditions under which the microorganisms exist, and microorganisms influ
ence the conditions prevailing in the environment. In any food envi
ronment, certain microbial species survive and become dominant.
Organisms that lack the ability to withstand stresses induced by an unfa
vorable environment will succumb. Microorganisms can respond rapidly
to changes in the growth conditions.
With optimum conditions for growth, some bacteria will reproduce
every 15 to 20 min. Although they may grow in conditions above or below
the optimum, the lag phase or the generation time may be longer (Fig.
4.1). If the stresses become too severe or increase in number, the organ
isms might not grow or might fail to survive.
The optimum conditions for microbial growth are influenced by the
enzyme systems. The enzymes are organic catalysts that increase the rate
of reactions. The rates of metabolic reactions are influenced by the envi
ronmental conditions that affect the activity of the enzymes.
stationary or
resting phase
./'
/"
/'
less than ideal ",
/
growth ",
logarithmic growth
phase / /'
./. ",/
./ ",/
/ //
. ",
./ ./
./ ."."
. .,.....
._-.,.....
Time
Figure 4.1. Bacterial growth curves. Under lessthan-optimum conditions, the lag
phase, generation time, or both may be extended.
The conditions that affect the metabolism and multiplication of mi-
croorganisms include nutrients, water, pH, inhibitors, oxygen, light, tem-
perature, and time. Since organisms are part of the environment and can
alter it, microbial interactions (the effect of one organism on another)
are important. The previous stresses (sublethal heating, freezing, or radi-
ation) that a microorganism has endured will affect its ability to cope
with the environment.
NUTRIENTS
All biosystems require certain chemicals and chemical reactions in
order to survive and reproduce. The microbial cell must be able to obtain
a source of energy and synthesize cellular protoplasm from its environ-
ment. Organisms require a source of carbon and nitrogen, growth fac-
tors, such as vitamins, minerals, and water. The ability of organisms to
utilize compounds and synthesize cellular components depends upon
the enzyme systems that the organism can make, according to its genetic
FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 103
code. Since the genetic code can change (mutate) there may be slight or
important differences between strains of a species of microorganism.
The substrate and other environmental factors affect the amount of en-
zymes produced by microorganisms. Some enzymes of microorganisms
are listed in Table 4.1 and are discussed more fully in Chapter 9.
Energy and Carbon Sources
In general microbiology a distinction is made between the types of
energy sources and carbon sources of microorganisms. The organisms of
primary concern in food microbiology are the chemoorganotrophs that
use organic compounds as their energy and carbon sources. By defini-
tion, these microorganisms are heterotrophic.
Nitrogen Source
Amino acids are needed to produce cellular proteins, including en-
zymes. A number of organisms, such as Escherichia coli, can utilize nitro-
gen in the form of nitrates or ammonia to produce amino acids. Other
organisms need one or several amino acids supplied to them in the sub
strate.
Other Growth Factors
Some organisms need growth factors, such as vitamins, purines, or
pyrimidines. For some fastidious organisms, metabolites must be sup-
plied as preformed molecules. In general, the Gram-positive bacteria reo
quire more preformed growth factors than do other microorganisms. Vi-
tamins function in coenzyme systems as listed in Table 4.2.
The obligate parasites, such as viruses, require a living organism to
supply the machinery to synthesize their components.
TABLE 4.1. SOME ENZYMES ASSOCIATED WITH MICROORGANISMS
Enzyme
Invertase
Maltase
Ribonuclease
Lactic dehydrogenase
Proteinases
Amylase
Lipases
Catalase
Urease
Substrate
Sucrose
Maltose
Ribonucleic acid
Lactic acid
Proteins
Starch
Fats
Hydrogen peroxide
Urea
Major Product
Glucose, fructose
Glucose
Nucleotides
Pyruvic acid
Peptides, amino acids
Dextrins, maltose, glucose
Glycerol, fatty acids
Water, oxygen
Carbon dioxide, ammonia
TABLE 4.2. FUNCTIONS OF VITAMINS
Vitamin
Biotin
Pantothenic acid
Folic acid
Riboflavin (B
2
)
Nicotinamide (B,,)
(Niacin)
Pyridoxine (B6)
Cob amide (Bd
Coenzyme
Biotin coenzyme
Coenzyme A
Tetrahydrofolate
Thiamin pyrophos
phate
Flavin mononu
cleotide (FMN)
Flavin adenine di-
nucleotide
Nicotinamide aden-
ine dinucleotide
Nicotinamide aden-
ine dinucleotide
phosphate
Pyridoxyl phos-
phate
Cob amide coen-
zyme, deoxyaden-
osyl (Bd
FunctionReaction
Carboxyl transfer, deamination, CO2 fixa-
tion
Acyl transfer or exchange
Transfer of one-carbon units, methylation,
purine, and pyrimidine synthesis
Accepts carboxyl groups from decarboxyl-
ation of keto acids; TCA cycle, lipid me-
tabolism
Dehydrogenation, electron transfer-oxida-
tions
Dehydrogenation
Dehydrogenation, energy generation
Dehydrogenation, hydrogen acceptor, syn-
thesis of fatty acids
Deamination, racemization, transamina-
tion, decarboxylation of amino acids
Transfer of methyl groups
It would be difficult to list the essential metabolites of specific micro-
organisms. There are shifts in the nutritional requirements of the strains
comprising a culture. Not only do species of organisms have different
essential metabolites, but strains within the species differ in their require-
ments.
Elements and Minerals
Certain elements or minerals found in cellular components are
needed in trace amounts by microorganisms. Somewhat larger amounts
of sodium, potassium, calcium, and magnesium are needed, as compared
to iron, copper, manganese, zinc, cobalt, and molybdenum.
Phosphorus and sulfur are also needed by microorganisms. Trace ele
ments enhance the activity of some enzyme systems and are needed for
the production of toxins and other secondary metabolites.
Foods as Nutrients
Foods contain carbohydrates, fats, proteins, vitamins, minerals, water,
and other factors which microorganisms need for growth. Since they are
derived from biological systems, foods vary in their composition. The
components of the soil can affect the chemical composition of plants and
the food derived therefrom. Variations in composition are also due to
geography, climate, season, maturity, processing methods, variety, and
market conditions. The type of diet eaten by an animal also can affect
the composition of the food derived from this source.
From their general composition, foods are classed as protein, carbo
hydrate, or fat. The protein foods are primarily of animal origin, the
carbohydrate foods of plant origin, and fats or oils are derived from ani
mals and plants.
Fresh meats contain essentially no carbohydrate (less than 1 percent),
since most is converted to lactic acid by glycolytic reactions during rigor.
The vitamin and mineral content of most foods is sufficient for the
growth of most organisms. The composition of foods influences the types
of microorganisms that will grow, as well as the products that are formed
during microbial growth and spoilage.
MOISTURE
Some microorganisms can remain alive in a dried condition but can
not carry out their normal metabolic activities or multiply without water.
Microorganisms can grow only in aqueous solutions. They cannot grow
in pure water or in the absence of water. Water dissolves more substances
than any other solvent. In this capacity, water is used to bring nutrients
into the cells and to dispel waste products. Water is involved in the chem
ical reactions that break down substrates to usable molecules. These reac
tions include the hydrolysis of the peptide bonds in protein, the ester
bonds in fats, and the conversion of polysaccharides to monosaccharides.
Water is a donor of hydrogen ions, contributes to the regulation of cellu-
lar pH and temperature, and is the lubricant of tissue (Beall 1983).
The water in a food is both bound and free. Bound water is held by
physical forces to macromolecules and is not available to act as a solvent
or to participate in chemical reactions. Hence, it is not available to micro-
organisms for metabolic activity.
When solutes are dissolved in water, the freezing point is lowered, the
boiling point is increased, the vapor pressure of the water is reduced,
and there is potential development of an osmotic pressure.
Water Activity
This is an index ofthe availability of water for chemical reactions and
microbial growth. Water activity (aw) is the ratio of the vapor pressure
(VP) above a material or solution (P) and that of pure water (Po) at the
same temperature. The values of water activity range from 0 to 1.
The VP of a pure liquid depends upon the rate of escape of molecules
from the surface. The escape of water to the air is measured by the equi
librium relative humidity (ERH). Thus, VP and ERR are related. When
pure water is altered by the addition of a solute, the concentration of
water is decreased and the rate of escape from the surface is diminished.
The water activity of a solution is defined in terms of VP and ERR by
the formula
Many systems have been discussed for calculating, predicting, or de
termining the water activity of various substances, including foods (Fa-
vetto et al. 1983; Ferro Fontan and Chirife 1981; Labuza et al. 1976; Nor-
tholt and Heuvelman 1982; Nunes, Urbicain, and Rotstein 1985; Stamp
et al. 1984; Teng and Seow 1981; Troller 1983a, 1983b; Wiebe et al. 1981).
Volatile compounds in a food may result in erroneous measurements of
aw when an electric hygrometer is used (Favetto et al. 1984). The a
w
of
food generally decreased as the temperature was raised from 23 to 27C
(Scott and Bernard 1983).
Water Activity and Microbial Growth
Microorganisms have a maximum, optimum, and minimum aw for
growth. Since the a
w
of pure water is 1.00, and microorganisms cannot
grow in pure water, the maximum or upper limit for microbial growth is
an aw somewhat less than 1.00. Some foods have aw values greater than
0.995, which are rounded to 1.00. However, microorganisms can grow in
these foods.
The prevention of microbial growth is of importance to food micro-
biologists. Hence, the minimum a
w
at which growth can occur has been
of most interest. In general, for growth, bacteria require a higher aw than
yeasts, and yeasts require a higher aw than molds. The minimum aw values
for the growth of various microorganisms are listed in Table 4.3. Even
different strains of the same species appear to have different minimum
aw for growth. The limiting aw for growth is influenced by other environ-
mental factors which should be at optimum values for the organism be-
ing tested if the absolute minimum water activity is to be determined. If
these factors are not optimum during testing, the resultant minimum aw
for growth of an organism will appear to be higher than the true value.
The solute used to lower the a
w
affects the minimum a
w
for growth of
microorganisms. Osmotolerant organisms grow at a lower aw when the
solute is sugar rather than salt. The reverse is true for halophilic or halo-
TABLE 4.3. APPROXIMATE MINIMUM WATER ACTIVITIES FOR GROWTH
OF MICROORGANISMS
Organism Minimum aw
Organism Minimum a
w
Most spoilage bacteria 0.90-0.91 Staphylococcus albus 0.88-0.92
Acinetobacter 0.95-0.98 S. aureus 0.83-0.92
Aeromonas 0.95-0.98 Streptococcus 0.92-0.98
Alcaligenes 0.95-0.98 Vibrio parahaemolyticus 0.94-0.98
Arthrobacter 0.95-0.98 Halophilic bacteria 0.75
Bacillus 0.90-0.99 Most yeasts 0.87-0.94
B. cereus 0.92-0.95 Osmophilic yeasts 0.60-0.78
Citrobacter 0.95-0.98 Most molds 0.70-0.80
Clostridium botulinum 0.90-0.98 Xerophilic molds 0.60-0.70
Type A 0.93-0.95 Aspergillus 0.68-0.88
Type B 0.93-0.96 A. glaucus 0.70-0.75
Type E 0.94-0.97 A. flavus 0.78-0.90
C. perfringens 0.93-0.97 A. halophilicus 0.68
Corynebacterium 0.95-0.98 A. niger 0.80-0.84
Enterobacter 0.95-0.98 Botrytis cinerea 0.93
Escherichia coli 0.94-0.97 Debaryomyces 0.87-0.91
Flavobacterium 0.95-0.98 Fusarium 0.80-0.92
Klebsiella 0.95-0.98 Hansenula 0.89-0.90
Lactobacillus 0.90-0.96 Mucor 0.80-0.93
Leuconostoc 0.96-0.98 Penicillium 0.78-0.90
Micrococcus 0.90-0.95 Rhodatoruia 0.89-0.92
M. roseus 0.90-0.93 Saccharomyces cerevisiae 0.90-0.94
Pseudomonas aeruginosa 0.96-0.98 S. rouxii 0.62-0.81
P. fluorescens 0.94-0.97 Xeromyces bisporus 0.60-0.61
Salmonella 0.93-0.96
tolerant microorganisms. Organisms tend to grow better in a substrate
in which the aw is altered by desorption rather than adsorption (Labuza,
Cassil, and Sinskey 1972).
The minimum aw for most bacteria is 0.90 to 0.91, except for S. aureus
and the halotolerant bacteria. The minimum aw for these latter bacteria
is listed as 0.75, which also is the approximate a
w
of a saturated NaCI
solution (Table 4.4). The growth of S. aureus at various a.,u levels is shown
in Figure 4.2. As the aw is reduced below 0.99, a reduced growth rate, as
well as less total growth is evident.
A very high aw may limit the growth rate. A typical curve relating the
growth rate to aw is shown in Figure 4.3. The optimum growth for most
organisms is slightly below 1.00, and for halophiles, or xerotolerant orga-
nisms, the optimum is lower. The halophile Vibrio costicola did not grow
at aw values above about 0.98 (Forsyth and Kushner 1970). The optimum
aw for the growth of Penicillium species varied from 0.93 to 0.98 (Hocking
and Pitt 1979).
TABLE 4.4. RANGES OF EQUILIBRIUM RELATIVE HUMIDITIES OF SATURATED
SOLUTIONS AT 20C
Chemical
LiBr
NaOHoH
2
0
LiCloH
2
0
K(C2Hs0 2)
CaCI26H20
MgCbo6H20
Cr03
Zn(N 03)26H20
K2C032H20
KN02
LiNOso3H20
Mg(N03ho6H20
Na2Cr2072H20
NaBro2H
2
0
NH.N03
Mg(C2H302)24H20
Range ofERH
6.6
7.0-8.9
10.2-15.0
20.0-23.0
32.3
32.5-33.6
35.0-38.7
42.0
43.0-44.0
45.0-49.1
47.0
54.9
55.2
55.3-59.3
64.9
65.0
Chemical
NaN02
KI
NaN03
NaCI
Na2S203
(NH')2S0
KCI
ZnSO.o7H20
BaCI
2
K2HPO.
KN03
NH.H2P04
N a2HP04012H20
K2S04
CuSO.o5H20
K2Cr20 7
Range ofERH
65.6-66.0
68.2-70.0
74.0
75.0-77.5
78.0
80.0-81.5
80.3-88.2
89.0-90.5
90.0-91.0
92.0
92.0-95.0
93.0-93.9
95.0-96.2
96.4-98.2
98.0
98.3
OTHER ASPECTS. Other aspects of water activity besides the mini-
mum activity for growth are important. These aspects include the germi-
nation of spores, toxin production, resistance to heat, and survival of
microorganisms.
Pitt and Christian (1968) listed the 0". requirements of thirty-six molds
for germination and for the formation of asexual and sexual sporulation.
Often a higher water activity is needed for sporulation than for germina-
tion and growth. Sexual sporulation usually requires a higher 0". than
does asexual sporulation. According to a review by Sperber (1983), the
aw requirement for bacterial sporulation is the same as for growth, but
germination usually can occur at lower 0". values.
The production of enterotoxin by S. aureus requires a higher 0". than
for growth (Smith et al. 1983). Higher 0". levels are needed for the produc-
tion of mycotoxins than for growth by several species of Aspergillus and
Penicillium (Beuchat 1981, 1983).
In general, lowering the 0". of the substrate increases the heat resist-
ance of microorganisms. However, at extremely low levels of aw, the resist-
ance may be decreased. Bacterial spores are most heat resistant at 0". val-
ues of 0.2 to 0.4.
Generally, the lower the 0"., the longer the organisms survive during
storage. The tolerance to low 0". values results from the ability of the
organism to accumulate compatible solutes within the cell. These solutes
include proline and 'Y-aminobutyric acid for bacteria, and polya1cohols
for fungi.
t-
Z
;:)
o
U
II
10
9
l&J 8
t-
c:t
..J
Q.
7
o
...J
6
5
4
o 20 40 60
HOURS
80 100
Figure 4.2. Growth of Staphylococcus aureus at various levels of water
activity.
Courtesy of Troller (1971).
109
EQUILIBRIUM RELATIVE HUMIDITY (%)
100 99 9B 97 96 95 94
1.8
a:
:;)
0
x
a:
w
Cl.
(fJ
z
52
(fJ
:>
i5
w
....
4
a:
X
....

a:
C)
1.00 0.99 0.98 0.97 0.96 0.95 0.94
WATER ACTIVITY (a
w
)
Figure 4.3. Effect of water activity on bacterial growth rate.
Water Activity of Food
The aw of food can be lowered by removing water (dehydration), by
adding solutes (sugar, salt), or by freezing. The usual aw levels of various
foods are listed in Table 4.5. Fett (1973) reported variations of a
w
of
0.003 to 0.01l, depending upon the system of measurement; other
researchers reported that different methods of measurement may vary
0.02 aw units (Labuza et al. 1976). Dewpoint hydrometer measurements
are reproducible to 0.003 aw and accuracy of 0.006 aw (Northolt 1979).
FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD III
TABLE 4.5. APPROXIMATE WATER ACTIVITIES OF SELECTED FOODS
Food
aU'
Food a
w
Fresh fruit or vegetables 0.97-1.00
Jelly
0.82-0.94
Fresh poultry or fish 0.98-1.00 Rice 0.80-0.87
Fresh meats 0.95-1.00 Fruitcake 0.80-0.87
Pudding 0.97-0.99 Flour 0.67-0.87
Eggs 0.97 Cake icing 0.76-0.84
Juices-fruit and vegetable 0.97 Molasses 0.76
Bread 0.95-0.96 Honey 0.54-0.75
White 0.94-0.97 Dried fruits 0.51-0.89
Crust 0.30 Chocolate candy 0.69
Cheese, most types 0.91-1.00 Rolled oats 0.65-0.75
Parmesan 0.68-0.76 Toffee 0.60-0.65
Cured meat 0.87-0.95 Caramels 0.60-0.65
Baked cake 0.90-0.94 Noodles 0.50
Refrigerated biscuit dough 0.94 Dried whole egg 0.40
Maple syrup 0.90 Biscuits 0.30
Salted egg yolk 0.90 Dried vegetables 0.20
Soft, moist pet food 0.83-0.91 Cereals 0.10-0.20
Nuts 0.66-0.84 Crackers 0.10
Jam 0.75-0.80 Sugar 0.10
Fresh foods, such as fruit, vegetables, meat, poultry, and fish have aw
values of 0.98 to over 0.99, which will allow the growth of most microorgan
Isms.
The rate of growth of bacteria is greater than that of yeasts or molds.
Hence, with foods of high aw, bacteria will outgrow the fungi (yeasts and
molds) and cause spoilage, while at aw values that restrict or prevent bac
terial growth, the fungi grow and become dominant. Exceptions to these
generalizations include fruits spoiled by fungi because of the acidity of
the product restricting bacterial growth. Products that have low Ow due
to sugar content Uams, jellies, or honey) will be subject to attack by os
mophilic yeasts, while products that contain high salt concentrations
(lowa,v) will be spoiled by halotolerant or halophilic bacteria. Dried foods
generally have a
w
values below 0.75. A safe aw level for storage is usually
considered to be 0.70 or less. In foods protected by low aw, enzymatic
changes can occur, but at a slow rate.
SORPTION ISOTHERMS. The moisture content of foods is often de
termined and reported. The relationship between moisture and a
w
values
is desirable. This relationship is described by a sorption isotherm that
has the shape ofa sigmoid curve (Fig. 4.4). Numerous mathematical equa
tions have been used to describe sorption isotherms (Aguerre, Suarez,
and Viollaz 1983; Ferro Fontan et al. 1982; King et al. 1983).
The curve may be an isotherm of adsorption (a moistening process),
or an isotherm of desorption (a drying process). The isotherms vary with
EQUILIBRIUM RELATIVE HUMIDITY (%)
o 20 40 60 80 100
o 0.20 0.40 0.60 0.80 1.00
WATER ACTIVITY (a
w
)
Figure 4.4. Typical sorption isotherm. 1Monomolecular film of water, no
growth of organisms; 2some added water to monomolecular film, slight
growth in upper range; 3water activity is sufficient for microbial growth.
temperature for different products. The adsorption and desorption iso
therms shown in Figure 4.4 have a hysteresis "loop." According to Troller
(1983a), equilibrium will occur, given sufficient time, so that only one
line is evident.
Three areas are apparent in most isotherms. In the lowest part of the
curve, the water is in a monomolecular layer on the food product, or so
called bound water, and is not available for biological activity. In the mid
dIe of the curve, additional layers of water are added to the monomolecu
lar film, and there is a rapid increase in a
w
with a moderate increase in
moisture content. In the upper regions of this section of the curve, some
water becomes available to support the growth of some molds and yeasts.
In the upper part of the isotherm, the moisture content increases rapidly
with comparatively small increases in water activity, and the food con
tains free water that is available for bacterial as well as fungal growth.
If food is not stored in moisture proof containers, it will equilibrate
with the relative humidity of the surrounding air according to its sorp
tion or desorption isotherm. The direction will be determined by the
moisture in the food and the relative humidity of the air. Dry foods may
pick up moisture to the extent that molds will be able to grow, while
foods with high a., stored in low humidity air may lose moisture, causing
shrinkage and other organoleptic changes (Labuza and Contreras
Medellin 1981). The rate of equilibration depends upon the relative hu
midity of the air, the initial a., of the food, and temperature.
In mixtures of foods such as cereals and raisins, or marshmallow top
ping on a cookie base, the moisture from one food will transfer to the
drier product. For raisins in cereals, this results in the cereal becoming
less crisp and the raisins becoming hard. Coating the raisins with bees
wax, oils, or sugar has been used to retard the transfer of moisture.
Since many of our foods are combinations of various components,
the a., values and isotherms of the ingredients should be considered in
order to avoid potential problems either with organoleptic quality or
with possible microbial growth. Although a product may have an a., not
conducive to growth of microorganisms, syneresis may occur, increasing
the a., in microenvironments, thus allowing microbial growth.
Case hardening, the phenomenon in which the moisture content of
the exposed surface is different from that in the unexposed portion, may
act as a barrier for moisture sorption or desorption. This may influence
the measurement of the a., of a food so that, although the determined a.,
of the food would appear in a safe range, the interior of the food will
support microbial growth.
Foods that can be eaten without rehydration but are microbiolog
ically stable without refrigeration are called intermediate moisture foods.
These foods are discussed in Chapter II.
pH
In many texts, pH is defined as the negative logarithm of the hydro
gen ion concentration. The pH is actually a function of apparent concen
trations or activity of the electrolytes in solution. Thus, pH is the negative
logarithm of the activity of the hydrogen ions. Since pH is usually deter
mined by means of a pH meter, it might be defined as a value calculated
from the electromotive force (emf) of an electromotive cell in which the
substance being tested is one of the electrolytes. In the food industry, the
determination and maintenance of pH are important in quality control
and processing requirements.
Effect on Microorganisms
Microorganisms have a minimum, optimum, and maximum pH for
growth. Most bacteria show optimum growth at a pH near 7.0, while oth
ers are favored by an acid environment, probably due to the inhibition
of other organisms, thus eliminating microbial competition. The acid for
mers (Lactobacillus and Streptococcus) can tolerate moderate acidity, while
proteolytic types (Pseudomonas) can grow in moderately alkaline sub
strates. In general, molds can grow at lower pH values than yeasts, and
yeasts are more tolerant of low pH values than are bacteria. Bacteria
usually grow faster than yeasts in a neutral or slightly acid substrate, but
at pH 5.0 or less, yeasts can compete with or outgrow bacteria.
Microorganisms can grow in a wide pH range (Table 4.6). The vari
ations in pH values for growth may be due to different strains of a species
or different species in a genus, the type of substrate, the acid or base
used to adjust the pH, or other factors. There is an interrelationship be
tween pH and other environmental factors.
Alteration of the pH of a substrate may relate indirectly to growth.
For example, the availability of metallic ions is altered. Although coexist
ing in the free form at low pH, magnesium and phosphorus form an
insoluble complex at higher pH values. In an alkaline medium, ferric,
zinc, and calcium ions become insoluble.
The pH of the substrate may influence cell permeability. At low pH,
the membrane becomes saturated with hydrogen ions, thus limiting pas
sage of essential cations. Conversely, at a high pH, saturation of the mem-
brane with hydroxyl ions will limit the passage of essential anions.
The toxicity of adverse pH values is due partly to the penetration of
undissociated molecules of acid or basic substances into the cells. At low
pH values, undissociated weak acids can enter the cell, then ionize and
alter the internal pH. In alkaline solutions, undissociated weak bases can
enter the cell. If the internal pH is changed to basic pH values, amino
acid transferase RNA is inhibited and protein synthesis is stopped. The
pH of the substrate influences the activity of enzyme systems and the
products of metabolism of microorganisms.
ALTERATION OF pH BY MICROORGANISMS. The environment in-
fluences microorganisms and the microorganisms influence the environ-
ment. During growth, metabolic products are formed. These can be
either acidic or alkaline, depending upon the substrate, the organisms
involved, and the time allowed for growth. The initial reaction of most
organisms is acidic because of the breakdown of carbohydrates and the
formation of organic acids. The alteration of pH by production of acids
is used in the food-fermentation industries. The lactic acid bacteria tend
TABLE 4.6 APPROXIMATE pH RANGES FOR MICROBIAL GROWTH
pH
Organism Minimum Optimum Maximum
Bacteria (most) 4.5 6.5-7.5 9.0
Acetobacter 4.0 5.4-6.3
Bacillus subtilis 4.2-4.5 6.8-7.2 9.4-10.
Clostridium botulinum 4.8-5.0 6.0-8.0 8.5-8.8
C. perfringens 5.0-5.5 6.0-7.6 8.5
C. sporogenes 5.0-5.8 6.0-7.6 8.5-9.0
Erwinia carotovora 4.6 7.1 9.3
Escherichia coli 4.3-4.4 6.0-8.0 9.0-10.
Gluconobacter oxydans 4.0-4.5 5.5-6.0
Lactobacillus (most) 3.0-4.4 5.5-6.0 7.2-8.0
L. acidophilus 4.0-4.6 5.5-6.0 7.0
L. plantarum 3.5 5.5-6.5 8.0
Leuconostoc cremoris 5.0 5.5-6.0 6.5
L. oenos 4.2-4.8
Pediococcus cerevisiae 2.9 4.5-6.5 7.8
Propionibacterium 4.7 6.2-7.0 7.5
Proteus vulgaris 4.4 6.0-7.0 8.4-9.2
Pseudomonas (most) 5.6 6.6-7.0 8.0
P. aeruginosa 5.6 6.6-7.0 8.0-9.0
Salmonella (most) 4.5-5.0 6.0-7.5 8.0-9.6
S. typhi 4.0-4.5 6.5-7.2 8.0-9.0
S. choleraesuis 5.0 7.0-7.6 8.2
Serratia marcescens 4.6 "6:0":'1.0 8.0
Staphylococcus aureus 4.0-4.7 6.0-7.0 9.5-9.8
Streptococcus lactis 4.1-4.8 6.4 9.2
Vibrio 5.5-6.0 9.0
V. cholerae 8.6
V. parahaemolyticus 4.8-5.0 7.5-8.5 11.0
Yeasts 1.5-3.5 4.0-6.5 8.0-8.5
Hansenula 4.5-5.5
Kluyveromyces 1.5-2.0
Pichia 1.5
Saccharomyces cerevisiae 2.0-2.4 4.0-5.0
S. rouxii 1.5 3.5-5.5 8.5-10.5
Molds 1.5-3.5 4.5-6.8 8.0-11.
Aspergillus niger 1.2 3.0-6.0
A. oryzae 1.6-1.8 9.0-9.3
Bot1ytis cinerea 2.5 7.4
Mucor 3.0-6.1 9.2
Penicillium 1.9 4.5-6.7 9.3
Rhizopus nigricans 4.5-6.0
to lower the pH by production of lactic acid, while proteolytic types such
as pseudomonads tend to raise the pH by production of ammonia or
other basic chemicals.
Bacillus coagulans grew in tomato juice, raising the pH from 4.5 to 5.0
(Anderson 1984). Molds growing on tomatoes raised the pH to levels that
allowed bacterial growth in the food (Mundt and Norman 1982).
SURVIVAL. The pH for survival of cells is somewhat different from
that for growth. Some organisms show greater survival at slightly acid pH
levels 5.6 to 6.5 and optimum growth at 6.8 to 7.2. Microorganisms can
survive at pH levels too acid or too basic for metabolism and growth. The
effect of pH on survival during heating is very evident.
pH of Food
The pH of a food, along with other environmental factors, will deter
mine the types of microorganisms that are able to grow and dominate,
and eventually cause spoilage, a desired fermentation, or a potential
health hazard. Besides the microbial aspects of the pH of foods, the acids
in foods play other important roles.
The food may be naturally acid, acids may be added, or acids may
be produced in the food by enzymatic action with or without microbial
growth.
The pH of a food is determined by the balance between the buffering
capacity and the acids or alkaline substances it contains. Since protein
has a high buffering capacity, protein foods have greater buffering than
do fruits or vegetables. This is important in the fermentations producing
lactic acid because the production of small amounts of acid in sauerkraut
or pickle processes will lower the pH significantly.
The approximate pH values for selected foods are listed in Table 4.7.
The pH values are only approximations because there is considerable
variation in the pH of some foods and the pH of food products can
change during ripening, processing, or storage. The pH values of various
foods were reported by Sapers, Phillips, and Divito (1984).
Foods have been categorized according to their pH as follows:
Highacid foods pH below 3.7
Acid foods pH 3.7-4.6
Mediumacid foods pH 4.6-5.3
Low or nonacid foods pH over 5.3
EGG WHITE. This is one of the most alkaline biological solutions. The
albumen of a freshly laid chicken egg is approximately pH 7.6. When the
egg is stored in air, the carbon dioxide from carbonic acid in the albumen
is released through the egg shell. When this occurs, the pH increases and
has been reported to reach levels of 9.4 to 9.7. Although the high pH
is desirable from a microbiologist's viewpoint, these high pH levels are
associated with thinning of albumen and a decrease in the strength of
the vitelline (yolk) membrane, or a general decrease in egg quality. Egg
quality is maintained at an albumen pH near 8.2, which is above the
TABLE 4.7. APPROXIMATE pH RANGES OF SELECTED FOODS
Food pH Range Food pH Range
Egg white 7.6-9.5 Cabbage 5.2-6.3
Shrimp 6.S-S.2 Turnip 5.2-5.6
Crab 6.S-S.0 Spinach 5.1-6.S
Scallops 6.S-7.1 Asparagus 5.0-6.1
Cod, small 6.7-7.1 Cheeses, most 5.0-6.1
Cod, large 6.5-6.9 Camembert 6.1-7.0
Catfish 6.6-7.0 Cottage 4.1-5.4
Soda crackers 6.5-S.5 Gouda 4.7
Maple syrup 6.5-7.0 Bread 5.0-6.0
Milk 6.3-6.S Carrots 4.9-6.3
Brussels sprouts 6.3-6.6 Beets 4.9-5.S
Whiting 6.2-7.1 Bananas 4.5-5.2
Haddock 6.2-6.7 Dry sausages 4.4-5.6
Cantaloupe 6.2-6.5 Pimientos 4.3-5.2
Dates 6.2-6.4 Tomato juice 3.9-4.7
Herring 6.1-6.6 Mayonnaise 3.S-4.0
Butter 6.1-6.4 Tomatoes 3.7-4.9
Honey 6.0-6.S
Jams
3.5-4.0
Mushrooms 6.0-6.5 Apricots 3.5-4.0
Cauliflower 6.0-6.7 Apple sauce 3.4-3.5
Lettuce 6.0-6.4 Pears 3.4-4.7
Egg yolk 6.0-6.3 Grapes 3.3-4.5
Corn, sweet 5.9-6.5 Cherries 3.2-4.7
Oysters 5.9-6.6 Pineapple 3.2-4.1
Celery 5.7-6.0 Peaches 3.1-4.2
Peas 5.6-6.S Rhubarb 3.1-3.2
Turkey 5.6-6.0 Strawberries 3.0-4.2
Chicken 5.5-6.4 Grapefruit 2.9-4.0
Halibut 5.5-5.S Raspberries 2.9-3.7
Beans, lima 5.4-6.5 Apples 2.9-3.5
Potatoes, Irish 5.4-6.3 Plums 2.S-4.6
Walnuts 5.4-5.5 Oranges 2.S-4.0
Pork 5.3-6.4 Cranberries 2.5-2.S
Beef 5.3-6.2 Lemons 2.2-2.4
Onions 5.3-5.S Limes 1.S-2.0
Sweet potatoes 5.3-5.6
maximum for the growth of many microorganisms. Storage in an atmo
sphere of CO2 or oiling the egg shell maintains the pH at an intermediate
level.
RED MEAT. The pH of living animal tissue is near neutral (pH 7.0-
7.2). The circulating blood brings nutrients and oxygen to the cells and
removes the waste products of metabolism. When an animal is slaugh
tered, blood no longer circulates, anaerobic conditions develop, and any
metabolic products accumulate. The inherent tissue enzymes ferment the
muscle glycogen to lactic acid, which lowers the pH. When glycogen sup'
plies are abnormally low in the muscle prior to slaughter, less acid is
produced, with a higherthannormal ultimate pH. The temperature at
which a beef carcass is held determines the rate of glycolysis and pH
changes.
Immediately after slaughter, the pH of most beef muscles is 6.9 to 7.2
(Hamm 1982). After 24 hr postmortem, most muscles from wellrested
animals have a pH of 5.4 to 5.6 (Currie and Wolfe 1980; Tarrant and
Sherington 1980). It is generally believed that pH 5.3 is the limit below
which glycolysis is inhibited, even though some glycogen may be present.
The pH of most pork muscles with normal glycolysis ranges from 5.3 to
5.8 (Warriss 1982).
It might be expected that a higher glycogen content is present in the
muscles of well fed, rested, unexcited animals than in starved, tired, or
stressed animals.
Preslaughter stress conditions, such as fighting among hogs, have
been associated with a rapid pH drop and low ultimate pH. This rapid
lowering of the pH while the muscles are still warm results in pale, soft,
exudative (PSE) pork. Preslaughter exercise or stress causes muscles to
be deficient in glycogen and results in ultimate higher pH values (above
pH 6.0). This meat is called dark, firm, dry (DFD) pork, or dark cutting
beef. With a higherthannormal ultimate pH, these muscles have a tend
ency to spoil more readily than do muscles with a lower pH.
Subjecting beef carcasses to an electrical current of various intensities
promotes tenderness of the muscles as well as a rapid drop in pH (Martin
et al. 1983). Also, a rapid pH reduction was noted when beef muscle was
pressurized (Elkhalifa et al. 1984).
The pH of commercial dry sausages was reported as ranging from 4.4
to 5.6 (Acton and Dick 1976). Fermentation is responsible for the lower
pH values of sausages as compared to fresh meat. The addition of acid
(acetic, lactic, citric) results in a low pH of pickled meats.
Microbiologically, a low pH in fresh meat is desired. The minimum
pH for the growth of most pseudomonads that spoil meat is 5.6. If meat
has an ultimate pH less than 5.6, it would be expected to have a longer
shelf life. On the other hand, higher pH values have been associated with
enhanced waterholding capacity, nutrition,juiciness, tenderness, and in
creased emulsifying properties and binding strength of meat, which are
desirable attributes.
CHICKEN. The pH of chicken muscle varies similarly to that of red
meats. Postslaughter chicken muscle is approximately pH 7.0, which is
reduced during glycolysis to 5.5 to 5.9. A rapid glycolysis at high temper
ature can result in tough meat. The pH of meat from heatstressed birds
was 5.46, from coldstressed, 5.51, and from control birds, 5.53 (Lee et al.
1976). This indicates that this type of stress has little influence on the
final pH of chicken meat.
SEAFOODS. The pH offish (7.0-7.3) is lowered during rigor to pH 5.5
to 6.5, depending on the species of fish and the initial amount of glyco
gen in the muscle. Fish muscle generally has less glycogen than poultry
or red meat.
The pH of cod was found to be lower at the end of the spawning
season, and large cod had a lower pH than small cod (Love et al. 1972).
They found that a low pH was related to gaping in cod. This is a condi
tion in which the tissue that holds the muscle segments together becomes
weakened and causes slits to appear across the fillets.
According to Love and Haq (1970), haddock has a lower pH than
whiting. These researchers postulated that the difference may be in their
eating habits. The whiting chases food, while the haddock tends to graze.
The haddock, being a less active fish, probably does not struggle as much
as a whiting when caught, so there would be greater carbohydrate reo
serve, which results in more lactic acid production postmortem and a
lower pH. Of all the fish for which data are available, the halibut has the
lowest ultimate pH and the best keeping quality.
The pH of canned crab is usually pH 6.8 to 7.4 but may be higher.
Motohiro and Inoue (1970) observed that the pH of fresh king crab was
6.8 for the hard shell and 7.4 for the paper shell intermolt stages.
A pH range of 7.1 to 8.2 was determined on brown shrimp obtained
from commercial fishing boats at landing (Cobb et al. 1973). The upper
limit, pH 8.2, may seem rather high, since pH 7.95 has been indicative
of shrimp spoilage. At the time of spoilage, the pH of brown shrimp was
found to be 7.3 to 8.5 (Cobb et al. 1973). This indicates that pH 7.95 has
little, if any, significance. At a pH of about 8.5, the bacterial numbers
were estimated at 10
5
per gram (Flores and Crawford 1973). This level of
microorganisms usually does not indicate spoilage.
FRUITS AND VEGETABLES. Fruits have a lower pH than do most
foods. Unripe fruit generally has a lower pH than ripe fruit. As the fruit
matures, the moisture and sugars increase, and the acids decrease or are
diluted. The ripening temperature influences the ultimate pH, generally
in a direct relationship. The pH of fruit influences not only the growth
of microorganisms, but also quality factors, such as softening and discol-
oration of the canned product. Since the pH is low, fruits are usually
spoiled by mold growth. The pH of the tomato is influenced by variety,
maturity, geographic area of growth, and storage conditions. Vegetables
generally have a higher pH than fruits and are therefore subject to bacte-
rial spoilage.
OXIDATION-REDUCTION POTENTIAL
When a substance is oxidized, it loses electrons, and these electrons
must be accepted by another substance which then becomes reduced.
The oxidation-reduction (OR) potential is a measure of the tendency of
a reversible system to give or receive electrons.
The OR potential is referred to in Chapter 2 in regard to the use of
reductase tests to estimate microbial numbers in foods. By use of indica-
tors, the OR potential of a system can be estimated. The indicator should
give a distinct color to the solution at the pH of the system being studied.
Unfortunately, some indicators behave as both OR and pH indicators.
They may give an intense color in an alkaline solution, but in neutral or
acid solutions yield only a dull tint. The usual method of measuring OR
is with a platinum redox electrode attached to a pH/m V meter.
Eh is a measure of intensity, not of capacity of a system. The capacity
is the poising ability, which acts in a manner similar to buffers in main-
taining pH at a constant value. A system is said to be poised if it resists
change in potential.
In microbial cultures, the simultaneous oxidations and reductions are
the sources of energy for cell processes. Since energy is needed by the
cell to function normally, OR reactions and OR potentials are important.
The broad classifications of organisms into aerobes and anaerobes,
as discussed in general microbiology, is based on their apparent toler-
ance to oxygen during growth_ Strictly aerobic organisms grow only in
the presence of free atmospheric oxygen. Strictly anaerobic organisms
survive and increase only in the absence of free oxygen. Facultative an-
aerobes can grow with or without free oxygen. Some facultative orga-
nisms prefer growth in atmospheric oxygen; others do not. Microaero-
philic organisms cannot multiply in either entirely aerobic or anaerobic
conditions. They grow best in a limited amount of free oxygen.
In discussing growth-limiting effects on anaerobic bacteria, Hentges
and Maier (1972) cited references to show that some obligate anaerobic
types of organisms failed to grow if exposed to oxygen in the air during
the manipulation of diluting and plating. These obligate anaerobes
rarely are reported in foods, probably because not enough care is taken
to find them. If they are such strict anaerobes, they probably cause few,
if any, serious problems in foods_
In one study, anaerobes were classed as intolerant, moderately toler-
ant, or tolerant, on the basis of their tolerance to atmospheric oxygen
(Rolfe et al. 1978). When exposed to oxygen, intolerant types survived
less than 2 hr, moderately tolerant for 4 to 8 hr, and tolerant anaerobes
at least 48 hr. Of the eleven anaerobic species the researchers tested,
Clostridium perfringens was the most oxygen tolerant.
From preliminary studies, Hentges and Maier (1972) stated that the
OR potential is unimportant regarding the multiplication of Bacteroides
species. When oxygen is excluded from the environment, Bacteroides are
unaffected by a high OR potential of the medium. Walden and Hentges
(1975) studied the growth and survival of Bacteroides fragilis, Clostridium
perjringens, and Peptococcus magnus at low and high Eh with and without
oxygen. Even at an Eh of - 50 mv these organisms did not grow in the
presence of oxygen. No inhibition was observed in the absence of oxygen
even at an Eh of + 325 mv. This indicates that oxygen, rather than a
positive potential, was the limiting factor for growth of these anaerobes.
Of these three organisms, P. magnus was the most sensitive to oxygen and
C. perfringens the least sensitive.
Other researchers reported that B. fragilis was not affected by an Eh
of + 300 mv in the absence of oxygen, but cell death occurred with oxy
gen at + 250 mv (Onderdonk et al. 1976). They suggested that dissolved
oxygen has an inhibitory effect on B. fragilis, which may be independent
of changes in Eh alone. Similar effects of oxygen at low Eh were noted
for sporulation and growth of Clostridium butyricium (Douglas, Ham
bleton, and Rigby 1973), and C. botulinum (Lund and Wyatt 1984).
Many anaerobic clostridia (c. botulinum, C. histolyticum, C. sporogenes)
do not need a negative Eh for growth. These organisms will grow at Eh
levels from + 85 to + 160 mv, especially in the absence of oxygen.
Due to complexities in maintaining and measuring OR potentials,
there'is not as much information on this parameter as on pH. Also, the
results vary considerably from one experiment to another, between
strains of species, and between different researchers.
Effect of Organisms on Eh
It has been well established that, as organisms metabolize, a lower Eh
value is produced in the medium or substrate. The lowering of the poten
tial has been ascribed to the consumption of oxygen or the production
of reducing substances.
With aerobic bacteria, there is a slight lowering of the OR potential
during the lag phase of growth due to the consumption of oxygen. As
the bacteria enter the log phase of growth, this oxygen usage increases
and causes a rapid drop in Eh. As the Eh becomes negative, the growth
rate of the bacteria decreases. An overall lowering of the potential is
between 400 and 500 mv.
At the beginning of growth, facultative anaerobes alter the Eh of the
substrate in a manner similar to aerobes, but the rate of reduction may
be somewhat slower. After the bacteria enter the log phase of growth, the
122 BASIC FOOD MICROBIOLOGY.
Eh decreases very rapidly. This overall potential drop may be from 700
to 800 mv or more. As the culture reaches the stationary phase, and as the
age of the stationary phase increases, the observed OR potential shifts to
more positive values.
Oxygen is removed from media prior to inoculation and growth of
anaerobes. As a result, the initial Eh is lower than that for aerobes or
facultative anaerobes. During the germination and emergence of spores
or the lag phase of growth, the Eh is lowered by 500 to 700 mv (Douglas
and Rigby 1974). There is little further reduction during the lag phase
of growth. Although the total reduction of Eh is generally less for anaer-
obes than for facultative anaerobes, a lower Eh is attained due to the
lower initial Eh of the substrate.
Redox Potential of Food
The OR potential of a food depends upon the composition (oxydiz-
ing and reducing agents present), the pH, the oxygen tension in the at-
mosphere, and the access that the atmosphere has to the food. Due to
the complications of determining the Eh of foods and the variations ob-
tained due to atmospheric influences, the OR potentials of only a few
foods are available.
Harrison (1972) stated that in a conglomerate mixture with different
systems, not in equilibrium, an overall redox potential cannot be con
ceived. It is evident that most foods are conglomerate mixtures. Regard-
less, there have been attempts to measure and compare the redox poten-
tial of foods. Living cells tend to have a low OR potential due to -SH
groups in animal products and ascorbic acid and reducing sugars in
plants. In dairy products, the redox potential is related to fat oxidation.
Thus, there is an inverse relation of OR potential and keeping quality.
The contamination of milk with cupric or ferric ions tends to increase
the OR potential, resulting in less shelf life of the product.
The OR potential of cheese depends on the type, with Emmenthal
(Swiss) cheese varying from - 200 to - 50 mv and Cheddar cheese from
+220 to +340 mv (Mossel and Ingram 1955).
The Eh of meat has been reported to range from -150 to + 250 mv.
There is an oxygen requirement for postmortem tissue. The myoglobin
of muscle can bind oxygen to form oxymyoglobin, or it can be oxidized
by oxygen to form metmyoglobin. To oxygenate the pigment requires
about 5 ttl of oxygen per gram of meat, while oxidation to metmyoglobin
requires about 13 ttl of oxygen per gram of meat. Other oxygen require-
ments are tissue respiration, lipid oxidation, tissue fluids with low oxy-
gen tension dissolving O
2
and bacterial demands. DeVore and Solberg
(1974) found that tissue respiration accounted for 80 percent of the oxy-
gen uptake of postmortem meat, while bacterial demands were insignifi-
cant.
The Eh of plant products varies from + 383 to + 436 mv for fruit
juices, + 74 mv for spinach, + 225 mv for barley, and - 470 mv for wheat
germ (Mossel and Ingram 1955)_ The Eh of cherries and peaches was
determined to be + 179 mv and + 175 mv, respectively (Ross, Bartlett,
and Hard 1953)_ The addition of ascorbic acid lowered the Eh of the
fruit_ The Eh of canned foods ranged from -18 mv for sliced carrots
packed in glass to - 446 mv for beef stew packed in cans (Montville and
Conway 1982).
The oxygen content of the atmosphere and the access of the atmo-
sphere to the food influences the OR potentiaL Large pieces of foods,
such as carcasses, have less total surface area than do smaller-sized foods.
Food in a deep vat has less surface exposed proportionately than does
food in shallow vats. Food at rest in a vat has a lower Eh than does liquid
being stirred or mixed. Food that is packaged in a material impermeable
to oxygen should have a lower Eh than unpackaged food_ Vacuum pack-
aging will reduce or prevent the access of free oxygen to the food.
Relationships of Food OR and Microorganisms
The relatively high Eh of fruit juices favors the growth of aerobic mi-
croorganisms. Since these products have a low pH, the aerobic yeasts and
molds are the predominant organisms that cause spoilage.
In meat, the surfaces exposed to the atmosphere allow the growth of
aerobic bacteria, but in deeper tissue, the Eh is lower so that anaerobes
can grow. In prerigor meat, the Eh is sufficiently high to prevent the
growth of anaerobic types, but during rigor, the Eh is reduced to the
extent that allows clostridia to grow_ The Eh of fresh raw milk inhibits
the growth of C. botulinum. The heating of milk lowers the Eh so that C.
botulinum can grow (Kaufmann and Marshall 1965).
Although molds are considered to be strictly aerobic, some types can
grow in low levels of oxygen_ One of the reasons that Penicillium roqueforti
can grow in the inside of Roquefort-type cheese is its tolerance for low
p02' Byssochlamys fulva grows in grape products at extremely low levels of
oxygen.
INHIBITORY SUBSTANCES
When you hear inhibitory substances in foods mentioned, you might
envision some chemical preservative added by the processor. However,
microbial inhibitors were present long before human beings appeared
on earth.
Action of Inhibitors
Generally, inhibitors affect microorganisms by acting on the whole
cell, cell wall, or cell membranes, by interfering with the genetic mecha-
nism of the cell, by interfering with the enzyme systems of the cell, or by
binding essential nutrients_
Many microbial inhibitors have been reported to be naturally present
in foods_ The factors involved with the growth of microorganisms, such
as pH, that have been discussed in this chapter, are partially due to chem-
icals naturally present in foods. Besides these, there are other inhibitors
in both animal and plant products.
Inhibitors in Animals Products
Microbial inhibitors have been found in egg white, milk, and various
tissues from animals.
EGG WHITE. The growth of bacteria in egg white is limited by the
presence of lysozyme, enzyme inhibitors (such as ovomucoid), avidin,
conalbumin, and a high pH (Table 4.8).
Lysozyme. In 1922, Fleming reported a lytic agent in egg white and other
biological systems, which was named lysozyme. This enzyme has been
referred to as muramidase, N-acetylmuramide glycanohydrolase, gluco-
hydrolase, mucopeptide glycohydrolase and {j-glucosaminidase. To help
alleviate confusion about the name of this and other enzymes, the Com-
mission on Enzymes has assigned numbers to the various enzymes. Ly-
sozyme is 3.2.1.17.
Lysozyme is found in egg white, milk, many tissues and secretions of
animals, some molds, and in the latex of various plants. This widespread
distribution has made it a very popular substance for study. Lysozymes
from different animal species and from different organs of the same ani-
TABLE 4.8. MICROBIAL INHIBITORS IN EGG WHITE
Inhibitor
Lysozyme
Ovomucoid
Conalbumin
( ovotransferrin)
Ovoflavoprotein
Avidin
pH
Lysis of cell walls (Grampositive bacteria)
Enzyme inhibitor
Chelates metal, ions, especially ferric ions
Sequesters riboflavin
Binds biotin
Alkaline conditions restrict growth, accen-
tuate iron chelation
Egg White
Solids (%)
3.5
11.0
13.0
0.05
mal differ chemically and immunologically, but they possess the same
type of biological activity. The lysozyme content varies not only between
species, but also between strains of the same species. On a dry weight
basis, chicken egg white contains about 3.5 percent lysozyme. This is the
most readily available source, and hence, the most studied of the lyso
zymes.
Bacteria display various sensitivities to the lysis of the cell wall by
lysozyme. Certain micrococci are rapidly lysed by 1 J.tg/ml of lysozyme.
Bacillus megaterium requires 50 J.tg/ml and B. cereus barely is attacked by 50
Itg/ml of lysozyme.
Gramnegative bacteria are rather insensitive to lysozyme because of
their lipoproteinlipopolysaccharide layer, which acts as a barrier pro
tecting the sensitive mucopolysaccharides of the cell. If the barrier is
damaged by certain treatments, the enzyme can penetrate to the muco
peptide layer and cause lysis, or partial lysis, of the cell wall. Starving
Gramnegative cells at an abnormal pH, treatment with polymyxin B, or
ethylenediamine tetraacetic acid, as well as reacting with complement, os-
motic injury or aerosolization can render these cells susceptible to lyso-
zyme action.
Enzyme Inhibitors. Enzyme inhibitors found in egg white are the ovomu-
coids, ovoinhibitors, and a ficin-papain inhibitor.
Rhodes, Bennett, and Feeney (1960) divided ovomucoids into four
classes according to their inhibitory activities. Some ovomucoids, such as
the chicken and goose, inhibit trypsin. Another group inhibits chymo-
trypsin (golden pheasant). A third group (turkey) inhibits approximately
equal amounts of trypsin and chymotrypsin, and the fourth group in
cluded those from duck and emu, which inhibit twice as much trypsin as
chymotrypsin. A duck ovomucoid inhibited a commercial bacterial pro-
teolytic enzyme.
Matsushima (1958) discovered an inhibitor in egg white which he
termed ovoinhibitor. This substance inhibited fungal proteinase of an
Aspergillus and a bacterial proteinase prepared from a strain of Bacillus
subtilis.
There is no evidence that these enzyme inhibitors have any signifi
cant effect on microbial growth in egg white.
Avidin. Each mole of avidin combines with two moles of biotin. Thus,
organisms having a strict requirement for biotin as a nutrient are in-
hibited by this compound. The binding of avidin to bacterial cells has
been discussed in detail (Korpela et al. 1984).
Conalbumin. This protein comprises about 12 percent of the total solids
of egg white. It forms a stable complex with two ferric or ferrous ions
per molecule, and by this action it inhibits organisms that require iron
for growth. This includes Gram-negative and Gram-positive bacteria as
well as yeasts. It is evident that conalbumin does not destroy the microor-
ganisms but merely inhibits growth by extending the lag phase. Feeney
and Nagy (1952) postulated that dissociation of the complex would al-
ways result in traces of free iron which, when used by the organism,
would allow the release of further iron from the complex. This results in
slower-than-normal growth of the organism.
pH. The high pH established when carbon dioxide leaves egg albumen
inhibits the growth of many microorganisms.
It might be postulated that because of lysozyme, and inhibition of
Gram-positive bacteria, spoilage of eggs is primarily due to Gram-
negative types. In the egg, conalbumin is the main inhibitor of Gram
negative bacteria. Although it does not prevent growth, it does inhibit or
delay it. The albumen was more inhibitory at 39.5C than at 30C
(Tranter and Board 1984). It is important that microbial growth be in-
hibited, because it is necessary to have shell eggs in commercial channels
for the time needed for grading, packing, distribution, and retailing.
DAIRY PRODUCTS. Several substances may be present in milk that
inhibit microorganisms. These include antibiotics, pesticides, bacterial
viruses, sanitizing and cleaning compounds, and various natural inhibi-
tors.
The naturally occurring microbial inhibitors in raw milk include lyso-
zyme, cationic proteins, agglutinins or antibodies, leucocytes, lactenins,
lactoperoxidase, lactoferrin, and perhaps, fatty acids. Many of the natural
inhibitors in milk are heat labile and not found in pasteurized milk.
Of thirty-seven samples of cow milk tested, only one contained lyso-
zyme at a level of 5 mg/kg (Carroll 1979). Only 10 percent of 200 samples
of camel milk inhibited one or more of six test organisms (Barbour et al.
1984). The lysozyme of these samples averaged 6.48 mg/kg.
The antibiotic nisin is produced by certain strains of Streptococcus
lactis, a common dairy organism, and may be present in small amounts
in raw milk and certain milk products.
Cationic Proteins. These proteins carry a positive charge at physiological
pH values and react with the anionic binding sites of cell walls and mem-
branes of bacteria (MacMillan and Hibbitt 1973). This causes an in-
creased permeability of the bacterial membranes, resulting in leakage
from the cells. Hibitt, Brownlie, and Cole (1971) isolated cationic proteins
from bulk milk samples. The antimicrobial activity was not destroyed by
heating to 70C for 30 min but was almost completely destroyed at
100C. These proteins have activity to both Gram-positive and Gram
negative bacteria.
Lactoperoxidase. Lactoperoxidase is the peroxidase enzyme in milk. Peroxi
dases catalyze the oxidation of molecules, with peroxide (usually hydro
gen peroxide) as the oxygen source. For antibacterial effects, besides per
oxidase and H
2
0
2
, the presence of thiocyanate (SCN-) is required. Reiter
and Harnulv (1984) suggested adding minute amounts of H
2
0
2
and
SCN- to milk to enhance lactoperoxidase effect and the shelflife of milk.
The action on cells is greatest at pH 5.0 or less (Reiter et al. 1976).
According to the review of Reiter and Harnulv (1984), this system may
be bacteriostatic or bactericidal. Bacterial enzymes are inhibited, the cy
toplasmic membrane is damaged, and synthesis of protein, DNA, and
RNA are inhibited.
Lactoferrin. This is an ironbinding protein in milk that acts on bacteria
similar to conalbumin in egg white. Normal milk contains 0.1 to 0.3 mgl
ml. This is increased from four to ten times in mastitic milk and in colos
trum (Harmon et al. 1975). Antibodies in milk enhance the activity of
lactoferrin (Dolby and Honour 1979; Spik et al. 1978). The addition of
citrate reduces the inhibitory effect of lactoferrin (Bishop et al. 1976).
Fatty Acids. Fatty acids may be inhibitory or stimulatory, or they may have
no effect on microorganisms. The action depends upon the type of or
ganism, the type and concentration of the fatty acid, the pH of the growth
medium, or the presence of certain other compounds. Shortchain fatty
acids are about equally effective toward either Gramnegative or Gram
positive cells. Fatty acids with six or more carbon atoms are more inhibi
tory to Grampositive cells than to Gramnegative cells. Longchain fatty
acids cannot penetrate the lipopolysaccharide layer of Gramnegative
cells.
The mode of action and the type of inhibition depend upon the fatty
acid and the concentration. At low concentration, some fatty acids are
stimulatory, while at higher concentrations they are inhibitory to the
same organism. The toxic effect is, in general, bacteriostatic, but for cer
tain organisms the fatty acids may be bactericidal at high concentrations.
The bacteriostatic effect may be due to the blockage of adsorption of
essential nutrients. With extended incubation, the cells seem to over
come this difficulty, and have, at times, shown total cell growth greater
than when no fatty acids were present. Besides affecting the permeability
of the cell membranes and inhibiting transfer mechanisms, fatty acids
inhibit the action of enzyme systems.
Although fatty acids are found in various foods containing fats, they
have been of special concern to the dairy industry because of their preva
lence in butter and cheese. The amount of free fatty acids increases dur-
ing the aging of cheese. Some common fatty acids are listed in Table 4.9.
TABLE 4.9. SOME COMMON FATTY ACIDS
Common Name
Formic acid'
Acetic acid
Propionic acid
Butyric acid
Caproic acid
Caprylic acid
Capric acid
Lauric acid
Myristic acid
Palmitic acid
Stearic acid
Arachidic acid
Crotonic acid
Palmitoleic acid
Oleic acid
Linoleic acid
Linolenic acid
Arachidonic acid
Systematic Name Chemical Formula
Saturated Acids
Methanoic acid HCOOH
Ethanoic acid CH,COOH
Propanoic acid CH,CH.COOH
Butanoic acid CH,(CH.).COOH
Hexanoic acid CH.(CH
2
).COOH
Octanoic acid CH.(CH2)6COOH
Decanoic acid
CH.(CH2)"COOH
Dodecanoic acid CH.(CH2)IOCOOH
Tetradecanoic acid CH.(CH2)'2COOH
Hexadecanoic acid CH.(CH2h.COOH
Octadecanoic acid CH.(CH2)'6COOH
Eicosanoic acid
CH3 (CH.h"COOH
Unsaturated Acids
trans2Butenoic acid
gHexadecenoic acid
cis-9-Octadecenoic acid
cis-9, cis-12-0ctadecadi-
enoic acid
9,12,15-0ctadecatrie-
noic acid
5,8,11,14-Eicosatetraenoic
acid
CH,CH=CHCOOH
CH
3
(CH
2
),CH = CH(CH
2
),COOH
CH
3
(CH
2
),CH = CH(CH
2
),COOH
CH.(CH
2
MCH2CH = CH).
(CH
2
),COOH
CH
3
(CH.CH = CH),(CH.),COOH
CH.(CH2MCH.CH = CH).(CH.laCOOH
'Formic, acetic, and propionic acids are included because they are listed as fatty acids in
some publications, although others consider butyric acid to be the simplest fatty acid_
TISSUE FOODS. Various natural microbial inhibitors have been found
in tissue foods (meat, poultry, fish). Lysozyme, discussed in relation to
inhibitors in egg white, is found in various tissues. Tolerances for antibi-
otic residues in meats have been established by the World Health Organi-
zation. Even though these inhibitors are at low levels, some microorga-
nisms may be inhibited.
A living animal has defense mechanisms to inhibit the invasion of
microorganisms. These include various antibacterial substances as well
as natural and immune antibodies. Although, after slaughter, the mecha-
nism for producing these materials is lost, those substances already pro-
duced in the tissue will remain. The extent to which they have an antibac-
terial function depends primarily upon their stability.
Tissues from cattle (brain, spleen, heart, kidney, and liver) possess
anti staphylococcal substances. Basic polypeptides with antibacterial
properties have been extracted from tissues (thymus, spleen, and thy-
roid). Synthetic basic polyamino acids are active against both bacteria
and viruses. Spermine and spermidine are polyamines found widely dis-
tributed in animal tissues and are inhibitors of many types of microorga-
nisms (Bachrach and Weinstein 1970; Fair and Wehner 1971). The anti
bacterial action of basic polyamino acids is thought to be disruption of
normal cell functions due to their combining with components of the
cell wall.
Some hormones are bacteriostatic. Progesterone (progesterol) is anti
bacterial to Grampositive organisms, and diethylstilbestrol (DES) has a
bactericidal effect on Staphylococcus aureus (Yotis and Baman 1969) and
Candida albicans (Yotis and Haan 1978). Deoxycorticosterone inhibits
Grampositive bacteria, yeasts, and molds. but not Gramnegative bac
teria.
Plant Products
Since the discovery of antibiotics, there have been several surveys in
which crude plant extracts or juices were tested for antibacterial activity.
The extracts of several thousand plant species have been tested and, de
pending upon the plants surveyed and the test organisms used, from 30
to 50 percent of the extracts showed some antimicrobial activity. Juices
and extracts from cabbage, carrot, green beans, celery, chicory, cucumber,
chard, okra, rhubarb, corn, and turnip have shown antimicrobial activity
to one or more microorganisms.
To be fair, it should be mentioned that some extracts have shown
stimulatory effects. Pea medium is used to detect heat-stressed anaerobes
in canned foods_
Several microbial inhibitors (flavanols, tannins, enzyme inhibitors,
lectins, cyclopropene fatty acids, phytic acid) found to be naturally pres-
ent in certain seeds, are discussed in a review by Halloin (1983). Although
they do have inhibitory properties, these compounds either impart unsat-
isfactory properties to foods, are toxic, or may enhance carcinogenic
properties of other compounds. These and some other selected micro-
bial inhibitors are listed in Table 4.10.
Besides those inhibitors, lysozyme is found in some plant products,
but at low levels. Enzyme inhibitors are found in various plant products.
Gossypol, found in cotton seeds, inhibits Gram-positive bacteria, yeasts,
and molds but has little or no effect on Gramnegative bacteria. The to
mato contains tomatin or a-tomatine, which inhibits various fungi and
bacteria. Anthocyanin pigments in cocoa possess antibacterial proper-
ties. Tannins comprise a group of natural phenolic compounds. English
walnut meats, carob pods, sorghum grains, malt, mint, and peanuts in-
hibit microorganisms due to the presence of tannins. There is evidence
that tannins interfere with enzyme activity, as well as absorbing on the
cell surface and altering the permeability of the cell wall.
Benzoic acid and its salts are used as food preservatives and are found
TABLE 4.10. MICROBIAL INHIBITORS NATURALLY PRESENT IN PLANTS OR FOOD
FROM PLANTS
Inhibitory Agent
Anthocyanin pigment
Acetaldehyde
Dimethoxyisoflavone
Purothionins
Juglone
Protease inhibitor
Trypsin inhibitor
Enzyme inhibitor
Lectins
Lipids
Free fatty acids
Tannin(s)
Phenolic
Lupulone
Humulone
Isohumulone
Methylxanthines
Cinnamaldehyde
Citral
Perillaldehyde
Citronellal
Extract
Phytic acid (binds zinc)
H ydroxycinnamates
p-coumaric acid
ferulic acid
p-methoxy cinnamate
Caffeic acid
Chlorogenic acid
Antiviral
Plant or Food
Fruits
Fruit tissue
Peanut
Wheat endosperm
Pecan
Barley
Corn
Bean
"Seeds"
Wheat germ
Peas
Cocoa
Peanuts
Mint
White potato
Hops
Cocoa beans
Oils (essential)
Carrot root
Soybeans
Fruits, vegetables
Grapes
Curcurma zedoaria
Chicory
Grape juice
Grape juice, apple juice,
tea
Organism Inhibited
Bacteria (enzymes)
Yeast
Aspergillus flavus
Pseudomonas, Xanthomonas,
Erwinia amylovora, Corynebac-
terium
Fusicladium effusum
Aspergillus
Fungi
Fungal cellulase
Virus
Trichoderma viride
Fungi
Aspergillus parasiticus
Antiviral
A. parasiticus
Gram-positive bacteria
Fungi
Saccharomyces cerevisiae, Escher-
ichia coli, Staphylococcus au-
reus, Bacillus cereus
S_ cerevisiae
Trichophyton rubrum
Aspergillus niger, S. cerevisiae
Fusarium nivale
Bacteria
Human enterovirus "inactiva-
tion"
Enteric virus
Poliovirus
naturally in many berries. Cranberries are especially prominent for con-
taining benzoic acid. Other compounds in cranberries with antimicrobial
properties were described by Chu, Clydesdale, and Francis (1973).
Dallyn and Everton (1970) found that some vegetable oils contain a
sporistatic or sporicidal agent. They believe that a peroxide precursor of
a carbonyl is involved in the antimicrobial activity.
Pectin is a component of plant products. Although pectin shows no
inhibitory properties to microorganisms, a product of hydrolysis of pec-
tin, ex methyl D-galacturonate, inhibits the growth of many Gram-negative
dysentery bacteria_
Olives contain oleuropein, a phenolic compound_ Juven, Henis, and
Jacoby (1972) reported that oleuropein was inhibitory to species of Lacto-
bacillus, Leuconostoc, yeasts, and molds_ An ethyl acetate extract of oleuro-
pein reveals two components (glycoside oleuropein and its phenolic agly-
cone), which have antibacterial activity_ Fleming, Walter, and Etchells
(1973) found that oleuropein was not inhibitory to lactic acid bacteria,
but two hydrolysis products, the aglycone and elenolic acid, were inhibi-
tory for four species of lactic acid bacteria_ The acid hydrolysate of oleuro-
pein showed inhibitory properties to species of Lactobacillus, Pediococcus,
Leuconostoc, Staphylococcus, Bacillus, Salmonella, Pseudomonas, Erwinia, and
Xanthomonas, but no inhibition of the yeasts that were used_ The inhibi-
tion of growth of Lactobacillus plantarum by oleuropein aglycone and elen-
olic acid also was detected by Federici and Bongi (1983)_ The inhibition
is due to membrane damage and leakage of cellular constituents_
Homogenates and extracts of onions and garlic have antimicrobial
activity_ It is thought that, when the bulbs are crushed, enzymes react on
compounds containing cysteine sulfoxide and form inhibitory thiosulfi-
nates_ One of the active compounds is allicin (thio-2-propene-1-sulfinic
acid-5-allyl ester)_ It is not known whether its action is due to enzyme
inhibition or to membrane disruption (Bogin and Abrams 1976)_ Of the
factors in onion, the lachrymatory factor (thiopropanal-S-oxide) had the
maximum inhibitory activity to spores of Aspergillus parasiticus (Sharma
et aL 1981)_ The inhibitory action was lost by heating, drying, aeration,
agitation, or prolonged storage_ Neither garlic nor onion oil inhibited
toxin production of Clostridium botulinum types Band E (DeWitt et aL
1979)_ Garlic extracts reportedly inhibit the growth of Cryptococcus neofor-
mans and Candida albicans_ Aqueous extracts inhibited the growth of Bacil-
lus cereus (Saleem and AI-Delaimy 1982)_ Higher microbial counts of on-
ion and garlic products are obtained when 0_05 percent K
2
SO:
J
is added
to the dilution water_
Fruits contain various organic acids that inhibit the growth of micro-
organisms_ A natural isoflavone found in some fruits has antifungal activ-
ity (Fukui et aL 1973)_ The essential oils of citrus fruits are found in the
peel and can be recovered from this waste product_ The disinfectant
power of lemon and orange oil against spore-bearing organisms is re-
portedly greater than that of phenoL Orange oil at 0_1 percent and 0_2
percent was found to inhibit both Gram-positive and Gram-negative bac-
teria (Subba, Soumithri, and Rao 1967)_ At 0_2 to 0_3 percent orange and
lemon oil suppressed growth of Aspergillus parasiticus and aflatoxin pro-
duction (Alderman and Marth 1976)_
Several spices or herbs and especially their essential oils inhibit var-
ious microorganisms (Shelef 1983)_ M ycotoxigenic molds were inhibited
by cloves, allspice, anise seed, eugenol extracted from cloves, thymol
from thyme, and anethol from anise seed (Hitokoto et aL 1980), by cloves,
cinnamon, mustard, allspice, and oregano (Azzouz and Bullerman 1982),
by o-methoxycinnamaldehyde from cinnamon (Morozumi 1978), and by
various essential oils (Benjilali et aL 1984).
Low levels of oregano or cloves stimulated acid production of Lactoba
cillus plantarum and Pediococcus cerevisiae, while higher concentrations in
hibited acid production (Zaika and Kissinger 1979,1981). Lactic acid bac
teria developed a resistance to the herbs oregano, rosemary, sage, and
thyme (Zaika, Kissinger, and Wasserman 1983). Grampositive bacteria
were more sensitive than Gram-negative bacteria to the spices and herbs
tested (Shelef, Jyothi, and Bulgarelli 1984).
Eugenol from oil of cloves was more effective than vanillin, an alco-
holic extract of the vanilla bean, in inhibiting medically important yeasts
(Boonchird and Flegel 1982). According to Davis and Cooks (1982), eu
genol is also a constituent of nutmeg.
Apparently there are inducible resistance responses in plants to
pathogenic bacteria (Caruso and Kut: 1979; Goodman 1978), which might
carryover to the fruit.
Summary
There are many natural microbial inhibitors in foods. These inhibi-
tors give the living animal or plant some protection from invading para-
sites. The food products, being an extension of the animal or plant, have
a residual amount of these agents. Most of these natural compounds in-
hibit Gram-positive more than Gram-negative organisms. Perhaps this is
one reason that spoilage is more often associated with Gram-negative
organisms.
The microbial inhibitors naturally present in foods may maintain the
food in a satisfactory state for transportation, processing, and distribu-
tion.
PROTECTIVE BARRIERS
Some foods are protected from direct contact with microorganisms
by a natural cover or barrier. Examples of barriers are the shell and shell
membrane of eggs, the testa of seeds, and the cuticle of intact plant
organs.
Egg Shell and Membranes
An invisible, natural, proteinlike film on the egg shell, called the cuti
cle, or bloom, is considered by some to be the first line of defense against
microbial penetration of the egg. The second physical barrier is the egg
shelL Inside the shell are two membranes, the outer and inner shell memo
branes, which are the third and fourth barriers to microbial penetration.
The structure of the egg and egg shell is shown in Figures 4.5 and 4.6.
CUTICLE. Washing of eggs and the use of abrasives to remove dirt
disrupt the cuticle layer and allow easier penetration by microorganisms.
There is a diversity of opinion on the effect of washing eggs on the result
ant contamination of the interior. The cuticle tends to crack and deterio
rate with time. Hence, it is not much of a barrier for stored eggs.
EGG SHELL. The egg shell is not a homogeneous structure. It consists
of an organic framework of fibers and an interstitial substance of inor
ganic materiaL The two main layers of the shell are the outer or spongy
layer and the inner or mammillary layer. The outer layer is thicker and
contains most of the minerals.
The shell contains from 6,000 to 8,000 microscopic pores. These
pores allow the exchange of water vapor and gases between the contents
of the shell and the outer atmosphere. The pores vary in size, with ex
tremes being reported as 1.6 to 7.47 p.m. The average pore size is prob
ALBUMEN
SHELL
Figure 4.5. The parts of an egg.
Courtesy of USDA.
YOLK
GI,. iul disc (Bludod,,.)
Latebll
Lltht yolk Ilyer
Oark yolk Ilyer
Yolk (Vit,lIine) .,.brut
MEMBRANE
Air etll
Out" shell .e.brln,
Inn" .h,1I bllne

"", / \',
I CUTICLE
2. SPONGY LAYER
:: } 3. MAMMILLARY LAYE"
_ ___ ---=- 7'.-- --. ___.. 4. SHELL MEMBRANE
-- - ,
'" ,
,''' !
5. MAMMILLA (MAMMILLARY KNOB) 6. PROTEIN MATRIX MATERIAL FORMING
CORE OF THE MAMMILLA
Figure 4.6. Magnified radial section through an egg shell.
Courtesy of USDA.
ably between 20 and 45 p.m. The size of most pores will allow the passage
of many microorganisms. Even yeast cells can be forced through pores,
and mold mycelia grow through the pores. There is a linear relationship
between shell porosity and infection of the eggs.
Shell weight and shell thickness do not correlate with penetration of
microorganisms. However, thin shells have a tendency to crack more
readily than thicker shells. Eggs with damaged shells are more subject to
penetration of bacteria and egg spoilage.
If the temperature ofthe egg is higher than the surroundings, a condi-
tion that exists when the egg is laid, there is a greater possibility of pene-
tration and, as the temperature differential increases, the potential for
infectivity increases. As the egg contents cool, they contract, which results
in a higher pressure outside the shell than inside. This pressure differen-
tial will force microorganisms through the pores of the shell. For further
information about the egg shell, refer to Board (1980) and Tung, Gar-
land, and Gill (1979).
Moisture on the egg shell, such as occurs during washing of eggs or
when a cold egg is brought into a warm and humid room, increases the
potential for microbial infection of the egg.
SHELL MEMBRANES. The outer shell membrane is in contact with
the egg shell, and the inner shell membrane, sometimes called the egg
membrane, is in contact with the albumen. They are composed mainly
of protein fibers (fibrin and mucin) strengthened with an albuminous
cementing material. The membranes have been considered effective bac
terial filters. The inner membrane is more effective than the outer memo
brane because of its closely knit fibers. Organisms inoculated onto the
shell surface are found on the membranes almost instantly, but micoor
ganisms inoculated into the air cell and onto the inner membranes are
not recoverable from the albumen for several days. This apparent reten
tion of bacteria on the inner membrane may be partly due to conalbumin
in the albumen, since adding iron to the inoculum or to the interior
albumen of the egg hastens the penetration of the bacteria through the
inner membrane. Wedral, Vadehra, and Baker (1971) found no differ
ence in the permeability of the inner shell membrane before and after
passage of either Pseudomonas aeruginosa or Salmonella typhimurium, indicat
ing that no enzymatic activity is needed for penetration. With the condi
tions of their experiment, both bacterial species were able to penetrate
from the shell surface through the inner membrane within two hours. By
36 hr after shell inoculation, all except one egg showed contaminated
albumen. Although there are no pores, per se, in the membranes, there
are apparently openings between the intermeshed fibers similar to a
maze, which allow passage of bacteria through the membranes.
Even if bacteria can penetrate these barriers, they are confronted with
the antimicrobial mechanisms of the albumen before they can attack the
nutrients in the yolk.
Barriers in Plant Products
Foods such as fruits, vegetables, grains, and nuts contain a peel, skin,
or hard surface that covers the interior food and protects it from micro
bial attack (Halloin 1983). The effectiveness of these barriers is evidenced
by the growth of mold primarily on the stem end of fruit, where injury
to the cover may occur during picking. Also, when injury occurs to the
barrier by insects, birds, or bruising, the microbial population in that
area is high and rotting of the fruit occurs there, before spreading to the
remainder of the fruit.
In the section on inhibition, the presence of essential oils in the peel
of citrus fruit is discussed. There are inhibitors in the waxlike coating on
many fruits. Cabbage leaves contain wax esters on their surface. The wax
like coating of apples increases during storage, the extent of the increase
depending upon the cultivar.
The protective coverings are not only microbial barriers, but also
help control diffusion of materials into and out of the fruits or nuts. The
sale of nuts in the shell has remained fairly constant, while purchases of
shelled nuts have increased. Removing the nuts from the shell subjects
them not only to microbial contamination but also to oxidative rancidity
of the fatty material and other deteriorative changes.
The seed coat and peri carp of grains, which allow the long term stor
age of these foods, were important in establishing human civilization.
According to Halloin (1983), the seed coat is the most important compo
nent in the resistance of seeds to deterioration.
TEMPERATURE
Since water, as the solvent, is necessary for microbial growth, the tem
peratures that will support microorganisms are limited to those at which
water is a liquid. Pure water freezes at oDe, but the addition of solutes
lowers the freezing point. The exact minimum temperature for microbial
growth is difficult to determine, because the solutes that are added to
prevent freezing of water below -lODe may inhibit the microorganisms.
Water boils at lOOoe and is no longer acceptable for growth at that
temperature. The boiling point of water is increased by adding solutes
or by increasing the pressure above atmospheric. By increasing the pres
sure in a chemostat, a culture of Bacillus caldolyticus grew at 105e
(Heinen and Lauwers 1981). With further increased pressure and higher
boiling points, bacteria might grow at much higher temperatures.
The absolute highest and lowest temperatures for growth remain un
known, primarily because of problems of incubation at extremely high
or low temperatures.
Temperature is one of the most important environmental factors that
regulates the growth of microorganisms. Temperature is related to the
ability of an organism to grow and to survive. The environmental temper
ature also has an effect on cell size, metabolic products such as pigments
and toxins, nutritional requirements, enzymatic reactions, and the chem
ical composition of cells.
Ranges for Growth
Each organism has a mInimUm, optimum, and maximum temper
ature for growth. The minimum and maximum temperatures are those
beyond which the organism ceases to grow. The optimum temperature is
more difficult to describe, since it may be the optimum for total cell yield,
rate of growth, rate of metabolism, respiration, or the production of
some metabolic product. Usually the optimum temperature is based on
the rate of growth.
In general microbiology, the microorganisms are usually divided into
three arbitrary classes: psychrophilic (low temperature), mesophilic (me
dium or middle temperature), and thermophilic (high temperature). Psy
chrophiles and thermophiles are divided into facultative and obligate
types.
The temperature ranges for the various types are listed in Table 4.11.
Microbiologists tend to disagree on the exact ranges of growth for the
different types. The fact that some organisms grow at low, some at inter
mediate, and others at high temperatures is more important than the
exact temperature for these ranges. The approximate temperature
ranges for the growth of selected microorganisms are listed in Table 4.12.
One should remember that the growth temperature depends upon the
strains of a species and on the chemical and physical properties of the
substrate.
In general, organisms can survive and show some growth at temper
atures considerably lower than the optimum temperature. When the tem
perature is increased above the optimum, there is usually a very rapid
decline in the growth rate. The maximum temperature is usually only a
few degrees (5
0
-12C) above the optimum. Above the maximum temper-
ature, growth ceases and the life of the cell is in jeopardy.
PSYCHROPHILES AND PSYCHROTROPHS. There are many defini
tions of psychrophilic organisms. One of the simplest definitions is that
psychrophiles grow well at OOC. They should produce a visible colony at
that temperature in seven, ten, or fourteen days. This definition does not
consider optimum, minimum, or maximum temperatures. Perhaps some
distinction should be made between those organisms with a maximum
temperature of 20C or less and those that can grow at a higher maxi-
mum. Morita (1975) suggested that psychrophiles have an optimal tem-
perature for growth at about 15C or lower, a maximum at about 20
o
e,
and a minimum at ooe or lower. Organisms that grow at low temper-
atures but do not meet the definition are called psychrotrophs. The term
TABLE 4.11. APPROXIMATE TEMPERATURE RANGES OF GROWTH
FOR ARBITRARY CLASSES OF MICROORGANISMS
Temperatures roC)
Minimum Optimum
Psychrophilic -15-5 10-30
Obligate -15-0 10-20
Facultative -5-5 20-30
Psychrotrophic -5-5 25-30
Mesophilic 5-25 25-40
Thermophilic 35-45 45-65
Obligate 40-45 55-65
Facultative 35-40 45-55
Maximum
20-40
20-22
30-40
30-40
40-50
60-90
70-90
60-80
TABLE 4.12. TEMPERATURE RANGES OF SELECTED MICROORGANISMS
Temperature (OC)
Microorganism Minimum Optimum Maximum
Bacteria
Acetobacter 5 42
Aeromonas 0-5 25-30 38-41
Bacillus cereus 10 47-50
Brevibacterium 5 42
Clostridium 0-45 60
C. botulinum 3-10 30-40 42-45
C. perfringens 15-20 30-40 45-51
C. putrefaciens 0 20-25 30
C. thermosacchamlyticum 43-45 55-62 70-71
Escherichia coli 3-10 37-41 48-50
Lactobacillus 5 30-40 53
Leuconostoc 10 20-30 40
Micmcoccus 10 25-30 45
Moraxella -1-2 30 41-42
Pmpionibacterium 2-3 30-37 45
Pmteus 10 37 43-45
Pseudomonas -7-4 20-30 31-43
P. aeruginosa 8 42
P. fluorescens 0-6 20-25 40
Salmonella 5-10 35-37 46-49
Staphylococcus 5-10 35-40 46-48
S. aureus 5-10 35-39 44-48
Streptococcus c?'emoris 25-30
Sfaecalis 5-10 37 49-51
S.lactis 10-15 25-30 40
S. thermophilus 20 40-45 52
Vibrio 10-37
V. parahaemolyticus 3-13 35-37 42-44
Xanthomonas 0-5 25-31 40
Yersinia enterocolitica 0-4 37
Molds
Aspergillus fumigatus 30-40
Botrytis cinerea -1 20 30
Cladosporium -5--8
Penicillium rubrum 25-28
Rhizopus stolonifer 5 25
Yeasts
Candida 0 29-48
C. lipolytica 5 25 35-40
Hansenula 37-42 50
Saccharomyces 0-7 20-35 40
Torulopsis
0 17-25 30-35
psychrotroph was suggested by Eddy (1960) to apply to microorganisms
capable of multiplying at SoC and below, regardless of the optimum tem
perature. This definition does not include the minimum or maximum
temperature. Eddy's definition of psychrotroph is essentially that of a
psychrophile, except that the temperature has been increased from 0 to
5C. There are few, if any, obligate psychrophiles in foods. Most microor
ganisms that grow in food at low temperature are psychrotrophic.
The spoilage of refrigerated food (meat, milk, eggs) is generally due
to psychrotrophic growth. Psychrotrophs are found in many genera and
include aerobes and anaerobes.
The pseudomonads are an important group of psychrotrophs. Some
members of the genus can grow at 4C, while others grow at 43C. Over
all, the optimum temperature is somewhere between 20 and 30C. P.
fluorescens grows at 4C or less, but not at 41C. The usual range for P.
aeruginosa is 8 to 42C. The optimum temperature for pigment produc-
tion of P. jluorescens is 20C. The diameter of colonies of P. jluorescens in-
creases at a more rapid rate at 30C than at 25C or less (johnson et al.
1970).
MESOPHILES. The mesophiles are organisms that grow in the middle
temperature range. We can define mesophiles as those organisms with
an optimum temperature of 25 to 45C. There are two groups of organ-
isms in the mesophilic range. The saprophitic organisms have an opti
mum temperature of 25 to 30C, and potential pathogens have an opti-
mum between 35 and 45C.
THERMOPHILES. These organisms have been defined as growing at
high temperatures, with an optimum of 45C or higher. The information
in Table 4.11 shows the minimum temperature for thermophiles some-
where between 35 and 45C, the optimum between 45 and 65C, and
the maximum between 60 and 90C. Since the minimum temperatures
for thermophiles overlap the higher mesophilic range, the thermophiles
have been divided into facultative and obligate types. The facultative
thermophiles can grow in the mesophilic range of 37C or less, but the
obligate thermophiles cannot grow at 37C.
ENUMERATION. If the arbitrary temperature classes have any mean-
ing, methods for determining the organisms should follow the defini
tions. The incubation condition for psychrotrophs is listed as 7C for ten
days (APHA 1984).
Juffs (1972) compared the extremes of the British Standards Institute
method of incubating at 5 to 7C for seven to ten days to determine
psychrotrophs. The high extreme (7C for ten days) is the same as the
APHA (1984) method.Juffs found that 7C for seven days or 5C for ten
days gave counts that were not significantly different. However, counts
from plates incubated at 7C for ten days were significantly higher, and
those using 5C for seven days were significantly lower.
The incubation period of seven or ten days becomes impractical as
a control measure for perishable refrigerated foods such as milk. By
the time the count is obtained, the product is consumed or may have
developed defects. As a continuing control procedure, it is of value by
revealing potential problems. To obtain a psychrotrophic count sooner,
incubation at 21C for 25 hr yields counts similar to the standard psy
chrotrophic method (Oliveria and Parmellee 1976).
Comparing the incubation temperature of 7C to the definitions of
classes, it fits neither the OOC given for psychrophiles nor the 5C given
for psychrotrophs. Since the term psychrotroph has been substituted for
psychrophile, and using the APHA plating system, we might define psychro
trophs as those organisms that grow at 7C and produce a visible colony
in ten days.
The mesophilic count can be related to the standard plate count, or
aerobic plate count. Incubation temperatures of 25 to 37C have been
used. The 37C temperature indicates the potential pathogen popula
tion better than the lower temperature; however, since spoilage organ-
isms are a problem and higher counts are obtained, a temperature of
32 or 35C is used (APHA 1984).
Thermophiles are enumerated by incubating the culture at 55C
2C for 48 to 72 hr (APHA 1984).
RELATIVE IMPORTANCE. The relative importance of each of the
temperature classes (psychrophiles, psychrotrophs, mesophiles, and ther-
mophiles) depends upon the field of microbiology. Much of our food
supply is held in cold storage, which makes psychrotrophs important as
potential spoilage organisms.
Psychrotrophic microorganisms are ubiquitous in nature. Although
they are favored by cold, they are not confined to temperatures of 5C
or less. Psychrotrophs are in fresh and salt water, in soil, and in or on
nearly all raw food. Stokes (1968) reported that psychrophiles constituted
35 to 95 percent of the bacterial population on meats and 67 percent of
the population on fresh chicken. At low temperatures (0-5C), carbo-
hydrate fermenters generally are inhibited and acids are not formed. Pro-
teolytic and lipolytic organisms grow at low temperatures, resulting in
food defects.
Mesophilic species can be found in nearly every genus of organisms
important in foods. Although spoilage of foods by me sop hiles is signifi-
cant, there is more concern with the mesophilic organisms that cause
foodborne illness. These include species of Salmonella, Staphylococcus, Clos-
tridium, Shigella, and Bacillus.
Since the rate of growth of microorganisms and enzyme activity is
higher in the mesophilic range than in the psychrophilic range, spoilage
of food occurs sooner.
Thermophiles can cause spoilage whenever foods are held at 50 to
70C. This may occur during heating of the food while cooking, process
ing, or pasteurizing. Their growth in hot syrup may cause undesirable
effects in sugarprocessing plants. In some tropical countries, thermo
philes-not psychrotrophs-are the important spoilage organisms.
The generation time is shorter for thermophiles than for either psy
chrophiles or mesophiles when each is grown at its optimum temperature.
This results in a more rapid spoilage of food by thermophiles.
The thermophiles may be more important than is presently realized.
Since food is not normally held at temperatures conducive for growth of
thermophiles, they have not been given the attention that has been allo
cated to the other classes. Due to the rapid growth and metabolism at
high temperatures, the new biotechnology is examining thermophiles for
direct use in fermentations or as a source of enzymes (see Chapter 9).
Effect of Organisms on Temperature
Just as the environmental temperature alters the growth of microor
ganisms, the growth of microorganisms alters the temperature. When the
organisms metabolize organic compounds, they not only grow and multi
ply, they also produce heat as a byproduct. In a compost pile, the temper
ature may rise to a level at which only thermophilic bacteria can grow.
In the production of compounds by microbial fermentation, it is often
necessary to use a cooling system in the fermenter to maintain the tem-
perature so that the maximum amount of desired product is developed.
GASEOUS ATMOSPHERE
The type of gas in the atmosphere surrounding the food may deter-
mine the types of organisms that become dominant. Oxygen in the atmo-
sphere will favor the growth of aerobic types. The lack of oxygen, or a
vacuum, will allow facultative anaerobes to become dominant.
Microorganisms vary widely in their tolerance to carbon dioxide. In
a CO2 atmosphere, the growth of some microorganisms is completely
suppressed, while others are less affected. The lag period for growth of
spoilage organisms on beef is increased in a 10 percent CO
2
atmosphere.
Also, these organisms need a higher minimum water activity for growth
in 10 percent CO2 as compared to the normal atmosphere (0.033 percent
CO2). On the other hand, it is well documented that the presence of low
concentrations (1 percent or less) of CO
2
stimulates oxygen uptake of
bacteria. The amount of stimulation varies with bacterial species.
According to King and Nagel (1975), CO
2
inhibits the synthetic proc-
ess of Pseudomonas aeruginosa, since resting cell metabolism is not affected_
MICROBIAL INTERACTIONS
Microorganisms can inhibit or stimulate the growth of one another.
Although pure cultures are usually used in laboratory studies, mixed cul-
tures are found in nature and on food products_ Since the main goals of
all living forms are self-preservation and perpetuation of the species, the
microorganisms have to compete for food and the other necessities. As
a result, there are various actions and reactions (or coactions) that may
be harmful or beneficial. Burkholder (1952) listed nine possible coac-
tions when organisms are in a mixed population:
Predation. In this relationship, the strong predators damage the weak
prey.
Parasitism. The weak parasite benefits at the expense of the strong
host.
Commensalism. The coaction results in the weak benefiting, and the
strong is unaffected.
Amensalism. The opposite of commensalism. The strong benefits and
the weak is unaffected.
Allotrophy. In this relationship, the strong feeds the weak.
Allolimy. The strong starves the weak.
Symbiosis. In symbiosis, there is mutual aid, with both organisms
benefiting from the relationship.
Synnecrosis. There is mutual conflict resulting in the death of both
organisms.
Neutrality. Neither organism benefits or loses from the relationship.
There is no coaction between them.
Systems for determining interactions of microorganisms have been
reported (MacFarlane and Makrides 1982; Nordbring-Hertz 1983).
Metabolism
The growth of microorganisms depends on the chromosomally deter-
mined primary metabolism. The loss of primary metabolism results in
the death of the cell. Secondary metabolism is not essential for the
growth and multiplication of microorganisms. Both primary and second-
ary metabolism produce metabolic products. Quite often the production
of secondary metabolites is induced by the presence of plasmids. The
plasmids are composed of extrachromosomal nucleic acids that can be
eliminated from the cell by various treatments and, under acceptable
conditions, can be transferred from one cell to another. Simple and rapid
methods for isolation and preparation of bacterial plasmids have been
described by Anderson and McKay (1983) and Summerton, Atkins, and
Bestwick (1983). By using plasmids to transfer genetic information, we
may be able to develop more useful organisms than we have now.
The products of metabolism may stimulate the growth of other organ
isms. A strain of Staphylococcus aureus that required thymine and trypto
phan was able to grow in mixed cultures or with culture filtrates of
Pseudomonas aeruginosa (Gadbois, De Repentigny, and Mathieu 1973). The
Pseudomonas supplied the thymine and tryptophan essential for the
growth of the S. aureus.
U sing a chemically defined medium that supported the growth of the
yeast Saccharomyces cerevisiae, but not Proteus vulgaris, researchers found
that both organisms grew in mixed culture (Shindala et al. 1965). A niacin
like factor, produced by the yeast, made it possible for the bacterium to
grow. When niacin was added, the Proteus was no longer dependent upon
the yeast and promptly outgrew it.
Several bacteria isolated from bananas stimulated the germination of
the conidia of Colletotrichum musae on this fruit (McCracken and Swin
burne 1980). On an agar medium deficient in L-cysteine, Legionella pneu-
mophila did not grow, but when Flavobacterium breve was present, the Legio-
nella grew as satellites around the Flavobacterium colonies (Wadowsky and
Yee 1983).
Although these citations show one organism aiding another or-
ganism, the dominance of one species can result in the utilization of the
nutrients so there is little or none left for other species.
pH. Microorganisms can alter the pH upward or downward, and
thereby influence the growth of other microorganisms. The lactic acid
bacteria (lactobacilli, streptococci, pediococci, leuconostoc) inhibit sev-
eral bacteria (both spoilage types and foodborne pathogens) and viruses
partially by acid formation with low pH and also by some undetermined
factors (Abdel-Bar and Harris 1984; Collins-Thompson, Wood, and Be-
veridge 1983; Gilbert et al. 1983; Moon et al. 1982; Rao and Pulusani
1981; Reddy and Ranganathan 1983; Ross 1981; Talon, Labadie, and Lar-
pent 1980). Montville (1982) reported that, in a model system at pH 4.4,
growth of Bacillus licheniformis raised the pH sufficiently to allow growth
and toxin production by C. botulinum. However, he found no toxin in
canned tomatoes with elevated pH values.
INHIBITORS. The production of microbial inhibitors by microorgan
isms is well known. Besides the chemicals called antibiotics, there are
other metabolic compounds that have antibioticlike or inhibitory charac
teristics. Several enzyme inhibitors are produced by microorganisms, but,
according to Umezawa (1982), most of them have no significant antimi
crobial activity. Some microbially produced inhibitors are listed in Table
4.13.
The pseudomonads produce several antibiotic substances, including
pyocyanine and pyrrolnitrin. Species of Pseudomonas influence the
growth of strains of Vibrio parahaemolyticus, S. aureus, Bacillus, as well as
some molds and yeasts (CollinsThompson, Aris, and Hurst 1973; Goat
cher and Westhoff 1975; Hemming et al. 1982; Scannell et al. 1972; Van
denbergh et al. 1983).
Strains of species vary in their interactions with other organisms. In
comparing the interactions of four strains of Streptococcus cremoris and
four strains of S. lac tis, it was found that two strains of S. cremoris domi
nated the four S. lac tis, one strain of S. cremoris was compatible with the
S. lac tis, and one strain of S. cremoris was dominated by the four S. lactis
strains (Reddy et al. 1971).
Bacillus species interact with other microorganisms. B. subtilis pro
duces an antibiotic called subtilin. This polypeptide has a marked action
against a wide range of Gram'positive, acidfast, and certain Gram
negative bacteria. Depending on the concentration, subtilin is bacterio
static or bactericidal. Barr (1975) found ten antimicrobial metabolites
produced by a strain of B. subtilis. Some of these were antibacterial, while
others were antifungal. Bacillus species isolated from mud inhibited the
growth of C. botulinum type C (Graham 1978). A thermophilic species of
TABLE 4.13. SOME TYPICAL MICROBIALLY PRODUCED MICROBIAL INHIBITORS
Inhibitory Substance Organism
Organism Produced Inhibited Reference
Serratia marcescens Prodigiosin E. coli, B. subtilis, Kalesperis, Prahlad,
Enterobacter aero and Lynch
genes, S. aureus, P. (1975)
aeruginosa
P. aeruginosa Amino acid anti Bacillus sp. Scannell et al.
metabolite (1972)
Saccharomyces Glycoprotein Torulopsis glabrata Bussey and Skipper
cerevisiae (1976)
Lactobacilli Hydrogen peroxide Pseudomonas sp. Price and Lee
Bacillus sp. (1970)
Proteus sp.
C. perfringens Unknown C. botulinum Smith (1975)
S.lactis Nisin C. botulinum Scott and Taylor
(1981)
Bacillus not only provided some protection of mushroom beds from the
mold Chaetomium olivaceum but also increased the yield of Agaricus bisporus
(Tautorus and Townsley 1983). A B. subtilis controlled the onion white
rot organism, Sclerotium cepivorum (Utkhede and Rahe 1983).
Volatile metabolites of certain strains of bacteria were able to inhibit
the growth, sporulation, and mycotoxin production of Penicillium and
Aspergillus species (Barr 1976).
Aflatoxin, a substance produced by the mold Aspergillusflavus, inhibits
various Bacillus species (Lillehoj and Ciegler 1970). Also, fusariotoxin
T2 produced by Fusarium inhibits the growth of Saccharomyces cerevisiae
and S. carlbergensis (Schappert and Khachatourians 1983).
Certain strains of Saccharomyces cerevisiae produce a toxin that kills sen
sitive strains of this yeast (Wickner 1983; Young 1981).
Antagonism among microorganisms may be due simply to competi
tion for space or nutrients. One organism may gain an advantage over
another because of its motility (Lauffenburger and Calcagno 1983) or to
environmental effects (temperature, moisture) favoring one organism
over the others.
Fatty Acids. These substances are discussed as inhibitors naturally present
in foods. They also are formed by microorganisms during metabolism.
The growth of Clostridium botulinum is inhibited on surfaceripened
cheese due to the formation of fatty acids by Brevibacterium linens. Salmo
nella gallinarum is inhibited by Leuconostoc citrovorum due to the acidic pH
and production of acetic acid (Sorrells and Speck 1970).
Hydrogen Peroxide. The mechanism of hydrogen peroxide (H
2
0 2) forma
tion by streptococci was described by Anders, Hogg, andJago (1970). The
amount of H
2
0
2
that accumulates varies among strains. The addition of
catalase or ferrous sulfate to milk prevents peroxide accumulation and
results in an increased rate of acid production by the lactic streptococci
(Gilliland and Speck 1969).
Organisms such as the micrococci produce catalase. It was reported
by Nath and Wagner (1973) that in the presence of a Micrococcus species,
the growth of, and acid production by, six cultures of lactic acid bacteria
is stimulated. The stimulation is greater than that observed with the addi
tion of catalase, indicating that factors besides hydrogen peroxide reo
moval are involved.
Hydrogen peroxide produced by Streptococcus mitis and Lactobacillus
acidophilus, in combination with a peroxidase and a halide, has viricidal
activity to polio virus (Klebanoff and Belding 1974).
BACTERIOCINS. The bacteriocins are bactericidal substances pro
duced by various species of bacteria. Although considered antibiotics,
they are not like the classical antibiotics since they are macromolecular,
including or consisting of polypeptide or protein, and they generally act
on strains of the same, or closely related, species (Vidaver 1983).
The colicins have been studied the most extensively. They are found
in E. coli and other Enterobacteriaceae. There are over twenty types of
colicins. In some respects, they act similarly to phage. They can be in
duced by UV light, similarly to prophage. In some cases, they are held
intracellularly, and when released, the cell is lysed.
When bacteriocins attach to the receptor sites of a sensitive cell, one
or more things may occur, depending upon the bacteriocin and the cell.
One of the effects is the inhibition of macromolecular synthesis, such as
proteins, RNA, and DNA (Konisky 1982). Some colicins inhibit only pro
tein synthesis, while others inhibit all three. Inhibition of respiration may
occur with some colicins, while others may block the function of per
meases. Interference with the formation of ATP, or enhancement of its
breakdown, has been reported. Leakage of cellular constituents has been
reported but this may be due to secondary reactions and not caused di
rectly by the bacteriocin. Colicins disrupt transport mechanisms of
amino acids, which results in a lack of protein synthesis.
Bacteriocins of Grampositive organisms generally have a wider spec
trum of activity than those produced by Gram-negative cells.
Parasites
Two of the parasites of bacteria are the bacterial viruses or bacterio-
phages and the bacterial genus, Bdellovibrio.
Some phages have a limited number of microbial species that are sus-
ceptible hosts. In a species of bacteria there may be susceptible and resist-
ant strains of bacteria. For other phages, there may be several species
and even different genera of bacteria that contain susceptible cells. For
instance, some pasteurella phages also attack strains of Salmonella and
Shigella. The limited hosts for phages makes it possible to use phage typ-
ing to differentiate bacteria.
The Bdellovibrio resemble the virulent bacteriophages in their ability
to lyse bacterial cells. However, unlike the phages, these microorganisms
are actively motile (a single flagellum), small (0.25-0.4 p.m wide and 0.8-
1.2 p.m long), vibroid, Gram-negative bacteria. The formation of plaques
(holes in a host lawn due to lysis of cells) is usually developed in 12 to
24 hr by phage, but Bdellovibrio plaques are visible only after two to four
days, and they enlarge up to six days of incubation. Host-independent
Bdellovibrio populations have been grown, which has not been possible
with phage. About one in a million cells is host independent, and these
host independent cultures can revert to hostdependent varieties at
about the same rate.
SUBLETHAL STRESS
During the processing of foods, microorganisms can be subjected to
various physical or chemical stresses. These include treatments such as
heat, freezing, drying, osmotic effects, irradiation, and various chemicals.
To maintain the quality attributes of the food, the treatments may be
minimal and the effect on the microorganisms is of secondary consider
ation. In other cases, the treatment may be an attempt to control certain
organisms, but others may be affected. These treatments that do not kill,
but damage or injure the cell, are called sublethal.
The damages or injuries to the organism due to sublethal treatments
are called lesions. As a result of the injuries, the growth capabilities of
the organisms may be altered, both in the food and on media during
enumeration. The stressed cells may have an extension of the lag phase,
more exacting growth requirements, or increased sensitivity to inhibitors
or to selective agents in media. The longerthannormallag phase can be
called the recovery period, during which the damage to the cell is reo
paired. Although the cells usually can recover in a noninhibitory growth
medium, they can repair malfunctions in media in which growth is not
evident. In other words, growth is not needed for repair of the lesion.
It has been stated that injured organisms that normally cause food
borne illness or food spoilage, when in a substrate conducive for repair,
can, after repair, be involved in these unacceptable activities. However,
in most populations with injured cells there are also some cells with no
injury. Hence, given sufficient time for multiplication, the uninjured cells
can cause foodborne illness or spoilage, without any assistance from the
repaired cells.
Heating
In several processes (scalding, blanching, pasteurization, cooking, and
spray drying), the food and associated microbial flora are subjected to
heat. Since none of these processes are designed to obtain a sterile prod
uct, some organisms may be killed, many damaged, and others unaffected
by the treatment.
Heatinduced lesions include damage of the cytoplasmic membrane,
with leakage of cellular components, alteration of metabolic capabilities
Qf the cell, impairment Qf enzyme activity, and degradatiQn Qf ribQsQmes
and ribQnucleic acid.
Heat-induced damage results in the IQSS Qf ability to. grow in cQndi-
tiQns in which nQrmal cells can grQw. N Qrmal S. aureus cells can grQw in
media with 7.S percent NaCI, but heat-damaged cells cannQt tQlerate 4
percent salt (SmQlka, NelsQn, and Kelley 1974). Repair, with the return
Qf salt tQlerance, Qccurs withQut grQwth in either a dilute synthetic Qr a
rich cQmplex medium (Hurst and Hughes 1981). The pH range fQr Qpti-
mum grQwth is mQre narrQW fQr heat-stressed cells than fQr nQrmal cells.
The additiQn Qf catalase Qr superoxide dismutase to. tryptic SQy agar pro-
tected heat-stressed cells frQm hydrQgen perQxide (Bucker and Martin
1982). This results in higher CQunts Qf sublethally injured cells (Egan
1979). For increased recQvery Qf injured E. coli cells in fQQd samples,
McDQnald, Hackney, and Ray (1983) suggested adding 3, 3'-thiQ-diprQpiQ-
nic acid to. supplement media fQr the repair process.
The temperature Qf incubatiQn befQre and after heating influences
the viability Qf E. coli (Katsui et al. 1982). They suggested that the fluidity
Qf the cell membrane was invQlved in this reactiQn. The repair Qf the
nucleQid structure Qf E. coli cells after heat shQck (SOC fQr S min) is
different than that after mQre severe heat treatments (PellQn 1983).
All SQlid media were more inhibitQry to. heat-stressed cells Qf Salmo-
nella typhimurium than were the corresPQnding liquid media (Mackey and
Derrick 1982). HQwever, the additiQn Qf either catalase Qr pyruvate to.
nutrient agar resulted in increased recQvery and cQmparable grQwth to.
that in nutrient brQth.
Yeasts heated in water near the maximum temperature fQr grQwth are
essentially undamaged, but the presence Qf glucQse induces leakage Qf
cell CQntents (Hagler and Lewis 1974). Calcium iQns tempQrarily protect
the yeasts against glucQse-induced leakage. This indicates damage to. the
cytQplasmic membrane. If heated in the presence Qf glucQse, injury af-
fecting endQgenQus respiratiQn is irreversible.
Yeasts have been enumerated Qn acidified (to. pH 3.5) media; hQwever,
when twelve species Qf yeast were heat stressed, the IQwest Qptimum pH
fQr any Qf these was pH 6.8 and the maximum Qptimum was as high as
pH 10 fQr two. yeasts (N elsQn 1972). KQburger (1972) fQund that mQst
retail fQQd samples yielded maximum counts Qf yeasts and mQlds when
the medium was adjusted to. pH 8. FQr enumeratiQn Qffungi, the additiQn
Qf antibiQtics to. media, rather than acidification, to. cQntrQI bacteria, is
nQW the usual prQcedure. The effects Qf variQus chemicals in media Qn
recQvery or inhibitiQn Qf heat-stressed S. cerevisiae were discussed by Beu-
chat (1982). CQnner and Beuchat (1984) fQund that variQus species Qf
heat-stressed yeasts CQuid nQt repair their irUuries when eXPQsed to. rela-
tively low levels of essential oils of plants (allspice, cinnamon, clove, gar
lie, onion, oregano, savory, thyme).
Heat injury can occur in the spores of Bacillus and Clostridium. The
germination system of B. subtilis is impaired by heating. Primary activa
tion of spores at 90C for 60 min, reduction of the incubation temper
ature by 10 to 15C or addition of CaCh and sodium dipicolinate, in
creases germination and colony formation of heatdamaged spores. The
addition of lysozyme to enumeration media improves the recovery of
severely heated Clostridium spores. Bacterial spore injury and recovery
were reviewed by Foegeding and Busta (1981). Michels and Kagei (1983)
recommended using trypticase soy agar supplemented with cysteine,
methylene blue, and egg yolk emulsion for the enumeration of heat
damaged spores of Clostridium sporogenes.
Cold Effects
Freezing of cells can cause an apparent increase not only in nutri
tional requirements but also to sensitivity to selective agents used in me
dia. Cells in the logarithmic growth phase are highly susceptible to cold
shock and show an apparent loss in viability, which can be recovered by
incubation with magnesium ions at 30C. They gradually lose their ca
pacity to recover if kept in cold magnesiumfree buffer. Loss of viability
due to cold shock is apparently due to damage of DNA (Sato and Taka
hashi 1970). The lethal effect of cold shock depends on the exposure
time to low temperature, the growth phase, and physiological condition
of the cells, the substrate, and the type of cell.
Drying
It is well established that organisms in dried foods may have meta
bolic injuries that impair the proliferation of the cells in selective media
containing inhibitors in a concentration well tolerated by normal cells
of the same species. These stressed cells can recover their tolerance to
inhibitors if allowed to recuperate or repair in a nonselective medium.
Dried organisms are stressed by freezing (freezedrying or lyophiliza
tion), aerosolization (spraydrying), or heat (roller, drum, tunnel or spray
drying), as well as existing in an environment of very low water activity.
The initial physiological state, the composition of the food, the loca
tion of the organisms in the food, the processing history, storage condi
tions, and the method of rehydration, will influence the survival and reo
covery of organisms from dried food.
Freezedried cells have altered permeability. Freezedried E. coli are
susceptible to antibiotics that do not affect normal cells. Also, there is
leakage of RNA from cells stressed by freeze drying. The alterations in
the stressed cells are reversible, since during or immediately after recov
ery of cellular permeability, the damaged RNA, as well as metabolic dam
age, is repaired (Sinskey and Silverman 1970). After freezedrying, resyn
thesis of cell wall and membranes and the reestablishment of transport
mechanisms are needed before cellular growth occurs. Many freezedried
cells require pyruvate, hematin, and menadione for recovery. The effec
tiveness of the three compounds during repair of the cells is additive. It
is believed these compounds may aid in reestablishing the permeability
barrier of the cell.
Besides the need for nonselective nutrients in the recovery medium,
due to the need for rehydration, the temperature at which this is per
formed is important. Ray, Jezeski, and Busta (1971a) recovered a higher
number of cells at 15 to 25C, but there was an earlier initiation of
growth and more rapid growth if rehydration was done at 35C. The rate
of repair is reduced by lowering the temperature of the recovery medium
from 35C to lOoC and is extremely low at 1C (Ray, Jezeski, and Busta
1971b).
Usually dried food is prepared for the analysis of microorganisms by
rapidly blending the food with a sterile diluent. However, a more gradual
system of rehydration such as soaking the food in diluent at a 1:1 ratio
for 30 to 60 min allows the microorganisms to adapt to a normal mois
ture level. Rapid rehydration can cause lethal or sublethal injury to the
dried cells. This results in a lower count of cells when the sample is plated
onto a selection medium (Ting and Banwart 1985).
Chemical Inhibitors
Since preservation processes cause injuries to microorganisms, per
haps chemical inhibitors may do likewise. Besides chemical preservatives,
organisms on equipment surfaces are exposed to cleaning and sanitizing
agents. If these organisms remain on the equipment, they may contami
nate the food; if injured, they may be difficult to enumerate.
The action of physical factors can be readily altered and the microor
ganisms tested for injury, but it is more difficult to eliminate chemicals
if they are sorbed to the cell or are inside the cell. In these cases, even
dilution may not eliminate the chemical effect on the cell, and death can
result.
Injury by acid and recovery of Salmonella bareilly was reported by Blan
kenship (1981). For recovery, it was indicated that synthesis of protein
and RNA and electron transport are needed. The exposure of E. coli to
hydrogen peroxide or to low levels of cadmium causes single-strand
breakage in DNA (Ananthaswamy and Eisenstark 1977; Mitra and
Bernstein 1978)_ The transfer of injured E. coli to a cadmium-free liquid
medium resulted in recovery without significant synthesis of DNA (Mitra
1984)_
Chlorine injury to coliforms (E. coli) was manifested as inhibition of
uptake of metabolites, reduced adhesion and ability to colonize the small
intestine, and inhibition in selective media for enumeration (Camper
and McFeters 1979; Graham and Brenniman 1983; Walsh and Bisson-
nette 1983).
Ultraviolet Light
The ultraviolet portion of the spectrum includes radiations from 150
to 390 nm. When living cells are irradiated with this light, some may be
"killed" and others may be mutated. The UV wavelengths (around
260 nm) most active in producing either of these effects are those most
readily absorbed by the nucleic acids. Both lethal and mutagenic effects
of ultraviolet light can be partially reversed by repair or reactivation_
Although the ultraviolet light may be absorbed by many cellular com-
ponents, absorption by the nucleic acids results in damage to cellular
mechanisms for division. Repair of DNA in the light is called photoreacti-
vation. Reactivation of UV-irradiated cells also can occur in the dark
(dark repair, dark reactivation, or excision repair).
Various compounds have been given credit for activation or protec-
tion of irradiated cells. One such, the enzyme catalase, breaks down the
microbial inhibitor, peroxide, that is formed during irradiation. Another
action of ultraviolet light is the inactivation of disulfide enzymes due
to disruption of cystine_ Inactivation of enzymes can affect microbial
growth but not necessarily cause death.
Nalidixic acid (Eberle and Masker 1971), coumarin, pyronin Y, 6, 9-
dimethyl 2-methylthiopurine and caffeine (Grigg 1972; Koukalova and
Reich 1981) inhibit or block repair of UV-treated cells_
Other Repair Systems
Microoganisms can repair damage by gamma rays (Foegeding and
Busta 1981; Yatvin, Wood, and Brown 1972), fluorescent and photo light
(Eisenstark and Ruff 1970), as well as water stress (McFeters, Cameron,
and LeChevallier 1982; Rhodes, Anderson, and Kator 1983). The injury
and repair of these sublethal treatments are similar to those discussed
for other treatments.
Summary
Various treatments as discussed in this section can cause lllJury to
microorganisms. Often these injuries can be repaired, but the treatment
rendered to the organism can be lethal depending upon the extent or
amount of treatment. A truly lethal injury cannot be repaired, but micro
organisms given a sublethal treatment, under certain conditions, can reo
cover, grow, and multiply. In some instances, the treatment appears to
kill the cells, since they are not able to multiply. However, they may still
be alive, since they are metabolizing and even growing, such as the fila
mentous forms of E. coli.
The sublethal injuries are important since, without repair, the organ
isms are not able to grow on selective media used in enumeration of a
particular type. Microorganisms in a stressed condition are more suscep
tible to inhibition and death by other stress factors.
OTHER FACTORS
Environmental conditions, such as pressure, surface tension, surfaces,
light and photosensitizers, and pesticides, as well as colloidal characteris
tics, emulsion structure, and concentration of nutrients, might help deter
mine the microorganisms that are able to metabolize, grow, reproduce,
and spoil food products or result in a public health hazard. Compared
to the other factors listed, these are of minor consequence in foods.
INTERACTIONS OF FACTORS
In a few instances, only one factor causes stresses on microorganisms.
The interations of these factors are important in foods.
Water Activity and Nutrition
The minimum a
w
for growth of a microorganism depends upon the
nutrients present in the growth medium. Christian (1955) showed that
the range of a
w
for growth of Salmonella oranienburg was appreciably
smaller in a chemically defined glucosesalts medium than in a nutrient
broth. The addition of small amounts of proline and four other amino
acids to the glucosesalts medium stimulated the growth of S. oranienburg
at 0.97 aw and permitted growth down to 0.96 au,. Addition of vitamins
showed further stimulation, and growth occurred at 0.95 a,u- Thus, the
minimum a
w
levels for microorganisms should be determined in a me
dium with ample nutrients for the growth of the organism tested.
Since foods differ in their nutrient content, microorganisms may be
able to grow in some foods at lower au, than required in other foods.
Water Activity and Temperature
The greatest tolerance to low aw occurs at the optimum temperature.
As the temperature is changed from optimum, the range of au, is reduced
in which spore germination and growth occur. Studying the growth of
fungi on jam, Horner and Anagnostopoulos (1973) found the interaction
of aw and temperature had a significant effect.
For Salmonella survival and multiplication, there was a close correla
tion between au, and storage temperatures of meat and bone meal (Liu,
Snoeyenbos, and Carlson 1969). The relationships of aw and temperature
as well as pH and antifungal agents on growth of Aspergillus flavus and A.
parasiticus were determined by Holmquist, Walker, and Stahr (1983).
Water Activity and pH
As the pH is increased or decreased from the optimum, the minimum
aw needed for growth is increased. Decreasing the pH of the growth me
dium increases the minimum au, at which Clostridium botulinum spores will
germinate and initiate growth (Baird-Parker and Freame 1967).
pH and Temperature
Outgrowth of spores of Clostridium botulinum type E occurred at
15.6C and a pH of 5.4 to 5.6. When the temperature was lowered to
5C, a pH of 6.2 to 6.4 was needed for outgrowth to occur (Emodi and
Lechowich 1969).
The optimum pH for B. subtilis spore germination is markedly
changed by altering the incubation temperature (Ishida, Ishido, and Ka-
dota 1976). The optimum pH was 7.4 at 37C and 5.4 at lOC.
Eh and pH
There is a definite relationship between the pH and the functional
OR systems in bacterial cultures. Staphylococci are more acid tolerant
aerobically than anaerobically (Barber and Deibel 1972). Most staphylo-
coccal strains initiate growth and produce detectable enterotoxin at pH
5.1 when grown aerobically. In anaerobic conditions, most strains fail to
produce enterotoxin below pH 5.7. When held at 5C, Brochothrix grew
aerobically at pH as low as 5.4, but anaerobically no growth was evident
below pH 5.8. It grew readily at pH 6.0 or higher (Campbell et al.
1979).
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5
Sources of Microorganisms
The microbial flora of a food consists of the microorganisms associated
with the raw material, those acquired during handling and processing,
and those surviving any preservation treatment and storage.
Since these microorganisms do not arise by spontaneous generation,
they must contaminate the food at some stage of production, harvesting,
handling, processing, storage, distribution, or preparation for consump'
tion. Most foods are subjected to many potential sources of microorgan
isms.
Why should we be concerned with sources of contamination? Primar-
ily so that we can control contamination and keep the microbial load on
or in the food as low as possible. By doing this, we obtain a longer shelf
life for the food; we hope that this reduces the chance of microbial food-
borne illness when the food is ingested. By keeping the contamination
low, we can more easily control or eliminate the microorganisms with
food-preservation techniques.
The potential sources of contamination are soil, water, air, plants,
feed or fertilizer, animals, human beings. sewage, processing equipment,
ingredients, product to product, and packaging materials.
Microorganisms can be exchanged between these sources. For exam-
ple, animals contaminate the soil with fecal material. Then rain washes
the microorganisms into the creeks and rivers. This water may be used
for irrigation and contaminate plants used for food. Thus, although wa-
ter is the carrier, the microorganisms originally come from animals.
For some foods, it is difficult to determine how many of the organisms
in the flora are contaminants and how many are the result of multiplica-
tion on or in the food.
SOIL
Soil is the natural habitat of many microorganisms which, at times,
may be found there in high numbers. The microbial density is greater
nearer the soil surface than at deeper levels. The types and numbers of
microorganisms vary with the type of soil (sandy, clay), as well as with
165
environmental conditions. The environment is changing constantly, es
pecially moisture and temperature.
Growth of Microorganisms
The microbial growth in soil is limited to areas of organic material.
These areas include the roots of plants, plant debris falling onto the soil,
animal carcasses, fecal deposition by animals, and dead microorganisms.
Besides the animal carcasses on the soil surface, dead earthworms, in
sects, and other small animals are in the soil.
The growth of microorganisms is influenced by the chemical compo
sition of the materials undergoing decomposition, the rate of decomposi
tion of the chemical constituents of these substances, and the environ
mental conditions. Fats, waxes, and lignins may gradually accumulate in
the soil because of their relative resistance to decomposition. This resid
ual, called humus, is subject to slow and gradual attack by a variety of
microorganisms. The main factor affecting multiplication is nutrient
depletion. In general, the soil environment is not a good medium for the
growth of microorganisms. However, they tend to survive.
Number and Types of Microorganisms
The microbial numbers can vary from a few organisms in sandy and
desert soil to as many as 10
10
/g in fertile soil. Due to the usually poor
environmental conditions, microorganisms in soil are in some form of
resting stage or in a reduced state of metabolic activity. Spores of Bacillus
and Clostridium are quite prevalent in some soils. Generally, bacteria out
number other types of microorganisms. Those that are common in food
and found in soil include Acinetobacter, Alcaligenes, Arthrobacter, Bacillus,
Clostridium, Corynebacterium, Flavobacterium, Micrococcus, Pseudomonas, and
Streptomyces. Yeasts are prevalent in the soil of orchards and vineyards.
Molds, especially spores, are found in some soils (Christensen, Raper,
and States 1978; McKenzie and Taylor 1983).
The natural habitat of Clostridium botulinum types A and B is soil. C.
botulinum type E was not found in cultivated soil or woodlands (Huss
1980). Thermophilic sporeformers that can spoil canned foods are found
in soil, usually in low numbers.
Enteroviruses survive sewage treatment and can be found in soil on
which treated wastewater is discharged. The viruses are adsorped to soil
particles, which makes them relatively resistant to inactivation. Another
important factor that influences inactivation is temperature. At 4C,
poliovirus persisted at least 180 days in saturated soil (Yeager and
SOURCES OF MICROORGANISMS 167
O'Brien 1979a). No infectivity was found in dried soil, regardless oftem
perature. The loss of infectivity was due to irreversible damage including
dissociation of viral genomes and capsids as well as degradation of viral
RNA (Yeager and O'Brien 1979b). Poliovirus survives in soil longer in
winter than in summer (Tierney, Sullivan, and Larkin 1977).
Other microorganisms become inactivated in soil by predators (Cas
ida 1980, 1983), bacteriolytic enzymes (Hemelt, Mares, and Upadhyay
1979), or toxins (Lynch 1982). In dry organic soil Ustilago spores remain
viable for one year, but in wet organic soil they should remain viable for
less than one week (Andreis 1980). St. John and Matches (1982) reported
survival of C. perfringens for at least twentyfour weeks in tilled soil.
Contamination of Food
Microorganisms in the soil can contaminate tubers or root crops by
direct contact. Also, dirt is blown by the wind or is splashed by rain fall
ing onto the soil, so that the dirt can contaminate crops such as strawber
ries, beans, cabbage, or peas that grow near the ground level. The micro
bial numbers and types on crops are influenced by the degree of
contamination of the soil in which they are grown.
Mechanical harvesting has increased the amount of soil contamina
tion as well as breakage of fruits and vegetables. Cereal crops are contam
inated mainly during harvesting.
Marine sediments have microbial counts in the range of 10
4
to 109/g.
The numbers are usually higher near the shore than at deeper levels. The
bacteria found in these sediments include Aeromonas, Bacillus, Chromobac
terium, Citrobacter, Escherichia, Pseudomonas, and Vibrio. These sediments
serve as a source of microorganisms for water as well as for fish and
shellfish. During trawling along the bottom with nets, the sediment is
disturbed so that it contaminates the fish or shellfish that are caught in
the nets.
WATER
Water is a potential source of microbial contamination of food. Rain
contains microorganisms that are washed from the air. As the water lands
on the ground, it is further contaminated by soil microorganisms. In the
ocean, organisms are in greater abundance near the shore than in reo
gions distant from land. The dumping of wastes such as sewage and the
runoff from animal feedlots result in considerable microbial contamina
tion of waterways with enteric types of bacteria.
Types of Microorganisms
The genera of bacteria that tend to be part of the "normal" flora of
water include Pseudomonas, Flavobacterium, Cytophaga, Acinetobacter, Mor-
axella, Aeromonas, Corynebacterium, Streptococcus, Klebsiella, Alcaligenes, Bacil-
lus, and Micrococcus. Also, enteroviruses are quite resistant to the treat-
ment given to sewage and therefore can be found in water supplies.
Escherichia coli is used as an indicator of fecal pollution of water in
temperate climates. This organism supposedly dies rapidly in the rela-
tively hostile environment of rivers due to low temperature, sunlight,
toxic chemicals, and a lack of nutrients. However, some reports indicate
that the coliforms and E. coli seem to maintain their population
(Coleman et al. 1974; Goodrich et al. 1970). Water is still the main carrier
of organisms that cause gastroenteritis in humans. From 1971 to 1982,
there were 387 outbreaks of gastroenteritis caused by consumption of
contaminated water in the United States_ Of these, 150 outbreaks were
caused by municipal water supplies (CDC 1983).
Food Contamination
Water contacts food during production, harvesting, and processing.
If the water used for irrigation of various crops is contaminated or is
sewage effluent, the fruits and vegetables can be potential health hazards
(Rosas, Baez, and Coutiiio 1984; Tamminga, Beumer, and Kampelmacher
1978; Tierney, Sullivan, and Larkin 1977; Van Donsel and Larkin 1977).
Seafoods are harvested from water. The microorganisms in the water
contaminate the surface, gills, and intestinal tract of fish and shellfish.
The occurrence of fecal coliforms in fish is a reflection of the pollution
level of their water environment. The skin of Atlantic salmon was ana-
lyzed by Horsley (1973). The predominant genera were Cytophaga, Flavo-
bacterium, Moraxella, and Pseudomonas. Other organisms found were Acineto-
bacter, Bacillus, Aeromonas, Vibrio, coryneforms, members of the families
Enterobacteriaceae and Micrococcaceae.
When bivalve mollusks feed, they filter large quantities of water and
concentrate bacteria and viruses that are present in the water environ-
ment. Shellfish are normally found in water near the shore. This water
is subject to contamination of runoff water carrying soil microorganisms
and sewage outfalls. The accumulation and concentration of these micro-
organisms from the water is of particular concern when they are poten-
tial pathogens such as salmonellae and human enteric viruses.
Potential pathogens in the drinking water of animals can be a health
hazard to the animals and to people who work with the animals. These
organisms from infected animals also can contaminate the carcass during
the slaughter operations.
During harvesting, water may be used for hydrocooling of vegetables.
Irrigation or surface water that is polluted with sewage or animal wastes
can serve as a source of inoculation with various organisms, including
potential pathogens. The water can redistribute organisms already on
the vegetables from high levels of contamination or spoilage to other
areas so that all of the vegetables are contaminated. Since many vegeta
bles and fruits are eaten raw, the use of untreated water to wash these
foods can serve as a vehicle of transmission of pathogenic organisms.
Ice is used to cool and maintain coolness in a variety of fresh foods.
As the ice melts, the organisms associated with it are passed on to the
foods and vice versa. The reuse of ice is neither a recommended nor an
accepted procedure, since this ice is contaminated. Chen and Chai (1982)
reported that icemelt drainage in fishholds of fishing vessels contained
bacteria at levels of 10
7
to 10
9
/ml.
The use of water in a food processing plant can be a source of micro
organisms for contaminating food. Water is the food industry's number
one raw ingredient. Water enters into the processing of most foods for
cleaning equipment and processing areas, for washing food, for convey
ing (fluming) food, or as an ingredient.
The safety of water that is used in foods is of major importance. How-
ever, although considered to be safe for drinking (potable), municipal
water supplies are not always acceptable for food processing. This is be-
cause of the presence of food-spoilage microorganisms as well as chemi-
cals that may produce objectionable odors and flavors in food. The
organisms that tend to be present in municipal water supplies are
psychrotrophs such as pseudo monads which can grow in water with a
limited nutrient content. Hence, when a food processor stores water in
small tanks, the growth of the pseudo monads may result in microbial
levels of 10
5
to 10
6
per milliliter.
In the dairy industry, these psychrotrophs are especially troublesome.
The wash water may contain up to 10
3
psychrotrophs per milliliter and
be one of the most important sources of microbial contamination. Psy-
chrotrophs canse spoilage of refrigerated milk and milk products.
In poultry-processing plants, water is used in the scald tank for wash-
ing of the carcass and for cleanup of the building and equipment. There
has been much speculation that water from the scald tank is a source of
contamination of the carcass. This is due to the fact that the dirt from
the head, feet, and feathers, as well as feces (due to defecation at death)
contaminate the scald water. The temperature of the water (53 to 61C)
can kill many pathogenic and spoilage types but is not sufficient to kill
all of the microorganisms that are present, and thermophilic types can
grow. Bacterial counts of the scald water range from 10
2
to 10
6
per milli-
liter. When the birds enter the scald tank, they may be taking their last
breath and the heart may be pumping. The contaminated water then
can contaminate the internal portions of the bird through the lungs and
vascular system. The addition ofNaOH to raise the scald water pH to 9.0
effectively reduced the level of bacterial population (Humphrey, Lan-
ning, and Beresford 1981).
Ice water used to cool the carcasses after processing can be a source
of microorganisms that can penetrate deep into the skin surface (Thomas
and McMeekin 1982).
In food-canning operations, water is used to cool the cans of food
after heat processing. Due to the heat and expansion of metal, the seams
and seals on the cans are under stress, and leakage can occur. Hence, it
is important that the cooling water be maintained with as few microor-
ganisms as possible. Methods of monitoring this water for microorgan-
isms have been described (Rey et aL 1982). Thompson and Griffith (1983)
found clostridial spores at low levels in about 10 percent of their samples
of cooling water. Since no further processing occurs after cooling, any
contamination of the food by microorganisms that can grow in the
canned product may cause spoilage or, if pathogenic, foodborne illness.
Water is used as an ingredient in many foods. In these cases, the water
is a direct source of microbial contamination.
With the present shortages and high cost of water, there is an effort
to reduce water consumption and to recycle water in the food-processing
plant. Caution must be used so that contamination or high microbial
loads on foods do not result from these pratices.
AIR
Today, the main concern with air pollution is with chemicals (carbon
monoxide, hydrocarbons, soot, fly ash) rather than with biological agents.
Nature is the major contributor of not only the chemical pollutants but
of biological agents such as plant cells, animal hair, pollen, algae, proto-
zoa, bacteria, yeasts, mold spores, and viruses. Food is subjected to air-
borne contamination until it is sealed in a package.
Types and Numbers of Microorganisms
There is no natural or normal microflora of air, which is contami-
nated from various sources. Generally, mold spores are more prevalent
than are other microorganisms. The main source of microorganisms, es-
pecially mold spores, is decaying plant materials near the ground surface.
Contamination of the air is caused by gusts of wind picking up the micro-
organisms or spores_ Compared to mold spores, there are relatively few
yeast cells in the atmosphere, and these are mainly near ground level.
One would expect yeasts to be prominent in the air of orchards and
vineyards. However, even here, they may comprise only 25 percent of the
fungal population of air.
There are many sources of microorganisms for contamination of the
air. Liquids are aerosolized by spraying, splashing, the bursting of bub-
bles, by forcing through a small orifice, or by vibrations. Trickling filters
used in sewage-treatment plants produce aerosols by the spraying action
and by the splashing of liquid sewage. The application of wastewater to
land by spraying under pressure causes aerosolization of organisms.
Teltsch et al. (1980) devised equations to predict aerosolized pathogenic
microorganisms downwind from a wastewater spray or aeration site.
The types of organisms in air are often associated with the type of
activity in the area. Downwind from a sewage-treatment plant, Pereira
and Benjaminson (1975) found species of Klebsiella, Bacillus, Flavobacter-
ium, Streptococcus, and Micrococcus. Streptocci are found near dairies, and
yeasts are found near bakeries and breweries. The microbial flora of the
air in a food processing plant reflects the sanitary condition of the plant
unless the air is purified. Incinerators, if not operated properly, can be
a source of aerosols that may contain infectious microorganisms.
Human beings shed organisms and also produce aerosols during talk-
ing, coughing, and sneezing. The microbial load of the air is proportional
to the numbers of persons in an enclosed space, their activity, and the
rate of air circulation. Although humans might not contribute signifi-
cantly to the microorganisms in outdoor air, they can be a significant
source for air in a processing room.
Gerba, Wallis, and Melnick (1975) reported that bacteria and viruses
become airborne when a toilet is flushed. These organisms settle out on
surfaces throughout the bathroom.
Thus, air is subjected to a number of sources of microorganisms (de-
caying matter, water, soil, human beings, animals, sewage). The numbers
and types of organisms present in the air or atmosphere depend upon
many factors, such as tendency to settle out and maintenance of viability.
There is a considerable variation of the microbial load of air. It has
been suggested that country air in the summer contains an average of
10,000 fungal spores/m3. Lacey (1973) suggested the level may reach 10
8
/m
3
during haymaking or harvesting. The handling of moldy grain or hay
indoors can result in a level of 10
9
spores/m3. One study found more
bacteria over an alfalfa field than over bare soil (Lindeman et al. 1982).
At 46 m downwind from a wastewater spray irrigation site there were
10
4
bacteria/m
3
(Bausum etal. 1982). Clark, Rylander, and Larsson (1983)
reported a level of 10
5
bacteria/m
3
in both poultry and swineconfine
ment buildings. Lenhart, Olenchock, and Cole (1982) stated that the at
mosphere in a poultry. processing plant contained bacteria that might be
a potential health hazard to the workers.
Lacey (1973) listed several genera of fungi as being present in rural
air, with Cladosporium predominating. This dominance was also reported
by Ogunlana (1975). Other researchers have reported that species of
yeasts, Aspergillus, Penicillium, Cladosporium, Alternaria, Helminthosporium,
and Fusarium are the most dominant fungi in corn dust (Hill et al. 1984).
The air near the earth is more contaminated than at higher altitudes,
although the microbial population at 2,000 to 3,000 meters is more stable
than that at lower altitudes. Air over land is more contaminated than
air over the ocean, although wind currents can carry micoorganisms for
several hundred miles out to sea. The upper atmosphere over the ocean
is more contaminated than air near the ocean surface.
Clouds often contain high levels of bacteria and fungal spores. Turbu
lent air tends to carry high numbers of cells aloft. Temperature inver
sions can affect the microbial load at various altitudes. Rain and snow
tend to wash the microflora from the air. The atmosphere is more con
taminated in the summer than in the winter.
Survival
Although Dimmick, Wolochow, and Chatigny (1979) reported that,
with special conditions of growth and aerosolization, bacterial cells in air
can divide, this has not yet been shown for naturally occurring microor
ganisms in air.
The stability of microorganisms in air is affected by the relative hu
midity, temperature, oxygen, solar factors including ultraviolet radiation,
and chemical components. Spores of molds and bacteria retain their via
bility better than do vegetative cells. Also, capsules help protect cells in
the atmosphere.
Aerosols of microorganisms have been produced in the laboratory
and the viability determined. Aerosolization damages the cytoplasmic
membrane and at least some of the associated transport mechanism (Ben
bough et al. 1972).
The stability of bacteria in aerosols is similar to that of viruses. There
is rapid death during aerosolization, followed by a less rapid death rate.
The method of recovery and enumeration influence the apparent effect
of relative humidity and oxygen on survival of bacteria in aerosols.
When air is sampled downwind from a source of contamination,
there is a significant decrease in the microbial load as the distance from
the source increases. This is because the organisms settle out onto var
ious surfaces, the organisms lose viability, and the air is contaminated by
less contaminated air. However, bacterial spores were found to travel in
air from near the Black Sea to Sweden (Bovallius et al. 1978).
Sunlight reduces the number of viable organisms in air. By analysis
of air near a spray irrigation system, Teltsch and Katzenelson (1978)
found higher counts at night than during the day.
Food-Processing Operations
The contamination of air in foodprocessing plants was reviewed by
Heldman (1974). The effect that air has on the microflora of food de
pends upon the level of contamination of air and the time of contact of
air with food.
Aerosols are produced in food processing plants by spray washing or
spray cooling of food, by highpressure sprays used in cleaning, by flood
ing of floor drains, by mixers and motors, and by the operation of various
other equipment. Workers in the area produce aerosols. The movement
of equipment, supplies, and people in a food plant causes turbulent wind
currents that increase the microbial load of the air.
There is considerable variation in the microbial load of air in various
areas of a processing plant. In clean areas there are very few organisms
in the air. In areas in which live animals are handled or raw products are
brought into the processing operation, the microbial load can be quite
high.
One method that is used to control the microbial load in the air of a
processing plant is to move air from clean areas to dirty areas or by using
positive air pressure in clean areas. With positive air pressure, if a door
is opened, air flows out of a room and outside air does not come in.
Fresh air entering the clean areas is filtered to remove dirt as well as
some microorganisms.
ANIMAL AND PLANT FOOD
Animal and plant food can serve as a source of potential chemical
and biological contamination of food.
Microorganisms in animal feed can contaminate the feet, hide, hair,
and feathers of animals. Consumption of the feed adds organisms to the
digestive tract. Those organisms that survive the rigors of digestion, upon
elimination, can contaminate exterior portions of the animal. When the
feed contains potential pathogens such as salmonellae, these may cause
illness in the animal, invade the body and locate in lymph nodes, contam
inate the carcass during slaughter, as well as contaminate external por-
tions of the animal (MacKenzie and Bains 1976)_
Plant food may contain organisms that can contaminate the surfaces
of plants and associated human foods. When animal or human wastes
are used as fertilizer, pathogens from these sources can contaminate the
plant and associated foods_ Since fruits and vegetables are eaten raw, they
can be a source of foodborne illness. In countries that use night soil as
a fertilizer for food crops, washing of the food is suggested. The decom-
position of plant tissue can cause an increase of potential toxin-forming
molds in fields (Griffin and Garren 1976).
Although pathogens may be present in manure or sewage used to
fertilize crops, sunlight tends to eliminate microorganisms from the
plant parts (Bell, Wilson, and Dew 1976; Jones 1976).
PLANTS
Plants are contaminated by microorganisms from several sources
(dirt, water, air, fertilizer, animals, and humans). Once contaminated, cer-
tain microorganisms can grow on the plant surfaces and plant pathogens
can attack their host plants. The microbial flora on plant surfaces varies
with the kind of plant.
One study found a total count of 10
9
/g including 10
7
coliforms, 10
8
psychrotrophs, 10
7
streptococci, 10
5
yeasts, and 10
6
molds per gram on
wild rice (Goel et al. 1970).
Pseudomonas species are quite prevalent on vegetables. Kominos and
colleagues (1972) isolated P aeruginosa from tomatoes, radishes, celery,
carrots, endive, cabbage, cucumbers, onions, and lettuce. They believe
that raw vegetables are a source of this organism contaminating patients
in hospitals. Various bacteria, including pseudo monads, can adhere to
and colonize the surface of leaves (Ercolani 1978; Hildebrand, Alosi, and
Schroth 1980; Leben and Whitmoyer 1979).
The flowers of fruits are inhabited by many genera of yeasts, includ-
ing the ascospore formers Saccharomyces and Hansenula, as well as the im-
perfect Torulopsis, Candida, Rhodotorula, and Kloeckera. In some fruits such
as grapes, the yeasts might not be present on the flowers but are found
on the ripe fruit.
A Pseudomonas and an Arthrobacter introduced into open flowers of
soybean plants were isolated from 24 of 177 resultant beans pods (Leben
1976).
There is evidence that not only the surface but also the interior tissue
of plants can be contaminated. Vegetables can harbor fecal streptococci
within unopened pods, heads, and other structures. Meneley and Stan-
ghellini (1974) found that 44 percent of apparently healthy cucumbers
contained bacteria internally. Enterobacteria (Proteus, Citrobacter, and
Enterobacter) were present in 10 percent of the cucumbers.
Thus, live plants can serve as a source of microorganisms. When the
plant dies, the decaying vegetation becomes an important source of air-
borne, waterborne, and soil microorganisms, which then contaminate
plants the following year.
ANIMALS
Animals have a normal or natural microflora that is established very
early in life. Besides this micro flora, animals tend to harbor the types of
organisms found in their environment, since they are contaminated by
soil, water, air, feed, and excreta.
Animals harbor organisms that can cause food spoilage or foodborne
disease. Organisms are found in animals' gastrointestinal tract, nasal pas
sages, cutaneous lesions, and on the skin, feet, hair, or feathers on the
outer surface. These organisms are readily transferred to the edible por
tions of the animal during processing.
Wild animals contaminate growing crops and stored products. One
of the means by which viruses are transmitted to plants is by insects (Cou-
driet, Kishaba, and Carroll 1979). Insects, birds, and rodents destroy the
protective covering on foods, so that not only do they contaminate the
food, they also make the food more susceptible to microorganisms that
cause spoilage. These animals also carry potential human pathogens that
may be transferred to the foods they contaminate (Kapperud 1975; Stek
1982).
Flies, much more so than many other types of common food plant
insects, are a serious potential carrier of disease-producing organisms.
Flies pick up organisms on their hairy legs and feet. They then contami
nate human food by walking or leaving their excreta on it. Flies have a
part in the spreading of Salmonella, Shigella, Vibrio, Escherichia, and other
diseasecausing organisms as well as foodspoilage types.
The muscle tissue of most live, healthy animals is essentially sterile.
In a diseased state or after slaughter (or after harvesting in the case of
fish), bacterial invasion of the muscle tissue can occur. Most of the micro
organisms in tissue are thought to result from surface or intestinal con
tamination or from processing operations. Vanderzant and Nickelson
(1969) removed muscles from slaughtered animals aseptically. The maxi
mum number found in any sample was 1,400 per gram in beef. Of sixty
five samples analyzed, forty four did not yield isolates when incubated at
37C. Hence, it is evident that even after slaughter, a majority of muscle
tissues are sterile.
One of the sources of microorganisms in the interior portion of ani
mals is the lymph nodes. These nodes tend to filter out bacteria from the
lymphatic system. Microbial counts of 105/g have been obtained from the
lymph nodes, with many types of organisms being present. Both cattle
(61 percent) and sheep (14 percent) at slaughter contained Salmonella in
lymph nodes (Samuel et al. 1981). Of fortytwo hog carcasses passed by
federal meat inspectors, Brown and Neuman (1979) found mycobacterial
infection of the lymph nodes.
The microbial flora that grows on refrigerated beef is composed
mainly of soil organisms derived from the hide, hair, and hooves of the
animals. The hides of animals may contain from 10
3
to over 10
9
microor
ganisms per square centimeter. Although they do not penetrate the intact
skin, these microorganisms are a source of contamination of the edible
portions of the carcass during processing.
The predominant organisms in the intestinal flora of both animals
and humans are obligate anaerobes such as species of Bacteroides and Pep
tostreptococcus. They may reach levels of 10
10
to lOll per gram. The faculta
tive anaerobes may reach levels of 10
7
to 10
9
per gram of intestinal con
tents. These include coliforms, enterococci, and lactobacilli. The natural
habitat of the human pathogen Salmonella is the intestinal tract of animals
as well as of humans. Other organisms, such as fungi and viruses, can be
found in the intestinal tract. There is a possibility that microorganisms
can pass through the intestinal wall into other organs and tissues. Viruses
were found in chicken tissues that appeared normal (Robertson, Wilcox,
and Kibenge 1984).
During life, the intestinal wastes can contaminate the outer surface
of the animal. During slaughter and processing, the sphincter muscle be
comes nonfunctional, and also the intestines can be punctured acciden
tally. In either case, the contents and associated organisms can contami
nate the carcass.
The circulatory system is sterile except for the occasional invader or
during an infection. Although a majority of samples of hemolymph from
the horseshoe crab contained no detectable microorganisms, 27 percent
did reveal low numbers of bacteria (Brandin and Pistole 1985).
The respiratory system acquires microorganisms from the air during
breathing. The nose filters out many microorganisms, and it tends to be
the most contaminated part of the system. The respiratory system may
serve as a passageway for microorganisms to contaminate internal tis
sues.
The surface of fish may contain 10
2
to 105 bacteria per square centi
meter and the intestinal contents vary from 10
4
to 10
7
organisms per
gram. These microorganisms reflect the condition of the environment
from which the fish are obtained (Austin 1982). This is especially evident
for shellfish (Ellender et al. 1980; Larkin and Hunt 1982; Metcalf et al.
1980; Perkins et al. 1980).
Milk in a healthy udder has few microorganisms, if any. However,
aseptically drawn milk usually ranges from less than 500 to 5,000 and
occasionally, up to 10,000 organisms per milliliter (Thomas, Druce, and
Jones 1971). The flora is predominantly coagulasenegative staphylo-
cocci, micrococci, and corynebacteria. Infection of the bovine mammary
glands is called mastitis. With this condition, counts of 10
5
or more per
milliliter of milk have been found. A variety of organisms can cause mas-
titis, and organisms such as coagulase-positive staphylococci, coliforms,
and streptococci, will dominate in the milk. Aseptically drawn milk con-
tains mesophiles, but few psychrotrophs or thermophiles. When milk is
obtained normally, it is contaminated by bacteria in the teat canal and
on the surface of the teat. The surfaces of the four teats, even after wash-
ing with disinfectant and drying, had a mean colony count of 10
6
bacteria
(Thomas, Druce, and Jones 1971). Perhaps this is due to the strong attach-
ment of bacteria to the teat surfaces (Notermans, Firstenberg-Eden, and
Van Schothorst 1979). Hibbitt, Benians, and Rowlands (1972) found an
average of 22,500 viable microorganisms per teat canal. McKinnon, Cous-
ins, and Fulford (1973) analyzed milk obtained normally from twenty-five
cows and found bacterial counts from 44 to 11,400 per milliliter, which
is similar to the so-called aseptically drawn milk referred to by Thomas,
Druce, and Jones (1971). Due to microorganisms normally present in the
teat canal, the first drawn milk contains more organisms than the last
portion from any particular quarter of the udder.
When an egg is laid, it is essentially sterile. Some bacteria (salmonel-
lae) and avian viruses may invade the ovaries of hens and contaminate
the yolk before the egg is formed. Even for those few eggs that might be
contaminated, the bacterial number is relatively low. Most of the contami-
nants on the egg shells are of intestinal origin. Other sources of orga-
nisms are nesting material, feather dust, the feet and body of the bird, as
well as subsequent handling and storage. Eggs obtained from poultry
flocks on wire have less bacterial contamination than do eggs from flocks
in houses with floor litter. This is because nesting material, including
feces, has less chance of coming into contact with the shell.
HUMANS
A human embryo develops in a relatively sterile environment. At
birth the baby is subjected to a massive invasion of microorganisms from
the mother, other people, bedding, air, food, water, and other materials.
The skin is the most available part of the body for colonization after
birth. Staphylococci are predominant on normal infant skin (Carr and
Kloos 1977). The colonization is followed by microbial invasion of the
nose, oral cavity, throat, and the respiratory, digestive, and urogenital
tracts. Many microorganisms are merely transients, while others become
established as a normal, perhaps permanent, flora. Besides the microbial
population on or in a human, clothing can be contaminated by external
sources or by the person, and then serve to pass the microorganisms on
to the person or to food products.
The skin is never free of bacteria, and the dirtier the skin, the greater
the contamination. Normal human skin contains a relatively stable mi
croflora. However, the numbers and types of microorganisms vary from
person to person, from site to site, and from day to day. There are differ-
ences in the environment at various places on the body. Some areas are
too dry and, in other areas, the pH is not satisfactory for bacterial growth.
The palm of the hand has primarily surface organisms, whereas the fore-
head has both surface and subsurface flora, but mainly subsurface. Sur-
face organisms consist of transient and normal resident types, while sub-
surface organisms are primarily normal resident types.
Washing the skin removes most of the transient microorganisms, but
it is practically impossible to remove all of the normal flora. Some micro-
organisms associate with the sweat glands, sebaceous glands, and hair
follicles, where they not only can grow but also are difficult to remove.
The predominant bacteria on the skin are staphylococci, corynebacteria,
and propionibacteria. Also, species of Micrococcus, Bacillus, Alcaligenes,
Pseudomonas, Enterobacter, Klebsiella, Proteus, Escherichia, and Citrobacter are
quite prevalent (Adams and Marrie 1982; Sunga, Heldman, and Hedrick
1970). S. aureus is found more often on the hands and face than on other
parts of the body. The organism is associated with the nose and is spread
when people handle their faces and noses. S. aureus is associated with
infections such as acne, pimples, and boils.
Humans are a source of airborne microorganisms as well as an impor
tant source of food contamination through handling of food. The hands
are subject to contamination by considerable numbers of bacteria, most
of which are unable to multiply on the hands and usually die. However,
these transient bacteria are passed on to food products when they are
handled. Many of the transient bacteria result from the handling of foods
(DeWit and Kampelmacher 1982; Seligmann and Rosenbluth 1975).
Workers in meat- and chicken-processing plants had rather high levels of
E. coli, and several had salmonellae on their hands.
The hair covering the skin is a potential source of microorganisms.
There is no normal flora of the hair, but hair acts as a carrier to retain
and shed organisms. Beards, sideburns, and mustaches may be especially
bad if worn in a food-handling area. Barbeito, Mathews, and Taylor
(1967) found that, although washing with soap and water reduced the
level of contamination of beards, it did not eliminate microorganisms
and toxins from them. If a person grows a beard to hide acne or pimples,
or a nasal carrier has a mustache, these hairy growths will merely serve
for continued dissemination of S. aureus. Besides the microorganisms that
may be shed into foods, the hairs and hair clippings may contaminate
the food.
Clothing is directly associated with humans and can serve as a carrier
to contaminate foods with human microflora or can contaminate people
with environmental microorganisms. Laundering does not necessarily re-
move all of the microorganisms from clothing. The effectiveness of laun
dering depends upon the type of organism, fabric, temperature, deter
gent, antibacterial agents, bleaches, flushes and rinses, drying, ironing,
and final packaging. Viruses tend to adsorb to the fabric and are difficult
to remove. Microorganisms tend to remain viable in wool longer than in
cotton. Synthetic fabrics require less drastic laundering, allowing greater
microbial survival. Microorganisms tend to survive storage for a shorter
time on these fabrics than on either cotton or wool.
During laundering, microorganisms are transferred among the cloth
ing in the same wash load, and it is conceivable that microorganisms can
carryover from one load to the next. Even dry cleaning does not assure
the sterility of clothes.
The gastrointestinal tract may be considered a hollow tube within the
body, but the internal portion is outside the body. The gastrointestinal
tract is important as a potential source of organisms, as well as the pri-
mary site of action of foodborne organisms causing illness (gastroenter
itis). Factors that can influence the flora of the gastrointestinal tract in-
clude the diet, antibiotics or other chemotherapeutic agents, and certain
diseases.
Although enormous numbers of microorganisms are ingested with
food and with mucus from the respiratory tract, most do not survive in
the digestive system.
In the normal stomach, the microbial flora increases and decreases.
The ingestion of food or mucus causes a rise in numbers. The flora is
reduced by acid and digestive enzymes and by emptying of the stomach
into the duodenum. There are usually low numbers of organisms in the
normal stomach, duodenum, jejunum, and upper ileum. The pH of the
stomach varies among individuals. Higher pH values will allow the sur
vival of more organisms. Foods tend to react with stomach acids and raise
the pH. Also, some microbial cells are imbedded in food particles, which
protects them from the acid.
Normal peristalsis, bile, and immunoglobulins tend to remove organ-
isms from the upper small intestine. However, drugs such as morphine
slow peristalsis; this and several other conditions can result in coloniza
tion (Gracey 1979). Some organisms are able to adhere to the epithelial
cells of the small intestine by at least two systems (Kleeman and Klaen
hammer 1982). Most of the organisms that cause foodborne illness must
attach to the epithelium and become established in the intestine, where
they produce toxins (see Chapter 6).
As the cells move down the intestines, the conditions improve for
microbial growth. Active multiplication begins in the lower small intes
tine and continues through the large intestine. Bacterial levels of more
than 1010/g of contents are attained in the colon and excreta. The redox
potential varies from - 150 mv in the duodenum to - 250 mv in the
colon (Holdeman and Moore 1972). This low level in the colon favors
anaerobic types of organisms.
The fecal flora of infants is composed primarily of species of Bifido-
bacterium. Other organisms include the anaerobic Bacteroides as well as
coliforms, enterococci, lactobacilli, and staphylococci (Mitsuoka and
Keneuchi 1977). In the adult, Bacteroides surpasses the anaerobic bifido-
bacteria. These two groups range from 10
9
to 10
10
cells per gram of feces.
The eubacteria and anaerobic lactobacilli are also at levels of 10
9
per
gram of feces. The remaining organisms consist of enterobacteria, enter-
ococci, lactobacilli, clostridia, bacilli, veillonellae, pseudomonads, yeasts,
molds, and viruses (Haenel 1970). The relationship of viruses to the fecal
microflora has not been examined thoroughly. Enteric viruses are pres-
ent in the intestines and feces of apparently healthy people.
Speck, Calloway, and Hadley (1970) tested the effect of diets on the
fecal flora. Their results indicated that a change in diet influences the
fecal microflora. The type of diet affected the aerobic population more
than the anaerobic microflora. A protein diet supported a higher aerobic
population than did either a carbohydrate or fat diet. Haenel (1970)
found no dramatic differences in the intestinal microbial flora with meat-
egg, milk-vegetable, vegetarian, or raw vegetarian diets. Even the con-
sumption of 1 kg of Bulgarian yogurt per day failed to dramatically alter
the microbial flora in most individuals. Conn and Floch (1970) fed eight
to twenty capsules, each containing five (10
10
) viable Lactobacillus acidophi-
Ius for seven to ten days. They found no significant increase in fecallacto-
bacilli, but they did notice a decrease in total anaerobic bacteria. Similar
results were reported by Paul and Hoskins (1972). A comparison of Sev-
enth Day Adventists revealed few statistically significant differences in
the fecal flora of vegetarians and nonvegetarians (Finegold et al. 1977).
Although some reports indicate that diet does influence the fecal flora,
in a review, Hentges (1980) stated that there are minor and inconsistent
changes in fecal flora composition in response to dietary alterations.
People with acute diarrhea may show the following features of intesti-
nal dysfunction: (1) bacterial colonization of the small intestine with de-
fective microbial clearing systems; (2) malabsorption of fat, carbohydrate,
and vitamin B12; (3) fluid accumulation due to alterations in electrolytes
and water transport; and (4) alteration of the mucosal morphology. Those
abnormalities are not necessarily manifested in all persons with acute
diarrhea or in all cases of diarrhea.
Mendes and Lynch (1976) found fecal organisms on various surfaces
in restrooms (door handles, flush handles, tap handles, toilet seat, floor).
The tap handles were more contaminated than the door handles. The
wash basin overflow was more contaminated than the floor and the area
under the rim of the toilet. They believed that their findings showed that
a person with Salmonella or Shigella infection could contaminate various
surfaces in a washroom or toilet. These areas could conceivably serve as
a source of contamination for other people.
Since people are carriers of pathogenic types of microorganisms, they
are especially hazardous when handling processed (cooked or pasteur-
ized) products that may be held for short periods and eaten with no fur-
ther treatment.
The hands of workers have been cited as increasing the microbial
load of many products_ Poultry products show increased counts when the
carcass is hand transferred from the picking to the eviscerating line, dur-
ing eviscerating, cutting up the carcass, and deboning by hand. In the
shellfish industry, the hand shucking of oysters and clams, shelling of
shrimp, and picking of crabmeat from the shell are associated with in-
creased bacterial counts. Slicing, weighing, and hand packaging of meat
products increase the contamination through human handling of the
product. The unpacking, trimming, sorting, and repackaging of fresh
produce (fruit and vegetables) provide an opportunity for contamination
of the food with human pathogens. Fresh produce may be eaten raw,
which makes contamination a potential hazard.
The carelessness of humans is an important cause of microbial con-
tamination of foods. Failure to properly clean and sanitize equipment,
failure to wash one's hands, working and handling food with an infec-
tion, poor personal hygiene, lack of care in handling of food, making
unsatisfactory alterations of equipment, and failure to keep foods at the
proper temperature are some of the careless things that people do that
can increase the microfloral load of foods.
Some shoppers have been seen opening containers of foods. This is
a deplorable practice that can cause contamination of the food with re-
sultant spoilage or a health hazard.
During the analysis of foods, microbiologists can be a source of con-
tamination (Denny 1972).
SEWAGE
Animal manure or, in some cases, human wastes are used as a fertil-
izeron crops. These biologically produced substances contain microorgan-
isms, including human pathogens. When added to soil, these pathogens
may survive for periods sufficient to contaminate the harvested crop.
In rural areas, septic tanks are used. Quite often these are not prop
erly installed and do not operate effectively, and raw sewage leaks into
the soil. Salmonellae are quite prevalent in raw sewage. Processing in
sewage treatment plants lowers the incidence, but even at 99 percent reo
duction, if the original level of salmonellae is high, they remain in the
effluent and sewage sludge (Farrah and Bitton 1984; Langeland 1982;
Nabbut, Barbour, and AINakhli 1982; Yaziz and Lloyd 1982).
Enteric viruses survive the treatment process, may remain infective
when bound to solids, and can contaminate soil and groundwater when
sewage sludge is applied to agricultural land (Goyal, Keswick, and Gerba
1984; Larkin et al. 1978; Ward and Ashley 1980).
EQUIPMENT
During the Industrial Revolution, machines were developed to do
most of the work of humans. Hence, there is less contact with food by
people and more contact by machines and equipment.
Thousands of small pieces of equipment, such as knives, cutting
boards, and bins, are used in food processing or handling establish-
ments. Although most equipment is metal, some parts may be made of
rubber or plastic. In some cases, cardboard is used in boxes used to hold
harvested fruits or vegetables. Cardboard or paper is used as packaging
material for bulk shipment and storage of various ingredients.
Metal processing equipment does not support the growth of microor-
ganisms. It has no natural or normal microbial flora. Yet, food-processing
equipment is one of the major sources of contamination of foods.
Equipment may be cleaned and sanitized, but this does not mean that
it is sterile. Even on washed, visibly clean surfaces, the survival and
growth of bacteria are possible. Even visibly clean surfaces may have food
deposits or films that provide microenvironments acceptable for survival
and growth of microorganisms. The potential for microbial buildup on
equipment is enhanced when equipment is improperly cleaned and sani-
tized. If food residues are visible on cleaned equipment, one can be as-
sured of a potentially high microbial level.
Pitted surfaces or poorly soldered joints are places in which foods
can lodge on the equipment. During the course of the daily operation,
bacterial growth will occur in these food films and deposits. This then
serves as a source of contamination when food contacts these surfaces.
It is well established that microorganisms adhere to surfaces. Strepto-
coccus thermophilus adheres to stainless steel during the pasteurization
process (Driessen, DeVries, and Kingma 1984). However, it is easier to
remove soil and bacteria from stainless steel than from rubber or plastics.
Although stainless steel is more expensive than some other materials, it
pays for itself through savings in cleaning, as well as durability and prod
uct quality. When flexibility is needed, such as in milking machines or in
chicken-picking machines, rubber or plastic becomes an essential part of
the equipment.
Gilbert and Maurer (1968) surveyed equipment in shops and cafes.
Swabbings from a meat-slicing machine revealed bacterial loads of 10
6
to 107/cm
2
The cleaning methods used were ineffective in reducing the
bacterial loads to an acceptable level. When we consider that most out-
breaks of foodborne illness are due to contamination of the food in
foodservice units or in the home, it should be obvious that the equip-
ment in the home and foodservice institutions needs more attention.
Meat
The contamination of meat by equipment begins with slaughtering
the animal. Contaminants from the knife used to cut the artery may circu-
late through the body during the bleeding process. Humane slaughter by
immobilizing the animal with CO
2
or electric stunning before bleeding
helps reduce the heart action and hence, contamination.
During the slaughtering operation, the carcass contacts various equip-
ment surfaces. Various pieces of equipment and humans can cause the
spread of Salmonella throughout the slaughter operation (Smeltzer,
Thomas, and Collins 1980a, b; Stolle 1981). When the carcass is cut up
and packaged for retail, further transfer of organisms is made from the
carcass surface by means of saws, knives, slicers, and cutting blocks. These
increased surfaces with their associated juices support large populations
of bacteria.
The grinding of meat further increases the surface area, releases
juices, and distributes the original surface bacteria throughout the meat.
Equipment may be involved in the distribution and increased counts of
the product. Meat grinders are highly contaminated with millions of bac-
teria (Dempster 1973).
Poultry
During processing there may be a tenfold increase in the microbial
count on the skin of poultry carcasses. Some of the increase is due to
contaminated scald water and to human factors. The equipment used in
processing the birds plays a role in contaminating the carcasses. At every
stage of the processing operation (Fig. 5.1) there is ample opportunity
Slaughter (shackle, stun, sever throat)
-I Bleed I
~ ~ +
I Spray Wash I+-'E---I Defeather 1+-(---1 Scald I
t
1 Singe I-I Cut
hocks and vent 1 ~ I Eviscerate I
+
~ I Spray Wash \-1 Remove head and neck \
..
I Package and weigh I-I Refrigerate or freeze
Figure 5.1. Poultry-processing operation.
for microbial contamination (McMeekin and Thomas 1979; Thomas and
McMeekin 1980).
Schuler and Badenhop (1972) sampled seventeen equipment sites on
two sampling days in thirteen poultry-processing plants. On only five of
the pieces of equipment was the average bacterial count less than
1,000/cm
2
on both sampling days prior to start of the operation. Sites of
contamination were the flexible fingers on the picking machine (11,000
to 99,000/cm
2
), the hock cutter (7.000 to 97,000/cm
2
), the transfer belt be-
tween dressing and eviscerating lines (5,000 to 56,000/cm
2
), lung gun
(3,500 to 73,000/cm
2
), and cut-up belt (2,000 to 35,000/cm
2
).
The picker fingers are difficult to clean properly, and the contamina-
tion of these fingers increases significantly during plant operation. There
is increased contamination of poultry carcasses during feather removal,
which can be due to leakage from the vent and the bacteria from the
feathers contaminating the skin via the flexible rubber fingers.
Further processing (cutting up the carcass and deboning) can increase
the microbial level of poultry meat (Denton and Gardner 1981). Sources
of organisms include both equipment and humans.
Milk
Since milk is a liquid, it is in contact with some type of equipment or
surface from the time it is removed from the cow until it is packaged.
Unsatisfactorily washed and sanitized milking and milk-handling equip-
ment constitute the main source of bacteria in milk at the farm level.
The bacteria in farm milk tanks include many psychrotrophs and few
thermodurics (MacKenzie 1973; Thomas, Druce, and Jones 1971). Ther
modurics are more prevalent than psychrotrophs in pipeline milking
plants (MacKenzie 1973).
Psychrotrophs can grow and cause spoilage of refrigerated milk, but
few thermodurics grow below 10C. Pasteurization of milk destroys most
of the psychrotrophs. Contamination of pasteurized milk by psychro
trophs from poorly cleaned pipes and filling machines can result in spoil
age of the refrigerated milk.
Seafood
When fish are caught they are subjected to contamination from fish
boxes (plastic, wood, or metal) and other fishholding systems on the
ship. However, there are few, if any, bacteria in the flesh of fish before
filleting and subsequent handling.
Filleting may be done manually or by filleting and skinning machines.
When fish are filleted by hand, the knives and cutting boards provide a
source of microorganisms, and filleting machines become contaminated
by the surface microflora on the fish, which is transferred to the flesh of
the fillet. The bacterial count on the filleting equipment varies from 10
3
to 10
8
bacterialcm
2
When the bacterial load on the surface of the fish is
high, there is a higher bacterial count on the filleting boards. Gloves
worn by the filleters often have a higher bacterial load than the incoming
fish.
Fruits and Vegetables
The microbial load of fruits and vegetables increases during harvest
ing and processing. Peas aseptically removed from the pods are essen
tially sterile. When the peas are separated by a viner, the bacterial count
may reach 10
6
/g. The microbial load of most vegetables is reduced by
washing and by blanching. After blanching, the bacterial load increases
due to contamination by equipment and may attain levels of 10
4
to
105/g. The equipment causing contamination includes conveyor belts,
hoppers, and filling machines.
Splittstoesser (1973) stated that the source of most organisms on fro
zen vegetables is contaminated equipment surfaces. His data show that
corn cutters, French bean slicers, spinach choppers, and inspection belts
for peas significantly increased the microbial level of these vegetables. In
one instance, he found that moving peas to the freezer by an airlift (to
conserve water) resulted in significantly more bacterial contamination of
the peas than when they were flumed.
INGREDIENTS
The quality of a processed food is influenced by the quality of the
ingredients. Although ingredients may constitute a small part of the total
food, they may add a substantial number of microorganisms. Hence,
specifications, including acceptable microbiological levels, are needed
for the purchase or production of ingredients.
Spices or seasonings are often the source of high microbial numbers.
Spices are parts of plants such as dried seeds, buds, fruit or flower parts,
bark, or roots, usually of tropical origin. These seasonings and flavors
may contain over 10
8
aerobic bacteria per gram. Also, they contain aero
bic and anaerobic spores. A sample of ginger contained 48 million total
bacteria, 12 million yeasts and molds, 26 million aerobic sporeformers,
and 0.72 million anaerobic sporeformers (Tjaberg, Underdal, and Lunde
1972). Pepper is usually highly contaminated with bacteria and fungi.
Flannigan and Hui (1976) reported that fourteen of twenty spices con
tained Aspergillus jlavus and four of these spices supported growth of this
mold and the production of aflatoxin. Mean standard plate counts of
over 10
6
per gram were obtained for black pepper, ginger, and paprika
(Julseth and Deibel 1974). These spices also contained over 10
6
bacterial
spores per gram. Psychrotrophic spores were absent in herbs and spices
analyzed by Michels and Visser (1976). A nationwide survey of ten spices
and herbs revealed less contamination at retail than had been reported
for these ingredients at import (Schwab et al. 1982). Some samples of all
ten spices and herbs had an aerobic plate count over lOG/g. However,
many samples had less than 100/g. Thus, the geometric means of individ
ual spices and herbs ranged from 1,400 for cloves to 820,000 for pepper.
Thermophilic bacteria, usually as spores, are added to foods with in
gredients such as spices, starch, flour, and sugar. Thermophilic spores
are important when added to canned foods. The higher the level of heat
resistant spores, the greater the chance that some may survive the heat
treatment and be a potential spoilage problem.
The total aerobic count of flour sampled at the mills is generally in
the range of 10
2
to 10
3
/g (Graves et al. 1967). They reported levels of total
aerobic thermophilic spores from 0 to 1,200 per 10 g of flour, and al
though thermophilic flat sour spores were not present in 10 g of most
flour samples, one 10g sample contained 730 flatsour spores.
Since sugar is a potential source of spores, the National Canner's As
sociation has set limits of aerobic thermophilic flat-sour and anaerobic
thermophilic spores when this ingredient is to be added to canned foods.
Eskin, Henderson, and Townsend (1971) suggested that infections of
confectionery items by osmophilic yeasts are due to contaminated nuts,
fruits, chocolate, flour, or flavoring syrup. Bakery products can be con-
taminated by the ingredients in the doughs or by adding materials to the
baked food. Icing and nuts are added to already baked sweet rolls. The
toasting of almonds and pecans is not sufficient to assure sterility. These
products may contain 10
4
to 10
5
bacteria and 10
4
yeasts and molds per
gram.
Batters used in the breading of seafoods, onion rings, or other prod-
ucts are a potential source of high levels of microorganisms. Batters used
in the manufacture of breaded onion rings had a total bacterial count of
2,500 to 8,500,000/g (Maxcy and Arnold 1972). There were 110 to 32,000
coliforms per gram of the batters they examined.
Since ingredients are an important source of microorganisms, food
processors must establish acceptable microbiological limits for these sub
stances. Then sufficient testing is needed to assure the processor that the
ingredients are acceptable and, if they are used in the manufacture of a
food, adjustments of the process are needed to take care of any excessive
bacterial load.
PRODUCT TO PRODUCT
There is an old saying that one' bad apple can spoil the barrel of
apples. This is very true, because the spoilage organisms from the bad
apple can contaminate and cause spoilage of surrounding apples until
all of the apples are spoiled.
In this example, the transfer of the organisms can be disastrous be
cause they are known to cause spoilage of apples. Although the transfer
of microorganisms from one food to another can occur directly if the
foods contact each other, more often it occurs through contamination of
wash water, equipment, or handling.
Researchers have reported that the shelf life of vacuum-packed sliced
cooked meats would be improved if the meats were handled and sliced in
an area strictly separated from raw cured meats (Mol et al. 1971).
People handling both cooked and raw foods can transfer microor
ganisms from the raw to the cooked product. With no further treatment,
this is a potential health hazard. The cook who cuts up a raw chicken on
a cutting board that is then used for cutting up raw vegetables for a salad
may transfer Salmonella from the raw chicken to the raw vegetables. The
meat department in a retail store may use the same knife and block to
cut fish, cold meat, chicken, beef, and other types of food. Some of these
foods may be eaten with no further treatment or only a minimal heating.
A potential health hazard can result from this. Crosscontamination of
food is one of the ten main factors that contribute to foodborne illnesses.
PACKAGING
Packaging is a potential source of contamination. There are many
studies concerning the effect of different types of packaging materials on
the shelf life of food. The possibility that the packaging material might
contribute to the flora is not mentioned. This is probably because the
number of flora on most foods is much greater than that on packaging
material. In their survey of microorganisms on poultry processing equip
ment, Schuler and Badenhop (1972) reported the total bacterial count of
bulk containers for icepacked poultry. The counts ranged from 290/4 in.
2
(U/cm2) to 9,770/4 in.
2
(376/cm
2
). They stated that the holding of boxes
overnight led to contamination by dust, rodents, and birds.
Korab (1963) tested one-trip glass bottles used for carbonated bever-
ages. He reported that a majority of bottles had no bacteria, yeasts, or
molds when they were received at the beverage company, but about 6.5
percent had more than fifty bacteria per bottle. Storage of uncovered
bottles for one week increased the microbial contamination. Storage out-
side resulted in more contamination than storage inside, and the contam-
ination increased with storage time. Compared to returnable and reused
bottles, the onetrip bottles have very low microbial counts.
Plastic materials are used for packaging many food products. During
manufacture and handling, electrostatic charges can occur on the plastic.
These charges attract airborne material such as dust and microorganisms
(Baribo, Avens, and O'Neill 1966). Although plastics are essentially ster-
ile when made into packaging material, they may become contaminated
if not handled in an acceptable manner. The static charges add to the
problem of maintaining plastic free of microbial contamination.
There was a time when containers used for shipping poultry were
reused to pack fresh vegetables for shipment. Due to the potential for
contamination of the containers by salmonellae from the poultry and
then the transfer of these pathogens from the container to the vegetables,
this practice was halted.
Packages serve as a protective covering to limit or prevent microbial
contamination; however, they do not prevent microbial growth. It is im-
portant that preparation of the food for packaging is designed to limit
or prevent microbial contamination prior to packaging. Also, the pack-
age must be durable enough to maintain its integrity during storage and
distribution.
REFERENCES
Adams, B. G., and Marrie, T.]. 1982. Hand carriage of aerobic Gramnegative rods by
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Foodborne Agents
Causing Illness
6
Throughout our lifetimes we are subjected to risks and hazards of all
kinds. The food supply in the United States is one of the most abundant,
nutritious, and safest on earth. However, there is no absolute degree of
safety, not even for the food we consume.
Foods should be safer today than in the "good old days," due to the
knowledge we have gained of bacteria and sanitation, as well as to in
creased regulations. However, due to large scale, highspeed food pro
cessing, alteration of traditional processing methods resulting in less con
trol of microorganisms, proliferation of heatandeat convenience foods,
and nationwide distribution with increased potential for mishandling, it
is possible for outbreaks of foodborne illness to occur that involve many
people. This is evident by the five-state outbreak of salmonellosis that
occurred in 1985. The number of reported outbreaks and cases fluctuates
from year to year, but since 1967 the number of cases per 100,000 people
has tended to increase. Part of this increase may be due to more complete
reporting of foodborne illness, and part may be due to an actual increase
in the number of cases of such illness.
TYPES OF FOOD HAZARDS
The agents that cause human illness and can be transmitted by foods
are bacteria, viruses, fungi, parasites, chemicals, and toxins naturally
present in plants and animals. Bryan (1973) listed approximately 200 eti-
ologic agents of foodborne illness.
In 1961, the Communicable Disease Center (since renamed the Cen-
ters for Disease Control, or CDC), became responsible for maintaining
records and reporting foodborne illnesses in the United States. It is well
recognized that all outbreaks or cases of foodborne illness are not re-
ported to the CDC. However, the data collected by the CDC are the best
that we have. Annual summaries of foodborne 'illnesses have been pub-
lished since 1966. The confirmed etiologies of fooJborne outbreaks for
1981 (CDC 1983b) are listed in Table 6.1. In 1981, ::lS well as in other years,
bacteria were involved in most of the outbreaks and cases.
195
TABLE 6.1. ETIOLOGY OF CONFIRMED FOODBORNE DISEASE OUTBREAKS,
CASES, AND DEATHS IN THE UNITED STATES IN 1981
Outbreaks Cases Deaths
Etiology No. %
No.
%
No. %
Bacterial
Bacillus cereus 8 (3.2) 74 (0.9) 0 (0.0)
Campylobacter jejuni 10 (4.0) 487 (5.6) 0 (0.0)
Clostridium botulinum 11 (4.4) 22 (0.3) 1 (3.1)
Clostridium perJringens 28 (11.2) 1,162 (13.4) 2
(6.3)
Salmonella 66 (26.4) 2,456 (26.8) 21 (65.6)
Shigella 9 (3.6) 351 (4.1) 0 (0.0)
Staphylococcus aureus 44 (17.6) 2,934 (33.9) 1 (3.1)
Streptococcus Group A 2 (0.8) 307 (3.5) 0 (0.0)
Streptococcus Group D 1 (0.4) 24 (0.3) 0 (0.0)
Vibrio cholerae nonOI 1 (0.4) 4 0.1) 0 (0.0)
Vibrio parahaemolyticus 2 (0.8) 13 (0.2) 0 (0.0)
Yersinia enterocolitica
2 (0.8) 326 (3.8) 0 (0.0)
Other 1 (0.4) 48 (0.6) 0 (0.0)
TOTAL 185 (74.0) 8,208 (93.2) 25 (78.1)
Chemical
Ciguatoxin 15 (6.0) 152 (1.8) 3 (9.4)
Heavy metals 2 (0.8) 4 0.1) 0 (0.0)
Monosodium glutamate 2 (0.8) 4 0.1) 0 (0.0)
Mushroom poisoning 11 (4.4) 25 (0.3) 3 (9.4)
Scombrotoxin 7 (2.8) 67 (0.8) 0 (0.0)
Other 14 (5.6) 75 (0.9) 0 (0.0)
TOTAL 51 (20.4) 327 (3.8) 6 (18.8)
Parasitic
Giardia lamblia 1 (0.4) 61 (0.7) 0 (0.0)
Trichinella spiralis 7 (2.8) 70 (0.8) 1 (3.1)
-
TOTAL 8 (3.2) 131 (1.5) 1 (3.1)
Viral
Hepatitis A 6 (2.4) 128 (1.5) 0 (0.0)
TOTAL 6 (2.4) 128 (1.5) 0 (0.0)
Confirmed Total 250 (100.0) 8,794 (100.0) 32 (100.0)
SOURCE: CDC (1983).
Not all of the diseases that may be transmitted by foods can be de
scribed thoroughly in this text. Therefore, those organisms and illnesses
that have been reported most frequently are stressed.
Definition of an Outbreak
A foodborne disease outbreak is defined by the CDC as an incident
in which two or more persons experience a similar illness, usually gastro
intestinal, after ingesting a common food, and epidemiological analysis
implicates the food as the source of the illness. For botulism or chemical
poisoning, one case constitutes an outbreak (see Figure 6.1).
/
,-----,
INITIAL REPORT
OF ILLNESS
Two or more
persons ill
j
Not compatible
with food borne
outbreak
Acute
gastroenteritis
Botulism,
trichinosis,
chemical, etc.
Compatible with
food borne outbreak;
same time & symptoms;
common food consumed
/\
List on
Register;
No outbreak
investigation
r-------,
Interstate consumer
product; notify
regulatory agent
/\
...--------,
Beef or poultry
report to USDA
Other interstate
products report
to FDA
J
OUTBREAK INVESTIGATION
(Including notification of
appropriate local, state, or
federal agencies)
Epidemiology Laboratory Environmental
Describe disease Food samples Hygiene
-Define persons -Human specimens -Describe food
-Incriminate food -Environmental events
L'W"" I po'"'
CONTROL
Figure 6.1. A scheme for handling food borne disease complaints, to be imple-
mented by state and local health departments.
Courtesy of CDC.
197
A microbial foodborne illness may result from ingesting a food con
taining either pathogenic microorganisms or a toxin or poison. When a
pathogenic microorganism is the etiologic agent, the illness is called an
infection. If a toxin or poison is the causative agent, the illness is called
a food intoxication or food poisoning.
Epidemiology
Epidemiology attempts to identify the cause and the mode of trans
mission of infections and to suggest and evaluate methods for controL
The diagnosis of the specific disease is important for treatment and
controL With a known etiology, acceptable therapy can be prescribed,
dangers from handling patients with infections can be avoided, and the
patient can be informed of the possible course of the illness.
Confirmed etiologies are those in which laboratory evidence is ob
tained and fulfill specific criteria of the CDC. The present reporting sys
tem involves many people and agencies. If the affected people do not
seek medical help, if the doctor does not report the illness, or if there
is no further investigation to confirm the cause of the illness, it is not
recorded.
The data recorded by the CDC showed that bacterial agents accounted
for 92 outbreaks and 3,270 cases in 1976. This increased to 185 outbreaks
and 8,208 cases in 1981 (Table 6.1). It has been estimated that less than
10 percent of the actual outbreaks and cases are reported.
For the United States, estimates as high as 10 or 20 million cases a
year have been made. Of the reported outbreaks, only about 50 percent
have a confirmed etiology.
The data in Table 6.1 show that two bacterial agents, staphylococci
and salmonellae, account for 44 percent of the outbreaks and more than
60 percent of the cases. The salmonellae accounted for more than 65
percent of the deaths due to foodborne agents. The three most promi
nent agents are salmonellae, S. aureus, and C. perfringens, not only in the
United States but also in Canada (Todd 1983a), the Netherlands (Beckers
1982), England and Wales (Roberts 1982), and Europe in generaL The
most commonly reported cause of foodborne illness in India is staphylo
coccal (Hobbs 1982).
Other important bacteria have been C. botulinum and Shigella, with B.
cereus showing an increase. Recent additions are Vibrio parahaemolyticus,
Yersinia enterocolitica, and Campylobacter jejuni. Pasteurized milk was the ve
hicle for Listeria monocytogenes (Fleming et aL 1985).
Cholera is an important illness worldwide, spread by poor sanitation
and contaminated food. There are illnesses, such as traveler's diarrhea,
in which symptoms resemble those of several foodborne diseases. The
CDC defines traveler's diarrhea as an acute intestinal illness that devel
FOODBORNE AGENTS CAUSING ILLNESS 199
ops one or more days after arrival in a foreign country. Enterotoxigenic
E. coli is the major agent, but other bacteria (Shigella, salmonellae), para
sites (Giardia, Entamoeba), and viruses (rotavirus, reovirus, parvovirus) may
be involved (Dupont 1981; Morgan et al. 1984).
Diarrheal syndromes are much more prevalent in countries in which
sanitation is lacking and health resources are less available than in the
industrialized nations. Nichols and Soriano (1977) estimated that 2.7 mil
lion children under five years of age die each year from diarrhea. Of
these deaths, 76 percent occur in Asia, and 13 percent in Africa.
Foods Involved
Various foods are involved in foodborne illness (Table 6.2), but meat
and meat products are the vehicles for transmission of a major share of
foodborne illnesses. Precooked roast beef and ham are frequently reo
TABLE 6.2. FOODBORNE DISEASE OUTBREAKS BY VEHICLE OF TRANSMISSION,
1977-1981
Year
Vehicle 1977 1978 1979 1980 1981 Total
%
Beef 27 14 20 17 34 112 4.4
Pork 8 10 10 5 7 40 1.6
Ham 10 12 10 8 9 49 1.9
Sausage 10 2 0 5 6 23 0.9
Other meat 10 8 8 12 3 41 1.6
TOTAL MEAT 65 46 48 47 59 265 10.4
Poultry products 14 7 13 24 34 92 3.6
Shellfish 12 16 9 17 9 63 2.5
Other fish 22 29 31 53 28 163 6.4
TOTAL SEAFOOD 34 45 40 70 37 226 8.9
Milk 0 2 0 2 9 13 0.5
Ice cream 4 2 2 2 5 15 0.6
Other dairy 4 4 3 5 4 20 0.8
TOTAL DAIRY 8 8 5 9 18 48 l.9
TOTAL ANIMAL
FOODS 121 106 106 150 148 631 24.8
Baked foods 12 5 4 9 12 42 1,6
Fruits and vegeta
bles 9 6 9 15 11 50 2.0
Salads, miscella
neous 23 20 19 19 21 102 4.0
Mushrooms 32 1 4 0 11 48 l.9
Chinese food 17 7 3 3 1 31 l.2
Mexican food 12 9 6 10 5 42 l.6
Other food 41 23 21 28 35 148 .5.8
Unknown 169 304 289 371 324 1457 57.1
TOTAL 436 481 461 605 568 2,551
SOlJRCE: Data from CDC Annual Summaries.
ported as vehicles of salmonellae and S. aureus, respectively. Poultry and
poultry products are a source of salmonellae, C. perfringens, S. aureus, as
well as other organisms.
Before pasteurization, milk was involved in outbreaks of many dis
eases. Unfortunately, milk may be contaminated after pasteurization or
may be used raw. Many of the illnesses caused by fishery products are
due to ciguatoxin, scombrotoxin, or paralytic shellfish poisoning. Foods
that contain these toxins are from an unsafe source. Fishery products
also are vehicles for clostridia and Vibrio species as well as parasites.
Canned foods are vehicles for botulinum toxin. If the container leaks,
the foods may be the vehicle for other agents that cause illness. Chinese
foods, another common source, have been studied as well (Bryan et al
1982).
Place of Mishandling
When a processor of foods is involved in a foodborne disease out
break, there is a great potential for many cases because of the widespread
distribution of the defective product. Fortunately, the processor is in-
volved in relatively few outbreaks (Table 6.3). Most outbreaks are caused
by the mishandling of foods in foodservice establishments (Bryan 1982).
The home is also an important place for such mishandling to occur. One
can assume that there are very few cases per outbreak for those occurring
in the home. The CDC no longer determines the place of mishandling,
but rather the place of consumption (CDC 1983b).
Contributing Factors
Various factors contribute to outbreaks of foodborne illness. The
main ones are improper holding temperatures (failing to properly refrig-
erate food), inadequate cooking, contaminated equipment (failure to
clean and disinfect kitchen or processing plant equipment), and poor
TABLE 6.3. PLACES IN WHICH FOODS WERE MISHANDLED, 1968-1978
Place
Food-processing establishment
Food-service establishment
Home
Unknown, unspecified, or not applicable
TOTAL
SOURCE: Data from CDC Annual Summaries.
No. of
Outbreaks
171
1,707
639
1,704
4,221
%
4.1
40.4
15.1
40.4
TABLE 6.4. CONTRIBUTING FACTORS CAUSING FOODBORNE ILLNESS,
1977-1981
Year
Factor 1977 1978 1979 1980 1981 Total
%
Improper holding tempera 168 150 115 184 181 798 39.5
ture
Inadequate cooking 67 53 53 41 62 276 13.7
Contaminated equipment 58 45 51 47 70 271 13.4
Food from unsafe source 23 16 7 23 40 109 5.4
Poor personal hygiene 87 63 70 80 110 410 20.3
Other 46 32 24 20 34 156 7.7
TOTAL 2,020
SOVRCE: Data from CDC Annual Summaries.
personal hygiene (Table 6.4). Other factors that contribute to foodborne
illness include preparing food a day or more before serving, with im
proper holding and reheating, crosscontamination (from raw to cooked
products) and adding contaminated ingredients to previously cooked
food without reheating (Bryan 1982; Roberts 1982: Todd 1983b). After
foods have been contaminated, the main danger is allowing them to reo
main at a temperature that permits the growth of potentially hazardous
microorganisms.
Most of these problems could be controlled with only a little effort
on the part of food handlers, whether in a processing plant, a restaurant,
a cafeteria, or a home. The tremendous turnover of food workers makes
effective training difficult. However, since many outbreaks occur because
of carelessness at home, everyone might benefit from training in food
handling and personal hygiene. A course in the sanitation of food han
dling could be offered in high school. According to Feachem (1984), the
incidence of diarrhea can be reduced by 14 to 48 percent by simply wash
ing contaminated hands with soap and water. Food microbiologists
should set a good example for others to follow.
Symptoms and Severity
The symptoms of the illnesses are variable. However, diarrhea, nau
sea, vomiting, and abdominal cramps are evident in most foodborne ill
nesses.
Usually, each day some 8 to 10 liters of water enter the intestines of
adults. The source of this fluid includes ingested liquid (food and water),
swallowed saliva, and secretions from the stomach, pancreas, liver, and
intestinal glands. Normally, about 90 percent of this liquid is resorbed
from the small intestine, and of the remaining liquid, about 90 percent,
is absorbed from the colon, so that only about 100 to 200 ml of fluid
passes out of the colon. Most of the excess water in the body is removed
by the kidneys. Perspiration and respiration account for the remaining
loss of water from the body.
Either malabsorption or excessive secretion of water and electrolytes
into the intestines can result in diarrhea. Researchers have defined diar
rhea as the occurrence of four or more unformed stools within one day,
together with one or more other symptoms (abdominal cramps, nausea,
vomiting, fever) (Dupont et al. 1980). Mild forms of diarrhea may result
in only a few hundred milliliters of extra fluid per day passing out of the
colon. However, in severe cases, up to 1 liter per hour may be expelled.
This excessive loss of water causes dehydration, which can result in
death. Other effects, especially in very young children, include malnutri
tion and growth retardation.
Fortunately, in most cases, the illness is not severe and the patient
recovers in one or a few days. However, twenty five deaths from diarrhea
occurred in the United States in 1981 (Table 6.1). Although salmonellae
were involved in most of the deaths, the mortality rate was higher for
botulism (4.5 percent) than for salmonellosis (0.9 percent).
Diarrhea has various causes. These include the ingestion of certain
drugs such as antibiotics and antimetabolites, excessive laxatives, herbal
teas, sugar alcohols in dietetic foods and beverages, various "health"
foods, certain carcinomas, and from modification of a diet or from anx
iety caused by traveling. Additional factors are listed in Table 6.1.
BACTERIAL DISEASES
Bacterial foodborne illness can result from toxins produced in the
food before consumption (S. aureus, C. botulinum) or by the organism
either infecting cells or producing toxins in the intestinal tract after in
gestion. Once in the intestinal tract, the organism must contact and ad
here to the cells of the epithelium (Archer 1984; Hill 1985; Smith 1984).
Otherwise, they will be removed by peristalsis and the movement of the
intestinal contents, as well as by mucociliary action.
Once attached to the cells, some organisms may invade the cells and
others produce enterotoxins; in either case, the water secretion into or
adsorption from the intestines may be affected.
For epidemiology of an outbreak and the prevention of foodborne
illness, both microorganisms and their toxins must be detected. Although
there are basic similarities in the various methods, there are also specific
differences.
Staphylococcal Intoxication
This illness accounted for over 17 percent of the outbreaks and al
most 34 percent of the cases of reported foodborne illnesses in the
United States in 1981 (Table 6.1). However, the actual extent of this ill
ness is not known.
CHARACTERISTICS OF THE INTOXICATION. The characteristics
of an illness can aid in determining the causative agent in an outbreak
of foodborne illness.
Incubation Period. The incubation period of a typical outbreak usually
ranges from 30 min to 8 hr, with most illnesses occurring 2 to 4 hr after
ingestion of the suspect food (Fig. 6.2).
Symptoms. Not all of the people eating a suspect meal become ill, and not
all ill people experience the same symptoms. The severity of the symp
CI)
W
20
15
CI) 10

()
5
r-

I-
-
I-
-
I- -
I- -
I- -
f-- - l-
I-
l-
f--
l-
f--
f--
f--
l-
f--
l-
I-
1
r-
II n II I
o
012345678
INCUBATION PERIOD (HOURS)
Figure 6.2. Depiction of an outbreak of staphylococcal in
toxication. Incubation period.
Courtesy of CDC.
TABLE 6.5. PERCENTAGE OF ILL PERSONS EXPERIENCING SPECIFIC
SYMPTOMS DUE TO STAPHYLOCOCCAL INTOXICATION
Outbreaks
Symptoms 2 3 4
Nausea 76 100 50
Vomiting 70 44 100 77
Diarrhea 19 67 100 82
Abdominal cramps 71 71 58 15
Chills
25 46
Headache 42 6 41
Prostration 63
Weakness 68
Leg cramps
Muscle soreness 5
Collapse 9
Hypotension 1
Fever
25 7 21
- = Not reported.
SOURCE: Data from CDC Morbidity and Mortality Weekly Reports.
5
41
76
73
16
35
73
11
24
toms varies with the concentration of enterotoxin in the food, the
amount of food consumed, and the susceptibility of the individual. The
principal symptoms listed in Table 6.5 are nausea, vomiting, abdominal
cramps, and diarrhea.
Duration and Therapy. Symptoms of the illness usually subside after one
or two days. The illness is rarely fatal. Due to the sudden onset and short
duration of the illness, treatment usually is not needed. However, hospi
talization is required in cases in which shock, dehydration, and extensive
vomiting have occurred. In these cases, therapy includes replacing lost
fluids and electrolytes. According to Holmberg and Blake (1984), 10 per
cent of the victims seek hospital care.
ETIOLOGIC AGENT. This illness is called an intoxication because the
etiologic agent is an enterotoxin. Payne and Wood (1974) listed six
known staphylococcal enterotoxins (A, B, C, D, E, F), (SEA, SEB, SEC,
SED, SEE, SEF) based on serological reactions. Although SEC) and SEC2
react with the same antibody and are often lumped together as SEC, they
also react with distinct minor antibodies. The presence of a third SEC,
enterotoxin C3, has also been reported (Reiser et al. 1984).
Enterotoxin A is the most frequently encountered enterotoxin in
food poisoning outbreaks in the United States. In New Zealand, Jarvis
and Harding (1972) found enterotoxins C and D to be more prevalent
than SEA. SEB rarely is involved in food poisoning outbreaks in the
United States.
FOOD BORNE AGENTS CAUSING ILLNESS 205
Properties of the Enterotoxins. Staphylococcal enterotoxins are simple pro
teins with a molecular weight between 25,000 and 35,000. They are read
ily soluble in water and salt solutions.
The heat stability is an important characteristic of staphylococcal
enterotoxins. Enterotoxin B is more heat resistant than A or D. The heat
resistance is influenced by the medium in which it is heated (composi
tion, pH). In foods, the enterotoxins are not completely inactivated by
normal cooking, pasteurization, or other usual heat treatments. Tatini
(1976) stated that thermal processing cannot be relied upon to inactivate
these toxins. Further, he found that heated toxins had greater biological
activity than unheated toxin when tested at the same dose level.
Action of Enterotoxins. Researchers used monkeys to study enterotoxin
induced emesis (vomiting) (Elwell et al. 1975). Their results indicate that
enterotoxin is not absorbed from the intestine. Orally ingested enter
otoxin is thought to mediate emesis by acting on sites in the intestine.
This stimulus apparently is transferred by the vagus and sympathetic
nerves to the vomiting center, which is part of the central nervous system.
The vomiting center somehow induces retroperistalsis of the stomach
and small intestine, resulting in emesis. The exact systems involved in
the vomiting syndrome have not been determined. The known facts and
hypotheses have been discussed and described elsewhere (Robins
Browne 1980; Thompson and Malagelada 1982; Van Miert et al. 1983).
Since the nervous system is involved, it has been suggested that the enter
otoxins should be called neurotoxins. However, neuronal binding of SEA
in the intestinal tract was not demonstrable (Beery et al. 1984).
The action of staphylococcal enterotoxins in the diarrheal syndrome
is not known. Staphylococcal enterotoxin shows an affinity to the walls of
the stomach and the small and large intestines. If sufficient enterotoxin is
present in consumed food, it causes inflammation and irritation of the
lining in the stomach and intestinal tract. Working with flounder intes
tine in vitro, the data of Huang, Chen, and Rout (1974) suggested that
enterotoxin stimulates active sodium and chloride secretion. Others
found that enterotoxin B did not interfere with water absorption in the
guinea pig ileum, but that staphylococcal delta toxin did inhibit absorp'
tion in the jejunum and ileum (Kapral et al. 1974).
Amount of Enterotoxin Needed for Illness. There are no definite data concern
ing the minimum amount of enterotoxin needed to cause symptoms in
a human. Gilbert (1974) listed estimates that ranged from 0.015 to 0.357
p,g of enterotoxin per kilogram of body weight. Besides body weight, indio
viduals vary in their sensitivity to enterotoxins.
FOODS INVOLVED. Various foods have been involved in staphylococ
cal intoxication since 1974 (Table 6.6). More than 35 percent of the out
TABLE 6.6. FOODS INVOLVED IN STAPHYLOCOCCAL INTOXICATIONS 1976-1981
Years
Food 1976-1978 1979-1981 Total
%
Beef 3 5 8 4.5
Ham 22 19 41 23.3
Pork 1 1 2 1.1
Other meat
5 7 12 6.8
Poultry 7 16 23 13.1
Shellfish 1 0 1 0.6
Other fish 0 1 1 0.6
Milk
0 1 1 0.6
Other dairy 1 2 3 1.7
Bakery products 3 10 13 7.4
Fruits and vegetables 1 1 2 1.1
Salads 13 13 26 14.8
Other foods
6 6 12 6.8
Unknown 11 20 31 17.6
TOTAL 74 102 176
SOURCE: Data from CDC Annual Summaries.
breaks involved red meats, with 23 percent being caused by contami
nated ham. Fresh meats are involved less often than cured meats since,
being perishable, they are refrigerated. Cured meats are not as perish-
able and may be mishandled by being allowed to remain at room temper-
ature. Also, it is difficult for S. aureus to compete with the microbial flora
on fresh meat as compared to that on cured meat.
The poultry products involved are usually either barbecued or in
salads. Other salads (potato, macaroni, and tuna fish) have been impli-
cated in more than 14 percent of the outbreaks. Bakery products contain-
ing custard or cream are important offenders.
In many outbreaks, the food is cooked, then contaminated with S.
aureus during handling and held at a temperature for growth and produc-
tion of enterotoxin.
THE ORGANISM (S. AUREUS). S. aureus is described briefly in Chap-
ter 3. These Gram-positive cocci produce colonies that may be white or
have pigment ranging from yellow to orange. The color is influenced by
growth conditions as well as strain variation.
In both aerobic and anaerobic conditions, S. aureus produces acid
from mannitol, glucose, lactose, and maltose_ Aerobically, many carbohy-
drates are metabolized with the production of acid, but acid is not pro-
duced from arabinose, cellobiose, dextrin, inositol, raffinose, rhamnose,
or xylose.
Typical S. aureus strains produce a-toxin, coagulase, and a heat-stable
nuclease_
SOURCES. Some reports call S. aureus ubiquitous because it is so wide-
spread (air, dust, clothing, floors, water, sewage, and insects). The princi-
pal source of S. aureus is the human nose, although it is found on the
skin, especially on the hands, in infected wounds, burns, boils, pimples,
acne, in nose and throat discharges, and in feces_ The primary site on
the hands is the fingertips, which relates to the habit of handling one's
nose with the fingers. The extent of nasal carriers is difficult to deter-
mine, but surveys have shown the carrier rate to vary from 6 percent to
over 60 percent of the population_ People associated with hospitals tend
to have a higher carrier rate than the normal population_
Animals are a source of S. aureus. Most of the strains of S. aureus iso-
lated from animals tend to have characteristics different from those asso
ciated with people (Devriese 1980; Kibenge, Wilcox, and Perret 1982).
The organism is found in food-processing operations (Harvey, Patterson,
and Gibbs 1982; Notermans, Dufrenne, and Van Schothorst 1982), and
at a high level on and in healthy food handlers (Holmberg and Blake
1984).
GROWTH. Researchers believe that 10
5
to 10
6
cells of S. aureus per
gram of food must generally be present before the production of enter-
otoxin reaches a level that can cause intoxication. Due to the normally
low numbers in food, multiplication must occur. By knowing and under-
standing the factors affecting the growth of S. aureus, we can control the
growth, enterotoxin production, and outbreaks of staphylococcal intoxi-
cation. The general factors affecting the growth of S. aureus are described
in other sections of this text. S. aureus is a relatively poor competitor, and
various bacteria can inhibit or outgrow it. This inhibitory interrelation-
ship of other bacteria with S. aureus is important in preventing toxin pro
duction in foods. It may be a primary reason for certain foods to be less
involved than others in outbreaks of staphylococcal intoxication. In
foods with aw of 0.90 to 0.95 or with 5 to 10 percent salt, S. aureus can
dominate because most other bacteria cannot grow.
Strains oflactic streptococci (S. lac tis and S. cremoris) inhibited S. aureus
inoculated into milk prior to cheese making (Ibrahim 1978). Since cheese
has been a vehicle of staphylococcal enterotoxin, it is evident that micro-
bial competition cannot be relied upon to always inhibit growth and
toxin production of S. aureus. Also, it has been suggested that some com-
petitive organisms may degrade the enterotoxins.
Obviously, S. aureus is able to grow on or in foods that have been
involved in staphylococcal intoxication. Some outbreaks have occurred
by holding food at room temperature for less than 4 hr. Longer incuba-
tion times increase the risk. The behavior (growth or inhibition) of S.
aureus has been reported for various cheeses (Koenig and Marth 1982;
Magrini, Chirife, and Parada 1983), pumpkin pie (Wyatt and Guy 1981),
potatoes (Tamminga et al. 1978), and meat (Lui ten , Marchello, and Dry
den 1982). The maximum temperature for growth of S. aureus was in
creased by the addition of salt, monosodium glutamate, or soy sauce
(Hurst and Hughes 1983).
Sublethal treatments and other factors (pH, a
w
, Eh, temperature)
discussed in Chapter 4 act to control growth and enterotoxin production
of S. aureus. Growth was not observed at aw 0.85 or below, pH 4.3 or less,
or at 8C or less (Notermans and Heuvelman 1983). Enterotoxin produc
tion required higher a
w
, pH, or temperature than that needed for growth
but was mainly inhibited by the effect of a
w

When warm or hot foods are refrigerated, a long time may be needed
to cool the food sufficiently to prevent growth of S. aureus. Foods with
gravies or sauces cool slower than do those without, and the substrate is
more readily available for growth of organisms.
TOXIN PRODUCTION. S. aureus produces several toxins, including
those listed in Table 6.7. The main difference between exotoxins and
endotoxins is not whether they are outside or inside the cell, but rather
their structure. Exotoxins are proteins with little or no nonprotein resi
dues. Endotoxins are primarily polysaccharide and lipid complexes (lipo
polysaccharides). Food microbiologists are concerned with the enterotox
ins, which, by definition, are exotoxins, since they are protein as well as
being found free from the cell.
Not all strains of S. aureus are enterotoxigenic. Schroeder (1967) esti
mated that only 4 percent of the staphylococcal strains in milk were capa
ble of producing enterotoxin. In a survey of S. aureus in meat, dairy prod
ucts, and other foods, Payne and Wood (1974) found that 125 of 200
strains (62.5 percent) produced enterotoxins. Wieneke (1974) found a
somewhat higher ratio of enterotoxigenic strains in cooked food than in
TABLE 6.7. SOME TOXINS OF STAPHYLOCOCCUS AUREUS
Toxin
a-toxin
J3-toxin (phospholipase C)
'Y-toxin
I)-toxin
Hyaluronidase
Staphylococcal coagulase
Staphylokinase
Leukocidin
Epidermolytic
Enterotoxins (A, B, C, D, E, F)
Toxic shock toxin
Action
Hemolytic, dermonecrotic, lethal
Hemolytic
Hemolytic
Hemolytic, enteric
Spreading factor
Coagulates plasma
Fibrinolytic
Kills leucocytes
Exfoliation
Emetic
Lymphoid hyperplasia
raw food. The higher ratio may be due to people contaminating cooked
foods with human strains. Such strains are more frequently enterotoxi
genic than are strains from animal or other sources.
The production of SEA results from a chromosomal gene (Mallonee,
Glatz, and Pattee 1982; Pattee and Glatz 1980; Shafer and Iandolo 1978b).
Shafer and Iandolo (l978a) reported that SEB production of strains S6
and 277 is determined by a chromosomal gene or genes and suggested
that this might be the case for many enterotoxigenic strains of S. aureus.
Dyer and Iandolo (1981) proposed that SEB production is dependent on
at least two unlinked genes. Altboum, Hertman, and Sarid (1985) reo
ported that elimination of plasmid pZA10 from strain 6344 caused the
loss of SEB and SEC] production. According to these researchers, the
plasmid can be transferred and confer toxigenicity to other S. aureus
strains, and chromosomal DNA can integrate the plasmid.
The conditions necessary for growth and enterotoxin production
have been reviewed (Smith, Buchanan, and Palumbo 1983). In general,
toxin production occurs in a more narrow range of environmental char
acteristics than those discussed for growth in Chapter 4.
The relationship of growth of S. aureus and SEB production was reo
ported by Markus and Silverman (1969) and is shown in Figure 6.3. The
rate of synthesis of SEB is greater than that for SEA, so higher concentra
tions of SEB are obtained. Tweten and Iandolo (1983) suggested that a
precursor of SEB is bound to the cell membrane. At some stage, mature
SEB is formed and released by the membrane into a specialized compart
ment in the cell wall. From there, it is released to the exterior environ
ment.
A constant dissolved oxygen (DO) level of 100 percent stimulated
growth, but enterotoxin production was not observed (Carpenter and
Silverman 1974). A DO of 10 percent yielded a higher level of enter
otoxin than did a DO of either 100 percent or 50 percent.
Aerobically, certain strains of S. aureus produce enterotoxin at a pH
of 4.8, but anaerobically, no enterotoxin is found at pH 5.4 (Barber and
Deibel 1972). Their results indicate that biological acid production can
not be relied upon to inhibit S. aureus in fermented sausage, and they
recommended chemical acidulation. Since the toxin is produced at a
higher level aerobically than anaerobically, they suggested sampling aero
bic portions of foods for the toxin. Mixing and crosssectional sampling
merely dilute the toxin with anaerobic portions of the food, which con
tain little or no toxin.
S. aureus (A100) grew and produced enterotoxin in precooked bacon
stored aerobically either at 37C and a minimum aw of 0.84, or at 20C
and an a
w
of 0.89 (Lee, Silverman, and Munsey 1981). Anaerobically, the
organism required a minimum aw of 0.90 at 37C, and 0.94 at 20C.
200
160
E
...... 120
01
~
a:l
Z
X 80
o
b
It:
LLI
!z
LLI
40
TIME
BACTERIA
ENTEROTOXIN B
Figure 6.3. Growth and Enterotoxin B production of S. aureus.
courtesy of Markus and Silverman (1969).
en
::i
en
Z
ct
(l)
It:
o
1 0 ~
It:
LLI
a:l
::i
:;)
z
(l)
o
..J
The effect of sodium chloride on growth and enterotoxin production
was reported by McLean, Lilly, and Alford (1968) and is shown in Figure
6.4.
METHODOLOGY. Foods or other samples are examined for S. aureus
and the enterotoxins to demonstrate postprocessing contamination, to
determine a potentially hazardous product, or to confirm a causative
agent in a foodborne illness. Since S. aureus is sensitive to heat and sani
tizing agents, the presence of the organism or its toxins in processed food
or on equipment generally indicates poor sanitation or handling. Post-
600
3000
500
2500
u;

z
:;)
a::
400
2000
w

lII:
.....
Z
>

g
300
1500
is

as
0
a::
a::
:;)
w

z
200 w
1000
100
500
o 1--__ ....... ___ .......... __ ---'1.....-__ --'-__ --'"' ... 0
o 2 4 6 8 10
% SODIUM CHLORIDE
Figure 6.4. Effect of salt concentration on the production of enterotoxin by S.
aureus.
courtesy of McLean, Lilly, and Alford (1968).
processing contamination usually is due to human contact or to improp
erly sanitized food contact surfaces.
The methodology involved in conjunction with S. aureus includes the
detection and enumeration of the organism; the characterization of the
organism by testing for coagulase, nuclease, and other enzymes; serologi
cal reactions; phage typing and the qualitative and quantitative analyses
for the enterotoxins.
Detection and Enumeration of the Organism. Many media have been devel
oped and suggested for the direct plating and enumeration of S. aureus.
The FDA (1978) method uses Baird-Parker agar_ This medium contains
potassium tellurite as a selective agent and egg yolk and tellurite as differ-
ential agents_ On this medium, S_ aureus colonies appear circular, smooth,
convex, moist, gray to jet-black, frequently with an off-white margin, due
to reduction of the tellurite to elemental tellurium_ They are surrounded
by an opaque zone and an outer clear zone due to the reaction on the
emulsified egg yolk_ When touched with an inoculating needle, the colo-
nies have a buttery to gummy consistency_ There may be variations to this
description_ Typical colonies are selected for further study_
A collaborative study revealed that Baird-Parker (BP) agar is satisfac-
tory for recovery of cells stressed or injured by heat or other processing
conditions (Baer et al. 1975)_
, Baird-Parker agar, modified by substituting pig plasma for egg yolk,
uses the coagulase reaction for differentiation and is as effective as the
original agar for growing stressed S_ aureus cells (Becker, Terplan, an9
Zaadhof 1983; Idziak and Mossel 1980)_ The addition of bovine fibrin-
ogen to plasma-modified BP agar was found to be effective in detecting
S. aureus and also determines the nuclease reaction (Beckers et al. 1984;
Chopin et al. 1985).
Characterization_ Colonies typical of S. aureus on an agar surface are se-
lected for further testing and characterization. These tests may include
various fermentations of carbohydrates, the presence of coagulase, heat-
stable nuclease or lysozyme, and determining the resistance to chemical
inhibitors or antibiotics (Baker 1984). None of these tests, or a combina-
tion of them is an absolutely reliable indicator of enterotoxin formation
by the organism.
The coagulase test is considered the most reliable single test for dif-
ferentiating potentially pathogenic S. aureus. However, not all coagulase-
positive strains produce enterotoxins. On the other hand, there are re-
ports that coagulase-negative strains produce enterotoxin or have been
involved in cases of staphylococcal intoxication (Lotter and Genigeorgis
1975). Some enterotoxigenic strains of staphylococci found to be coagu-
lase negative on first isolation, become coagulase positive after subcultur-
ing several times, and some strains may lose the ability to produce coagu-
lase but remain enterotoxigenic.
The coagulase reaction in clotting blood serum was reviewed by Tager
(1974). According to this review, there are three main reactions, ending
with the conversion of fibrinogen to fibrin and formation of a clot. Ac-
cording to Baird-Parker (1972), over 90 percent of the strains of S. aureus
produce a coagulase. Several tests have been suggested and evaluated to
detect the presence of coagulase. A tube test is used to determine free
coagulase, and a slide test detects a clumping factor. These are distinct
entities. A latex slide agglutination test to determine clumping factor and
protein A simultaneously was described by Essers and Radebold (1980)
and reported to be as accurate as the tube test and more accurate than
the slide test by Doern (1982).
Although other organisms may produce nucleases, the heat stability
of S. aureus nuclease is unique. It is generally agreed that the enzyme
loses but little activity by boiling for 30 min. It retained 10 percent of its
activity after heating at 100C for 180 min, 120C for 34 min, or l30C
for 16 min (Erickson and Deibel 1973).
The basic test for staphylococcal nuclease was described by Lachica,
Genigeorgis, and Hoeprich (1971). Since then, several suggestions for im
provement have been made (Ibrahim 1981; Pham and Davis 1979; Stad
houders, Hassing, and Galesloot 1980). However, other staphylococci
produce thermostable nuclease (Gudding 1983). These nucleases can be
distinguished from each other by seroinhibition, the inhibition of
nuclease activity by specific antibodies (Gudding 1983; Lachica, Geni-
georgis, and Hoeprich 1979).
The thermostable nuclease test might be used as a screening method
for foods to detect the possible presence of high populations of S. aureus
or enterotoxin (Tatini et al. 1975; Tatini, Cords, and Gramoli 1976). This
system seemed to be applicable to certain foods, but was of questionable
value for others. Ibrahim and Baldock (1981) always detected nuclease in
cheese that contained enterotoxin.
Medwid and Grant (1980) questioned the value of the thermonuclease
test for foods containing high numbers of Streptococcus faecalis subsp. lique-
jaciens, since this organism inactivated the enzyme.
Phage Typing. Some bacteriophages are very specific for the cells they will
lyse, while other phages have a wider spectrum of susceptible cells. Obvi-
ously, for phage typing of S. aureus, those phages that have a limited spec-
trum of susceptible cells are desirable.
The International Committee on Nomenclature of Bacteria estab-
lished a Subcommittee on Phage Typing of Staphylococci in 1953. The
Staphylococcus Reference Laboratory of the British Public Health Labo-
ratory Service in London became the International Reference Center for
the subcommittee. This subcommittee established a set of phages for rou-
tine testing of S. aureus. In 1961, it became the World Health Organiza-
tion Centre for Staphylococcus Phage Typing. A description of the sys-
tem used for phage typing was reported by CDC (1976b) and Parker
(1972).
Phage typing is of particular help in the epidemiological study of
staphylococcal intoxications or infections. Various strains isolated dur-
ing an outbreak from patients, foods, and food handlers can be tested for
their phage patterns. This information makes it possible to differentiate
between a strain responsible for the outbreak and unrelated strains.
Smith (1972) stated that there are only two good reasons for typing
S. aureus, either by phages or antisera. The main reason is for epidemio
logical investigations, and the second reason is to give a label to the or
ganism for research work.
Enterotoxin Detection. The detection of enterotoxin is important. If an out
break occurs and isolates of S. aureus are obtained from a food, the enter
otoxins produced can be compared to the enterotoxin causing the intoxi
cation. In one outbreak involving dried milk, no S. aureus isolates were
detected. In this case, the organisms multiplied, produced the toxin, and
then were killed by heat during the processing. In cases such as this, the
food can be analyzed for the enterotoxin.
The principal methods for detecting enterotoxins can be separated
into biological and serological systems.
A biological system can be some living entity or part thereof. The
best living entity to determine enterotoxin activity is a human. However,
humans are not always readily available. Also, the amount of enterotoxin
cannot be determined, because people vary in their sensitivity to enter
otoxin.
Animals such as monkeys, chimpanzees, dogs, pigs, cats, and kittens
have been tested as biological systems. Monkeys and chimpanzees can be
given the enterotoxin orally, whereas the other animals are injected
either intraperitoneally or intravenously. Because vomiting is the first
symptom to occur and is the most readily observable reaction to enter
otoxin, animals such as rodents that do not have a vomiting mechanism
are not used. Pigeons, frogs, tropical fish, nematodes, or various protozoa
or bacteria show no obvious reaction with enterotoxins. Tissues includ
ing human intestinal cells, rabbit gut segments, chicken embryos, tissue
cultures (HEp-2 and HeLa cells) and isolated enzyme systems have been
tested for assay of enterotoxin. Although crude enterotoxins have shown
some effect on some of these systems, the purified toxins have revealed
little or no effect.
Since monkeys or chimpanzees can be fed the enterotoxin orally, they
are the preferred test animal. The monkey is not as sensitive to enter-
otoxin as humans are and the sensitivity of chimpanzees is somewhere
between the sensitivities of humans and of monkeys. Another disadvan-
tage is that these animals are expensive to maintain and, as they are used
to assay enterotoxin, they tend to develop a resistance (a limited immu-
nity) to the toxins. Thus, their usefulness for enterotoxin detection is
limited. A direct skin test on sensitized guinea pigs reportedly has a sensi-
tivity of 10 to 100 pg of SEB per milliliter of prepared food sample
(Scheuber et al. 1983).
Tests using animals were necessary before the enterotoxins were puri-
fied and serological tests could be developed_ It is evident that the animal
tests are still important for the detection of any new types of enterotox-
ins, or testing the toxicity of isolated compounds, or a particular chemi-
cal portion of a toxin_
Serological systems that have been suggested for assaying enterotox-
ins include Ouchterlony double-diffusion plate (Ouchterlony 1968), Ou-
din single-gel diffusion tube, Oakley double-gel diffusion tube, micro-
slide (Casman and Bennett 1965), polyvalent single radial immuno-
diffusion (Meyer and Palmieri 1980), hemagglutination inhibition, re-
versed passive hemagglutination (Yamada, Igarishi, and Terayama 1977),
immunofluorescence, radioimmunoassay and enzyme-linked immuno-
sorbent assay (ELISA) tests_
The Ouchterlony system was adapted for use on a glass slide by Wads-
worth_ A modification of this microslide method was used by Casman
and Bennett (1965) to detect enterotoxin in foods. Bennett (1971) stated
that 0.005 J-tg of enterotoxin A per gram of cheese was detectable. In
order to determine low levels of enterotoxin, procedures of extraction,
separation, and concentration are needed (Bennett and McClure 1980).
According to a collaborative study reported by Bennett and McClure
(1976), the microslide gel double-diffusion test has a high degree of speci-
ficity, is simple, and has good reproducibility in the identification of
enterotoxins. This system has been adopted as official by the AOAC.
The various diffusion tests have been used with varying results. The
radioimmunoassay (RIA) technique reportedly can detect as little as 0.1
ng/ml of SEA and 0.5 ng/ml of SEB (Areson, Charm, and Wong 1980).
However, the radioactive label deteriorates and should be replaced after
about two months. Radioactive waste is created. The system also requires
expensive equipment and scarce and expensive purified toxins.
To overcome some of the problems of the RIA, Saunders and Bartlett
(1977) suggested an enzyme immunoassay (EIA). Using an EIA, Kauffman
(1980) stated that it was sensitive to 2 ng SEA/ml. He considered this
method to be sensitive and precise enough to serve as an alternative to
the RIAThe FDA guidelines for acceptable enterotoxin detection is 1 to
2 ng/g of food_
Procedures using ELISA now have the greatest potential for the detec-
tion of recognized enterotoxins. First, a measured amount (100 J-t1) of
purified enterotoxin is added to wells in a polystyrene plate. After suffi-
cient time at 4C to allow the toxin to bind to the plastic, the liquid is
removed and the wells filled with serum albumin to block any residual
binding sites on the plastic. Then the wells are emptied, washed with
buffer, and known antitoxins, to which an enzyme such as peroxidase or
alkaline phosphatase is conjugated, are added_ After about 2 hr, the wells
are aspirated to remove the liquid and washed to remove excess conju-
gated antitoxin. Then a substrate is added which the conjugated enzyme
can change to a colored compound. If there is no enterotoxin, there is
nothing to which the antitoxin can react. It, along with the enzyme, are
removed by washing, and there will be no color reaction when the sub
strate is added. It is evident that the amount of enterotoxin is related to
the amount of enzymeconjugated antitoxin that remains, and that is reo
lated to the intensity of color that is formed by the enzyme acting on the
substrate for a specified time. The testing of known amounts of enter
otoxin is used to establish the color intensities produced in a specified
time, so that the ELISA is a quantitative procedure. Extensive purifica
tion of the toxin in the sample is not needed for the ELISA system. There
are several variations to this basic system.
Using a competitive ELISA, SEA, SEB, or SEC concentrations of 0.1
ng or less were measured (StifflerRosenberg and Fey 1978). Four ver
sions of the ELISA were compared by Fey, Pfister, and Ruegg (1984). They
stated that the sandwich with labeled antibody was the best, with a sensi
tivity of 0.1 ng of enterotoxin per milliliter. To detect enterotoxin in
food, the ELISA is as sensitive as the RIA (Freed et al. 1982; Kuo and
Silverman 1980) and more sensitive than the microslide technique (Lenz
et al. 1983).
Injection of an antigen into an animal results in the production of
antibodies. A single B cell produces a single antibody specific for a single
antigenic determinant. However, the B-cell population is polyclonal in
nature and produces heterogeneous, polyclonal antibodies even when
a highly purified antigen is injected. If the B cells were a monoclonal
population, a large amount of single antibody would be produced. It is
difficult to culture individual B cells. However, when B cells are fused
with myeloma (tumor-producing) cells, the resultant hybridoma combines
the antibody production of the B cell and the reproduction of the mye-
loma cell. The hybridoma can be cultured and antibody harvested from
the culture supernatant, or it can be obtained from the ascites fluid of a
host animal bearing a hybridoma tumor.
The monoclonal antibodies produced by the hybridoma are very spe-
cific for a particular antigenic determinant. However, monoclonal anti-
bodies do not readily cross-link when binding to antigen. Cross-linking
is important in diagnostic tests, which depend on precipitation reactions,
such as the gel-diffusion tests. Also, if two or more antigens share a
common determinant recognized by the monoclonal antibody, cross-
reactions can occur.
The above is a rather simplistic description of monoclonal antibody
production. More complete descriptions of production or use can be
found in many publications, including articles by L0vborg (1984), Sher-
man (1984), and van Hell and Helmich (1984). Monoclonal antibodies
have been produced for staphylococcal enterotoxins (Edwin et al. 1984;
Meyer et al. 1984; Thompson, Ketterhagen, and Bergdoll 1984).
Although the biological activity of the enterotoxins is not measured
by these tests, Bergdoll (1970) stated that correlation between the two is
adequate to justify the use of serological tests to analyze for enterotoxins.
Chang and Dickie (1971) interpreted their observations with enterotoxin
B to mean that antigenic sites and toxin sites are probably not the same.
They did not speculate on whether this would invalidate the serological
tests as methods for the analysis of the enterotoxins. Other researchers
reported that heating in gelatin caused both immunological inactivation
and biological inactivation of SEB, indicating a possible relationship
(Notermans et al. 1983).
CONTROL OF STAPHYLOCOCCAL INTOXICATION. It should be
relatively simple and easy to prevent staphylococcal intoxication. The
obvious control measure would be to keep the organisms out of foods.
For those S. aureus that do invade food, methods to destroy them or to
prevent their growth and enterotoxin production can be employed. If
toxin is produced, then destruction of the toxin is needed.
Prevent Contamination. It is impossible to keep all foods free from S. aureus
due to the ubiquitous character of the organisms. However, it would seem
possible to use common sense measures to keep the contamination low.
Humans are the main reservoir of the organism. The health, hygiene,
and work habits of food handlers can influence the level of contamina
tion of foods. People who are sick should not be allowed to handle food.
They contaminate the food not only with S. aureus, but also with other
disease organisms. People with colds, sinusitis, or other respiratory dis
turbances are good sources of S. aureus, as are boils, pimples, acne, and
infected cuts. It is not possible to segregate all carriers of S. aureus, since
a sizable portion of the population may be involved. However, those
people who are obviously contaminators of food should be removed
from foodhandling areas.
S. aureus has been isolated from knives and slicers during investiga
tions of outbreaks of food poisoning. Foodhandling equipment should
be designed so that it can be properly and effectively cleaned and sani
tized. The cleaning and sanitizing of equipment are discussed in Chap
ter 9.
Prevent Growth and Toxin Production. The main control of S. aureus is to
hold the food at a temperature unsatisfactory for growth. There is little
or no growth below 4C or above 46C.
Toxin production occurs in a narrower temperature range than cellu
lar growth. Troller (1976) suggested a range of 10 to 45C. Greater
amounts of enterotoxin are produced in the range of 33 to 38C than
at either higher or lower temperatures.
Obviously, to warm the product for serving, or to cool leftover heated
food, the temperature of the food must go through the growth range.
This range must be traversed as quickly as possible. Three hours has been
suggested as the maximum time allowed. To facilitate cooling of leftover
food, the food should be placed in shallow layers in shallow pans. The
deeper the food, the longer it will take to cool. The warm food should
not be held at room temperature to cool, but should be placed in the
refrigerator. This means that the refrigeration system must be adequate
to cool these potential extra loads. The refrigerator cannot be jammed
full of hot food, since no refrigerator can handle such a condition.
In fermented sausage products, S. aureus can be controlled by lower
ing the pH with starter cultures or chemical acidulation (Daly et al. 1973;
Raccach 1981). A combination of a culture and acidulation or chemical
inhibitor is more effective than either treatment alone. Adding solutes
or drying of foods to lower the a
w
below 0.85 should prevent growth and
enterotoxin production of S. aureus.
Dipping poultry carcasses in a 5 percent potassium sorbate solution
reduced the growth of inoculated S. aureus (To and Robach 1980). In
model systems, concentrations above 3 percent were needed to inhibit
50 percent growth of S. aureus (Lahellec, Fung, and Cunningham 1981).
Butylated hydroxyanisole (BHA) was reported to be lethal to S. aureus
at 100 {tg/ml (Degre and Sylvestre 1983). Although Parada, Chirife, and
Magrini (1982) found some growth inhibition of S. aureus in a model sys
tem by 100 {tg/ml of either BHA or butylated hydroxy toluene, neither
was effective in delaying growth in a cheese spread (aw of 0.976).
Destroy S. aureus, Inactivate Toxin. Heating is the principal method used
to kill S. aureus cells as well as other organisms. Although heat may kill the
cells, if enterotoxin has been produced, the toxin may persist, because, in
a food, the enterotoxin is more heat stable than the organism.
Not only is the temperature important, the time of exposure is also
important. The temperature and time needed to kill an organism depend
on factors such as the heat resistance of the organism, the number of
cells present, the age of the cells, the type offood or suspending medium,
the ingredients added to the food, the pH, a"" and previous treatment of
the food or organism (stress conditions). Thermal processing is described
elsewhere in the text, so only a brief preview is given here.
To help describe the heat resistance of an organism, D and z values
are used. The D value is used to describe the time temperature relation
ship to the killing of an organism. A lD is the time needed at a specified
temperature to reduce the number of cells by 90 percent, or, conversely,
10 percent of the cells survive this treatment. The z value is a measure of
the effect of a change in temperature on the resistance of an organism.
It is the of or c required for the thermal resistance to change by a factor
of lO. Most of the thermal evaluations are reported in of; however, when
possible, the of have been converted to C.
Angelotti, Foter, and Lewis (1961) determined the D values of various
organisms in foods. In custard heated at 60C, the D value for S. aureus
196E was 7.82 min, while for S. aureus Ms 149 it was 7.68 min. This means
that if 100 cells of S. aureus Ms 149 were present per gram of custard,
heating for 7.68 min at 60C would reduce this number to 10. In another
7.68 min, there would be only 1 cell per 10 gram and after another 7.68
min, only 0.1 of a cell per gram, or 1 cell per 10 grams of custard would
be viable. The importance of aseptic methods to keep the original popu
lation as low as possible should be quite evident. In chicken a la king,
the respective D values for S. aureus 196E and Ms 149 were 5.37 and 5.17
min. This indicates that the organisms are more susceptible to heat in
chicken a la king than in custard, since the D values are lower for the
organisms in chicken a la king. Other D values for S. aureus are listed in
Chapter 12.
It is relatively easy to kill S. aureus by normal pasteurization or cook
ing procedures. The problem lies in recontamination of the heated food
with S. aureus. Deboning poultry or slicing of ham after cooking furnishes
opportunities for recontamination from the hands of food handlers.
With the destruction or reduction of the indigenous flora during heating,
the recontaminating cells of S. aureus are not competitively inhibited. If
this recontaminated food is allowed to remain at temperatures for
growth of S. aureus, enterotoxin production may occur, resulting in a po-
tential outbreak of staphylococcal intoxication. To reduce the occurrence
of staphylococcal intoxications, it is important that strict sanitation and
hygiene be followed anywhere that food is handled.
Heating is not a practical means to eliminate enterotoxins from food,
because of the heat stability of these agents (Fig. 6.5). Calcium hypochlo-
rite at 50 to 200 {tg/ml detoxified SEB-contaminated water (Meyer, Hin-
terberger, and Korte 1977). Using chlorine to detoxify food might not be
acceptable, but it may be effective for surface decontamination.
Botulism
From 1899 through 1976, there were 1,875 reported cases of botulism
in the United States. Since 1976, cases of botulism have been classified
into four categories (foodborne, infant, wound, and undetermined).
From 1977 through 1980, there were 170 cases of foodborne botulism,
171 cases of infant botulism, and 5 cases of wound botulism reported in
200
100
~
50
_ 40
U)
I.IJ 30
~
::::)
~ 20
2
10
!
5
4
3 - NEGATIVE
2 + POSITIVE (AT LEAST 1.0 J.l0
ENTEROTOXIN A)
100 104.4 110.5 115.5 121.1
TEMPERATURE c
126.6
Figure 6.5. Thermal inactivation of staphylococcal enterotoxin.
Courtesy of Hilker et al. (1968).
the United States (CDC 1979, 1981; Gunn and Terranova 1979; Morris
and Hatheway 1980).
Although the number of cases of botulism is comparatively low, from
1899 to 1976 there were 992 deaths, for a case fatality rate of 52.9 per
cent. The case fatality rate for 1977 was reported as 6.3 percent (CDC
1979a) or 8.2 percent (Gunn and Terranova 1979). The case fatality rate
was 5.2 percent, 0, and 16.7 percent for 1978, 1979, and 1980, respec
tively. This rate has decreased considerably since 1950 (Fig. 6.6). The avo
erage outbreak involves only two or three people. The largest outbreak
reported in the United States occurred in Michigan in 1977. This out
break involved 59 people, but there were no deaths. Home-processed
food was the source of the toxin.
100
90
80
70
60
a:
W
m
50
::E
::::l
Z
40
30
20
10
0
1880'61 '62
CASES
DEATHS
,..,.---
".,..,-----------.... ,----
~ ..."
...... ~
*
'83 '84 '86 '88 '87 '88 'S8 '70 '71 '72 '73 '74 '76 '78 '77 '78 '78 '80
* NOT AVAILABLE FOR 1979 AND 1980
Figure 6.6 Foodborne botulism-death-to-case ratios, by 10-year periods, 1899-
1973.
Courtesy of CDC.
ETIOLOGIC AGENT. Botulism is a neuroparalytic intoxication caused
by neurotoxins produced by toxigenic strains of Clostridium botulinum.
Eight antigenically distinct toxin types (A, B, C], C
2
, D, E, F, and G) are
recognized. Sometimes types C) and C
2
are grouped as C, but they are
serologically distinct. Types A, B, E, and F cause botulism in humans.
Although types C and D have been reported as causing illness in humans,
the involvement is rare and uneventful. Types C and D affect animals.
Type C is especially involved with birds and type D with cattle. Swine are
moderately to highly resistant to most of the toxins.
The number of outbreaks caused by types A, B, E, and F in the United
States from 1899 to 1976 is shown in Figure 6.7. Type A toxin is the main
cause of botulism in the United States, followed by types Band E. Only
one outbreak of foodborne illness has been attributed to type F toxin.
26
a:
24
4(
W
>-
22
a:
20
W
Q.
18
t/)
W
16
t/)
4(
U
14
u.
12
0
a:
10
W
m
8
::E
:::J
6
Z
Z
4
4(
w
::E
2
0
UNKNOWN
TYPE A
TYPE B
TYPE E
TYPE F
1899' 1900-09 1910-19 1920-29 1930-391940-491950-59 1980-691970-77
*1-YEAR PERIOD
.* 8-YEAR PERIOD
10-YEAR PERIODS
tlNCLUDES ONE OUTBREAK OF 58 CASES, 1977
Figure 6.7. Number of cases of botulism, by type of toxin.
Courtesy of CDC.
No human cases of type G have been reported in the United States, but
the organism has been isolated from necropsy samples of four adults
and one infant in Switzerland (Sonnabend et al. 1981). Type G toxin was
demonstrated in the serum of three of these cases.
Most of the type A outbreaks occur in the western states, while type
B is predominant in the east and central United States. This correlates
with the finding that type A spores predominate in the soil in the west,
while type B spores predominate in the east and central areas. In Europe,
type B botulism is the predominant type, which correlates with the occur-
rence of type B spores in the soil. Although not prominent in the contig-
uous fortyeight states of the United States, type E botulism has ac-
counted for more than 40 percent of the outbreaks in Japan, Canada,
and the Scandanavian countries, and for almost all of the cases in Alaska.
It is generally agreed that for foodborne botulism, the C. botulinum
grows in the food and produces the neurotoxin, which is then ingested
orally. However, for wound botulism, the organism produces the toxin
in the infected wound, in vivo. Infant botulism apparently results from
the ingestion of the organism with subsequent intraintestinal production
of the toxin. Hence, there may be an unusual circumstance in which the
toxin might be formed in the intestinal tract of adults after ingestion of
the organism or its spores.
During the logarithmic growth phase, the toxin accumulates intrace1-
lularly, with small amounts found extracellularly. Beyond the logarithmic
phase of growth, by cell lysis, the toxin is released to the extracellular
medium.
Properties of the Toxins. The toxins are simple proteins that are water solu-
ble, heat sensitive, and acid stable. On a molar basis, the toxins produced
by C. botulinum are the most lethal natural products known. Since the
toxins are protein, they are antigenic. Various molecular weights ranging
up to 900,000 have been reported. According to Sugiyama (1980), these
large compounds are complexes of toxins and nontoxic material. The
molecular weights range from 128,000 to 170,000 depending upon the
type of toxin and culture. The toxins are dichains composed of two
single-chain polypeptides of different length combined by a sulfide link-
age. Proteolytic organisms form these dichains naturally. For nonproteo-
lytic strains, trypsinization of the toxin components produces similarly
linked dichains of the pep tides. This has been called "activation." The
presence of two dissimilar components was shown for type C
2
toxin (Iwa-
saki, Ohishi, and Sakaguchi 1980; Ohishi 1983b). Neither component
manifested the original toxicity, but when mixed together and trypsin-
ized, the original toxicity was restored.
Action of the Toxins. Except for wound or infant botulism, the preformed
toxin is considered to be ingested orally. To react with and affect the
nerves, the toxin must traverse the barrier of the gastrointestinal tract
and be transported to the susceptible nerves. The action on the nerves
causes neuromuscular blockage, paralyzing the muscles.
The type of food in the GI tract can affect the toxin and its action.
The food might protect the toxins from the enzymes or other destructive
actions, such as by stomach acids. Food increases or decreases the secre-
tion of digestive juices. The food might combine with the toxin, forming
larger particles less able to penetrate the intestinal wall. Foods may affect
the rate of peristalsis, which increases or decreases the time the toxin is
in an area of the intestine affording the greatest opportunity for penetra-
tion of the wall.
The major site for absorption of the toxin is the small intestine (Bon-
ventre 1979; Ohishi 1983a). Perhaps only a small amount of the ingested
toxin is absorbed, but Bonventre (1979) estimated that about lO 11 mole-
cules of toxin reaching the peripheral nerve endings is sufficient to cause
symptoms of botulism in adults. Also, the large intestine may be an abo
sorption site, since, according to Lamanna (1968), intrarectal instillation
of toxin into monkeys and rabbits caused death, although the animals
lived longer than when the toxin was given orally. The different toxins
are absorbed at different rates (Sugii, Ohishi, and Sakaguchi 1977).
That the toxin is "wasted" in the alimentary tract is evident from work
with animals. When type C toxin is administered to mink intraperi-
toneally, only 50 MLD (mouse lethal dose) causes death. Oral administra-
tion of 4,000 MLD causes death, but with 2,500 MLD, the mink survive.
With pigs, the oral to intravenous ratio of amount of toxin needed to
cause death is 16,700:1 (Smith, Davis, and Libke 1971). Data from various
outbreaks indicate that not all of the toxin penetrates the intestinal wall,
and that it can be present in the intestines for a prolonged period.
The toxin passes with the lymph through the thoracic duct and is
dumped into the bloodstream. It is carried by the vascular system to the
nerves. Lamanna (1968) reported that when the lymph system was pre-
vented from emptying into the bloodstream, animals fed botulinum
toxin did not develop symptoms of botulism.
At the nerves, the toxin attaches to the presynaptic terminals of cho-
linergic nerves, and interferes with the release of acetylcholine at the
myoneural junctions (Sellin 1981; Sugiyama 1980). The failure of im-
pulses to be transmitted across the nerve fiber junctions results in paraly-
sis of the muscles, which are controlled by the nerves.
CHARACTERISTICS OF THE INTOXICATION. Since the toxins af-
fect the nerves, this illness differs from most other foodborne illnesses.
Incubation Period. After food contaminated with botulinum toxin is in-
gested, the usual time for symptoms or signs of botulism to appear is 12
to 48 hr (CDC 1983b). However, this time may vary from 2 hr to 8 days,
depending upon the amount of toxin ingested, the type of toxin, the
resistance of the individual, and perhaps even the type of food. Gener-
ally, if the incubation period is less than 24 hr, the person will be more
severely affected, and death is more likely than if the incubation period
is longer.
Attack Rates. Although botulinum toxin is the most potent natural poison
known, not everyone who consumes contaminated food will acquire
symptoms or succumb to the toxin. This has been related to the amount
of contaminated food ingested. However, there have been reports that
merely tasting a food and spitting it out resulted in intoxication and
death. This led to the supposition that toxin could be absorbed directly
from buccal exposure. The contention of Lamanna, Hillowalla, and AI-
ling (1967) is that the normal swallowing reflex transfers the toxin from
the mouth to the GI tract.
Symptoms and Signs. The cardinal features of botulism are as follows:
1. Fever is absent but may develop if a complicating infection occurs.
2. Mental status is normal. Patients may be anxious or agitated and
some are unusually drowsy; however, in the absence of secondary
complications, patients are responsive.
3. The pulse rate is normal or slow, but tachycardia may occur if hy
potension develops.
4. Although vision may be blurred, numbness and paresthesias are
absent, and a sensory deficit does not occur.
5. Neurological manifestations are symmetrical.
The symptoms and signs from cases reported to the Centers for Dis-
ease Control are listed in Table 6.8. The first symptoms of illness are
gastrointestinal. Nausea or vomiting, substernal burning or pain, abdom-
inal distension, and decreased bowel sounds may occur. Some cases have
initial transitory diarrhea but subsequently become constipated. These
symptoms and signs may mislead physicians to diagnose the illness as
appendicitis, bowel obstruction, or even diaphragmatic myocardial in-
farction. The mucous membranes of the mouth, tongue, and pharynx
may be red, dry, and painful, which might be diagnosed as pharyngitis.
Sometimes other illnesses are diagnosed as botulism. Investigation of
438 suspect botulism outbreaks by CDC (1974a) revealed that only 75
were actually botulism, and the rest were due to other disorders.
The neural symptoms usually begin with the eyes and face, and paral-
ysis progresses downward to the throat, chest, and extremities. When the
diaphragm and chest muscles become fully involved, respiration is not
possible. Death is due to asphyxiation. This usually occurs in three to six
days. In nonfatal cases, complete recovery may take several months. Men-
tal processes are usually clear during the illness.
Therapy and Duration. Prompt clinical, epidemiological, and laboratory ef-
forts are required because each hour is critical to the survival of a patient
with botulism. The patient should be hospitalized for treatment.
The treatment of botulism can be separated into three parts: remov-
ing unabsorbed toxin from the alimentary tract, neutralizing toxin with
antitoxin, and treating the symptoms, such as respiratory distress.
The sooner the antitoxin is administered, the better the chances for
recovery. The reduction of the mortality rate in recent years has been
attributed to the prompt treatment of the patients and administration of
antitoxin. In some cases, guanidine hydrochloride is used as an adjunct.
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This chemical seems to compensate for neural effects of the toxin. The
use of germine in combination with guanidine is beneficial in the treat
ment (Cherington, Soyer, and Greenberg 1972).
The treatment of symptoms is especially important with respiratory
difficulty or failure. Tracheotomy of the patient is used to assist in
breathing. In a severe case, a mechanical respirator may be needed to
maintain breathing.
With very little exposure to the toxin, the patient may remain asymp-
tomatic or develop symptoms with little distress that pass in a few days.
With increased amounts of toxin and development of respiratory failure,
so that a respirator is needed, it may take several months for the patient
to recover. Partial paralysis may persist for six to eight months (Bryan
1973).
One reason given for the lower mortality rate in Europe compared
to the United States is that type B is prevalent in Europe and type A in
the United States. Type A toxin is known to bind rapidly to tissue. In one
case, 24 hr between ingestion and treatment was sufficient for the type
A toxin to adhere irreversibly to the patient's myoneural tissue (Dillon
et al. 1969). The antitoxin reacts with free toxin. When the toxin is irre-
versibly bound, administration of antitoxin has little effect on the recov
ery of the patient. Muscles injected with type A toxin were paralyzed up
to seven days, while type B caused paralysis for less than five days (Sellin,
Thesleff, and Dasgupta 1983).
Although immunization of people could be accomplished, the rare
occurrence of botulism makes widespread immunization impractical. Im-
munization is recommended for laboratory or other personnel who are
exposed to the toxin.
FOODS AS VEHICLES OF THE TOXIN. Usually botulism is associ-
ated with foods that have been given a preservation treatment, stored for
some time, and consumed without appropriate heating. The preservation
treatment in these foods is inadequate to destroy the spores that were
present in the food.
The foods involved in botulism outbreaks in which the toxin type
was determined are listed in Table 6.9. In more than 54 percent of the
outbreaks, vegetables were the vehicle of the toxin. Fish, fruit, and condi-
ments were other important vehicles, whereas meat, poultry, and dairy
products have been involved rarely in botulism. Home-processed foods
accounted for the majority of the outbreaks (72 percent), while commer-
cially processed foods were involved in less than 10 percent of the out-
breaks. Unknown vehicles accounted for slightly less than 20 percent of
the outbreaks (Table 6.10). Although commercially processed foods have
been involved in fewer outbreaks than home-processed foods, the inci
TABLE 6.9. FOODS INVOLVED IN BOTULISM OUTBREAKS IN WHICH THE TYPE
OF TOXIN WAS DETERMINED, 1899-1977
Botulinum Toxin Type
Food Group A B E F A+B Total
%
Vegetables 115 31 1 2 149 54.4
Fishery products 11 4 25 40 14.6
Fruit 22 7 29 10.6
Condiments 17 5 22 8.0
Beef 6 1 8 2.9
Dairy products 3 2 5 1.8
Pork 2 1 3 1.1
Poultry 2 2 4 1.5
Other 8 3 3 14 5.1
TOTAL 186 56 29 1 2
%
67.9 20.4 10.6 0.3 0.7
SOURCE: CDC (1979b).
dents which involve commercial foods are publicized, while those caused
by home-packed foods rarely are mentioned. The reason is the wide-
spread distribution of the commercial packs. If 500 cans of home-packed
corn are contaminated, chances are only one of these will be involved
in botulism in one family. If 500 cans of commercially canned corn are
contaminated, they could cause botulism in 500 families. So far, this has
not happened in the United States.
The outbreaks of botulism due to commercially packed foods since
1960 caused some health "experts" to suggest that families should pro-
cess food at home. Data certainly do not support the theory that home
canning reduces the number of botulism outbreaks.
THE ORGANISM. The genus Clostridium is divided into four groups
according to spore formation and gelatin liquefaction. Clostridium botuli-
num is in Group II. In this group, the spores are terminal and gelatin is
hydrolyzed.
The species includes a heterogeneous group of strains that are di-
vided into types A through G based on the antigenic neurotoxins that
are produced. These seven types are divided into four groups according
to their deoxyribonucleic acid homologies and biochemical, physio-
logical, and serological characteristics. The members of these groups are
as follows: group 1, type A and proteolytic strains of types B, C, D, and
F; group II, type E and non proteolytic strains of types Band F; group III,
non proteolytic strains of types C and D; and group IV, type G (Buchanan
and Gibbons 1974).
The optimum temperatures for growth are 30 to 40C for group 1,
25 to 37C for group II and 30 to 37C for groups III and IV
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organisms. Group I organisms digest milk, group II coagulate milk with
a soft curd but do not digest it, group III organisms do not change milk,
and those in group IV digest milk slowly. Other differences and simi
larities were described by Lynt, Kautter, and Solomon (1982).
Toxigenic Strains. Apparently most strains of C. botulinum are capable of
producing a toxin; however, this ability may be lost due to laboratory
cultivation, and non toxigenic clostridia have been isolated from both mao
rine and terrestrial environments.
Inoue and Iida (1970) suggested that production of toxin by C. botuli
num is influenced by prophages in the cells. By curing the cells of the
phage, some toxigenic strains lose the ability to produce toxin. With rein
fection by phage, the cells regain toxigenicity. It has been established that
the phage type can determine the toxin type that is produced and not
all lysogenic phages will confer toxigenicity on the infected cells (Hari
haran and Mitchell 1976; Oguma, !ida, and Inoue 1975). Most of the non
toxigenic strains that were converted to the toxigenic state reportedly
were not stable to serial transfer through cooked meat medium (Oguma
1976).
Some strains of C. botulinum cured of their prophages by researchers
remained toxigenic (Eklund et aL 1970). The researchers suggested that
the organisms might carry more than one prophage, or alternatively that
not all C. botulinum toxins are induced by bacteriophage.
The studies of phage inducement of toxin formation primarily have
used type C and D strains. The relationship of phage to toxigenicity of
strains causing human botulism has not been evaluated sufficiently.
Sources. The organism is widely distributed in nature. It occurs in soil
apparently throughout the world. The type of C. botulinum depends upon
the locality. The factors that affect the distribution of the different toxin
types are not known. Healthy animals may be carriers of types of
organisms to which they are immune, thus serving as reservoirs for reo
plenishing the organisms in the soil and spreading the organisms to var
ious places.
Bottom sediments of marshes, lakes, and coastal ocean waters contain
C. botulinum. The primary type in marine environments in northern areas
appears to be type E; however, other toxin types of the organisms also
are found. Type E is common in the intestine of fish taken from certain
waters. The organism has been isolated from the environment of trout
farms and from trout grown in these establishments (Cann and Taylor
1982) and in a salmon hatchery (Eklund et aL 1982). The organism does
not multiply in living fish, but it does multiply in aquatic vegetation and
bottom deposits. In marshes, the growth of algae reduces the oxygen con
tent of the water to a level that allows C. botulinum to multiply.
Since the organism is widely distributed in nature, it is evident that
food, especially vegetables and fish, can be contaminated during produc
tion, harvesting, or processing. C. botulinum has been reported in com
mercial honey (Huhtanen et al. 1981; Kautter et al. 1982; Midura et al.
1979), corn syrup (Kautter et al. 1982) and liver sausage (Hauschild and
Hilsheimer 1983). Fortunately, if present, the spores are usually in low
numbers, and they can germinate only if the conditions of microenviron
ments of the food are favorable. After germination, sufficient time is
needed for growth and toxin production.
GROWTH AND TOXIN PRODUCTION. The foods involved in out
breaks of botulism obviously present an environment or microenviron
ments that allow the growth of C. botulinum.
Various minimum and maximum temperatures have been reported
for growth and toxin production. Eklund, Wieler, and Poysky (1967) reo
ported growth and toxin production of a strain of type B C. botulinum at
3.3C. The maximum temperature of growth for types A and B is about
48C. Type E can grow in a range from 3.3 to 45C. Growth and toxin
production of type E is less pronounced at 37C than at either 30 or
22C (Ajmal 1968). A higher number of cells developed at 30 than at
22C, but no difference was found for toxin production, indicating that
the amount of toxin produced per cell was greater at 22 than at 30C.
When incubated at 4C, growth and toxin in four
to five weeks. '\,,'
Type A (14 strains) and proteolytic type B (13 strains) did not multi
ply, but decreased in number during holding in cooked meat medium at
100C for eighteen weeks (Smelt and Haas 1978). At 12C some strains
formed toxin in three to four weeks. At 4C, non proteolytic type Band
type F cultures grew in tryptone glucose yeast extract broth, but not in
crabmeat (Solomon, Kautter, and Lynt 1982). For growth in crabmeat, a
temperature of 12C was needed.
At pH 4.8, the growth of C. botulinum was detected in only one food
(pineapple rice pudding) of the many tested (Townsend, Vee, and Mercer
1954). No growth was found at any pH below 4.8. Several other reports
have confirmed that C. botulinum fails to grow below pH 4.8 (BairdParker
and Freame 1967; Huhtanen et al. 1976; Ito et al. 1976; Segner, Schmidt,
and Boltz 1971). These and similar reports have been the basis for the
FDA setting an upper limit of pH 4.6 for acid foods. These require less
heat treatment in the canning process than do those foods over pH 4.6.
However, growth and toxin production have been detected at pH 4.3 to
4.6 when media with high amounts of protein in suspension were used
(Smelt et al. 1982; Tanaka 1982).
In some outbreaks of botulism, the pH values of the foods involved
were below that regarded as necessary for growth and toxin production.
One explanation is that there are pH gradients in some foods so that
some microenvironments are acceptable for growth and toxin produc
tion. Another possibility is that the growth of other organisms can result
in acceptable microenvironments (Anderson 1984; Montville 1982; Od
laug and Pflug 1979).
The effect of salt on the growth and toxin production varies with the
types and strains of C. botulinum. An aqueous phase level of 5.5 percent
salt in homogenized cod was sporostatic to two strains of C. botulinum
type E (Boyd and Southcott 1971). In lumpfish caviar, no toxin was
formed at water phase salt concentrations of 5.56 percent or greater
(Hauschild and Hilsheimer 1979).
C. botulinum is an anaerobic organism. The clostridia lack cyto
chromes, cytochrome oxidase, catalase, and peridoxase, so the oxidation
reduction potential (Eh) must be low for growth to occur (Lund and Wy
att 1984). C. botulinum type E inoculated into meat and fish produced
toxin under both aerobic and anaerobic conditions on incubation (Ajmal
1968). There are microenvironments in these foods which have low
oxidation-reduction potentials favorable for the growth of this organism.
Sugiyama and Yang (1975) inoculated spores of C. botulinum into fresh
mushrooms, which were then packaged and stored at 20C. The respira-
tion of the mushrooms removed the free oxygen and allowed the spores
to germinate and the cells to produce toxin in three to four days. The
mushrooms appeared to be edible. No toxin was detected in mushrooms
held at 4C.
Since vacuum packaging in gas-impermeable plastic produces anaero-
bic conditions, it was reasoned that this practice might encourage the
growth of C. botulinum and result in toxic foods. The rate of toxin produc-
tion is higher in vacuum-packaged fish than in fish packaged without
vacuum (Pace and Krumbiegel 1983). The slowest rate of toxin produc-
tion is in unpackaged products. Research on vacuum packaging of raw
fish has revealed that when the fish are stored at lOoC or less, spoilage
is evident before toxin is detected (Eyles and Warth 1981). This safety
margin is reduced if storage is at 20C. Flushing the package with CO
2
may increase the risk of botulism, since CO
2
tends to inhibit spoilage
organisms but enhance the germination of C. botulinum spores and cellu-
lar growth. However, 100 percent CO
2
delays toxin production (Doyle
1983). Vacuum-packaged and modified atmosphere-packaged foods must
be stored below 4C to assure safety (Notermans, Dufrenne, and Keijbets
1981; Stier et al. 1981).
The interaction of microorganisms is both stimulatory and inhibitory
to growth of C. botulinum. The growth of other organisms tends to reduce
the Eh of the growth medium, which may then allow growth of C. botuli-
num. Some organisms, such as yeasts, may produce growth factors favor
able for C. botulinum.
The growth of C. botulinum in foods can cause a foul, putrid odor
which should serve as a warning to the consumer. In many of the out
breaks of botulism, either a patient or an asymptomatic participant
stated that the food had an off.odor or flavor, but these warning signs
were ignored. Some people have a high tolerance to off.flavors or off
odors. In some foods (smoked, spiced, fermented), slight off.flavors or
off.odors are difficult to detect.
There are reports that toxin is present in spores (sporebound toxin)
of C. botulinum (Booth et al. 1972; Yamakawa, Nishida, and Nakamura
1983). Phagocytosis releases the spore toxin, which has the same lethal
effect as other free toxins.
METHODOLOGY. The methodology of C. botulinum involves the detec
tion and enumeration of the organism, the characterization of the or
ganism, or qualitative or quantitative determinations of the neurotoxins.
Detection and Enumeration. Due to the hazards involved, before working
with C. botulinum, a person should be protected by suitable toxoids.
The demonstration of botulinum toxin in a food implicated in a botu
lism outbreak is often all that is done. However, the detection and isola
tion of C. botulinum from the food furnishes confirmatory evidence. Also,
methods of detection, isolation, identification, and enumeration are ba
sic in determining the distribution of the orgamism in nature.
A system for detecting the organism is described in the Bacteriological
Analytical Manual (FDA 1978). The medium for detection of C. botulinum
is anaerobic egg yolk agar or liver vealegg yolk agar.
Characterization. There is a widely held view that no organism isolated
from a suspected case of botulism should be identified as C. botulinum
unless it is toxigenic. Therefore, the outstanding characteristic of a strain
of C. botulinum is the toxin it produces.
Once an organism has been isolated and found to be a Grampositive
rod that forms spores and is anaerobic, other tests can be made. These
include digestion of gelatin, milk, and meat; fermentation of various car
bohydrates; indole production; hemolysis; and lecithinase and lipase ac-
tivity. Various biochemical reactions of C. botulinum types are listed in
Table 6.11. However, there may be disagreement on some of these reac-
tions. Others are listed by Dezfulian and Dowell (1980).
It is evident from this information that biochemical tests can be con-
sidered reliable only to a limited extent in determining or distinguishing
C. botulinum. This is because different toxin types of C. botulinum, or even
strains within toxin types, represent quite distinct metabolic groups.
TABLE 6.11. REACTIONS OF CLOSTRIDIUM BOTULINUM
Proteolytic N onProteolytic
Types Types
A,B,C,D,F B,C,D,E,F G
Digestion of
Gelatin
+" + +
Casein
+ +
Meat
+
N
Acids produced from
Glucose
+ +
Fructose
+ +
Mannose
+
Lactose
Salicin V
Other tests
Indole
Urease
Nitrates reduced
Lipase
+ +
Hemolysis
+ + +
.,+
= positive reaction; - = no reaction: V = variable reactions; N = insufficient data.
Serological Reactions. Besides the toxins, the organism has three sources of
antigenic substances: the flagellar antigens (H), the somatic antigens (0),
and the spore antigens. The flagellar antigens show a narrow range of
specificity, and the somatic antigens are often common to all members
of a species or type. The spore antigens are type specific and are dissimi
lar to either the flagellar or somatic antigens of the vegetative cell (Solo
mon et al. 1969).
Toxin Detection. The Bacteriological Analytical Manual (FDA 1978) describes
a procedure for detection of preformed toxin in food and for detection
of toxins produced by organisms in the food with an enrichment proce
dure.
An extract is analyzed for toxin by intraperitoneal injection of mice.
The injected mice are observed over a period of 92 hr. Death indicates
the presence of toxin. The mouse neutralization test for toxin has been
accepted as official by the AOAC.
Serological techniques, as described for detection of staphylococcal
enterotoxin, have been tested for the analysis of botulinum toxins. The
ELISA system has been adapted for type A (Notermans, Dufrenne, and
Van Schothorst 1978), type B (Kozaki et al. 1979), type E (Notermans,
Dufrenne, and Kozaki 1979) and type G toxin (Lewis et al. 1981). Cross
reactivity and nonspecific reactivity are problems not only for the ELISA,
but also for other serological systems for analyzing for botulinum toxins.
Dezfulian and Bartlett (1984) produced specific type A antitoxin by selec-
tive suppression of the immunological response of inoculated rabbits to
unwanted antigens and subsequent immunization with a toxoid_ Even
then, there was some cross-reaction of the antitoxin with culture filtrates
of type B organisms_ Monoclonal antibodies to type C1 and D toxins were
produced and used in the ELISA to detect these toxins (Oguma et aL
1984)_
CONTROL OF BOTULISM. For botulism to occur, the spores or cells
of toxigenic C. botulinum must be present in the environment and gain
access to the food; viable cells or spores must remain in the food after
processing; and the food must have an environment favorable for germi-
nation and outgrowth of the spores and growth of the vegetative cells to
produce the toxin; then the food must be eaten cold or with insufficient
heating to destroy the toxin_
Therefore, the principal methods of controlling staphylococcal intox-
ication also are important in the control of botulism. Quite simply, we
can prevent contamination of the food, prevent growth and toxin pro-
duction, destroy the organisms or toxin, and not eat suspect foods.
Perhaps the best method of controlling botulism is to heat the food
during processing to a temperature that will destroy the spores of toxi-
genic C. botulinum. However, there are many foods, such as cured meat
products, that would suffer organoleptically if these heat treatments were
used. For these foods, additives are needed that will inhibit toxin produc-
tion by C. botulinum contaminants.
Prevent Contamination. Generally, C. botulinum gains access to food as a
soilborne or dustborne contaminant. Vegetables are harvested from the
soil or in close proximity to the soiL Washing of vegetables will remove
some soil and associated organisms, but it is difficult to remove all
organisms by this method.
Fish and marine animals feeding in waters that contain C. botulinum
can be contaminated. Most of the contamination is in the intestines of
fish_ Proper cleaning and eviscerating procedures result in minimal con-
tamination of the fish flesh_
High levels of contamination are more difficult to destroy than are
low levels or, ideally, no spores. Therefore, sanitary practices are very
important.
Prevent Growth and Toxin Production_ Foods that do not have an environ-
ment conducive to the growth and toxin production of C. botulinum will
not become toxic.
One of the simplest methods to inhibit the growth of microorganisms
is to hold the food at temperatures below which growth will occur. Since
certain strains of C. botulinum can grow and produce toxins at SoC or less,
normal refrigerator temperatures cannot be relied upon to control toxin
formation in foods. Freezing does not destroy the toxin or the spores of
C. botulinum, but as long as the food is frozen, germination and growth
of the vegetative cells with toxin production does not occur. Frozen foods
should not be allowed to thaw and remain in that condition for extended
periods. C. botulinum does not grow at an Ow of 0.93 or less. Foods dried
below this level should not become toxic (there is the possibility that
toxin could be formed prior to drying).
A chemical added to cured meats is sodium nitrite. This chemical is
important in color fixation of cured meat, as well as in inhibiting growth
and toxin formation by C. botulinum. Various other chemicals have been
tested for inhibition of the organism. These chemicals and nitrites are
discussed in Chapter 11.
During processing, the destruction of microbial competitors, the reo
moval of air by heating (lowering the redox potential), and the destruc
tion of cellular tissues with the release of cellular fluids, favors the growth
of C. botulinum.
Destruction ofC. botulinum. Since this organism produces spores, it is more
difficult to destroy than is S. aureus. The method used to destroy the
spores is thermal processing.
Most of the outbreaks of botulism have been caused by consumption
of toxic heatprocessed food. Thus, it may seem odd that heating is the
only presently acceptable method of destroying the spores of C. botuli-
num. The spores of C. botulinum vary in their heat resistance. Type E
spores are the least resistant and thus are most easily destroyed by heat.
Due to outbreaks of botulism from eating contaminated fish, it was
recommended that during smoking, the fish be held at 82.2C for 30
min. Pace and Krumbiegel (1973) examined commercially smoked fish
and found that 0.9 percent to 2.0 percent contained C. botulinum type E
spores, although the fish had been subjected to 82.2C for 30 min.
Various results of heat processing are obtained undoubtedly because
of the difference in heat resistance of various strains of type E spores.
One group of researchers determined the thermal-death-time of five
strains of type E spores in fish paste (Crisley et al. 1968). The D values in
this medium at 80C varied from an average of 1.6 min to 4.3 min for
the five strains.
Boiling (l00C) is not recommended to destroy spores of type A or
B, since several hours would be required. Processing of low-acid foods is
accomplished at a temperature of 121.1 C. A 12D thermal process (see
Chapter 12) is considered essential for low-acid canned foods. With this
treatment, the probability of survival of C. botulinum spores is very re-
mote_
Of the clostridial spores, those of C. botulinum are included in the
most radiation-resistant group_ The spores vary in their resistance, with
type F generally considered to be the most resistant and type E spores
the least resistant, with types A and B intermediate_
A combination of radiation and heat has a synergistic effect on de-
struction of spores of C. botulinum. Spores that are exposed to low-level
radiation are more susceptible to heat inactivation than untreated
spores. The heat is also of value in halting enzyme action and inactivating
any toxin that might be present. Thus, pasteurization levels of radiation
followed by heating at values less than those presently required may be
beneficial in controlling C. botulinum. Heating before radiation does not
lower the radiation resistance, but radiation lowers the heat resistance of
the spores.
Inactivation of Toxin. Radiation pasteurization does not inactivate pre-
formed botulinum toxin, whereas heat pasteurization does. The heat sta-
bility of botulinum toxin is considerably less than that of staphylococcal
enterotoxin. The types of botulinum toxin vary in their heat resistance.
The time necessary to inactivate the toxins depends upon the amount of
toxin present, the temperature, and the substrate in which they are
heated. The toxins are more stable at acid pH levels and are most stable
about pH 5.0. Various times and temperatures have been recommended
for inactivating botulinum toxins (Bradshaw, Peeler, and Twedt 1979,
1981; Woodburn et al 1979).
Although the toxins are relatively heat labile, being inactivated in 2
min or less at 90C, this heat treatment usually is not suggested for de-
stroying toxins formed in canned foods. Bryan (1973) recommended that
before serving, canned food should be heated to boiling (about 100C)
and held for 5 min to 15 min.
The resistance to chemicals depends upon the type of toxin, temper-
ature, pH, and other substances in the medium. In water, the purified
toxin is sensitive to chlorine, bromine, or iodine. Free available chlorine
with a residual of 1 mg/L (1 ppm) will destroy at least 99.9 percent of all
the types of botulinum toxin in 5 min or less. Type E toxin is the most
resistant to destruction by chlorine.
Canned food that is not normal should not be consumed. In most
outbreaks of botulism involving canned food, one or more people noted
swelling of the can, bubbles in the food, off-odors, or turbid liquid. If the
food is suspicious, it should be returned to the store, if purchased, or
disposed of in a manner so that it is not consumed by animals or by other
people.
Clostridium perfringens Foodborne Illness
This illness has been called a food poisoning, an intoxication, a
foodborne illness, an infection, and an infective food poisoning. These
different designations are undoubtedly due to the fact that the release of
the toxin is different from that of S. aureus or C. botulinum. Large numbers
of the organism are associated with the illness. This is true for infections;
however, in this illness, a toxin is the causative agent.
The actual number of outbreaks and cases in the United States is not
known. Due to the rather mild symptoms and short duration of the ill
ness, medical help usually is not needed. Hence, most cases probably are
not reported to the CDC. Table 6.12 shows the number of confirmed
outbreaks and cases reported in recent years. The majority of the re-
ported outbreaks are caused by food consumed in mass-feeding establish-
ments. Outbreaks that occur in the home might not be reported because
of the type of illness and the small number of people involved.
CHARACTERISTICS OF THE ILLNESS. The illness is the result of a
sequence of events. The food is contaminated by the organism. During
cooking, the vegetative cells and the heat-sensitive spores may be killed,
but heat-resistant spores will survive. The heat experienced during cook-
ing may activate the spores to germinate.
If the food is held at a temperature allowing growth (100 to 50C),
the vegetative cells will multiply. A mean generation time of 10 to 12
min was reported for the organism in cooked poultry at 37C (Mead
1969). With this short generation time, the organism can increase 1,000-
fold in 2 hr.
The exact number of C. perfringens needed to cause the illness has not
been determined. However, over 10
1i
to 10
8
cells per gram of food can be
a potential health hazard. The organism must first pass through the low
pH of the stomach to reach the intestines. Organisms in the early lag
phase are least resistant, and resistance increases to a maximum at the
end of the growth phase. It is well documented that the acid in the stom-
TABLE 6.12. OUTBREAKS AND CASES CAUSED BY CLOSTRIDIUM PERFRINGENS
Year Outbreaks Cases Cases per Outbreak
1977 6 568 94
1978 9 617 68
1979 16 936 58
1980 25 1,463 58
1981 28 1,162 41
TOTAl. 84 4,746 56
SOVRCE: Data from CDC Annual Summaries.
ach combines with consumed proteins, causing an increase in the overall
pH. This effect of protein on the stomach acidity, and resultant protec
tion of the bacterial cells, may be the factor in the involvement of protein
foods in outbreaks of the illness.
When the remaining organisms reach the small intestine, they find
an environment acceptable for multiplication and sporulation. The spor-
ulating cells produce the enterotoxin, which is released during cell lysis.
The organisms may sporulate in some foods, but usually, by the time
the food is toxic, it is not palatable (Craven, Blankenship, and McDonel
1981 ).
Incubation Period. The interval of time between the meal and onset of ill-
ness for an outbreak of C. perfringens food poisoning is shown in Figure
6.8. In this example, the range is 2 to 29 hr, with a median of 13 hr. CDC
(1983b) listed the incubation period as 9 to 15 hr.
Attack Rate. Not everyone who eats the contaminated food becomes ill.
The reasons for the escape of some people are similar to those described
for staphylococcal intoxication and botulism. People with hyperacid
stomachs may be protected, due to the death of C. perfringens cells. Previ-
ous contact or any immunity apparently has little effect, if any, on acquir-
ing the illness when contaminated food is ingested.
The disease is not prevented in parenterally immunized animals that
possess neutralizing serum antibody against the toxin (N iilo 1971). In a
survey of human sera, a majority contained antibody against C. perfringens
enterotoxin (Uemura et al. 1974). They speculated that antibody could
be induced by the acute C. perfringens food poisoning or be due to the
continuous presence of the organism and small amounts of toxin in
asymptomatic people.
Symptoms. The symptoms reported in four outbreaks of C. perfringens ill-
ness are listed in Table 6.13. The two prominent symptoms of the illness
are diarrhea and abdominal cramps. Nausea and headache were fairly
important in two outbreaks but were of minor occurrence in the others.
Fever, vomiting, dizziness, and bloody stools may occur, but they are rare.
In young people, the symptoms are usually mild, but in elderly, ill, or
debilitated people, the illness can be severe. Although rare, death has
occurred in cases complicated by other illness.
Duration and Therapy. The illness is usually of short duration, from less
than 12 hr up to 24 hr. A few cases may persist for 48 hr. The illness is
followed by a complete and uneventful recovery. Although weakness may
be evident the day after symptoms begin, a person usually can return
to work or normal activity. Due to the rather mild symptoms and short
duration, usually no therapy is needed.
15
14
13
II) 12
10111
~ 10
~
08
0:7
~ 6
~ !5
Z 4
3
2
o
-
-
-
-
- -
-
- -
-
r-
I-
r-
I-
r-
i-
nn
l-
n
-
-
-
-
r-
I I I
I I I nn
I 2 3 4 !5 6 1 8 9 10 II 12 13 14 15 16 11 18 19 20 21 22 23 2425 26 2128 29 ~ 31
HOURS
Figure 6.8. Typical outbreak of Clostridium perfringens food borne illness-incuba-
tion period.
Courtesy of CDC.
Foods Involved. The foods involved in outbreaks of C. perfringens illness are
generally protein-type foods that have been boiled or lightly roasted or
meat and poultry stews, sauces, gravies, pies, casseroles, salads, and dress-
ings (Table 6.14). Usually the incriminated food is cooked one or two
days in advance, then refrigerated until reheated for serving.
When meat is cooked in bulk, the heat gain is slow, and subsequent
cooling is slow. Heating lowers the oxygen content of the food, providing
a more anaerobic environment for the clostridia to grow.
TABLE 6.13. CLOSTRIDIUM PERFRINGENS FOODBORNE ILLNESS: PERCENTAGE
OF PEOPLE REPORTING SYMPTOMS IN FOUR OUTBREAKS
Outbreak
Symptom 2 3 4
Diarrhea 82 85 89 91
Headache 40 4 44
Nausea 33 13 48 42
Fever 8 8
Bloody stools 7 1
Vomiting 6 16 11
Dizziness
2
Prostration 39
Chills 29
SOCRCE: Data from CDC Morbidity and Mortality Weekly Reports.
TABLE 6.14. FOODS INVOLVED IN OUTBREAKS OF CLOSTRIDIUM PERFRINGENS
FOODBORNEILLNESS
Food
Beef
Pork
Other meat
Poultry
Fishery products
Other, multiple, unknown
TOTAL
SOCRCE: Data from CDC Annual Summaries.
Number
19
2
2
8
2
13
46
%
41.3
4.3
4.3
17.4
4.3
28.3
ETIOLOGIC AGENT. The etiologic agent for the illness is an enter
otoxin. The enterotoxin protein is a structural part of the spore coat that
is overproduced in some strains of C. perfringens (Frieben and Duncan
1973). Synthesis of the spore coat is an early event in sporulation (Labbe
and Duncan 1977).
Two enterotoxigenic strains followed different patterns of produc
tion: one strain produced a high amount of toxin during 2.5 to 4 hr of
growth, with the level stabilized at 5 hr; the other strain revealed a rapid
increase of toxin after 4 hr, with a maximum level after 25 hr. Using the
ELISA, researchers detected levels 1 ng/ml to 10 ng/ml of enterotoxin
during vegetative growth even with an enterotoxin negative strain (Gra
num et al. 1984). They assumed that 1 ng/ml of the compound was
needed for sporulation. The 12 J-tg and 18 J-tg/ml produced by the enter
otoxigenic strains during sporulation was excess compound which hap
pens to be an enterotoxin. Most methods will not detect 1 ng/ml of enter
otoxin, but the ELISA is a very sensitive system (Bartholomew and
Stringer 1984). This low level of enterotoxin (l ng/ml) does not notice
ably affect biological systems. Hence, it is necessary for sporulation of
the cells to produce sufficient levels of toxin to cause enteric distress.
Properties of the Enterotoxin. The purified toxin is a protein. It is essentially
free of nucleic acids, lipids, and reducing sugars (Hauschild 1971). The
molecular weight is between 33,000 and 35,000, with an isoelectric point
of pH 4.3. Being protein, the toxin is antigenic, and only one type has
been detected, regardless of the producing strain of organism.
The heat needed to inactivate the enterotoxin is influenced by the
substrate (type, pH). According to N aik and Duncan (1978), the biological
activity is destroyed within 5 min at 60C. However, other researchers
reported that inactivating 12.5 J-tg/ml enterotoxin at 61C required 23.8
min for gravy and 25.4 min for buffer, both at pH 6.1 (Bradshaw et al.
1982).
The toxin is inactivated by pronase, but not by trypsin, lipase, chymo-
trypsin, or papain (Duncan and Strong 1969; Hauschild and Hilsheimer
1971).
Enterotoxigenic Strains. On the basis of the production of four major exo-
toxins (alpha, beta, epsilon, and iota), strains of C. perfringens are divided
into five groups, A to E (Table 6.15). This grouping makes it possible to
correlate strains with illnesses of people and animals.
The main food-poisoning strains are included in type A, although
type C strains were involved with a severe gastroenteritis in Germany
shortly after World War II, and in other countries since then (Hauschild
1973). Skjelkvale and Duncan (1975) isolated enterotoxin from type C
strains and Uemura and Skjelkvale (1976) reportedly isolated enter-
otoxin from a strain of type D. These enterotoxins are identical serologi-
cally, whether from type A, C, or D.
Not all strains of type A produce enterotoxin. Even organisms that
have been isolated from foodborne outbreaks seem to lose the ability to
produce the enterotoxin after transfer in laboratory media. Some
organisms do not sporulate, and hence fail to produce the enterotoxin.
Strains of C. perfringens that fail to produce fluid accumulation in li-
gated rabbit ileum or overt diarrhea when injected into the rabbit ileum,
do not cause illnesses when fed to human subjects (Strong, Duncan, and
Perna 1971).
Action of the Enterotoxin. Hauschild (1971) stated that the enterotoxin
causes increased capillary permeability, vasodilation, and excess fluid
movement into the intestinal lumen, resulting in diarrhea. Also, there is
an increase in intestinal motility. McDonel and Duncan (1977) reported
that ileal loops responded to purified enterotoxin, with net secretion of
fluid, sodium, and chloride, inhibition of glucose uptake, and substantial
sloughing of epithelial cells. Damage to the epithelial layer of villus tips
was evident, and even complete destruction of epithelial cells was ob-
served (McDonel et al. 1978). The primary site of action of the enter-
otoxin is the brush border membrane of the villus tip epithelial cells
(McDonel 1980). The enterotoxin is absorbed from the small intestine
and can be detected in lymph or blood (Ohishi, Yamamoto, and Saka-
TABLE 6.15. TYPES OF CLOSTRIDIUM PERFRINGENS
Types
A
B
C
o
E
Major Toxins Produced
alpha
alpha, beta, epsilon
alpha, beta
alpha, epsilon
alpha, iota
guchi 1981). The action is greatest in the ileum, less in the jejunum and
least in the duodenum.
Tests with ligated loops indicate that the toxin acts directly on the
intestine, since fluid accumulates only in inoculated loops and not in
adjacent control loops (see Fig. 6.9). There does not seem to be any inva
sion of the intestinal mucosa by the cells of C. perfringens, which is sugges
tive of a toxic action on the intestine.
THE ORGANISM. The cells in this species are anaerobic, Grampositive,
sporeforming, straight rods. They occur singly, in pairs and, rarely, in
short chains, are not motile, produce capsules, and liquify gelatin. This
species ferments many of the common carbohydrates. Acid is produced
in litmus milk (fermentation of lactose), but the clot is broken up by the
production of large amounts of gas, resulting in a stormy fermentation.
Sources. C. perfringens has been called ubiquitous, due to its widespread
distribution in nature. It is found in soil, dust, air, water, sewage, human
and animal feces, and on many food products.
The presence of C. perfringens in the intestines of humans and animals
is well established. People who practice poor hygiene or partake of com
munal meals tend to have a higher incidence of C. perfringens than do
people using good hygienic practices. The level of C. perfringens contami
nation normally is 10
2
to 10
4
organisms per gram of fecal material. How
ever, Nakagawa and Nishida (1969) reported that about onethird of the
positive samples had 10
3
or less, one third had 10
4
to 10
5
, and onethird
c (0)
50 (3.5)
30(2.0)
,0 (0)
30(3.0)
25 (1.5)
Figure 6.9. Reactions in ligated intestinal loops of two chickens. Numbers in
parentheses = micrograms of enterotoxin injected per fluid volume, in milli
liters; c = enterotoxinfree cell extract; s = saline.
Courtesy of Niilo (1974).
had 10
6
or more C. perfringens per gram from normal human intestines.
Others found that some healthy aged adults persistently excreted from
10
7
to 10
9
C. perfringens per gram of feces (Yamagishi et al. 1976). However,
these may not be enterotoxigenic types.
With the widespread distribution of C. perfringens in the environment,
and the fact that the organism can produce spores that are resistant to
adverse conditions, the organism is a logical common contaminant of
food.
Growth. Although C. perfringens is an anaerobe, it is aerotolerant. There
fore, strict anaerobic conditions are not needed for growth. The redox
potentials of meat products are favorable for growth of the micro
orgamsm.
Good growth occurs between pH 5.5 and 8.0, and no growth occurs
below pH 5.0 or above pH 9.0. At pH 5.5, vegetative growth occurred,
but not sporulation and enterotoxin production (Labbe and Duncan
1974). They reported the optimum range for enterotoxin production as
pH 6.5 to 7.3.
C. perfringens grows readily at temperatures between 20 and 50C,
with maximum growth between 37 and 47C. Mead (1969) found no
growth at either 15 or 52C. Reportedly, some strains show limited
growth at 15C. A long lag phase is a characteristic of growth at low
temperatures. Sporulation and toxin production were optimum at 37C
(Labbe and Duncan 1974). No sporulation or toxin production was noted
at 46C.
At aU! values less than 0.995, the rate of growth is reduced. The mini
mum aw for growth depends upon the solute, temperature, pH, and other
factors. The absolute minimum a
w
for growth appears to be 0.93 (Kang
et al. 1969). In this case, glycerol was used to lower the a
w

C. perfringens can grow in concentrations of curing salts (sodium ni
trite, and nitrate) considerably higher than those used in normal curing
operations (Gough and Alford 1965). Ando (1975) reported that with 2
percent NaCl, a level of 0.05 percent (500 ppm) NaN02 was needed to
completely prevent outgrowth of germinated spores of C. perfringens.
Lower concentrations permitted some outgrowth.
METHODOLOGY. The clinical and epidemiological pattern of C. per
fringens foodborne illness is so characteristic that it is almost diagnostic.
However, it is necessary to analyze appropriate samples in order to con
firm the presence of the organism. Because the enterotoxin is released
primarily during sporulation, and most of the sporulation occurs in vivo,
it is assumed that the analysis of food for the enterotoxin may be of little
value. In one study, however, researchers inoculated ground and auto
claved chicken meat with C. perfringens and analyzed it for enterotoxin
(AI-Obaidy et al. 1985). They reported a level of 0.76 p,g/g of enterotoxin
after incubation for 14 hr, indicating that the toxin might be detected in
foods involved in gastroenteritis due to C. perfringens.
To help establish C. perfringens as the cause of a foodborne outbreak,
three laboratory tests are suggested: (1) organisms of same serotype in
epidemiologically incriminated food and stool of ill individuals; (2) isola-
tion of organisms with same serotype in stool of most ill individuals and
not in stool of controls; or (3) demonstration of greater than 10
5
C. per-
fringens cells per gram of incriminated food, provided the food sample
was properly handled.
Detection and Enumeration ofC. perfringens. Food should always be analyzed
promptly. This is especially true for C. perfringens detection because un-
protected cells are sensitive to low temperatures. A buffered glycerol-
sodium chloride solution mixed in equal portions with a food and stored
in dry ice at - 56C protected cells of C. perfringens (Harmon and Placencia
1978). The number of C. perfringens was only slightly lowered by this treat-
ment.
For the enumeration of C. perfringens, tryptose sulfite cycloserine
(TSC) agar without egg yolk was effective (Harmon, Kautter, and Peeler
1971). This medium with or without added egg yolk emulsion is recom-
mended for enumeration of C. perfringens (AOAC 1985; APHA 1984; FDA
1978). The medium is differential because of the presence of sodium bi-
sulfite and ferric ammonium citrate. Bacteria that reduce sulfite to sul-
fide produce black colonies due to the formation of iron sulfide. Since
many organisms can reduce sulfite to sulfide, D-cycloserine is added to
select for clostridia.
For enumeration of this organism in foods, the MPN technique using
thioglycollate-cycloserine (Debevere 1979), iron milk (Abeyta 1983; Ab-
eyta, Michalovskis, and Wekell 1985; St. John, Matches, and Wekell 1982),
or rapid perfringens medium (DeBoer and Boot 1983; Erickson and
Deibel 1978) reportedly is simpler and more effective than direct plating
systems.
Confirmational Tests. C. perfringens is the only clostridial species that re-
duces sulfite, is nonmotile, reduces nitrate, and produces a stormy fer-
mentation in milk (Buchanan and Gibbons 1974).
Nitrate reduction and motility are determined by stab inoculation of
nitrate motility medium. It is recognized that all strains do not reduce
nitrate in this medium. However, when the medium is modified by add-
ing 0.5 percent galactose and glycerol, then good nitrate reduction is
obtained (Hauschild and Hilsheimer 1974).
Serology. Not all of the strains are typable with the presently available
antisera. The many problems associated with serotyping C. perfringens
were reviewed by Stringer, Turnbull, and Gilbert (1980).
Bacteriocins. The specificity of bacteriocins can be used to type microor
ganisms. Watson (1985) used fifty bacteriocins to type 802 isolates of the
organism. A greater proportion of those strains involved in foodborne
illness were bacteriocinogenic than were other isolates.
Toxin Detection-Enterotoxin. Although the determination of enterotoxin
may not be important epidemiologically, it is important when studying
the role of toxin in pathogenesis and the mechanisms of enterotoxic ac
tion. Various systems have been used for the assay of the enterotoxin.
Ligated intestinal loops have been used to assay for the enterotoxin
(Hauschild, Hilsheimer, and Rogers 1971; Niilo 1974). When toxin is in-
jected into a ligated loop, it causes fluid accumulation (Fig. 6.9). Measure-
ment of this fluid as compared to control loops is an indication of the
amount of toxin that is injected. Since it is inconvenient to assay toxin
in this manner, other methods have been devised.
Intradermal injection of the enterotoxin into rabbits and guinea pigs
causes erythema around the injection site in 1 to 2 hr, and reaches a
maximum in 18 to 24 hr. The diameter of this reddened area is related
to the concentration of enterotoxin (Niilo 1975). The reaction is distinc-
tive for the enterotoxins. According to Hauschild (1970), this system is
1,000 times as sensitive as the ligated intestinal loop, as well as being
more rapid and accurate.
Various mammalian cell lines have been tested. African green mon-
key kidney (Vero) and dog kidney (MDCK) cells are sufficiently sensitive
to assay for the enterotoxin (Giugliano, Stringer, and Drasar 1983; Mc-
Donel and McClane 1981).
The immunodiffusion methods listed for assaying S. aureus enter-
otoxin can be used to assay for C. perfringens enterotoxin. According to
Skjelkvale and Uemura (1977), both the reversed passive hemagglutina-
tion and counterimmunoelectrophoresis assays are rapid and reliable
systems. The radioimmunoassay detects enterotoxin at a level of 1 ng/ml
(Stelma et al. 1983).
Various ELISA systems are specific and sensitive for C. perfringens
enterotoxin. In one study, as little as 25 ng/ml of enterotoxin was de-
tected with an indirect ELISA, while 1 ng/ml was detected with a four-
layer sandwich ELISA (McClane and Strouse 1984). Other researchers
have detected 5 ng/g of enterotoxin in feces with a sandwich ELISA (Bar-
tholomew et al. 1985).
CONTROL OF C. PERFRINGENS FOOD POISONING. The three gen-
eral methods for control of this foodborne illness are (1) limit or prevent
contamination of the food; (2) prevent or inhibit growth of the organism;
or (3) destroy the organism. Any combination of these methods also may
be used. Since most, if not all, of the enterotoxin is produced in the
intestinal tract, systems to prevent production or to destroy the toxin are
not applicable_
Limit OT PTevent Contamination. Due to the ubiquitous character of the or-
ganism, preventing contamination of food cannot be relied upon as a
means of control. However, this does not mean that we should ignore
good sanitary practices_
PTevent Gmwth. The presence of a few C. peTfTingens cells in a food product
is not considered to be a health hazard. The hazardous level is some 10
6
to 10
8
cells per gram of food. The prevention of germination of spores
with outgrowth and multiplication of the vegetative cells is the only prac-
tical method to control the illness caused by C. peTfTingens_
One simple method of control is to cook and serve the food without
an extended holding period. If this is not possible, refrigeration of the
food in small quantities for quick cooling will inhibit growth. The mini-
mum temperature of growth is about 15C. Any acceptable refrigerator
should be able to maintain this temperature unless it is overloaded. The
lower the temperature of the refrigerator, the sooner the food will be
cooled. As the temperature approaches DoC, the population tends to de-
cline, since the vegetative cells are not stable at such low temperatures.
Even the spores appear to be damaged at low refrigerator or freezer tem-
peratures.
The maximum temperature for growth is 50 to 52C. Above 52C,
no growth should occur, and the vegetative cells may die. There was
greater than a 99 percent reduction in the number of C. peTjTingens on
cooked beef cubes when held at 53.3C (Brown and Twedt 1972).
Destmy the OTganism. Vegetative cells of C. peTfTingens are destroyed by thor-
ough cooking, but heat-resistant spores can survive_ Subjecting the spores
to sublethal temperatures stimulates germination_ The heat resistance of
the spores varies from strain to strain; hence, heating to a certain temper-
ature is not required, since the killing effect is dependent upon the strain
of organism that is present.
If heat-resistant spores are present, it is not possible to heat foods
sufficiently to inactivate all of them without damaging the organoleptic
properties of the food. It must be assumed that there are surviving spores
in the cooked food, and the food must be kept hot until it is served_
If any food is cooked one day for serving the next, and even if it is
refrigerated, it should be reheated prior to serving to kill the vegetative
cells resulting from germination of spores and possible further multipli-
cation_ To be safe, foods such as gravies should be reheated by boiling for
10 to 15 min_ and roasts or poultry should be reheated to a temperature
sufficient for inactivation of C. perfringens (Roy, Busta, and Thompson
1981).
Gamma radiation will destroy spores of C. perfringens. Clifford and
Anellis (1975) calculated 12D values for eight strains of C. perfringens
spores. These values, divided by 12, revealed D values ranging from 130
to 350 Krad.
Salmonellosis
All members of the genus Salmonella are potentially pathogenic for
humans as well as for vertebrate animals. The transmission of the disease
is usually from animals to humans by the ingestion of food of animal
origin. Direct transmission is also possible from human to human, from
human to animal, and from animal to human (Fig. 6. 10). Diseases or
infections naturally transmitted between vertebrate animals and humans
are called zoonoses.
The illness caused by salmonellae can be divided into four syndromes
which may occur individually, simultaneously, or consecutively in the
course of an infection. These syndromes are the carrier state (convales-
cent or asymptomatic), enteric fever (typhoid or paratyphoid fever), gas-
troenteritis (food infection), and septicemia (characterized by a brief fe-
brile illness or a prolonged or relapsing illness with localized lesions).
Acute gastroenteritis, caused by nearly all serotypes of Salmonella, is
the most frequent syndrome encountered. It is of primary importance to
food microbiologists.
ANIMAL
BYPRODUCTS
FEED
ANIMALS
/HUr
N
FOOD .... HUMAN
Figure 6.10. Cycles of infection for salmonellosis.
According to CDC (l983b), salmonellosis accounted for 26.4 percent
of the reported outbreaks, 26.8 percent of the cases, and 65.6 percent of
the deaths due to foodborne illness in 1981. Only the number of cases
of S. aureus intoxication surpassed salmonellosis in these categories. The
reported isolations of Salmonella from humans (1968-1980) is shown in
Figure 6.11. Most isolations occur during the warm months. The number
of reported isolations each year appears to increase.
The increase in the number of cases has been attributed to better
diagnosis, laboratory procedures, and reporting. However, in those areas
in which diagnosis and reporting have always been adequate, increases
have occurred in the number of outbreaks and cases of salmonellosis.
The prevalence of salmonellosis is increasing, although much energy has
been expended to control this illness. This increase of Salmonella in both
human and animal sources is associated with national and international
commerce in animals, feeds, and foods; with largescale, intensive animal
raising; and with increased use of ready-to-serve or heat-and-eat foods.
It has been estimated that only 1 to lO percent of the actual number
of cases are reported. There may be over 2,000,000 cases of salmonellosis
each year in the United States. Many cases are mild and no professional
care is solicited_ Salmonellae may be the major cause of bacterial gastro-
enteritis and foodborne illness in developed countries.
The economic loss due to Salmonella was estimated to be at least $300
million annually in the United States (Foster et al. 1970). The cost in-
cludes medical care, hospitalization, lost time and income through ab-
sence from work, death of animals, decreased production of animals, loss
or reduced value of contaminated products, testing and control proce-
g
"
..
'"
"'

'"
"'
"'
I
;!,
f/l
"'
I-

0
!!)
4000
3500
3000
2500
2000
1500
1000
500
1968 1969 1970 1971 1972 1973 197-4 1915 1976 1977 1878 1871 11180
DATE or REPORT TO CDC
Figure 6.11. Reported number of isolations of salmonellae from humans by month,
1968-1980.
dures, and recall of products from market channels. A more recent esti-
mate (CDC 1978b) is that the annual cost for salmonellosis is $1.5 billion
for medical expenses alone. Depending on the circumstances, the aver-
age cost per case ranged from $277 to $4,548 for 12 outbreaks (Todd
1985). The median cost per case was $769. At this rate, 2 million cases
cost more than $1.5 billion per year in the United States.
More deaths are caused by salmonellae and salmonellosis than by bot-
ulism. The death-to-case ratio is rather low, with a case fatality ratio of
0.26 percent from 1962 to 1968 (CDC 1971) and 0.15 percent for 1974
(CDC 1975). In 1981, the number of deaths jumped to 21 for a case-to-
fatality ratio of 0.86 percent (CDC 1983b). Most deaths occur in very
young or old people.
CHARACTERISTICS OF THE ILLNESS. Salmonellosis has been con-
sidered to be an infection caused by the action of the organism in the
intestine. However, there is evidence that a toxin or toxins may be in-
volved.
Incubation Period. In thirty-four random outbreaks reported by the CDC,
the range for the incubation period varied from 1 hr to eight days. The
usual incubation period is reported as 6 to 48 hr (CDC 1983b).
Symptoms. The symptoms of nine random outbreaks reported by CDC
are listed in Table 6.16. The symptoms and their severity depend on the
number of organisms and the serotype of Salmonella, as well as the resist-
ance of the host. The most reported symptom is diarrhea, followed by
abdominal cramps, fever, nausea, vomiting, chills, and headache.
TABLE 6.16. SALMONELLOSIS: PERCENTAGE OF PEOPLE EXPERIENCING
SYMPTOMS IN RANDOM OUTBREAKS
Outbreak
Symptom 2 3 4 5 6 7 8 9
Diarrhea 87 100 100 96 93 75 93 96 95
Diarrhea (bloody) 4 5 5
Abdominal cramps 70 47 70 81 86 82 76 66 57
Fever 68 82 85 62 80 48 97 43
Nausea 53 69 80 62 69 70 52 38
Vomiting 53 82 80 40 40 62 26 54 19
Chills 38 54 70 79 52 86
Headache 36 66 60 65 63 29
Dizziness 42
Muscle aches 70 95
"- = Not reported; however, the symptom might have existed.
SOURCE: Data from CDC Morbidit,r and Mortality Weekly Reports.
Duration. In normal, healthy adults, the gastroenteritis usually lasts for
only two to three days, but if further infection occurs, the illness may
persist for months or years, and it may result in death. Thomas and Mog
ford (1970) stated that the duration of infection was longer than two
months in nearly 25 percent of the cases, and intermittent excretion of
the organisms was observed in 20 percent of the cases. Children are more
susceptible than adults to prolonged excretion of the organism.
Therapy. The majority of the cases need no therapy. When hospitalization
is required, an attempt is made to stabilize loss of fluids and prevent
dehydration, as well as to maintain the electrolyte balance.
The administration of broad spectrum antibiotics usually gives no ap
preciable benefit; the salmonellae become resistant to antibiotics. Treat
ment with antibiotics can cause the asymptomatic carrier state to become
an active case (Saroglou and Bisno 1978).
Eating yogurt was suggested as part of the therapy for the salmo
nellosis outbreak involving 2 percent milk in 1985. Although yogurt did
not prevent salmonellosis in rats, it did significantly reduce the mortality
as well as other effects (Hitchins et al. 1985a, 1985b).
FOODS INVOLVED. Various foods have been the vehicle for transmis
sion of salmonellae. Most of these foods are of animal origin or contami
nated by foods of animal origin, as shown in Table 6.17.
The involvement of meat and meat products continues to increase
and accounted for more than 50 percent of the known foods from 1977
to 1981. During 1977, there were several outbreaks and more than 180
TABLE 6.17. FOODS INVOLVED IN SALMONELLOSIS, 1977-1981, NUMBER
OF OUTBREAKS
Year
Food 1977 1978 1979 1980 1981 Total
%
Beef 11 3 4 4 13 35 14.9
Pork 2 7 2 2 1 14 6.0
Other meat 1 3 1 0 0 5 2.1
Poultry 6 4 3 7 9 29 12.3
Eggs 0 0 2 1 0 3 1.3
Fishery prod 0 0 0 0 2 2 0.9
ucts
Dairy products 4 4 2 3 5 18 7.7
Bakery products 1 0 1 1 1 4 1.7
Salads 3 2 2 3 2 12 5.1
Other, unknown 13 22 27 18 33 113 48.1
TOTAL 41 45 44 39 66 235
SOURCE: CDC Annual Reports.
cases of salmonellosis related to the consumption of precooked roast
beef. These, as well as outbreaks in 1975 and 1976, stimulated action by
the USDA to require specific times and temperatures for heating beef
roasts (USDA 1983).
Poultry and poultry products have been implicated in many out
breaks of salmonellosis. Since the USDA required the pasteurization of
egg products, these have been eliminated as a significant vehicle for sal
monella organisms. The failure to cook chicken and turkey sufficiently
to eliminate the natural contamination is the usual contributing factor
causing salmonellosis.
Raw milk has been the vehicle for salmonellae causing salmonellosis
throughout the world (Bryan 1983; CDC 1984a; Reilly et al. 1983; Tacket
et al. 1985a). All milk should be pasteurized before human consump
tion, but even pasteurized milk has been implicated in outbreaks of sal-
monellosis (Bryan 1983; CDC 1984b). A widespread outbreak in 1985,
affecting over 16,000 people in several states, involved pasteurized milk.
This incident should make us aware that pasteurization systems and
proper procedures for handling pasteurized milk are needed to prevent
salmonellosis and perhaps other illnesses.
Apple cider involved in an outbreak was produced from apples that
had fallen onto the ground on which animal waste had been used as
fertilizer. Although the apples were washed, sufficient salmonellae re-
mained to cause illness. S. eastbourne was isolated from the environment
in which cocoa beans were processed (CDC 1974b), apparently causing
the contamination of chocolate candy which was involved in a wide-
spread outbreak. No source was determined for S. napoli, which was iso-
lated from chocolate bars involved in several cases of illness (Greenwood
and Hooper 1983).
ETIOLOGIC AGENT. The mechanism by which the salmonellae cause
gastroenteritis has not been fully explained. Perhaps more than one sys-
tem is involved. The heating of food to destroy salmonellae results in a
safe product. Hence, it was believed that salmonellosis was an infection
that required high numbers of the organism to cause the illness. How-
ever, the heating needed to kill the organism also might inactivate any
heat-labile toxins produced by the organisms.
The number of ingested Salmonella needed to cause an infection var
ies with the strain of the organism and the characteristics of the individ-
ual ingesting the organisms. Healthy adult males can ingest 10
5
to 10
7
cells (depending upon the strain of the organism) before symptoms of
the illness occur. However, person-to-person transmission in hospitals in-
dicates that only a few organisms are needed to cause illness. Blaser and
Newman (1982) reviewed eleven outbreaks and calculated that, in six of
these, less than 1,000 ingested organisms causerl the illness. In a series
of cases involving chocolate bars, it was estimated that an infective dose
was roughly fifty organisms (Greenwood and Hooper 1983). The very
young, aged, debilitated, and undernourished, and individuals with other
illness are more susceptible than normal healthy individuals to infection
by Salmonella.
The pH of the gastric juice can have a marked effect on salmonellae
(Giannella, Broitman, and Zamcheck 1971). Healthy gastric juice with low
pH (1-2) kills small numbers of salmonellae, but when it is deficient (pH
3.0 or higher), an otherwise harmless number of cells may survive.
When salmonellae reach the small intestine, they can live and multi
ply. The extent of multiplication depends on factors such as the peristal
tic rate, attachment to the mucosa, the composition of the intestinal flora,
the ingestion of various therapeutic agents (such as antibiotics), exposure
to radiation, and presence of irondeficiency diseases.
Attachment of the salmonellae to the intestinal mucosa is considered
to be essential for the organisms to remain in the intestine and cause
illness. Although invasion of the intestinal epithelium is needed for ex
cess secretions (Giannella 1979), invasion, per se, is not sufficient to
cause salmonellosis (Formal, Hale, and Sansonetti 1983). The results of
rectal biopsies of twentytwo patients varied from normal mucosa to
edema, hemorrhages, and sloughing (Day, MandaI, and Morson 1978).
Some strains of salmonellae are known to cause mucosal damage and
increased fluid output, some cause minimal mucosal damage and in
creased fluid, while others cause damage and no increase in fluid output.
Salmonella infection causes lesions in the ilea of pigs (Wilcock 1979) and
calves (Wray and Sojka 1978).
Researchers in one study suggested that S. typhimurium causes fluid
secretion by altering the sodium and chloride transport systems (Giannella
et al. 1975). When infected, the intestinal mucosa showed an increase in
adenyl cyclase activity, which results in ileal secretion of fluids. In are
view, Smith (1977) suggested that an extracellular product is involved,
because the microvilli degenerate before coming into contact with the
bacteria.
Only four of thirteen strains of Salmonella tested produced a positive
reaction in the rabbit ligated gut loop when living organisms were used
(Sakazaki et al. 1974). When culture filtrates were tested in the ligated
loops, eleven of thirteen strains of Salmonella gave a positive reaction.
The researchers contended that the culture filtrates contained enter
otoxic activities.
The rabbit ileal loop model was used to detect enterotoxin produced
by salmonellae (Sedlock and Deibel 1978). The serotypes that were tested
varied in their ability to produce the toxin, but even old stock cultures
maintained this trait. This indicates that the action is not due to a
plasmid.
A factor isolated from S. typhimurium caused cells of Chinese hamster
ovary (CHO) to elongate (Sandefur and Peterson 1977). This action is
indicative of an enterotoxin. They found that this effect was blocked by
antisera for cholera toxin and the B fragment of cholera toxin.
It is quite evident that most, if not all, strains of Salmonella produce
enterotoxins that elongate CHO cells and cause secretion in gut loop
tests (Baloda et al. 1983; Sharma, Singh, and Singh 1983). Jiwa and Mans
son (1983) and Alouf (1982) reported that both heat-labile and heat-stable
enterotoxins are produced by some strains of salmonellae_ Although the
enterotoxin usually reacts with antitoxin to cholera enterotoxin, one
study reported a salmonella enterotoxin that did not cross-react with
antisera to either cholera or heat-labile E. coli enterotoxins (Baloda et al.
1983).
Other researchers reported that, in salmonellosis, the cyclic adeno-
sine monophosphate (cAMP) is increased in epithelial cells in a manner
similar to that of cholera (Peterson et al. 1983). They stated that it was
not clear whether the increase in cAMP was due to enterotoxins, to pros-
taglandins formed during the inflammatory response to the bacteria, or
to both systems.
THE ORGANISMS_ The organisms in this genus are separated on the
basis of their somatic (0) and flagellar (H) antigens. The antigenic classi-
fication of Salmonella is known as the Kauffmann-White scheme. New spe-
cies or serotypes are continually being found and added to the many
already classified. In 1964, there were about 900 known serotypes. Now
there are more than 1,700 serotypes (CDC 1982a). The Arizona group
has been incorporated into the Kauffmann-White scheme for Salmonella
by Rohde (1979).
Although there are many serotypes, only about fifty cause nearly all of
the outbreaks and cases of salmonellosis. Each year, some ten serotypes
account for more than 60 percent of the isolations of Salmonella. The
ten most prominent serotypes isolated from humans for the years 1977
through 1980 are listed in Table 6.18. S. typhimurium is the most fre-
quently reported serotype isolated from both human and nonhuman
sources (Table 6.19). Also, S. typhimurium is the most frequently isolated
serotype in many other countries.
Growth Factors. Most strains of salmonellae can grow in a simple medium
consisting of ammonium nitrogen, mineral salts, and glucose. A few
strains need essential growth factors, primarily vitamins. The minimum
water activity for growth is between 0.93 and 0.96. The salt concentration
TABLE 6.18. MOST FREQUENTLY F.!EPORTED SALMONELLA SEROTYPES
ISOLATED FROM HUMAN SOURCES: RANKING (1-10) FOR YEARS 1977-1980
Year
Serotype 1977 1978 1979 1980
S. typhimurium" 1 1 1 1
S. heidelberg 3 2 3 2
S. enteritidis 4 3 2 3
S. newport
2 4 4 4
S. infantis 5 6 5 5
S. agona 6 5 6 6
S. saintpaul 7 9 7 7
S. montevideo 9 7 9 8
S. typhi 8 8 8 9
S. oranienburg 10 10 10
S.javiana 10
"Includes S. typhimurium var copenhagen.
needed to inhibit growth of salmonellae depends on the temperature
and other factors (Alford and Palumbo 1969). In ground pork, at pH 5.0,
stored at lOoe, a salmonella grew with 3.5 percent salt, but did not grow
at 5 percent salt. At 20C or 30C, salmonellae grew at the 5 percent salt
level. With ground pork at pH 6.5, growth occurred with 8 percent salt
added at 20C, or 30C, but no growth occurred at lOoe. The pH values
for growth of salmonellae are listed in Table 4.6.
TABLE 6.19. MOST FREQUENTLY REPORTED SALMONELLA SEROTYPES
ISOLATED FROM NONHUMAN SOURCES: RANKING (1-10) FOR YEARS 1977-1980
Serotype
S. typhimurium"
S. derby
S. panama
S. agona
S. manhattan
S. infantis
S. weltevreden
S. heidelberg
S. oranienburg
S. habana
S. montevideo
S. cholerasuis
S. worthington
S. tennessee
S. london
S. meleagridis
S. anatum
S. newington
"Includes S. typhimurium var copenhagen.
1977
2
1
3
4
8
6
10
9
5
7
1978
1
2
5
3
4
10
6
7
8
9
Year
1979 1980
1 1
3 2
5 3
2 4
5
4 6
10 7
6 8
7 9
10
8
9
10
Since salmonellae are pathogenic, they generally are considered to
be mesophilic. However, some strains appear to be psychrotrophic (see
Table 4.12). In general, these organisms require a temperature of 2 to
4C above the minimum reported in order to grow in food products.
Even then, at low temperatures, the more psychrophilic organisms tend
to outgrow the salmonellae. According to Tesone, Hughes, and Hurst
(1981), the addition of 0.2 molar NaCI raises the maximum temperature
of growth for some strains.
The interaction of other microorganisms naturally present in the
food with salmonellae can result in the inhibition of these potential path
ogens. There are numerous reports on the observed inhibition of Salmo
nella by lactic acid bacteria. Gilliland and Speck (1972) believed the antag
onistic action was caused by factors in addition to the acidic environment
created by the bacterial fermentation.
Biochemical Reactions. The main biochemical reactions of the salmonellae
are listed in Table 6.20. Even though a "+" or" -" reaction is listed, this
does not mean that all of the many serotypes and strains, types or vari
ants show that reaction. Subgenus I includes the more prominent salmo
TABLE 6.20. MAIN BIOCHEMICAL REACTIONS OF THE SALMONELLAE
Test or Substrate
Gas from glucose
Methyl red test
Indole production
VogesProskauer test
H"S production
Growth in Simmons citrate
Urease production
Gelatin liquefaction
Phenylalanine deamination
Growth in KCN medium (D)
Growth in malonate
Motility
Reduction of nitrate
Fermentation of:
Adonitol
Dulcitol
Inositol
Lactose
Maltose
Mannitol
Salicin
Sucrose
+
+
+
+
+
+
d
d
+
+
"+ = prompt, positive; X late and irregularly positive; (+)
different biochemical types; - = negative.
Subgenus'
III
+
+
+
+
(+)
+
+
+
+ or X
+
+
delayed positive; d =
nellae serotypes, and subgenus III consists of the organisms commonly
referred to as the Arizona group, now designated as S. arizona.
Motility. Although the organisms are said to be motile, there are mutants
that lose this ability, and two serotypes, S. pullorum and S. gallinarum are
not motile and do not possess flagella.
Sources. As a genus, the salmonellae are said to be ubiquitous. They are
worldwide and found in or on soil, air, water, sewage, animals, humans,
food, feed, processing equipment, and some plant products. Some sero-
types tend to be localized in a region or a country, but with national and
international travel and trade, the organisms are readily disseminated_
The natural habitat of the organisms is the intestinal tract of humans
and animals. Thus, it is logical that humans, animals, and their environ-
ments are the primary sources of Salmonella.
Few animals, if any, are born with intestinal contamination by Salmo-
nella.Just as human infants are highly susceptible to salmonellosis, so are
very young animals_ Some young animals may survive salmonellosis and
become temporary or long-term shedders of the organisms. However, it
is more likely that the animal is continually infected by some source of
Salmonella.
The sources of salmonellae for infection of domestic animals include
the parent stock, feed, water, rodents, wildlife, pets, humans, insects, arach-
nids, soil, and vegetation. The salmonellae can spread from animal to
animal during production and processing. They spread from animals to
people by direct interactions of humans and livestock, wild animals and
pets, as well as through poor food-handling practices and the consump-
tion of raw or undercooked foods of animal origin.
The relationship of Salmonella in feed to human salmonellosis is ap-
parent with S. agona_ Prior to 1971, the serotype was reported from hu-
man hosts in the United States only six times. It was isolated from im-
ported fish-meal on several occasions from 1970 to 1972_ Since these
isolations, it has been found in domestic animals, and since 1973 it has
ranked as one of the ten most prevalent salmonellae from humans (see
Table 6.18). This suggests the involvement of feed in a chain or cycle of
infection (see Fig. 6.10).
The role of free-living (wild) animals as a source of Salmonella was
thought to be quite important. Now it is thought that rodents, birds, and
other wild animals may be victims of their environment rather than the
source of the organisms. Even so, these free-living animals can transfer
the organism from infected areas to clean areas.
Since the waste materials from infected farms contain salmonellae,
the use of this material as fertilizer for crops or pastures can spread the
potentially infectious material to contaminate future animals. Rain fall-
ing on the farms, feed lots, or fields containing contaminated wastes can
wash the organism into streams and lakes. Besides the runoff from ani
mal quarters, sewage sludge and effluents from abattoirs and poultry
processing plants contain salmonellae. These polluted waters can cause
infection of farm animals and wild animals that drink from them.
Various pets are potential sources of salmonellae. They may contami
nate food or directly transmit these organisms to humans. Pet turtles are
an important source of Salmonella for transmission primarily to children.
An estimated 280,000 cases of turtleassociated salmonellosis occurred in
the United States in 1970 and 1971 (Lamm et al 1972). The interstate
shipment of turtles less than 4 inches long was banned by the FDA in
1975, because studies showed that 14 percent of the reported salmo-
nellosis in the United States was linked to pet turtles. However, some
turtles are still being sold in the United States, and some 3 or 4 million
are raised each year for export.
The direct transmission of Salmonella from animals to humans poses
a problem in zoos that have children's petting areas or other open areas
such as the aviary (Komorowski and Hensley 1973).
Although the cooking of raw meat and poultry will destroy salmonel-
lae, the raw foods are a source of contamination to the food preparer
and the kitchen equipment and can cross-contaminate cooked foods and
other foods that are eaten raw.
Fish and shellfish are contaminated by water that contains waste
products from humans and animals. Salmonellae were recovered from
oysters, clams, and crabs harvested from both the east and west coasts of
Florida (Fraiser and Koburger 1984). The east coast site was approved
for shellfish harvest, but 25 percent of the trapped crabs and more than
13 percent of the clams contained salmonellae. The west coast site was
an unclassified harvesting area, and the incidence of salmonellae in
clams was higher than 43 percent. The highest level of contamination
reported was 2.2 salmonellae per 100 grams of shellfish.
Fruits and vegetables are rarely involved in outbreaks of salmo-
nellosis. However, vegetables can be contaminated by organisms present
in animal wastes, or polluted irrigation water used on crops. Velaudapil-
lai, Niles, and Nagaratnam (1969) found that l.3 percent of the vegetables
sampled were infected with Salmonella, Shigella, or enteropathogenic E.
coli. Salmonella serotypes were found on celery, leeks, spinach, green
beans, and carrots. Wild olives contained S. sandiego. Samples of lettuce
(68.3 percent) and fennel (7l.9 percent) yielded one or more serotypes
of salmonellae (Ercolani 1976). Salmonellae were isolated from over 22
percent of the vegetable samples analyzed by Tamminga, Beumer, and
Kampelmacher (1978a). Other researchers found that four samples of
vegetables (radish, carrot, celery and spinach) offifty examined contained
salmonellae (Rude et al. 1984). They suggested that vegetables eaten raw
should be considered a source of salmonellae in outbreaks of salmo
nellosis. Further, raw vegetables may be a source of human parasites. Co
conut products are believed to be contaminated by salmonellae in the
soil. This contamination is transferred to the inner, edible portion duro
ing processing.
Humans may be one of the main reservoirs of Salmonella. If not a
source, humans can act as the vehicle for transfer of the organisms from
one area to another.
Food handlers or workers in animal by product plants often have a
high rate of carriage of Salmonella. Although they might not have an active
infection, the children of these workers have been observed to have
higher rates of gastroenteritis than children of other workers. This indio
cates that a worker who handles products that contain Salmonella can
bring the organisms home to the other members of the family.
Survival in Nature. The natural habitat of Salmonella is the intestinal tract
of animals. Excreted organisms are found in fecal material, food, and
various parts of the environment. To cause salmonellosis, they must be
able to survive in these unnatural surroundings.
Salmonellae in slurry (liquid manure) sprayed onto grass may survive
from eighteen days (Taylor and Burrows 1971) to thirty three weeks (Find
lay 1972). Jones, Hagler, and Hamilton (1977) found that the survival of
salmonellae in cattle slurry was influenced by the normal microbial flora.
In sterilized slurry, S. dublin multiplied and survived for more than 370
days, while in natural slurry, there was no multiplication and shorter sur
vival times. Survival time in a pasture is influenced by grass cover, sun
light, temperature, and rainfall.
Effluent from apparently healthy pigs was applied to land (Chandler
and Craven 1981). The salmonellae were detected for eight months from
soil and nearly two months from pasture. S. typhimurium survived in soil
for seven to fortytwo days, depending upon soil moisture and temper
ature (Zibilske and Weaver 1978).
Liu, Snoeyenbos, and Carlson (1969) found that the survival of salmo
nellae in meat and bone meal was related to water activity and holding
temperature. As water activity and temperature increased from low lev
els, the survival decreased. At an aw of 0.82 and temperature of 50C, a
5log reduction occurred in 72 hr. However, at 10 percent moisture (aw
about 0.55) at 4C, there was essentially no reduction in viable cells of S.
senjtenberg 775W for 20 days. Elevated temperatures, oxygen, and unsatu
rated fatty acids accelerate the death of S. oranienburg in fish meal (Lam
precht and Elliot 1974).
When fingertips were contaminated with 500 to 2,000 cells of S. an
atum, the cells could be recovered 3 hr later. Contamination, followed
by washing, eliminated levels of less than 1,000, but when 6,400 were
inoculated, they could be recovered 10 min after washing. Even fewer
than 100 cells of S. anatum inoculated onto fingertips infected samples
of corned beef and ham handled 10 min after exposure (Pether and Gil
bert 1971). This shows how Salmonella can be spread by workers handling
first contaminated products and then clean products.
Survival in Food. There is less destruction of inoculated salmonellae duro
ing pan drying than during spray drying of egg white. Pan drying is more
similar to the natural drying of materials. Once dried, the organisms can
survive for long periods at room temperature or below. Salmonellae were
isolated from dried eggs after fortyone weeks at 20C, and at sixtyfive
weeks when held at 2C.
Salmonellae can survive up to eleven weeks on vegetables held at 2
to 4C, and seven weeks at room temperature. In acid foods, survival is
relatively short, but if salmonellae survive freezing, such as in certain
fruits, they may remain viable for considerable periods. Salmonella can
survive at least thirteen months on frozen poultry held at - 21C. In ice
cream, the organisms can survive for several years.
Salmonellae inoculated into cold pack cheese food survived in excess
of twentyseven weeks at 4.4C, when no preservative (potassium sorb ate)
or acids were added (Park, Marth, and Olson 1970). Six Salmonella strains
were inoculated into sheep milk at 10
4
cells/ml and processed into Man
chego cheese (Medina, Gaya, and Nunez 1982). The organisms were abo
sent from all lots of eightweek Manchego cheese.
The cells of S. typhimurium tended to die when present in yogurt
(Rubin 1985). The presence of casein and an increased pH of yogurt pro
tected the cells from the inhibitory agent, lactic acid.
When inoculated into a beef.pork mixture at a level of 10
4
cells per
gram, S. dublin survived pepperoni processes and persisted after forty
two to fortythree days of drying (Smith et al. 1975). S. typhimurium was
more sensitive than S. dublin to the acid condition of Lebanon bologna
during processing and aging (Smith et al. 1975b).
Salmonellae inoculated into chocolate bars at a level of about 10
6
cells/g were viable after storage for nine months (Tamminga et al. 1976).
The reduction was greater in bitter chocolate than in milk chocolate. S.
typhimurium inoculated at a level of about 1,000/g of chocolate was not
detected in 55 g after fifteen months (Tamminga et al. 1977). S. eastbourne
survived better than S. typhimurium.
In a review, Bryan (1968) summarized the survival of salmonellae.
These organisms can survive in the low range of temperature and relative
humidity. According to references cited by him, they can survive in con
taminated earth and pasture for more than 200 days, cloth for 228 days,
plastic cover slips for 93 days, sweeper dust for 10 months, rodent feces
for 148 days, roach pellets for 199 days, poultry feces for more than 9
days, dried cattle feces for more than 1,000 days, on egg shells from 21
to 350 days, in dried whole eggs for more than 4 years and in meat salad
for 77 days.
BASIC METHODOLOGY. It is not the intent of this text to precisely
describe the procedures needed to obtain samples, or to detect or enu
merate salmonellae in the samples. For the procedures, the reader is reo
ferred to FDA (1978), AOAC (1985), APHA (1984), and USDA (1971). Sam
pIing and testing plans for salmonellae were reviewed by Olson (1975).
We are concerned primarily with the analysis of food samples, swabs
from food surfaces and water, and environmental swabs, although the
analysis of other samples may be required, even in a food microbiology
laboratory.
In many foods, the organisms have been subjected to debilitating or
sublethal processes such as freezing, desiccation, extremes of pH, heat,
osmotic pressures, or curing of ingredients during the manufacture or
storage of the product. When present, salmonellae usually comprise a
very small component of the total population. For most foods, there is
no tolerance for Salmonella. Even one Salmonella detected in a 25g sample
is considered to be adulteration of the food (exceptions are raw chicken
and meat). Hence, it is necessary to detect very low levels of these organ
isms. This small number of salmonellae, especially in comparison to the
larger number of other organisms, means that the presence of salmonel-
lae is unlikely to be demonstrated by direct plating on selective media,
and that an enrichment procedure in broth is needed.
The basic procedure for the examination of foods for Salmonella con-
sists of preenrichment and enrichment in broths, streaking and detection
on selective-differential agars, and then characterizing typical colonies to
confirm that they are salmonellae. Confirmation is done by means of
biochemical tests, serological typing and, for a few serotypes, phage typ-
ing. It is evident that a long time period is needed before results of any
kind are obtained. Hence, many attempts have been made to develop
rapid procedures. Besides the basic procedure, some of these rapid meth-
ods are discussed.
Preenrichment. The preenrichment phase is accomplished by incubating
the sample in a non inhibitory broth (usually in the ratio of one gram of
sample to nine of broth) for 18 to 24 hr at 37C. Shorter preenrichment
times (4 to 6 hr) have been suggested, but longer preenrichment times
(18 to 24, or 48 hr) have been more effective (D'Aoust 1981; Kafel 1981).
The recovery of salmonellae from dried food products may be improved
by reduced hydration systems (Andrews, Wilson, and Poelma 1983;
Poelma, Andrews, and Wilson 1984; Wilson et al. 1985).
Media that have been used for pre enrichment include lactose broth,
trypticase soy broth, reconstituted nonfat dry milk, nutrient broth, and
buffered peptone water.
Generally, processed foods that may contain sublethally injured sal
monellae are preenriched. Raw or heavily contaminated products are put
directly into a selective enrichment broth. The review by Andrews (1985)
pointed out that preenrichment increased the recovery of salmonellae
from these foods. However, for the examination of oysters, Miller and
Koburger (1984) reported greater recovery by direct enrichment in sele
nite cystine broth than by using a preenrichment broth.
Enrichment. The enrichment process is the creation of a special environ
ment which permits the growth or selection of the desired species or
group of microorganisms from a mixture of microorganisms with which
they are likely to be found. The procedure usually functions by furnish
ing conditions for the desired organisms to outgrow all others. Ideally,
an enrichment system for salmonellae should allow these organisms to
grow and inhibit the growth of all other organisms.
It has been difficult to develop the ideal enrichment medium. Since
there are some 1,700 serotypes of Salmonella, any selective agent is likely
to be inhibitory to one or more serotypes or to be ineffective in inhibit
ing the nonsalmonellae that are present.
Foods vary in their nutrient content, pH, inhibitory substances, and
other factors that may influence the growth of Salmonella. Therefore,
when a food is added to an enrichment medium, usually in a ratio of 1:9,
the characteristics of the medium are changed. Each food will alter the
medium in its own peculiar way. Therefore, to be effective, a different
enrichment medium should be designed for each food.
The enrichment broths commonly used in the United States are sele
nite cystine (SC), tetrathionate (T), tetrathionate brilliant green (TBG),
and TT. Rappaport's medium (with modifications) reportedly is an effec
tive broth for salmonellae enrichment (Fricker and Girwood 1985; Tong
pim et al. 1984; Vassiliadis et al. 1984). However, it has never received
much attention in the United States.
The enrichment broths are used as secondary enrichment of samples
that have been preenriched (usually 1 ml of sample from the preenriched
broth to 10 ml of enrichment broth), and as direct enrichment for raw
or heavily contaminated food. Most analytical procedures suggest adding
25 g of food sample to 225 ml of broth. For feed samples, 30 g is added
to 100 ml of broth. Due to multiple sampling of a food lot to assure the
absence of Salmonella, the analysis of larger quantities of pooled material
from these samples has been suggested.
Some foods have certain peculiarities that call for special procedures
for better detection of Salmonella. When gelatin is added in enrichment
media, the viscous suspension that results can interfere with Salmonella
analysis. Rose (1972) suggested adding gelatinase to the enrichment me-
dium. The enzyme was not inhibitory to salmonellae, and better recovery
was made from gelatin artificially inoculated with S. typhimurium.
When milk is analyzed, the acid produced causes formation of curds.
Salmonella trapped inside these clots of milk are not detected. The addi-
tion of trypsin aids detection by digesting the protein of the milk, but
apparently it does not injure the salmonellae.
The inoculated enrichment broth is incubated at either 37 or 43C
for 18 to 24 hr, and then a loopful is streaked onto an agar surface that
is selective and differential.
Isolation. Many more Salmonella-positive samples are found when two or
three agars are used. Due to the results of collaborative studies, research-
ers recommended retaining the use of bismuth sulfite (BS) agar, but rec-
ommended that salmonella-shigella agar and brilliant green (BG) agar be
replaced by Hektoen enteric (HE) agar and xylose lysine desoxycholate
(XLD) agar for detection and isolation of salmonellae (Sanders 1981).
Bailey, Cox, and Thomson (1983) reported that BG and HE were superior
to BS agar for recovery of Salmonella. Hawa, Morrison, and Fleet (1984)
used a dulcitol bile novobiocin agar for isolating salmonellae from
chicken carcasses.
It is recommended that BS plates be observed after 24 hr and, if no
typical salmonellae colonies are observed, the plates should be incubated
for another 24 hr before they are called negative.
With preenrichment for 24 hr, enrichment for 24 hr and BS incuba-
tion for 48 hr, a total of 96 hr (four days) is needed even for samples with
no Salmonella, before any decision can be made.
The procedure to be used (type of preenrichment medium or no pre-
enrichment, types of enrichment and plating media, as well as incubation
temperatures) is dictated somewhat by the type of sample being analyzed,
as well as the types of organisms and serotypes of Salmonella that might
be present.
It is difficult to compare media for the preenrichment, enrichment,
and isolation for Salmonella. If salmonellae are present in the sample at
a high level, almost any of the typical media can be used to detect them.
If the organisms are at a very low level, some of the samples for preen-
richment or direct enrichment may not contain any Salmonella. To over-
come these circumstances, many samples need to be tested and the re-
sults analyzed by an acceptable statistical system.
In all of the research that has been done, it is evident that the more
different types of broths and agars that are used, the greater is the chance
for recovery of Salmonella, especially if sublethally injured and at a very
low level, with various other organisms at high levels.
Biochemical Characterization. If no typical colonies are found on the isola
tion medium, this is satisfactory evidence that the sample is Salmonella
negative. If typical colonies are detected, this is presumptive evidence
that the sample may be Salmonella positive. For further characterization
of the organisms in these typical colonies, both biochemical and serologi-
cal tests are needed. Neither the biochemical nor the serological tests are
specific for Salmonella, and neither system will detect all the serotypes of
the genus.
Rapid and easily run methods have long been the desire of microbiol-
ogists. One method employed to get a quick reaction is to use a large
inoculum in a small amount of medium.
With the number of biochemical tests that need to be run, consider-
able time and material are required to prepare the media, inoculate, in-
cubate, determine the reactions, and classify the cultures. To cut down
on the time and cost, rapid methods, composite media, and simplified
test kits have been developed. Commercial preparations have the poten-
tial benefit of providing many laboratories with standard materials for
use in assessing bacterial reactions. These commercial systems include
the API 20E, Enteric-Tek, Enterotube II, Micro-ID, Minitek, Repliscan II,
and Sensititre systems.
These systems have certain things in common. They are all miniatur-
ized and rapid tests based primarily on reports in the literature by var-
ious workers. None of the systems agrees 100 percent with the conven-
tional tube method for determining reactions. The common opinion is
that these tests are acceptable for general use.
Serology. Serological considerations are used in the immunization of hu-
mans and animals for certain diseases caused by salmonellae, for testing
the serum of humans or animals for potential carriers of the organisms,
and for determining the serotype of an isolated test culture. The serologi-
cal reactions of the test culture are needed to supplement the informa
tion obtained from biochemical tests.
The salmonellae possess 0, H, and K antigens (Kauffman 1972). The
o (somatic) antigens are considered to be a constitutive part of the cell
wall, but they may extend beyond this barrier. They are a complex of
lipopolysaccharide (LPS) and protein. The detailed structure of the 0
antigen varies from strain to strain. This variation forms the basis for the
serological differences of the organisms. The 0 antigens are considered
to be heat stable, resisting boiling for 21;2 hr.
Salmonella can change in colony morphology form smooth to rough
(S--> R variation). These rough forms are caused by mutations that block
the synthesis of polysaccharides. Thus, they have no 0 side chains, lose
o agglutinating ability, and have a reduced virulence.
The H antigens, or flagellar antigens, are found only in motile cuI
tures. They are destroyed at 100C (heat labile), and by dilute alcohol or
acid. Flagella are composed of the protein flagellin, with a molecular
weight of about 40,000. Many species of salmonellae have two types of
flagellar antigens (phase 1 and phase 2). A single bacterium manifests
only one of these.
Phase 1 antigens are specific and are shared by only a few serotypes
of salmonellae, while phase 2 antigens are nonspecific and are common
to many serotypes.
Usually, only the 0 and H antigens are determined when typing a
culture, although the Vi antigen is sometimes of interest. The cultures
are typed according to the KauffmannWhite scheme. This system is out
lined by Kauffmann (1972) and Krieg and Holt (1984).
The specific H or 0 antigens are determined by cell genetics. Due to
mutations or phage conversions, the antigens can be altered. This may
account for the multiplicity of serotypes and the overlapping patterns of
antigenicity.
If there is an agglutination with the 0 antisera, it is not proof that
the culture is a salmonella. There are crossreactions with the Salmonella
o antisera and nonsalmonellae. Some strains of closely related orga
nisms, especially strains of E. coli, Citrobacter, Shigella, and Enterobacter,
either share certain somatic antigens or have antigens similar to those of
salmonellae (Refai and Rohde 1975). Other unrelated microorganisms
reportedly react with Salmonella 0 antisera (Aksoycan and Saganak 1977;
Corbel 1975).
The H antigens are determined by a tube test. A motile organism is
necessary for reaction with H antigens. Since S. pullorum and S. gallinarum
are not motile, they will not react. For the exact procedure for detecting
H antigens, the reader is referred to AOAC (1985) or FDA (1978).
The medium in which the organism is grown can influence the reac
tion with H antiserum (Banwart and Kreitzer 1972; Stamper and Banwart
1974).
Besides biochemical and serological tests, for epidemiological pur
poses phage typing, antimicrobial resistance patterns (resistotyping), and
plasmid typing may be used for characterizing salmonellae (Brunner et
al. 1983; Farrar 1983; Riley et al. 1983; Somerville, Nhlapo, and Alberts
1983).
OTHER METHODS. The conventional procedure requires consider
able material and time to detect Salmonella. Other tests have been devised
to obtain results more quickly and easily. Usually rapid tests result in a
loss of accuracy or precision. However, even with the conventional test,
not all of the Salmonella serotypes are detected in all cases. The results
obtained with a rapid test should at least compare favorably with those
obtained with the conventional test (FDA 1978).
To detect the low number of salmonellae in most foods, the sample
must be incubated in a growth medium to enrich the salmonellae prior
to analysis, even by the "rapid" methods.
HydrophobicCrid-Membrane Filtration (HCMF). After preenrichment and
enrichment, a portion of enrichment broth is passed through an HGM
filter (En tis et al. 1982). The filter is transferred to a selective agar surface
and, after incubation, observed for salmonellae. Presumptive colonies
must be evaluated biochemically and serologically as in the conventional
test. The HGMF system has been adopted by the AOAC.
Phage Systems. The use of bacteriophage has been suggested for detection,
characterization, and the typing of certain serotypes of salmonellae.
To detect S. typhosa (S. typhi) in soil samples, Kande1aki (1964) added
typhoid indicator phage to the sample. An increase in the phage titer
during incubation was evidence that S. typhi was present in the sample.
The phage titer increase detected more positive samples than did the
bacteriological plating system Kandelaki used.
For determining the character of colonies on selective agars, one
team of researchers (Cherry et al. 1954) suggested using bacteriophages.
According to them, the method is rapid, simple, and quite specific. They
used the 0-1 phage of Felix and Callow. This system was reevaluated by
We1kos, Schreiber, and Baer (1974). They tested 652 strains of Salmonella
and found that the phage (0-1) reacted with 640 (98.2 percent). Of 1,463
nonsalmonellae strains, only E. coli strains were lysed. Of 239 strains of
E. coli tested with 10
12
plaque-forming units/ml (PFU/ml), 14 (5.9 percent)
were susceptible to lysis. Gunnarsson, Hurvell, and ThaI (1977) found
that 98 percent of 5,287 strains of salmonellae were lysed by 0-1 phage.
They warned that monophasic strains of subgenus III and strains of sub-
genus IV usually are not sensitive to the 0-1 phage.
The increase in 0-1 phage was determined with high performance
liquid chromatography to detect salmonellae in milk and feces (Hirsch
and Martin 1983; Crane, Martin, and Hirsch 1984). A plaque system using
0-1 phage reportedly can detect salmonellae in milk in 24 hr (Hirsch and
Martin 1984). Since not all strains of salmonellae are affected by the
01 phage, Glidel and Fey (1981) suggested adding a G47 phage to the
0-1 phage to increase the number of strains that are lysed.
Immunoassays. There are many immunoassay systems, some of which are
discussed for the detection of S. aureus enterotoxin. The immunoassays
of interest in salmonellae detection are those using a labeled reactant
(usually the antibody).
For the radioimmunoassay (RIA), a radioactive substance is conju
gated onto one of the reactants. Although the RIA is a sensitive system,
there are problems (see S. aureus enterotoxin detection, page 215).
When a fluorescent compound is conjugated onto a reactant in sal-
monellae determinations, it is called the fluorescent antibody (FA) tech-
nique. This test has been adopted by the AOAC (Sanders 1975). After an
enrichment procedure, a direct or indirect FA test can be used.
For the direct test, the specific antibody is conjugated with a flit ores-
cent dye. When this conjugate is added to a solution containing homolo-
gous antigen, they will react, and the product will be fluorescent in ultra-
violet light.
For the indirect method, if the antibody is produced in a rabbit, rab-
bit serum is injected into another animal, such as a goat, to produce anti-
rabbit serum. This goat antirabbit serum is conjugated with the fluores-
cent dye. For the test, the antigen is reacted with the specific antiserum
produced in the rabbit. Then the conjugated goat antirabbit serum is
added, which will react with the antiserum produced in the rabbit.
The advantage of the indirect method is that, as long as the antibodies
are produced in a rabbit, only one conjugated antiserum is needed (goat
antirabbit). This conjugate will work in FA tests for clostridia, staphylo-
cocci, salmonellae, and any organisms, as long as the specific antiserum
to the antigens of the organism are produced in a rabbit.
Besides the requirement for a good fluorescent (ultraviolet) micro-
scope and well-trained personnel, the diagnostic sera used to detect sal-
monellae by the FA is expensive. The serum should react with all of the
serotypes of Salmonellae and not cross-react with the nonsalmonellae. This
type of serum is not yet available.
In many surveys, the FA procedure reveals more Salmonella-positive
samples than the conventional culture test. Various reasons for this have
been suggested. One proposal is that the FA is more sensitive than cultur-
ing. A more logical explanation is that cross-reactions with nonsalmonel-
lae by the FA antisera result in more apparently Salmonella-positive sam-
ples. Thus, the samples that appear positive with the FA technique must
be run through the conventional test for confirmation.
When an enzyme is conjugated onto a reactant, it is called an enzyme
immunoassay (EIA) (see S. aureus enterotoxin detection, page 215). For
salmonellae testing, this and the other immunoassays might be used to
detect the organisms as well as their enterotoxins.
For their EIA, Mattingly and Gehle (1984) stated that 10
6
Salmonellal
ml were needed for detection. This means that the salmonellae in the
samples must be enriched prior to analysis. However, the enrichment
period for the EIA has been shortened from that suggested for the con-
ventional test (FDA 1978)_ Of these immunoassays, the EIA is the newest
and most promising (Mattingly et aL 1985; Swaminathan, Aleixo, and
Minnich 1985)_ The ELISA has been used to detect salmonellae enter-
otoxins (Richter 1983)_
For all of these immunoassays, specific antibodies are needed that will
react with all of the salmonellae and not cross-react with nonsalmonellae_
Although many claims have been made regarding various antibodies, no
such antibodies are presently available and might never be found_ Even
the FDA (1978) test yields a few false negative samples, however, and
sometimes even a false positive sample_
Although these systems have some advantages over the conventional
method (FDA 1978), to study the organisms in an epidemiology of an
outbreak of salmonellosis, they must be isolated_
Deoxyribonucleic Acid (DNA) Hybridization Assay_ DNA is composed of two
parallel, complementary strands that are uniquely matched and held to-
gether by chemical bonds_ These strands can be separated_ The two com-
plementary strands, under specific conditions, can find each other and
rejoin or hybridize, even in the presence of other molecules_ Specific
strands of DNA, called probes, that are unique to salmonellae and report-
edly do not hybridize with other organisms are used in this assay_ These
probes make it possible to detect the presence of salmonellae in a com-
plex mixture of organisms that are present in a food sample_ It is neces-
sary to enrich the sample to obtain sufficient salmonellae for the assay
to work properly_
In the past, radioactive phosphorus (p32) was used as a label on the
probes to detect hybridization_ However, enzyme labels have been devel-
oped for this purpose (Olsiewski, Thalenfeld, and Engelhardt 1985)_ Us-
ing enzyme labels (rather than radioactive phosphorus) makes the assay
safer and more acceptable_
Reportedly, a properly formulated DNA probe will neither cross-
react with other organisms nor give a false positive result_ Other DNA
probes can be used with the basic system for the detection of various
organisms (Fitts 1985; Sayler et aL 1985)_
Comments_ At the present time, Salmonella methodology is confusing to
some_ There is more than one so-called official method, as well as many
other proposed tests, several of which are not listed or discussed in this
text
However, considering the many types of samples and the various com-
positions of foods, as well as the bacterial populations, and considering
that salmonellae, if present, are usually in very low numbers, perhaps
many different methods are needed_ Perhaps we should evaluate all of
the potential media and systems with each food to develop the best sys-
tem for each food_
The results of research have shown that, with the FDA (1978) method,
the more types of media that are used, the greater the likelihood that all
Salmonella-positive samples will be detected_ With nearly all of the so-
called rapid methods, only one medium is used_ Are these systems that
superior to using a selective agar isolation system? It seems doubtfuL Ob-
taining a false positive sample may increase the cost of food due to re-
examination, reprocessing or discarding the suspect food_ However, miss-
ing a Salmonella-positive sample with a resultant salmonellosis outbreak
can result in an even higher cost to the processor.
CONTROL OF SALMONELLA. The measures needed to control Salmo-
nella and salmonellosis are essentially the same as those needed for the
control of S. aureus and the clostridia (prevent contamination, prevent
growth, destroy the organisms). The number of cases of typhoid fever
due to S. typhi has been drastically reduced_ Proper sewage disposal,
protection and chlorination of communal water supplies, restricting the
harvesting of aquatic food from contaminated water, pasteurization of
certain products, sanitary control of food processing and sales establish-
ments, exclusion of typhoid carriers from food-handling occupations,
and immunization programs, have reduced the incidence of typhoid fe-
ver in the United States.
Since the prevalence of salmonellosis is increasing, it is evident that
these procedures are not effective in controlling this illness; other mea-
sures need to be incorporated_
There is an incentive for the food industry to market products with
no salmonellae. Section 402 of the Federal Food, Drug and Cosmetic Act
defines a food to be adulterated if it bears or contains any poisonous or
deleterious substance that may render it injurious to health, and if it has
been prepared, packed, or held under unsanitary conditions whereby
it may become contaminated with filth or whereby it may be rendered
injurious to health_ Foods containing Salmonella or other pathogens fall
within this definition.
The control of salmonellae in foods includes the acquisition of Sal-
monella-free raw materials, with processing, storage, and distribution un-
der conditions that prevent the increase of Salmonella and, ideally, a ter-
minal treatment of the food to destroy any salmonellae that may be
present.
Prevent Contamination. Because salmonellae are ubiquitous, keeping them
out of food might seem to be a hopeless task_ However, it is generally
believed that, with a sincere effort by all factions of feed and food pro-
duction, handling, processing, and preparation, the contamination of
food by salmonellae can be prevented or reduced.
A major cycle of infection is animal byproduct to feed, feed to ani
mals, and animals to food to humans (Fig. 6. 10). There are other cycles,
such as human to food to human or even human to human. If the cycle
of feed to animals to humans can be broken, the incidence of salmo
nellosis might be reduced significantly.
There should be Salmonellafree breeding stock, feed, and water, strict
sanitary practices, segregation of sick animals, and sale of only healthy
animals. Sanitation includes many facets, such as pest control, cleaning
and disinfecting housing, removal and disposal of waste, storing feed so
that it is not contaminated, preventing workers from carrying diseases
with them from one farm area to another, and keeping out visitors. There
are sanitary requirements for dairy farms, but other farming practices
are usually left to the farmer. However, Oosterom, Van Erne, and Van
Schothorst (1982) reported that using practical farming situations, even
with sanitary precautions, it was impossible to produce Salmonella-free
pigs.
To reduce or eliminate Salmonella from chickens, a system called com-
petitive exclusion shows promise (Blanchfield et al. 1984; Goren et al.
1984; Pivnick et al. 1982; Schneitz, Seuna, and Rizzo 1981). It is based
on colonization of the gastrointestinal epithelial surface by indigenous
organisms. Feeding or otherwise inoculating fecal, cecal, or anaerobic
cultures from adult fowl into the intestines of day-old chicks will allow
these bacteria to occupy the attachment sites. This deters or prevents
salmonellae from colonizing the intestines of the chicks. Competitive ex-
clusion also protected turkeys from salmonellae (Reid and Barnum
1983). The feeding of antibiotics to animals can alter the indigenous
flora, allowing salmonellae to colonize the intestinal tract (Barrow, Smith,
and Tucker 1984; Que and Hentges 1985).
For seafoods, the habitat in which such animals grow must be free of
Salmonella. The first step is to prevent untreated sewage or water from
entering streams, lakes, or oceans. Shellfish, such as oysters, are believed
to cleanse themselves of salmonellae and E. coli in 48 to 72 hr if placed
in clean water. At an initial level of 400 salmonellae per gram, oysters
contained no detectable cells after 2.5 days, whereas 900 cells per gram
required 3.5 days (Son and Fleet 1980). However, clean beds are needed
for the production and harvesting of shellfish, since the depuration pro-
cess cannot be relied upon for the elimination of all pathogens.
In the preparation of food, one of the important factors for spreading
Salmonella is the cross-contamination from raw to cooked food. Until such
time as raw poultry or meat products can be produced with no Salmonella,
we must assume that the organisms will be present. Handling of these
products and then handling ready-to-eat products transfers the bacteria
from the raw to the ready-to-eat products.
Thorough daily cleaning and sanitizing of the processing plant and
equipment can reduce the dissemination of salmonellae and other organ-
isms into the environment and onto foods. Testing for salmonellae
should be part of the control effort. If the organisms are in the plant
environment, there is a good chance they will be in the finished product.
Cleaning and sanitizing are discussed further in Chapter 10.
Foster (1969) pointed out that elimination of salmonellae from foods
would not necessarily eliminate human salmonellosis. We also need to
consider the human carrier as well as household pets. The human carrier
is important in the processing plant, in the food-preparation and serving
industry, and in the home.
Foods have been placed into five categories in terms of hazard to
human health (Foster 1971). No safe level of salmonellae has been estab-
lished, but it is evident that illness is more likely when the number in-
gested is increased. Three hazard characteristics were established for a
particular food. These are (1) the food or an ingredient is a significant
source of Salmonella; (2) there is no control step (heating) in the process
to destroy the organism; and (3) if the product is mishandled, growth can
occur, resulting in an increased hazard. The higher the number of hazard
characteristics of a food, the more potentially dangerous it is. A food
with no hazard characteristics is not considered dangerous, foods with one
or two are intermediate, and foods with all three are hazardous. The sus-
ceptibility of people varies, with infants, the aged, and the infirm the
most susceptible to Salmonella infection. With this aspect included, the
five categories are established:
Category I foods possess any of the hazard characteristics and are
intended for use by infants, the aged, or the infirm.
Categories II, III, IV, and V foods are for general uses.
Category II foods possess all three hazard characteristics.
Category III foods have two of the hazard characteristics.
Category IV foods have one hazard characteristic.
Category V foods have no hazard characteristics_
With these hazard categories, it is evident that fewer samples of foods
in category V need to be tested (if negative results are obtained), than
those in category I or II. Considering 25-g samples, it was proposed that
the food should be accepted if there are no positive samples in fifteen
samples (Category III, IV, or V), thirty samples (Category II), and sixty
samples (Category I) or, if no more than one positive sample is detected
in twenty-four samples (III, IV, V), forty-eight samples (Category II) or
ninety five samples (Category I). Thus, the lower the risk category num
ber and the greater the hazard, the greater the number of samples that
need to be analyzed and the lower the percentage of positive samples
that are allowed.
Prevent Growth. Although refrigeration at 4 to 5C will control the multi
plication of Salmonella, it will not improve the hazard characteristic, since
the food can be abused in market channels or during preparation and
serving. Foods should not be held between 10 and 50C. These temper
atures should be traversed as rapidly as possible during heating and
cooling.
The salmonellae do not grow in foods with naturally low pH (below
pH 4.0). Acids can be added to some foods or feeds to inhibit growth of
the organisms. Although salmonellae might not grow, they may survive
in some foods at a low pH.
Potassium sorb ate reportedly inhibits the growth of salmonellae (Rice
and Pierson 1982; To and Robach 1980). Sodium sulfite and metabisulfite
inhibit growth of Enterobacteriaceae in ground meat (Banks and Board
1982). Although sulfites are allowed in some foods in the United States,
they are not allowed in fresh red meats.
Destroy the Organisms. The destruction of the organisms is the best way to
make certain that they do not present a health hazard. It is preferable if
the destruction can be accomplished in the final package, so that recon
tamination does not occur during further handling.
Liquid propylene oxide was effective in destroying salmonellae in
meat and bone meal (Tompkin and Stozek 1974). The concentration of
propylene oxide needed for destruction of salmonellae depends on the
relative humidity, level of contamination, and time of exposure. There is
a residual limit of 300 j.tg/g in certain food ingredients.
Fumigation of feed (fish, meat, or bone meal) with formaldehyde gas
is an effective means of destroying salmonellae (Duncan and Adams
1972). The gas eliminates salmonellae to a depth of 1.91 cm of feed.
The chlorination of water is used to destroy S. typhi. This treatment
can aid in the control of salmonellae in food processing plants whether
chlorine is used in wash water, equipment rinse water, or cooling water.
The heating of foods is the principal system to eliminate salmonellae.
Pasteurization is used to control salmonellae in liquid egg products. This
is discussed in Chapter 11. Pasteurization of coconut at 80C for 8 to 10
min effectively killed salmonellae without affecting the product quality
(Schaffner et aL 1967). Another report stated that the present milk pas
teurization process (63.33C for 30 min or 71.67C for 16 sec) will inacti
vate salmonellae, as long as the number does not exceed 3 X 10
12
salmo
nellae per milliliter of milk (Read et aL 1968). It would be extremely
unlikely that this level of salmonellae would be attained without spoilage,
due to other bacteria.
Although bacteria have an increased heat resistance in dried prod
ucts, it is not impossible to eliminate salmonellae from dry animal feed.
Carrol and Ward (1967) found a combination of 87.8C for 10 min con
sistently reduced salmonellae to a nondetectable level in fish meal.
Van Cauwenberge, Bothaste, and Kwolek (1981) stored salmonellae
inoculated corn flour (10 and 15 percent moisture) at 49C. After 24 hr,
99.9 percent of the salmonellae cells were destroyed. However, heating
salmonellae inoculated poultry feed in a microwave oven (reaching a
temperature of 186C), in a hot air oven at 110C for 1 hr, or in flowing
steam for 10 min failed to consistently eliminate the salmonellae (Bur
dick et al. 1983).
The cooking of foods usually destroys any salmonellae that may be
present. S. senftenberg 775W is killed during the normal processing of
frankfurters in the smokehouse when the internal temperature of the
product reaches 7l.1C (Palumbo, Huhtanen, and Smith 1974). Heating
pepperoni to 60C or Lebanon bologna to 5l.7C eliminated 10
4
inocu
lated cells of salmonellae (Smith et al. 1975a, 1975b). Goodfellow and
Brown (1978) determined D values (time needed to destroy 90 percent
of the population) for salmonellae in a ground beef system. The D values
they reported were 5l.6C (61-62 min), 57.2C (3.8-4.2 min), and 62.7C
(0.6-0.7 min). These D values were used to establish the times and tem
peratures needed to cook roast beef to assure the death of any salmo
nellae.
Salmonellae are only moderately resistant to radiation treatment. AI
though the needed radiation dose varies with the type of product, condi-
tions ofradiation, and level of contamination, Thornley (1963) suggested
that 0.5 Mrad would reduce the number of salmonellae by a factor of 10
7

Ostovar, Pereira, and Gallop (1971) found that 0.5 Mrad was sufficient to
destroy salmonellae in smoked whitefish. Another team of researchers
reported that, for frozen meat, 0.6 Mrad would reduce Salmonella num-
bers by a factor of at least 10
5
(Ley et al. 1970). For decontamination
of herring meal, radiation doses of 0.8 to l.3 Mrad were recommended
(Underdal and Rossebo 1972). Mulder, Notermans, and Kampelmacher
(1977) suggested treating poultry carcasses with 0.25 Mrad to eliminate
salmonellae. More recently it was reported that, after treatment with 0.25
Mrad at 5C, the poultry might still contain salmonellae, but that, after
storage of this treated poultry at -18C for three months, no salmonellae
were detected (Mulder 1982). Pearce (1979) found that salmonellae were
quite stable in stored casein, but treatment with 0.5 Mrad eliminated
these organisms from the protein.
The low doses of radiation needed to eliminate salmonellae have no
apparent adverse effect on the flavor or nutritional quality of the food
or feed. It would seem to be a very useful process for animal feed, since
gamma irradiation can be accomplished after the feed has been packed
into impervious bags.
Shigellosis (Bacillary Dysentery)
This illness accounts for less than 10 percent of the reported out
breaks of foodborne illness in the United States. Since only a few states
report this illness to CDC, it is probably more widespread than the data
indicate. The predominant mode of transmission is human to human,
but contaminated water and food also are important.
Sometimes the illness is called the filth disease, because it is associ
ated with poor personal hygiene and sanitation. The illness is often
prominent in areas where there are large groups of people. Summer
camps, mental institutions, Indian reservations, and low socioeconomic
urban communities are the highrisk population areas. The increase in
infant day care centers has resulted in another high risk situation.
The mortality rate appears to be greatest in infants and in adults over
fifty years of age. Most of the cases are children under ten years, and the
highest attack rate is children in the one-to-four age group. This is prob-
ably due to their lack of training in personal hygiene at this young age.
In the twenty-to-twentynine age group, there is a higher rate of illness
in females than in males. This is believed to be due to women's having
closer contact with children, especially sick children, than do men of this
age group.
Although considered a disease of the poor, the affluent have acquired
the illness through travel to foreign areas, such as Mexico, Central Amer-
ica, and the Far East.
Shigellosis is primarily an illness of humans, but other primates, in-
cluding monkeys and chimpanzees, can acquire the disease (Rout et al.
1975). By using procedures such as starvation, folic acid deficiency, con-
trol of normal intestinal flora, and administration of opium to slow peri-
staltic action, experimental shigellosis has been established in mice and
guinea pigs (Nelson and Haltalin 1972; Maier and Hentges 1972). The
ligated small intestine of rabbits serves as an animal model for studying
shigellosis.
PATHOGENESIS. The pathogenesis of Shigella is rather complex. After
reaching the small intestine, the first requirement is the attachment to
and penetration of cells of the intestinal mucosa. If the cells cannot in-
vade, they are avirulent. After invasion, shigellae multiply and cause the
destruction of cells (Levine et al. 1973). This results in mucosal ulcera-
tion. Speelman, Kabir, and Islam (19S4) studied colonic lesions in thirty
three patients with shigellosis. Their findings indicated that the rectosig
moid area is the most frequently and most severely affected, and that
during the course of illness, the colonic lesions extend in a proximal
direction. In some cases, lesions extended to the splenic flexure, the dis
tal transverse colon, and the proximal transverse colon. In 15 percent of
the patients, pancolitis was evident.
Shiga toxin, an enterotoxin produced by S. dysenteriae 1, the most viru
lent Shigella, was purified by one group of researchers (Brown et al. 19S2).
The toxin was cytotoxic to HeLa cells, caused secretion in rabbit ileal
loops, and was lethal to mice. S. sonnei and S. jlexneri also produce toxins
that are antigenically and biologically related to Shiga toxin of S. dysenter
iae (Keusch and Jacewicz 1977; O'Brien et al. 1977). These three species,
as well as S. boydii, produce a toxin that causes morphological changes in
Chinese hamster ovary cells (Takeda, Okamoto, and Miwatani 1979).
The exact role of the enterotoxins in shigellosis has not been estab
lished, but there are various speculations as to their action (Fernandez
et al. 19S4; Formal, Hale, and Sansonetti 19S3; Mathias et al. 19S0). The
mechanism of the pathogenic action of shigellae and their toxins is not
known exactly. The illness may be an infection caused by the organism,
some form of intoxication, or a combination of these actions.
CHARACTERISTICS OF THE ILLNESS. The number of organisms
needed to cause the infection may be rather low, since persontoperson
transfer is the main mode of transmission. One study reported that as
few as ten organisms of virulent strains produced illness in human vol
unteers (Levine et al. 1973).
In most outbreaks of shigellosis there is an index case. One person
develops symptoms of the disease, and then one or two days later, others
became ill. If the index case is a food handler, a foodborne outbreak can
occur. An example of an outbreak is shown in Figure 6.12.
Incubation Period. The time from ingestion of the organisms until symp'
toms appear varies with the individual (health, age), number of organ
isms ingested, and virulence of the organism. CDC (l 9S3b) listed the
incubation period for shigellosis as 12 to 50 hr. Due to person to person
spread, it sometimes is difficult to determine the exact times for the in
fections.
Symptoms. The symptoms of shigellosis as listed in several outbreaks are
shown in Table 6.21. The main symptom is diarrhea. Infections associ
ated with mucosal ulceration usually cause more fever, abdominal pain,
and rectal bleeding than do toxin induced diarrheas.
In the United States, the disease is usually self.limiting and rather
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50
40
20
10
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X
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"0
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1 2 3 4 5 6 7 8
DAYS
Figure 6.12. Progress of a typical outbreak of shigellosis.
9 10
mild. However, the diarrhea may be severe; the drastic loss of fluids with
resulting dehydration is the major consideration. In young children the
disease may be very severe, or even fatal, due to dehydration and ex
traintestinal manifestations.
Duration and Therapy. The illness may last for 12 hr to three weeks, with
the average illness lasting from five to six days. The infection usually
persists beyond clinical recovery, and the duration of the convalescent
carrier state is commonly three to four times that of the duration of
symptoms.
Since very young patients may not understand the need for strict per-
sonal hygiene, drug therapy is often warranted to prevent secondary
spread. Food handlers should be considered potential spreaders of the
organism and should be given therapy. The type of drug used is depen-
dent on the sensitivity of the cultured strain and on the sensitivity of the
patient to various antibiotics.
TABLE 6.21. SHIGELLOSIS: PERCENTAGE OF PEOPLE EXPERIENCING
SYMPTOMS IN EIGHT OUTBREAKS
Outbreak
Symptom 2 3 4 5 6 7 8
Diarrhea 100 98 91 92 100 98 100 91
Bloody 6 6 II 9 23 6 5 24
Mucus 19 19 19
Tenesmus 31 54 17 31 54
Abdominal pain or
cramps 94 85 64 76 79 85 16 69
Chills 56 54 57 27 51 54
Fever 53 47 57 78 95 47 27 76
Nausea 41 59 47 56 59 35 57
Vomiting 33 27 21 53 49 27 13 48
Headache 66 43 29 51 66 57
Muscle ache 55 55
Weakness 71
SOCRCE: CDC Repoyts.
After recovery from shigellosis, an apparent state of immunity often
develops, but repeated infections may occur. Immunization of a popula
tion would not be practical, due to the low incidence of shigellosis. In
institutions or other areas of high incidence, immunization would be
beneficial.
Foods Involved. As might be expected, the foods that have been involved
in most outbreaks are those that are handled the most. These are salads
(potato, tuna, shrimp, macaroni, and chicken). The ingredients may be
clean, but during the preparation, the salad is contaminated by hand
manipulation or mixing. The organisms can multiply readily in moist
foods held at room temperature, so even a small inoculum can cause a
largescale outbreak of shigellosis.
THE ORGANISMS. The genus Shigella contains four species: S. dysenter
iae, S. jlexneri, S. boydii, and S. sonnei. They are separated mainly by sero
logical tests and DNA relatedness. Each of the four species contains sev
eral biotypes or serotypes. In the United States more than 60 percent of
the isolates are S. sonnei, and more than 25 percent are S. jlexneri. On a
relative basis, S. sonnei is decreasing and S. jlexneri is increasing in the
frequency of isolation. Typically, the shigellae are nonmotile, fail to fer
ment lactose, and are anaerogenic (do not produce visible gas from fer
mentation of carbohydrate).
Sources. The normal habitat is the intestinal tract of human beings and
other primates. Isolation from other animals is rare.
In a survey of market vegetables in Ceylon, Velaudapillai, Niles, and
N agaratnam (1969) found cabbage, leeks, spinach, beans, carrots, and
pumpkins contaminated with shigellae. They believed that the contami-
nation may have originated from the water used for irrigation, fertilizer
(manure), or from handling the vegetables at the market.
The main source of shigellae involved in outbreaks is people who are
symptomless carriers, or ambulant cases. The organisms can be isolated
for several weeks after recovery from the illness. Testing of healthy food
handlers for these organisms would be meaningless; however, a person
experiencing diarrhea should not be allowed to handle food.
METHODOLOGY. The methodology involved with the shigellae in-
cludes enrichment, plating, and differentiation to detect the organism.
Serological reactions, colicin typing, and DNA evaluation are aids to fur-
ther separate the shigellae. The use of colicin typing as an epidemiologi-
cal tool was questioned by Vlajinac and Krajinovic (1983).
Virulent and avirulent strains can be differentiated by feeding human
volunteers. A simpler method is based on the fact that keratoconjuncti-
vitis develops in the eyes of rabbits and guinea pigs when they are inocu-
lated with virulent strains of shigellae (Cross and Nakamura 1970). The
cytotoxic activity of Shigella toxins can be assayed with HeLa cells (Gentry
and Dalrymple 1980).
CONTROL. Foodborne outbreaks of shigellosis are caused by the mis-
handling of food_ A high standard of personal hygiene by food handlers
(washing hands after using the toilet, not handling food during illness or
a diarrheal symptom), and sanitation of the premises, with proper cook-
ing and refrigeration of foods, should help control shigellosis.
Escherichia coli Enteritis
Certain strains of E. coli cause enteric disease syndromes in people
and other vertebrate animals. In countries lacking basic sanitation, E. coli
is a prominent cause of enteritis. In most of the outbreaks in the United
States, the organisms are transmitted by water. Some outbreaks are food-
borne, although E. coli is not listed as a causative agent in Table 6.l.
In 1971, an outbreak due to enteropathogenic E. coli (EEC) in
imported cheese resulted in 387 reported cases. This was the first well-
documented United States outbreak in which food containing EEC af-
fected adults.
In a survey of infants and children with diarrhea, salmonellae or shi-
gellae were isolated from 42 percent of the patients, while enterotoxin-
producing strains of E. coli were found in 86 percent of the diarrhea
group and 41 percent of the control group (Rudoy and Nelson 1975).
This indicates that in enteric diseases of young people, E. coli may be as
important as salmonellae or shigellae.
ETIOLOGIES. The role of E. coli in enteric disease, especially the diar
rheal syndrome, is quite complex. Although all virulent strains can be
called enteropathogenic, there are classical enteropathogenic strains, in
vasive strains that cause illness similar to shigellae, strains that produce
enterotoxins, and a more recently recognized enterohemorrhagic E. coli
(Levine and Edelman 1984).
Classical enteropathogenic strains of E. coli that are neither invasive
nor produce enterotoxin (ST or LT) have been reported (Levine et al.
1978; Robins-Browne et al. 1982; Toledo et al. 1983). The colonization of
the intestines by enteropathogenic strains can result in moderate to se-
vere injury to the mucosa (Rothbaum et al. 1982; Ulshen and Rollo 1980).
It has not been established that mucosal injury causes the gastroenteritis.
The possibility that a toxin different from the conventional enterotoxins
is involved in the diarrheal syndrome has been proposed (Klipstein et
al. 1978). Other researchers have suggested that a shigalike cytotoxin may
playa role in enteropathogenic E. coli gastroenteritis (Cleary et al. 1985).
In a study of an enteropathogenic strain, Ryder, Kaslow, and Wells (1979)
found that it produced a heat-stable enterotoxin.
The invasive strains of E. coli cause a dysentery (fever, cramps, and
diarrhea containing blood and mucus) similar to that caused by shigellae
(DuPont 1978; Sakazaki et al. 1974). The invasive strains can be distin
guished by the development of keratoconjunctivitis when placed on the
guinea pig eye, or by the penetration of HeLa or HEp2 cells (Mehlman
et al. 1982).
Enterotoxigenic E. coli (ETEC) may form a heat-labile (LT) , a heat-
stable (ST), or both LT and ST toxins. The heat-labile toxin of E. coli is
readily destroyed at 60C. It is similar to cholera enterotoxin (cholera-
gen) immunologically, structurally, and functionally. Cholera antitoxin
reacts not only with choleragen and Salmonella enterotoxins, but also with
the LT of E. coli. Both choleragen and LT are composed of two subunits,
designated as A and B.
Acting alone, neither A nor B is toxic. For toxicity, the B subunit binds
with host cell membrane receptors and acts as the messenger for the A
subunit, which activates adenyl cyclase (Gill and Richardson 1980; Moss
et al. 1984). This alters the cyclic adenosine monophosphate metabolism
in intestinal cells, which induces an enhanced secretion of fluid and elec-
trolytes into the intestine, resulting in the diarrheal syndrome in the
same manner as cholera toxin.
In one study, researchers separated the A and B subunits of LT and
choleragen (Y. Takeda et al. 1981). Then they recombined them to form
hybrids of the two toxins. These hybrids revealed a toxicity similar to that
of the parent toxins. According to Clements and Finkelstein (1979), one
difference between LT and choleragen is that LT is plasmid mediated,
while choleragen is chromosomally mediated.
The heatstable (ST) enterotoxins of various E. coli strains have been
purified, and their properties determined. Heating at 100C for 1 hr has
no effect on biological activity (Robertson, Dreyfus, and Frantz 1983), but
the toxin is inactivated when heated at 121C for 15 min (Lallier, Lari
viere, and St. Pierre 1980). They reported that the maximum amount of
enterotoxin was produced after 7 hr of growth.
There may be more than one ST (Burgess, Mullan, and Newsome
1980). It has been observed that not all ST shows activity in the suckling
mouse assay. Those toxins showing activity in both the suckling mouse
and piglet assays have been designated as ST a. Other toxins having activo
ity in the piglet assay but not the suckling mouse assay have been called
ST
b

The ST molecule is a polypeptide with a molecular weight of less than
5,000. This means that it is a rather poor antigen. By coupling with a
protein carrier and using multiple booster doses, researchers have pro
duced antiserum (Frantz and Robertson 1981). The antiserum reacted
with STa from various strains of E. coli but not with ST
b
Frantz and Rob
ertson stated that STa from various strains of E. coli share at least one
antigen determinant.
In the lower small intestine, E. coli STa stimulates the enzyme guany
late cyclase, which increases cyclic guanosine monophosphate, including
secretion of fluids into the intestine (Argenzio et al. 1984; Dreyfus, Jaso
Friedmann, and Robertson 1984; Gianella and Drake 1979; Rao et al.
1980). The mechanism of the activity of ST b is unknown, but apparently
the increase in cyclic nucleotides such as GMP or AMP is not involved
(Dreyfus, Frantz, and Robertson 1983; Kennedy et al. 1984).
The ability to produce the enterotoxins is determined by plasm ids
(Echeverria et al. 1985; T. Takeda et al. 1981). Since the plasmids are
transferable, theoretically any E. coli might become enterotoxigenic.
However, only certain serotypes of E. coli tend to accept the plasmid
(Echeverria et al. 1982; Reis 1980). The plasm ids were successfully trans
ferred to Salmonella, Shigella, Citrobacter, Enterobacter, Klebsiella, Edwardsiella,
Serratia, and Proteus species (Neill, Twiddy, and Holmes 1983; Takeda et
al 1983).
There appear to be several steps in the development of E. coli gastro
enteritis. Normally, when a person ingests cells of E. coli they pass
through the stomach and small intestine and may colonize in the large
intestine. If the cells can adhere to the epithelial cells in the small intes
tine, they may remain for a sufficient period to multiply, and, if toxi
genic, to produce enterotoxin.
Reportedly, the enterotoxins are produced by the organisms in the
small intestine. However, they have been produced in laboratory media,
with the culture filtrates showing fluid accumulation in ileal loops.
Hence, it is possible that some enterotoxin is preformed in foods. No
one has reported finding E. coli enterotoxin in foods.
CHARACTERISTICS OF THE ILLNESS. Since there are enteropatho
genic, enteroinvasive, enterotoxigenic, and enterohemorrhagic strains of
E. coli, there is more than one type of illness.
Incubation Period. The incubation period is listed as 6 to 36 hr (CDC
1983b). In an outbreak of enteropathogenic organisms involving im
ported cheese, the mean incubation time was 18 hr, and the average dura
tion of symptoms was two days (Marier et al. 1973). For an outbreak in
volving invasive strains, the incubation period ranged from less than 24
hr to more than 72 hr, with most cases between 24 and 47 hr (Tulloch et
al. 1973). In an outbreak due to heatlabile enterotoxin, symptoms began
by 12 hr and peaked at 36 to 48 hr (CDC 1976a). In an outbreak involving
toxigenic E. coli, the incubation ranged from 3 to 166 hr, with a median
of 44 hr (Taylor et al. 1982).
Symptoms. The main symptom is diarrhea. In a foodborne outbreak due
to imported cheese (Marier et al. 1973), 88 percent of the people reo
ported this symptom. Other symptoms (with the percentage of people
experiencing the symptom) were fever (72 percent), nausea (71 percent),
cramps (66 percent), chills (38 percent), vomiting (35 percent), malaise
(34 percent), aches (28 percent), and headache (19 percent). With severe
diarrhea, dehydration can occur. Newborn infants are especially suscepti
ble to enteropathogenic E. coli. In an outbreak of invasive strains, 100
percent of the patients had diarrhea and 7 percent had bloody diarrhea,
similar to that with Shigella (Tulloch et al. 1973). Tenesmus was a symptom
in 96 percent of the patients. In another outbreak, the main symptoms
were diarrhea, nausea, abdominal cramps, headache, chills, and dizziness
(Snyder et al. 1984).
Duration and Therapy. The duration of the illness depends upon the sever
ity of the disease and the type of individual. The average illness persists
for three to four days, but in some cases, the diarrhea may persist for
fourteen days. Usually, coliforms are removed from the upper intestine
in seven to ten days, but in rare cases, they may persist for several weeks.
With severe watery diarrhea, fluids and electrolytes must be replaced.
Glucose tends to induce absorption of sodium and water; thus, treatment
with oral glucose-electrolyte solutions has been suggested (Klipstein and
Engert 1978).
Foods Involved. The only well-documented foodborne outbreaks in the
United States involved soft cheese. In other countries, outbreaks have
been associated with consumption of dairy products, salads containing
raw vegetables, meat and meat products, poultry, fish, and baked prod-
ucts.
Number of Organisms Needed. Although newborn humans and animals are
highly susceptible, adults are more resistant. In a survey by Rudoy and
Nelson (1975), EEC was isolated from 41 percent of a control group of
children without diarrhea. This indicates that, even if a few organisms
are present, they do not cause illness if the numbers are kept under con-
trol by the body defenses or by interactions with other organisms in the
intestine.
One study found that all of their human volunteers developed diar-
rhea 11 to 48 hr after ingestion of 10
6
to 1010 colony-forming units of E.
coli (Donta et al. 1974). The type and number of cells of E. coli and the
age and physical condition of the host determine the occurrence and the
severity of the illness.
NATURE OF THE ORGANISM. Escherichia coli is an important or-
ganism in the microbiology of foods. Besides being involved in gastroen-
teritis, the organisms in this species are considered to be indicators of
possible fecal contamination and can cause spoilage of some foods.
There are more than 160 serotypes of E. coli.
Sources. The main habitat of E. coli is the intestinal tract of humans and
animals. However, fecal contamination causes it to spread to other envi-
ronments, especially soil and water.
Researchers analyzing various foods of animal origin found enter-
otoxigenic E. coli in each type (Sack et al. 1977). They believed that these
foods are potentially important vehicles for these toxigenic organisms,
especially if the food is not handled properly.
EEC was detected in about 10 percent of 2,000 samples of cheese
(Fantasia et al. 1975). None of the foods examined from a village in the
Philippines (Echeverria et al. 1978) or Ethiopia (Jiwa, Krovacek, and Wad-
strom 1981) contained enterotoxigenic E. coli.
METHODOLOGY. There is no simple culture medium or system that
will differentiate pathogenic from nonpathogenic E. coli. After enrich-
ment and isolation of E. coli, it is necessary to characterize the organisms
with biochemical, serological, and other tests.
Invasiveness and Enterotoxigenicity. Laboratory models are available to de
tect and assay invasiveness or enterotoxigenicity of E. coli (Table 6.22).
Invasive strains are detected by their ability to penetrate the superfi
cial layers of the cornea and cause keratoconjunctivitis in the eye of the
guinea pig. The invasion of rabbit intestinal mucosa can be tested if the
culture causes keratoconjunctivitis.
The ligated intestinal loop test can be used to determine pathogenic
ity of E. coli. By using bacteriafree inocula, the reactions of enterotoxins,
assay of enterotoxins, and the effect of antibodies on the reaction can be
obtained. Generally, rabbit intestine is used, but other animal models are
acceptable.
In rabbit ileal loops, the LT causes maximum fluid accumulation in
about 18 hr, with the ratio of volume (ml) to length (cm) about 2.0:2.5.
To detect ST, the loops are examined at 6 to 8 hr, although some effect
of LT is evident at the time. To determine both LT and ST, the test mate
rial is introduced through the stomach with a tube or injected into the
intestine of a seventonine dayold rabbit. After 6 or 7 hr, the animal is
sacrificed, the entire GI tract is removed and weighed, the fluid is reo
moved and measured, and the ratio of volume to fluid is determined
(Gorbach and Khurana 1972). The rabbit as an animal model is relatively
expensive and the method is time consuming with both false positive and
false negative reactions (Thorne and Gorbach 1978).
An infant (suckling) mouse assay has been used to detect ST. The one
tofour day old mice are inoculated intragastrically. After 4 hr, the mice
TABLE 6.22. DETECTION OF INVASIVENESS AND THE ASSAY OF ENTEROTOXINS
OF ESCHERICHIA COLI
Property
Invasiveness
Heat-labile enterotoxin (LT)
Heat-stable enterotoxin (ST)
LT and ST
Test
Guinea pig eye-keratoconjunctivitis test (Sereney test)
Invasiveness of rabbit (or other animal) intestinal mu-
cosa
Plasmid-140 megadalton
Rabbit ileal loop (18 hr)
Skin-permeability test (rabbit)
Chinese hamster ovary tissue culture
Y-I mouse adrenal tumor cells
Seological tests (ELISA, Biken, latex particle, RIA)
Gene probe
Hamster-uterine growth
Suckling mouse
Rabbit ileal loop (6 hr)
Guanylate cyclase activation
Gene probe
ELISA
Suckling mouse test (6 hr)
Gas-liquid chromatography
are sacrificed and the ratio of intestinal tract plus contents weight to the
remaining body weight is determined. A ratio of greater than 0.83 indio
cates the presence of ST. Researchers reported, however, that the mouse
test was not sufficiently sensitive (Merson et al. 1980). Frantz and Robert
son (1981) found the assay to be impractical for screening large numbers
of isolates. They stated that it was fairly expensive and did not detect all
forms of ST. A dog assay detected more ST-producing strains than did
the mouse assay (N alin et al. 1978).
Rabbit skin tests have been used to detect LT_ The preparation is in
jected intracutaneously, followed 24 hr later by an injection of Evans blue
dye. Areas of induration and bluing are read 1 to 2 hr later. Young rabbits
show a better reaction than do old rabbits.
Certain cell lines can be used to assay for these enterotoxins. The LT
causes elongation of Chinese hamster ovary cells.
Originally flat Y-1 mouse adrenal tumor cells become rounded when
exposed to the enterotoxins (Gurwith 1977). Vero (African green mon-
key) cells appear enlarged, thick-walled, and refractile, with several fila-
mentous tendrils in response to E. coli LT (Speirs et al. 1977). The ST has
no effect on these three cell lines (Giugliano, Stavric, and Konowalchuck
1982). The Y- 1 test for LT and the suckling mouse test for ST were
adopted as official first action by the AOAC (Lovett and Peeler 1984).
Another team of researchers described a gas-liquid chromatographic
method that could detect and differentiate LT and ST (Brooks et al.
1984). The activation of guanylate cyclase was used to detect ST (Wald-
man et al. 1984). They claimed it was more sensitive than the suckling
mouse test. The increase in uterine weight after inoculation i.p. into cy-
cling hamsters was used to detect the presence of LT (Alleva and La-
manna 1984). Cholera toxin also gave this response.
Various serological tests similar to those used to detect staphylococcal
enterotoxins have been used for E. coli LT (Honda et al. 1981; Tsukamoto
et al. 1980; Yano et al. 1982), and ST (Giannella, Drake, and Luttrell
1981). A staphylococcal coagglutination test for LT was developed by
Brill, Wasilavskas, and Richardson (1979) and modified by later research-
ers (Honda et al. 1983a). Monoclonal antibodies have been developed
for both LT and ST (Belisle, Twiddy, and Holmes 1984; Brandwein et al.
1985).
Enzyme-linked immunosorbent assays (ELISA) have been developed
to detect LT (Honda, Sato, and Miwatani 1984; Merson et al. 1980; Ris-
taino, Levine, and Young 1983; Sack et al. 1980; Svennerholm and
Wiklund 1983) as well as ST (Klipstein et al. 1984; Thompson et al. 1984).
At the present time, the ELISA systems seem to be the most promising
for the detection of toxins as well as other antigenic materials.
Methods have been developed to detect enterotoxigenic E. coli (ETEC)
by means of DNA colony hybridization using DNA or gene probes (Mose
ley et aL 1980; Patamaroj, Seriwatana, and Echeverria 1983). According
to these authors, this system can detect ETEC in food or stools without
the need to enrich the sample. The use of this system for determining
ETEC among isolated organisms was approved as official first action by
the AOAC (Hill and Payne 1984).
CONTROL. The control of the illness is similar to that for other enteric
diseases. As far as foodborne illness is concerned, the number of cases
would not warrant the immunization of all humans in developed coun
tries but might be worthwhile for people at risk in developing countries.
Some data suggest that there is a natural development of immunity in
an endemic area (Black et aL 1981).
To prevent contamination, Bryan (1973) stated simply that we should
practice personal hygiene, prepare foods in a sanitary manner, protect
and treat (chlorinate) water, and dispose of sewage in a sanitary manner.
The easiest way to prevent growth of the organism is to provide an
unacceptable environment for E. coli.
Since apparently large numbers of cells are needed to cause illness
in other than newborn babies, simply cooking the food sufficiently pro
vides a safe product.
Bacillus cereus Gastroenteritis
Under normal circumstances, B. cereus is not considered to be a path
ogen. Foodborne illness due to B. cereus rarely is reported in the United
States. The number of reported outbreaks is much higher in European
countries and is one of the major causes of foodborne illness in Hungary.
There are two distinct syndromes caused by enterotoxins produced
by B. cereus. One enterotoxin causes an illness similar to that produced
by the S. aureus enterotoxin. The primary symptoms are nausea, vomit
ing, and abdominal cramps. Diarrhea may occur in the later stages of
some cases. The incubation period usually is less than three hr. A second
enterotoxin results in an illness similar to that caused by the C. perfringens
enterotoxin. In this case, the effects are primarily in the lower intestinal
tract, and the primary symptom is diarrhea. The incubation period is
about 10 to 12 hr.
In both syndromes, fever is absent, indicating that toxins are in
volved. Usually, the symptoms last about 12 to 24 hr, but some cases,
especially of the emetic type, may be severe. Due to the usually mild ill
ness, with short duration, people might not seek medical aid. Therefore,
most cases probably are undetected and unreported.
High numbers of B. cereus (10
6
to 109/g) have been associated with
foods involved in the illness. Due to multiplication of the cells in the
food before analysis, these numbers may be higher than when the food
was ingested.
THE ORGANISM. B. cereus is an aerobic, Grampositive, motile, spore
forming rod. It hydrolyzes esculin and starch, reduces litmus milk and
nitrate, and is catalase positive.
Since many cells (> 10
5
/g of food) are needed to cause foodborne
illness, the food must be abused sometime during preparation. In out
breaks in England due to rice, the rice was boiled and then allowed to
sit at room temperature for 12 hr to three days. The rice was either boiled
or fried before being served. The original heating was not sufficient to
kill the spores of B. cereus but could activate the spores to germinate.
Holding the rice at room temperature allowed the vegetative cells to mul
tiply to levels that caused illness.
Source. B. cereus is common in soil and dust, so it is logical that foods that
are readily contaminated by soil and dust will contain the organism.
Plant products (cereals, flour, starch, bakery products, spices), animal
products, and mixtures of ingredients (spaghetti sauce, pudding, soup
mixes, gravy mixes) can contain a few or many cells or spores of B. cereus.
There is a similarity between foods involved in illnesses due to B.
cereus and to C. perfringens. In either case, the food is prepared ahead of
time in large batches and is not properly cooled before reheating (if
needed) and serving. The reheating is not sufficient to destroy the cells.
METHODOLOGY. To detect B. cereus a spread plate system using
mannitolegg yolkpolymyxin (MYP) agar is suggested (FDA 1978). On
this medium, the organism produces pink colonies surrounded by a
dense precipitate (lecithinasepositive reaction). B. cereus is mannitol neg-
ative. Harmon, Kautter, and McClure (1984) found MYP slightly superior
to two other media (polymyxin pyruvate-egg yolk-mannitol-brom thymol
blue and trypticase-soy-polymyxin blood agars), but all three were satis-
factory for detection of B. cereus.
Selected colonies are isolated and the organisms tested for the Gram
reaction and biochemical tests. A method for differentiating B. cereus was
described by Harmon (1982) and was adopted by the AOAC as its interim
official first action.
ENTEROTOXIN. Turnbull (1981) discussed the various toxins of B. ce-
reus. Gilbert and Kramer (1984) compared the various properties of the
diarrheal and emetic enterotoxins. The diarrheal toxin is a protein with
a molecular weight of about 50,000, while the emetic toxin is a peptide
(not antigenic) and has a molecular weight of less than 5,000.
The diarrheal toxin can be detected by the rabbit ileal loop test or
skinpermeability system of either rabbits or guinea pigs. Monkey feeding
with observation of vomiting is the system used to detect emetic enter
otoxin.
CONTROL. The methods of control are the same as those for C. per
fringens. Keeping B. cereus out of the food would appear to be a difficult
task. However, proper holding temperatures (55C or higher, or below
lOC) for the food would prevent growth of the organisms. Multiplica
tion of the cells is needed to attain sufficient numbers to cause illness.
Streptococcal Infections
Group A (Streptococcal pyogenes) may be present in food. This organism
causes scarlet fever or septic sore throat. Besides a sore throat, symptoms
may include vomiting and diarrhea.
A betahemolytic group A streptococcus caused an outbreak of pharo
yngitis (CDC 1982b). The food involved was not determined. Outbreaks
of pharyngitis believed due to contaminated salads were reported by
CDC (l984c).
Although group A streptococci usually are involved, group G strepto
cocci occasionally cause pharyngitis. Among the possible symptoms are
nausea, vomiting, and diarrhea. An outbreak in 1979 involving chicken
salad was reported by Stryker, Fraser, and Facklam (1982).
A group C streptococcus contaminating homemade cheese caused fe
ver, chills, and vague constitutional symptoms (CDC 1983a).
When streptococcal foodborne infection is mentioned, the organisms
in group D, often referred to as enterococci, or fecel streptococci, are
considered to be the etiologic agent of the illness.
The reported number of outbreaks due to enterococci is not signifi
cant In cases in which enterococci have been involved, the incubation
period ranged from 2 to 30 hr. The symptoms included nausea, vomiting,
and diarrhea. Usually, the symptoms were milder than those in other
foodborne illnesses. The duration was usually only a few hours.
Some people believe that the enterococci do not cause foodborne
illness, and some are convinced that these organisms are involved in out
breaks. Other workers are unsure, but they realize that more convincing
proof is needed to relate enterococci to foodborne illness.
Illness Due to Vibrios
The genus Vibrio is discussed in Chapter 3. The two important species
causing gastroenteritis are V. cholerae and V. parahaemolyticus. Other Vibrio
species such as V. fluvialis (Tacket et aL 1982) and V. mimicus (Ciufecu,
N acescu, and Israil 1983) have been involved in causing diarrhea in hu-
mans_
V. CHOLERAE. This organism causes cholera in humans_ Generally,
the ingestion of 10
6
to 10
10
cells is needed to cause the illness_ The nor-
mally low pH of the stomach will kill unprotected cells, but they can pass
through the stomach if the pH is altered, such as by the ingestion of
antacids_ Those cells that pass into the small intestine must be able to
adhere to the mucosa on which they grow and produce an enterotoxin_
It is believed that the flagellum is associated with attachment, since non-
motile cells generally cannot adhere to the intestine_ From 24 to 48 hr
after ingestion, symptoms of cholera may occur. The symptoms include
profuse watery diarrhea, abdominal cramps, nausea, and vomiting_ In
severe cases, a liter or more of water may be lost each hour, resulting in
rapid dehydration, shock, and eventually, death_
The principal strains of V. cholerae that cause illness are in serogroup
0:1, although other strains have been involved_ The 0:1 serogroup con-
tains two biovars, the classical and the eltor (Krieg and Holt 1984)_ There
are two subtypes, Ogawa and Inaba_ A third subtype, Hikjima, has been
proposed_ Although the toxin is produced in response to a gene located
on the chromosome of the cell, not all 0:1 strains are enterotoxigenic.
The cholera enterotoxin (CT) and E. coli heat-labile toxin (LT) share
some antigenic properties_ The CT has a molecular weight of about
84,000_ It consists of five B subunits arranged in a ring in which there is
an A subunit. The B subunits combine to the membrane receptor, GM1
ganglioside, in the intestine, which then allows the A subunit to traverse
the cell membrane and activate the membrane-bound adenyl cyclase,
thereby causing an increase in cyclic adenosine mono phosphate (cAMP)_
This causes an inhibition of absorption of sodium and chloride at the
villus tip, which is accompanied by water being dumped into the intes-
tine_ There is no structural damage to the intestinal mucosa_ The pres-
ence of bile acids seems to enhance the excretion of water into the intes-
tine (Eidels, Proia, and Hart 1983; Lange, Hansson, and Lonnroth 1983;
Middlebrook and Dorland 1984)_
Cholera toxin is determined by methods similar to those used for E.
coli LT, such as the skin reaction (Kuroki 1981), CHO cells (Nozawa, Yo-
kota, and Kuwahara 1978), animal models (Richardson, Giles, and Kruger
1984; Spira, Sack, and Froehlich 1981), and immunological reactions
(Beutin et aL 1984; Holmes and Twiddy 1983; Ito, Kuwahara, and Yokota
1983; Shah, Kauffman, and Boutin 1982)_ A gene probe for detecting
enterotoxigenic strains was described by Hanchalay et aL (1985)_
Cholera is associated with poor sewage treatment and disposal, inade-
quate treatment of drinking water, and crowded conditions with poor
sanitation. Although it is important in Asia and Africa, it has been reo
ported only rarely in countries with proper sanitation. Cholera was not
reported in the United States from 1911 to 1973, when a single case oc
curred in Texas. In 1978, there were eight cases of cholera in Louisiana,
apparently caused by eating inadequately cooked crabs (CDC 1978).
Since 1978, cases involving V. cholerae have occurred in Florida and Texas
due to consumption of raw oysters and water, as well as foods contami
nated with the water (CDC 1981d; Johnston et al. 1983; Shandera et al.
1983). Non0:1 and toxigenic 0:1 V. cholerae were involved in these cases.
The various cases of cholera have increased interest in the presence
of V. cholerae in United States coastal waters. It has been reported in the
gulf coast from Texas to Florida (De Paola et al. 1984; Hood et al. 1983;
Roberts et al. 1982), the Chesapeake Bay (Colwell et al. 1981) and Califor
nia waters (Kenyon et al. 1984). The contamination of coastal waters
means that shellfish (oysters, clams) present the greatest risk when con-
sumed. Also, fish from these waters may contain V. cholerae.
V. PARAHAEMOLYTICUS. Although perhaps not as important as
cholera on a worldwide basis, the prevalence of this illness seems to have
increased. In 1968, V. parahaemolyticus was not listed as an etiological
agent of foodborne illnesses by CDC. In 1971, there were three outbreaks
with 370 cases and in 1972, six outbreaks with 701 cases. From 1975 to
1981 there was an average of two outbreaks per year.
The organism was recognized as a cause of illness in Japan during the
early 1950s. During the summer months, the organism accounts for a
majority of the reported foodborne illnesses in Japan.
The illness was thought to be unique to Japan. However, due to a
spreading epidemic of cholera in the early 1970s, techniques to isolate
vibrios were improved, and diarrheal stools were examined for vibrios.
It was discovered that V. parahaemolyticus was an important cause of food-
borne illness, not only in Japan, but also in the United States and other
countries.
The Illness. The main symptom is diarrhea. Other symptoms are listed in
Table 6.23.
The incubation period usually is 12 to 24 hr with a range of 4 to 96
hr. The usual duration is two to three days, but it ranges from a few hours
to ten days. If treatment is needed, fluid replacement and antibiotic med-
ication may be used.
Foods that have been incriminated in outbreaks in the United States
include steamed crabs, crab salad (made from canned crabmeat), raw
crab, processed lobster, boiled shrimp, roasted oysters, and raw oysters.
TABLE 6.23. VIBRIO PARAHAEMOL YTiCUS GASTROENTERITIS: PERCENTAGE
OF PEOPLE REPORTING SYMPTOMS IN FOUR OUTBREAKS
Outbreak
Symptom 2 3 4
Diarrhea 100 100 100 98
Nausea 46 63 51 76
Vomiting 33 59 38 74
Headache 33 46 32 25
Chills 45 71 37 10
Fever 28 34 17 26
Bloody diarrhea 3 5 1
SOURCE: CDC Reports.
The seafoods were inadequately cooked or refrigerated or there was
crosscontamination between cooked and raw products.
Besides these foods, in other countries the consumption of raw fish,
as well as such diverse foods as meat, eggs, cereal products, and vegeta
bles have been involved in outbreaks.
Etiologic Agent. The exact mechanism whereby V. parahaemolyticus causes
gastroenteritis has not been determined. When the organism is grown
on Wagatsuma blood agar (Miramoto et al. 1969), those strains isolated
from human cases exhibit a beta hemolysis, while most of the strains
from natural environments or seafoods are not hemolytic. This hemolytic
activity is called the Kanagawa phenomenon, with hemolytic strains be
ing Kanagawa positive (K +) and nonhemolytic strains Kanagawa negative
(K -). There is a close correlation of hemolytic activity and human patho
genicity.
To remain in the intestinal tract, the organisms must be able to attach
or adhere to the intestinal cells. The adherence to intestinal cells was
greater for K + than for K - strains (Carruthers 1977). In comparing K +
and K - cells, Gingras and Howard (1980) found no difference in adher
ence, while a second group of researchers (Reyes et al. 1983) found one
strain, a K-, which adhered to a significantly greater extent than the
other strains.
Invasiveness is a pathogenic factor. Both K + and K - strains pene
trated into the lamina propria of the ileum (Boutin et al 1979). They
suggested that the organism is capable of more than a superficial coloni
zation of the gut.
Craig (1972) stated that it seemed unlikely that the same molecule
would have hemolytic and enterotoxic properties. He suggested that an
enterotoxin is elaborated by the organism in the intestine. Other reo
searchers found a pathogenic strain that was not hemolytic (Sakurai et
al. 1974). They believed that a factor or factors other than a hemolysin
was responsible for the illness. Yet another team reported that antisera
to the hemolysin did not prevent accumulation of fluid in ileal loops
when challenged with living cells of either K + or K - strains (Honda et
al. 1983b). This would indicate that a substance other than the hemolysin
was responsible for this action.
A study by Bradshaw and others (1984) stated that there is no satisfac
tory evidence for a filterable enterotoxin, but that the invasive and adhe
sive traits substantiate the theory that the Kanagawa hemolysin is the
primary virulence factor.
It is not known how many cells need to be ingested to cause the ill
ness. Smith (1971) stated that 10
6
to 10
9
cells were needed, depending on
the condition of the stomach, type and quantity of food eaten, and
buffering capability of the ingested food. Accidental ingestion of 10
5
cells
caused symptoms in a laboratory worker. Using rabbit ileal loops, Twedt,
Peeler, and Spaulding (1980) found that concomitant inoculation of
other nonvirulent vibrios can increase the number of cells of V. parahae
molyticus needed to cause dilation of the loops.
Nature oj the Organism. V. parahaemolyticus is a Gramnegative, straight or
curved, facultatively anaerobic rod. It is halophilic, requiring 1 to 3 per
cent salt for growth. The cells grow in broth with 8 percent, but not 10
percent salt (Kourany and Vasquez 1975). The minimum aw is about 0.94.
Usually the pH range for growth is 5 to 11 (Twedt, Spaulding, and Hall
1969). The optimum is pH 7.6 to 8.6.
The organism is sensitive to cold temperatures, so the minimum for
growth is usually listed as 10 to 13C. The upper temperature is about
42 or 43C, with an optimum between 30 and 37C. The effect of low
temperature on the cells is related to the type of product and the temper
ature of storage. When exposed to 2C, the membrane is damaged (Van
den Broek and Mossel 1977). The presence of salt tends to protect the
cells from low temperature damage.
Smith (1971) stated that the organism has a very short generation time
of 9 to 11 min. The average generation time of four strains was 13.6 min
(Lee 1973).
The natural habitat is coastal and estuarine ocean water throughout
the world. It lives in sediment during the cold winter months, and, as the
temperature rises in late spring, the organism colonizes the water and
animal life. Direct relationships have been demonstrated between the
water temperature, abundance of the organism in the water, and out
breaks of vibrio foodborne illness. The organism has been isolated from
a variety of seafoods.
Methodology. V. parahaemolytieus, if present in a food, is usually in low num-
bers. Hence, an enrichment procedure using a broth such as glucose salt
teepol (GST) is used (FDA 1978). For chill-stressed cells, GST supple-
mented with magnesium or ferric iron salts increases recovery (MaLin
and Beuchat 1980). A loopful of the enriched sample is streaked onto
thiosulfate citrate bile salts sucrose (TCBS) agar (FDA 1978). On this me-
dium, the organism appears as round colonies 2 to 3 mm in diameter
with green or blue centers. The salt concentration (1 percent) and pH
(8.4), plus other inhibitors, suppress the growth of most organisms except
halophiles, such as V. parahaemolytieus. Sucrose-fermenting organisms pro-
duce yellow colonies. These include V. eholerae, some enterics and S.
aureus. Organisms besides V. parahaemolytieus that can form green colonies
include some vibrios, pseudomonads, and enterics.
A hydrophobic grid membrane filtration system using V. parahaemolyti-
eus sucrose agar was described by Entis and Boleszczuk (1983). They
claimed that significantly higher counts were obtained with their system
than with the FDA (1978) method.
Kourany (1983) found TCBS to be of dubious. value in certain cases
and suggested a specially supplemented trypticase soy agar for detection
of V. parahaemolytieus.
Since organisms besides V. parahaemolytieus can grow on the isolation
media, biochemical tests are needed to differentiate it from other vibrios
as well as other Gram-negative organisms.
The organism has 0, H, and K antigens. Serological typing schemes
using the K and H antigens have been devised (Beuchat 1982; Shinoda
et al. 1983).
Control. The control of V. parahaemolytieus foodborne illness should be rel-
atively simple. The organism is readily killed by heat. When inoculated
at a level of 10o/ml in shrimp homogenate, no survivors were found after
heating at 100C for 1 min (Vanderzant and Nickelson 1972). According
to CDC (1978a), for boiled seafoods, Louisiana law requires a minimum
of 7 mIll of boiling to ensure the destruction of pathogens. Delmore and
Crisley (1979) reported D values at temperatures from 49 to 55C for
nine strains of V. parahaemolytieus. At 55C, the D ranged from 0.03 to
0.24 min in sterile clam homogenate.
Although cooking seafoods will destroy the organisms, it is difficult
to change the habit in some cultures of eating raw seafood. The use of
radiation treatment of seafoods might be a solution.
Campylobacter
Prior to 1980, Campylobaeter was not listed as a causative agent of
foodborne gastroenteritis by CDC. G.jejuni was involved in five outbreaks
in 1980 and ten in 1981 (CDC 1983b).
Analysis of 8,097 fecal samples obtained from eight hospitals in var
ious areas of the United States yielded twice as many isolations of C. jejuni
as salmonellae (4.6 percent to 2.3 percent) (Blaser et al. 1983). Similar
findings were reported by other researchers (Drake et al. 1981; Svedhem
and Kaijser 1980). Stool samples of patients admitted to an Australian
hospital were examined for various potential pathogens (Cavanagh et al.
1980). The percent of stools containing these organisms was as follows:
C. jejuni, 13 percent; salmonellae, 20 percent; shigellae, 4 percent; and
parasites, 13 percent.
If C. jejuni is so prominent, why was it not listed as a cause of food
borne illness in the CDC reports prior to 1980? With the development
of simplified and selective methods to isolate C. jejuni from stools (Skir
row 1977), the number of confirmed cases has increased, and only con
firmed cases are listed by CDC. Although there are several species of
Campylobacter, the one most often associated with human distress is C.
JeJuni.
The organism is a Gramnegative, slender, curved to spiral rod. The
cells have a single polar flagellum and move with a corkscrewlike motion.
C. jejuni does not ferment or oxidize carbohydrates. Energy is ob
tained from amino acids or from intermediates of the tricarboxylic acid
cycle. It does not hydrolyze gelatin, casein, deoxyribonucleic acid, or es
culin. The organism is catalase and oxidase positive and reduces nitrate
to nitrite; most strains hydrolyze hippurate.
It is microaerophilic with 5 percent O
2
and 10 percent CO
2
optimal
for growth. The minimum temperature for growth is 32 to 35C and the
maximum is about 44 to 45C. At 42C it grows in 1.5 percent but not
in 2 percent NaCl. At 25C, 1.0 to 2.5 percent salt enhances the death
of the organism, and it is sensitive to 1.0 percent salt at 4C (Doyle and
Roman 1982a).
The minimum pH for growth generally is 5.3, but some strains grow
at pH 5.1 (Gill and Harris 1983) and one strain showed growth at pH 4.9
(Doyle and Roman 1981).
The illness is a typical gastroenteritis, with the dominant symptoms
being diarrhea and abdominal pain, usually with fever. Other symptoms
include nausea, vomiting, mucus or blood in the stool, headache, chills,
fatigue, backache, and dehydration. The illness may mimic appendicitis
(Chan, Stringel, and MacKenzie 1983; Pitkanen et al. 1983), and can cause
abortion (Gilbert et al. 1981). The illness may be severe in some patients.
Smith and Blaser (1985) reported on two fatalities associated with C. je
juni. The incubation period usually is 2 to 5 days but may range from 1
to 11 days. The duration of the illness usually is 1 to 3 days but may last
for three weeks. The organism may be excreted for over a year (Richard
son, Koornhof, and Bokkenheuser 1981). If the illness is severe, treat
ment with erythromycin and fluid replacement may be needed.
C. jejuni can invade the intestinal epithelium and cause damage to the
mucosa. In calves, AI-Mashat and Taylor (1980) found a thickening of
the wall of the ileum, inflammation of the jejunal and ileal mucosa, an
enlargement of mesenteric lymph nodes, as well as dilated capillaries in
the mucosa of the small intestine_ Invasion and lesions in the intestinal
tract also were noted with experimentally infected hamsters (Humphrey,
Montag, and Pittman 1985) and newly-hatched chicks (Welkos 1984).
Mills and Bradbury (1984) stated that little is known of the mecha-
nism of the pathogenicity of the organism. Since fever is a symptom, it
would seem to be an infection. However, the researchers have detected
toxin production in twenty-four of thirty-two strains of C. jejuni (Ruiz-
Palacios et al. 1983). Culture supernatants caused fluid secretion in rat
ileal loops, but not in rabbit loops or the infant mouse assay. It caused
elongation ofCHO cells. Cholera antitoxin prevented the toxic response,
indicating a serological similarity to cholera enterotoxin, E. coli LT, and
possibly the enterotoxins of salmonellae. These observations were con-
firmed by Klipstein and Engert (1984, 1985) and McCardell, Madden, and
Lee (1984).
Domestic and wild animals are the main reservoirs of C. jejuni. Hence,
foods derived from animal sources tend to be those involved as vehicles
for infection of humans by the organism. Drinking raw (untreated) water
is the highest risk factor (Hopkins, Olmsted, and Istre 1984). They listed
further risks (in descending order) as drinking raw (unpasteurized) milk,
having a cat in the home, and eating undercooked chicken. Besides the
ingestion of contaminated water or food, the organism can be trans-
ferred directly from animals to humans or from humans to humans (Nor-
krans and Svedhem 1982).
The exact mechanism by which milk becomes contaminated is not
known. However, milk from cows with Campylobacter mastitis might be
used. Also, according to CDC (1981e), up to 60 percent of healthy cows
shed Campylobacter in their feces. Without very careful handling, fecal
contamination of milk might occur. The incidence of C. jejuni in farm
bulk milk is about 1 to 2 percent (Lovett, Francis, and Hunt 1983).
Milk has been involved in several outbreaks of campylobacteriosis
(CDC 1981a, 1984d; Hudson et al. 1984; Potter et al. 1983; Wright et al.
1983). In a survey of raw milk, deBoer, Hartog, and Borst (1984) con-
cluded that there was no association between developing campylobac-
teriosis and consuming raw milk. They, as well as Doyle and Roman
(1982c), reported that C. jejuni survived longer in sterile milk than in raw
milk when held at 4C.
C. jejuni is quite prevalent in poultry-processing plants (LuechtefeId
and Wang 1981; Oosterom et al. 1983b; Wempe et al. 1983) and on the
carcasses of the processed poultry (Rayes, Genigeorgis, and Farver 1983;
Shanker et al. 1982). Fortunately, C. jejuni tends to die during refrigerated
holding so there is less contamination at the retail store than immedi-
ately after processing (Kinde, Genigeorgis, and Pappaioanou 1983)_ From
a survey of chicken livers, Barot, Mosenthal, and Bokkenheuser (1983)
concluded that most of the contamination with C jejuni occurred at the
processing plant.
The organism has been detected on red meats in processing plants
and at retail stores (Stern et aL 1985; Turnbull and Rose 1982)_ Koidis
and Doyle (1983) reported that C jejuni survived well in refrigerated
ground beef. The extent of survival depended upon the strain of the
organism. Conversely, Bolton, Dawkins, and Robertson (1982) and Oost-
erom et al. (1983a) found that cold storage reduced the level of C jejuni
on carcasses below detectable levels.
When food is contaminated, it is usually at a low leveL Due to temper-
ature and oxygen requirements, the organism generally does not multi-
ply in food. Hence, it is evident that low levels of contamination are suffi-
cient to cause illness in humans, although the exact number of cells
needed is not known. Only a low-level infection occurs during direct
transfer from animals or people to the recipient. A level of 90 C jejuni
was the minimum infective dose causing diarrhea in 90 percent of inocu-
lated chickens (Ruiz-Palacios. Escamilla, and Torres 1981).
Various methods have been used to detect the presence of C jejuni in
foods. Since a relatively low number of cells are in foods, an enrichment
procedure usually is needed. Selective enrichment broths are generally
a basal medium to which various supplements and antibiotics are added
to stimulate the growth of C jejuni and inhibit other organisms (Barot
and Bokkenheuser 1984; Doyle and Roman 1982a; Rogol et aL 1985;
Rothenberg, Stern, and Westhoff 1984). After incubation of the inoculated
enrichment broth in a microaerobic environment at 42C, a portion is
surface plated onto a selective agar surface_ In some cases, the sample
may be plated directly without enrichment. The selective agars tend to
be similar to the selective enrichment broths, with 1.5 to 2.0 percent agar
added for solidification (Fricker 1985; Moskowitz and Chester 1982; N g,
Stiles, and Taylor 1985; Stern 1982). After incubation microaerobically
at 42C, the inoculated surfaces are observed for typical colonies of C
jejuni.
These can be screened by microscopic observation (phase-contrast)
for motility and morphology, and then for oxidase and catalase-positive
characteristics. After these simple tests, other biological and biochemical
tests can be run (Lior 1984; Roop, Smibert, and Krieg 1984; Stern 1982).
Serological and other tests may be performed to further confirm the
presence of C jejuni (Bradbury et aL 1984; Harvey and Greenwood 1983;
Kosunen, Bang, Hurme 1984; Penner, Hennessy, and Congi 1983; Ten-
over et al. 1984, 1985; Wong et al. 1985).
The control of C jejuni enteritis is similar to that for salmonellae.
Foods derived from animal sources should be handled as though they
contain the organism. G.jejuni is not very heat resistant. In skim milk, the
D values for G.jejuni ranged from 7.2 to 12.8 min at 48C and 0.74 to 1.0
min at 55C (Doyle and Roman 1981). In ground beef, these values were
5.9 to 6.3 min at 50C and 0.2 to 0.35 min at 58C (Koidis and Doyle
1983). In autoclaved ground chicken, the D values ranged from 20.5 min
at 49C to 0.79 min at 57C (Blankenship and Craven 1982). Hence, pas
teurization of milk and cooking of beef and poultry should destroy any G.
jejuni in these products. Svedhem, Kaijser, and Sjogren (1981) suggested
heating food to at least 60C for 15 min to prevent infection. Gill and
Harris (1984) recommended minimal cooking of poultry at 190C for 20
min to eliminate G. jejuni.
The organism is more sensitive than salmonellae to gamma radiation
(Lambert and Maxcy 1984). Competitive exclusion in young chicks
worked similarly to that for salmonellae (Soerjadi-Liem, Snoeyenbos, and
Weinack 1984).
Brucellosis
The number of cases of brucellosis has generally decreased in the
United States. It has become a disease of people who handle cattle and
swine. This includes veterinarians, farmers, and packinghouse workers.
The workers in the kill department are most subject to contamination,
but workers handling the carcasses and meat also have become infected.
In 1975, there were twentyfour cases of brucellosis caused by the in-
gestion of unpasteurized dairy products, such as raw domestic milk and
imported cheese. The consumption of raw dairy products continues to
cause about 10 percent of the cases of brucellosis (Bryan 1983).
QFever
Coxiella burnetii causes Q fever, a rickettsial zoonosis, in humans. Cat-
tle, sheep, and goats are the usual reservoirs of the organism. Usually,
transmission to humans is by inhaling aerosols derived from contami
nated animal products (including excreta). The organism is a hazard to
people working in meat packing plants and handling raw meat (McKelvie
1980). Infected cows shed the organism in their milk. The pasteurization
of milk was adjusted so that the treatment inactivates G. burnetii.
Yersinia
Yersinia enterocolitica is a member of the family Enterobacteriaceae. For
most strains, the temperature growth range in nutrient broth is 1 to 44C
(Sutherland and Varum 1977), and, at 25C, the pH range for growth is
about 4.6 to 9.6 (Stern, Pierson, and Kotula 1980).
The organism produces several disease syndromes in people (enteri-
tis, terminal ileitis, mesenteric lymphadenitis, erythema nodosum, poly-
arthritis, septicemia, and metastatic abscesses in various organs). The gas-
troenteritis is characterized by diarrhea (which may be bloody),
abdominal pain, fever, and vomiting. The illness may last from two days
to several months. The illness may mimic appendicitis and, in some out-
breaks, appendectomies have been performed before yersiniosis was sus-
pected (Black et al. 1978; Shayegani et al. 1983). The organism has been
isolated from the appendices of patients (Fukushima et al. 1981).
Y. enterocolitica can be found almost anywhere in nature, but only cer-
tain serotypes are involved in human infection. These serotypes are prev-
alent in swine (Fukushima et al. 1983; Walker and Grimes 1985), and
have been isolated from dogs (Fukushima et al. 1984). Hence, although
the organism has been isolated from various animals and food products
derived from animals, these isolations are important only if they are sero-
types that can cause human yersiniosis.
The ability of the organism to grow at refrigeration temperatures,
makes refrigerated animal foods, such as milk, a potential hazard. Milk
that was either improperly pasteurized or contaminated after pasteuriza-
tion has been involved in various outbreaks (Black et al. 1978; CDC
1982c; Tacket et al. 1984).
A fermented soybean product, tofu, was a vehicle for outbreaks (Au-
lisio et al. 1983; Tacket et al. 1985b). Besides foods, the organism is found
in water and can be acquired by direct transfer to people from animals
or from other people.
Since fever and bloody diarrhea are symptoms of gastroenteritis, an
invasive type of infection similar to shigellosis would be suspected. A
plasmid is associated with invasiveness and other virulence factors (Kay,
Wachsmuth, and Gemski 1982).
Some strains of Y. enterocolitica produce an enterotoxin similar to
E. coli ST, which stimulates the activity of guanylate cyclase (Inoue et al.
1983; Rao et al. 1979). The results of Schiemann (1981) indicated that
the enterotoxin has no role in pathogenesis. Hence, the exact role of
invasiveness and enterotoxin needs further evaluation. Also, the extent
of involvement of persiniae in human illness is not known.
The methodology for Yersinia has been discussed by various research-
ers (Bercovier et al. 1984; Hill, Payne, and Aulisio 1983; and Sack 1984).
Listeria
The first human illness due to Listeria monocytogenes was recognized in
1929. The organism is present in soil and has been detected in many
species of animals. The infection of humans usually occurs orally, such as
with contaminated food, or possibly by direct contact with infected ani-
mals_ About 1 percent of humans excrete the organism_
The organism is a small Gram-positive rod with a tendency toward a
diplobacillary form_ Although it is aerobic, it grows better at reduced O
2
and increased CO
2
levels_ It is more motile at 25C than at 37C. The
cells can multiply at pH 9_0 and at 10 percent NaCl. It can grow at 4C,
which makes it a problem in refrigerated foods. It ferments a variety of
sugars, producing acid but no gas.
There are several manifestations of listeriosis, including septicemia,
(which in pregnant women can lead to abortion or stillbirth), endocar-
ditis, pneumonia, conjunctivitis, pharyngitis, cutaneous papules, and
pustules, urethritis, and meningitis. Septicemia may result in the inva-
sion of organs such as the spleen, liver, or adrenal gland. The generalized
infection may mimic other diseases such as typhoid fever or infectious
mononucleosis.
A rather large outbreak of listeriosis was described by CDC (1985a).
According to this report, the outbreak occurred in California due to con-
sumption of contaminated cheese, with eighty-six reported cases and
twenty-nine deaths. Of these twenty-nine deaths, there were eight neona-
tal deaths and thirteen stillbirths. The other eight were non-neonatal.
There are indications that forty-eight or more deaths may have occurred
in this outbreak.
There have been other recent outbreaks of listeriosis. In 1983, four-
teen persons died in Massachusetts after consuming pasteurized milk.
Although considered to be heat sensitive, the organism is believed to be
able to survive pasteurization by living parasitically within white blood
cells present in milk.
In August, 1985, soft cheese produced in Ohio was found to contain
L. monocytogenes and fortunately was recalled before any illness occurred.
Although most of the problems with Listeria have been associated with
the dairy industry, fowl are considered to be a natural reservoir of the
organism. Fortunately, most poultry products are cooked sufficiently to
kill the organisms before consumption.
Others
Although not listed as major causes of gastroenteritis, several organ-
isms produce enterotoxins. These include species of the genera Aeromo-
nas (Buchanan and Palumbo 1985; Goodwin et al. 1983; Jiwa 1983;
Ljungh, Eneroth, and Wadstrom 1982), Citrobacter (Wadstrom 1976),
Enterobacter (Klipstein, Engert, and Short 1977), Klebsiella (Klipstein, En-
gert, and Short 1977), and Kluyvera (Farmer et al. 1981). Clostridium difJicile
and its enterotoxin (toxin A) have been associated with a severe gastroin-
testinal illness in patients who have ingested certain antimicrobial agents
(Mulligan 1984; Thelestam and Florin 1984). Some strains of Clostridium
sordellii also produce an enterotoxin (Yamakawa et al. 1983). The extent
ofthe role of these organisms as agents of foodborne illness has not been
fully evaluated.
FUNGAL ILLNESSES
Fungi are a diverse group of organisms that include molds, yeasts,
rusts, smuts, and mushrooms. These organisms can cause many diseases
in plants, animals, and humans.
In humans, the fungi can cause mycoses, allergies, or toxicoses.
Mycoses are diseases resulting from the invasion of living cells by the
fungi. Allergies are diseases resulting from the development of hyper sen-
sitivity to fungal antigens. Toxicoses consist of illnesses due to ingesting
toxic fungal metabolites formed in the food (mycotoxicoses) and the
mycetisms caused by ingesting toxic fungal fruiting bodies_
Besides producing toxins, the fungi can degrade the food so that it is
deficient in certain nutrients, or the mycosis can upset the metabolism
of the animal so that a nutrient deficiency occurs_ Our concern is primar-
ily with mycotoxicoses.
Mycotoxins
Mycotoxins are secondary metabolites produced by fungi and can
cause unnatural or deleterious biological changes in plants, animals, hu-
mans or microorganisms. The mycotoxins may be contained within the
spore or fungal thallus, or they may be secreted into the growth substrate.
Mushroom poisoning was experienced by people in ancient times.
Cases of mushroom poisoning still occur in the United States and else-
where (Kendrick and Shimizu 1984).
Ergotism, or St. Anthony's Fire, was reported in the Middle Ages.
This illness is caused by eating cereals infected with Claviceps purpurea.
This fungus can grow and produce toxic alkaloids on wheat, barley, rye,
oats, and wild grasses. About forty alkaloids have been isolated from the
fungus. These alkaloids can cause constriction of peripheral blood ves-
sels with violent pain, intense burning, and gangrene of the extremities,
or they may cause convulsions and hallucinations, as well as abortion in
pregnant women_ Some of the ergot alkaloids are important therapeutic
agents (Esser and Diivell 1984; Rehacek 1984). The last major outbreak
of ergotism occurred in France in 1951. The maximum level of ergot
allowed is 0.1 percent dry weight in barley, oats, and triticale, and 0.3
percent in wheat and rye (USDA 1978).
Alimentary toxic aleukia (ATA), or septic angina, has occurred in the
Soviet Union at infrequent intervals. Symptoms of this illness include a
burning sensation in the mouth, stiffness of the tongue, and diarrhea,
nausea, vomiting, and perspiration. After these symptoms, there is a
quiescent period, followed sometime later by leukopenia, weakness,
hemorrhages of the skin and mucous membranes, necrotic areas in the
mouth, throat and skin, gangrenous pharyngitis, fever, and recovery or
death. The fatality rate varies from 2 to 80 percent.
This illness (ATA) was especially severe during World WarIl. The So
viets did not have farm labor to harvest the grain in the fall. When the
grain that was harvested during the following spring was consumed, it
caused outbreaks of AT A. Investigations revealed several toxin
producing fungi on ovewintered grain, with Fusarium predominating.
Also, during the period of World War II, rice that Japan imported
from other Asian countries was responsible for outbreaks of an illness
called "yellow rice disease," which caused several deaths. The illness was
associated with the invasion of rice by Penicillium islandicum, P. citrinum,
and P. citreoviride.
In 1929, Fleming described a substance from a Penicillium mold that
he called penicillin. During the search for other antibiotics, several sub
stances were found that are more toxic to animals and humans than to
microorganisms. Even with the finding of these toxic substances, there
was little activity in mycotoxin research until 1960, when over 100,000
turkeys died in England. An investigation revealed that the deaths were
due to peanut meal, in which Aspergillus jlavus had produced mycotoxins
called aflatoxins.
With an increased interest in fungal toxins, more than 150 species of
fungi nave been reported to be capable of producing substances that are
toxic. The number of mycotoxins has been reported as over 100 (Pollock
1983), about 200 (Wyatt 1980), and several hundreds (Stark and Demain
1980). New mycotoxins are found each year. Many genera of molds can
produce mycotoxins, but fortunately most molds produce no toxins.
Not all species of a mycotoxin producing genus, and not all strains of a
mycotoxin producing species, are toxigenic.
TYPES OF TOXINS. Apparently most mycotoxins do not affect hu
mans. However, those that might are listed in Table 6.24. A few, such as
aflatoxins, citrinin, luteoskyrin, patulin, penicillic acid, rugulosin, and
sterigmatocystin are carcinogens (Stark and Demain 1980). Although a
discussion of every individual mycotoxin is not possible in this text, it
seems desirable to investigate a few of those that might affect humans.
TABLE 6.24. SOME MYCOTOXINS THAT MIGHT CAUSE HUMAN ILLNESS
Mycotoxin
Aflatoxins
Citrinin
Cyclopiazonic acid
Ergotoxins (alkaloids)
Luteoskyrin
Ochratoxins
Patulin
Penicillic acid
Roquefortine
Rubratoxin
Sterigmatocystin
Tenuazonic acid
Trichothecenes
Deoxynivalenol (vomitoxin)
Fusarenonx
Neosolaniol
Nivalenol
Fusartoxin T-2
Zearalenone
Some Producing Organisms
Aspergillus jlavus, Aspergillus parasiticus
Penicillium citrinum, Penicillium vividicatum
A. flavus, Aspergillus tenuis, Penicillium cyclopium
Claviceps purpurea
Penicillium islandicum
Aspergillus ochraceus, P. viridicatum, Penicillium
verrucosum
Penicillium expansum, Penicillium patulum, Aspergillus
clavatus, Byssochlamys nivea
Penicillium martensii, P. viridicatum, P. cyclopium, Peni
cillium puberulum, Penicillium palitans
Penicillium roque forti
Penicillium rubrum
Aspergillus versicolor, Aspergillus nidulans
Alternaria tenuissima, Alternaria alterata
Fusarium graminearum (Gibberella zeae)
Fusarium nivale
Fusarium solani
F. nivale, Fusarium roseum
Fusarium tricinctum, F. solani, F. roseum
F. graminearum (G. zeae), F. tricinctum, Fusarium culmo
rum, Fusarium roseum
Aflatoxins. Investigations of the deaths of turkey poults in England, the
deaths of trout in a fish hatchery in the United States, and the deaths of
ducklings in Africa revealed that feeds containing a toxic substance were
involved. A feed constituent, peanut meal, and extracted fractions from
this meal showed a toxicity similar to that involved in killing the turkey
poults. Molds isolated from peanut meal included Aspergillus flavus. Frac
tions isolated from growth of A. flavus were found to be toxic and were
called aflatoxins.
A chromatograph of the extract of moldy feed was prepared and ob
served with ultraviolet light. Four fractions appeared. Two were blue and
two were green. The toxic substances were called aflatoxin B), B
2
, G), and
G2, the letters corresponding to the fluorescent color, and the numbers
denoting the relative mobility. Thirteen of these compounds are known
to occur in nature (Ciegler 1978). They all possess a coumarin nucleus
fused to a bifuran moiety. Eight aflatoxins (B), B
2
, B
2a
, MI, M
2
, PI, Ro, and
QI) have a pentene ring, while four (G
1
, G
2
, G
2a
, and G
ml
) have a sixmem
ber lactone ring. The other toxin was referred to as parasiticol (B3) (Cie'
gler 1975) and is similar to the B series, except that the pentene ring is
opened. Eight of these aflatoxins are shown in Figure 6.13.
Aflatoxin BI is the most abundant aflatoxin and is considered to be
the most toxic. When given orally to ducklings, the LDso in mg/kg for
I 1(6

B1
o
o
Furan
1(6

B
2a
11M

G
1

VJ
Coumarin
1M

G
2a
Figure 6.13. Formulae of aflatoxins. They contain a
coumarin molecule and bifuran ring.
some of the aflatoxins is 0.36 (BJ), 0.33 (MJ), 0.78 (GJ), 1.7 (B2)' and 3.5
(G2) (deWaart 1973). B2a and G
2a
are not toxic to ducklings, even when
given at very high levels. Thus, there may be justification for not calling
these substances toxins.
Aflatoxins can cause a response in microorganisms, cell cultures,
plants, and animals. There may be acute or chronic effects, depending
upon the dosage and the frequency of exposure to the toxins. The effects
of aflatoxins can be toxigenic, mutagenic, teratogenic, or carcinogenic.
The current concept is that BJ is activated by liver chromosomes to a
2,3epoxide before it can exert its carcinogenic and mutagenic effects in
animals.
The effect of aflatoxins on animals is influenced by the type of afla
toxin, species of animal, age, weight, health, and diet. Environmental
stresses may alter the susceptibility of the animal.
Very young animals are more susceptible to aflatoxicosis than are mao
ture animals. Species of animals vary in their resistance. In farm poultry,
ducklings are the most susceptible, followed by turkey poults, goslings,
and domestic chicks. For farm animals, the order from high susceptibility
to resistant is young pigs, pregnant sows, calves, fattening pigs, mature
cattle, and sheep.
Acute aflatoxicosis has been characterized by hemorrhage in tissues,
anorexia, hepatitis, and death of animals. The liver is the primary tissue
that is affected. However, the spleen, pancreas, kidneys, and other tissues
may be involved. Aflatoxin also causes fragility of the capillaries, so there
is a greater tendency for bruising (Huff et al. 1983). The clinical signs of
BJ toxicosis in goats included decreased feed consumption, some loss of
body weight, mucopurulent nasal discharge, dyspenea, coughing, leth
argy, icterus, diarrhea, and subnormal body temperature 24 to 48 hr be
fore death (Clark et al. 1984). They found that the clinicopathological
changes included increases in red blood cell count, packed cell volume,
hemoglobin concentration, serum bilirubin concentration and serum ac
tivities of aspartate aminotransferase, isocitric dehydrogenase, and or
nithine carbamyl transferase. At necropsy, goats fed aflatoxin BJ showed
evidence of ascites, pale livers, petechial hemorrhages, nasal discharge,
and icterus, as well as bile duct proliferation, hepatocytic karyomegaly,
hepatocellular degeneration, pneumonia, rhinitis, and proximal renal tu
bular nephrosis (Miller et al. 1984). Similar effects have been noted after
BJ was fed to other animals (Applebaum and Marth 1983; Ketterer et al.
1982; Wyatt et al. 1985). Reduced egg production was noted when BJ was
fed to pullets at 0.7 mg/kg body weight, or at higher levels (Exarchos and
Gentry 1982). The effect of aflatoxins on rats was reduced when cabbage
or cauliflower was included in the diet (Boyd and Stoewsand 1981; Boyd,
Babish, and Stoewsand 1982).
Aflatoxin is carcinogenic in ducklings, rainbow trout, rats, and ferrets
(Detroy, Lillehoj, and Ciegler 1971). They stated that some reports indio
cate that aflatoxin induces carcinomas in pigs, hamsters, guinea pigs,
mice, and sheep. A monkey developed a cancer due to aflatoxin inges
tion (Gopalan, Tulpule, and Krishnamurthi 1972). Although the carcino
genic activity is primarily in the liver, aflatoxin might induce tumor for
mation in other organs.
There is ample evidence that aflatoxins produce acute and chronic
illness in animals. What is their effect when ingested by humans? The
deliberate feeding of aflatoxins to human subjects has not been reported.
The only information concerning humans is from instances in which the
food, naturally contaminated with aflatoxins, has been consumed. Thus,
evidence for human aflatoxicosis is not based on scientific experiments.
Information accumulated after an incident is often incomplete.
Aflatoxin Bl was detected in specimens of stools, urine, liver, brain,
and kidney obtained from Thai children who died of encephalopathy
and fatty degeneration of the liver (Shank et al. 1971). In another out
break, caused by consuming moldy maize, 106 people died and 291
showed signs of hepatic dysfunction (Krishnamachari et al. 1975).
Attempts have been made to associate the high incidence of liver can
cer in humans in certain areas to the presence of toxigenic molds. New
berne (1974) listed evidence associating aflatoxins with cirrhosis of the
liver, as well as hepatoma, hepatitis, and Reye's syndrome. Stora, Dvor
ackova, and Ayraud (1983) further associated aflatoxin Bl with Reye's
syndrome and the deaths of five children. At autopsy, aflatoxin Bl was
detected in the liver at levels of 120 to 810 {tg/kg. They reported fatty
degeneration of the liver, kidneys, myocardium, and fibers of the striated
muscles. Wray and Hayes (1980) reported liver cancer in a patient whose
serum contained aflatoxin B1 In a study of the diets of ninety people
with liver cancer, and ninety controls in the Philippine Islands, research
ers found that the mean aflatoxin load per day was 440 percent greater
for people with liver cancer than for the controls (BulataoJayme et al.
1982). Dichter (1984) considered the current levels of aflatoxin exposure
from consuming peanuts and peanut products, and, on the basis of epi
demiological data of populations in Africa and Thailand, estimated some
fiftyeight cases of liver cancer annually in the United States. We are ex
posed to aflatoxins from other products and activities, so the rate may
be higher. There is surely sufficient evidence, if not from scientific exper
iments, that aflatoxins can cause human illness, including liver carci
noma.
Any food that becomes contaminated with spores of toxigenic A. fla
vus or A. parasiticus is capable of supporting growth of the mold and, if
held in an environment favorable for growth and toxin production, can
become a source of aflatoxin. Foods found to contain aflatoxins are listed
in Table 6.25. In the United States the main concern has been with afla
toxin on peanuts, corn, and cottonseed.
Peanuts can become contaminated with mold and aflatoxin during
growth, harvesting, or storage. Peanuts grown in soil used consistently for
peanuts tend to have a higher accumulation than when crops are rotated.
The type of soil and rainfall following dry weather influences pod split
ting of peanuts (Graham 1982). The infection of peanuts also is associ
ated with mechanical damage during or after harvesting. It is essential
that the harvested peanuts be dried to 8 percent or less moisture, and
stored under conditions so that the moisture content will not increase.
When contaminated peanuts are pressed to remove the oil, most of
TABLE 6.25. FOODS AND FEEDS FOUND TO CONTAIN AFLATOXIN
Almonds Meat
Almond paste Milk
Bakery products Millet
Barley Noodles
Beans Nutmeg
Beer Oats
Black pepper
Palm kernels
Brazil nuts Peas
Bread Peach seed paste
Capsicum pepper Peanuts
Cayenne pepper
Peanut butter
Cheese Peanut meal
Chili powder Pecans
Cocoa beans Pepper corns
Cocoa Pistachio nuts
Coconut Raisins
Coconut oil cake Rapeseed
Coffee beans Rice
Copra Rue
Corn Sesame
Corn grits Sheanuts
Cottonseed Sorghum
Cottonseed meal Soybeans
Cowpeas Soybean meal
Dried chili pepper Soy sauce
Dried fish Spaghetti
Dried milk Sunflower flour
Egg white Sunflower meal
Figs Sunflower seeds
Filberts Sweet potato
Flour Various leafy foods
Garlic Walnuts
Hazel nuts Wheat
Locust beans Wheat flour
Wine
the toxin remains with the meal cake, and only small amounts remain
with the oil. Clarification of the oil removes much of the residual toxin.
During refining, a hot alkali wash and bleaching treatment given the oil
removes most, if not all, of the remaining aflatoxin. Thus, peanut oil is
considered to be safe. However, the peanut cake or meal can be highly
contaminated.
Aflatoxin has been detected in corn prior to harvesting (Lillehoj et
al. 1984; Shotwell and Hesseltine 1983). There is a direct correlation of
insect damage to mold contamination and the presence of aflatoxin in
corn kernels (Lillehoj et al. 1980; Widstrom 1979).
Ninety percent of all corn harvested is used as animal feed, and the
remainder is used for breakfast food, grits, corn meal, starch, sugar,
syrup, and alcohol. There is little, if any, carryover of aflatoxin to the
main edible products of the wet milling process (starch, sugar, syrup).
However, the residuals (steepwater, gluten, germ) retain the toxin (Ben
nett and Anderson 1978). The aflatoxin is not carried through the distil
lation process for alcohol production (Lillehoj et al. 1978). It is found in
the spent grains that are used for animal feed.
The consumption of aflatoxincontaminated feed may result in the
deposition of the toxin in meat (Stubblefield et al. 1983; Trucksess et al.
1983), milk (Stubblefield et al. 1983), and eggs (Trucksess et al. 1983).
When dairy cows ingest aflatoxin -Bb about 2.2 percent (Patterson,
Glancy, and Roberts 1980), 1.6 percent (Price et al. 1985), or 0.6 percent
(van Dijk, O'Dell, and Bodine 1984) is converted to aflatoxin M
I
, which
is found in the milk. Aflatoxin MI is about as potent a toxin as BI (Brown
1982). Since milk is the primary diet of infants and young animals, the
presence of aflatoxin in this food can be especially hazardous. Aflatoxin
in liquid milk is concentrated about eight times when the milk is dried.
It was reported that aflatoxin MI from milk was found in cottage cheese
(Applebaum and Marth 1983), in yogurt and buttermilk (Wiseman and
Marth 1983) and in brick and Limburgerlike cheese (Brackett et al.
1982).
From the list of foods found to contain aflatoxin, it is evident that
there can be a carryover of aflatoxin from the field crop through process
ing to human food. An example of carryover is wheat to wheat flour and
then to bread, spaghetti, and noodles.
Citrinin. In 1931, a yellow compound was isolated from P. citrinum and
named citrinin (Fig. 6.14). Several species of Penicillium and a few species
of Aspergillus can produce this mycotoxin. Primarily, citrinin is a nephro
toxin, causing kidney degeneration and necrosis in various laboratory
animals, hogs, and poultry (Mehdi, Carlton, and Tuite 1984).
Citrinin has been found in various cereals and is thought to be in
volved in the yellow rice disease in the Far East. Although it does not
appear to be a carcinogen, it apparently increases tumor production in
laboratory animals by certain carcinogenic agents.
Cyclopiazonic Acid. This mycotoxin (Fig. 6.14) is produced by several Penicil
lium and Aspergillus species including P. camemberti, which is used in the
production of Camembert cheese, and A. flavus, a producer of aflatoxin.
LeBars (1979) detected low levels of this toxin in the crusts of eleven of
twenty samples, but not in the interior of four samples of Camembert
cheese. Also, the toxin was reported in twentyone of twentyseven sam
pIes of loose shell kernel fractions of peanuts at levels of 32 to 16,525
J.tg/kg and at lower levels in sound fractions (Lansden and Davidson
1983).
The effect of this toxin on humans has not been determined, but it
OH

CHs CHa
Citrinin
CHI
Cyclopiazonic Acid
COOH OH 0

CI
J---;f0
COXOH
Ochratoxin A
Patulin
Peniciliic Acid Roquefortine
o CHa H H H
OH II I I I I
OH
I

H H H H c-o
HO I I I 1/
c=c-c-C-C
1 1 I I
H H H H
Sterigmatocystin Zearalenone
Figure 6.14. Formulae of various mycotoxins.
was found to be mutagenic to two strains of Salmonella typhimurium using
the Ames test (Sorenson, Tucker, and Simpson 1984).
Luteoskyrin. This mycotoxin is produced by Penicillium islandicum. In reo
views by Mislivec (1981) and Stark (1980), it is listed as a carcinogen to
rats and mice. When given orally, it causes liver cirrhosis and hepatomas.
It binds to DNA and is mutagenic to yeast. In areas in which rice is a
major constituent of the diet, the presence of this toxin is associated with
a high incidence of liver cancer in humans.
Ochratoxin A. This is the most common and toxic member of a group of
related toxins (Fig. 6.14). It was first discovered as a product of Aspergillus
ochraceus. Also, it is produced by other aspergilli and penicillia (see Table
6.24). This toxin has been found in various grains (wheat, barley, oats,
corn), beans, peanuts, and as residues in animal carcasses. In hogs, the
residues are greatest in the kidney and declining amounts in lean meat,
liver, and fat (Madsen, Mortensen, and Hald 1982). In Denmark, hog car
casses are condemned if the ochratoxin residual in kidneys exceeds 25
!kg/kg.
The ingested toxin can cause various syndromes in many types of
animals. However, it affects pimarily the kidneys (nephrotoxin). The ef.
fect of ochratoxin A on humans has not been determined, but there may
be a relationship of renal disease of humans to the consumption of food
containing this toxin.
Patulin. This mycotoxin (Fig. 6.14) is produced by some 19 species of
fungi, including those listed in Table 6.24. Being produced by P. patulum,
it was given the trivial name patulin. Also, it has been named claviformin,
expansin, and clavicin. It inhibits a wide variety of bacteria and for a time
was used as an antibiotic. However, side effects of intestinal distress, vom
iting, and diarrhea ended its use as a therapeutic agent. The toxin is
mutagenic (Mayer and Legator 1979), terotogenic (Ciegler, Beckwith, and
Jackson 1976) and carcinogenic (Stark and Demain 1980).
This toxin has been found in fruit products, especially apple juice
(Brackett and Marth 1979a; Scott and Bullerman 1975). During the alco
holic fermentation of apple juice, patulin is converted to other chemicals
(Stinson, Osman, and Bills 1979).
PR Toxin. This mycotoxin is produced by Penicillium roqueforti, the mold
used in the production of Roquefort, blue, and Gorgonzola cheese. Scott
(1981) stated that it was one of the most acutely toxic metabolites of P.
roqueforti. It is mutagenic to bacteria and cytotoxic to various cell lines.
The toxin causes degenerative changes in the liver and kidney of rats.
When it was injected into rats, researchers observed breathing difficul
ties, motor incoordination, and flaccid paralysis (Polonelli et al. 1978).
Chen, Chen, and Wei (1982) concluded that the effects of PR toxin in
animals are increased capillary permeability and direct damage to the
lungs, heart, and kidneys. The effect on humans has not been deter
mined.
Although it is produced by P. roque forti, the toxin was not produced
in blue-veined cheese (Polonelli et al. 1978). The toxin is not stable in
cheese.
Penicillic Acid. This toxin is produced by species of Penicillium and Aspergil
lus including those listed in Table 6.24. According to the reviews of Misli
vec (1981) and Stark and Demain (1980), it causes malignant tumors in
rats at the site of subcutaneous injection. It is cytotoxic to various cell
cultures. The ingestion of a sufficient amount of penicillic acid can in
duce liver necrosis in mice and fatty liver degeneration in quails. The
effect on humans has not been determined. According to Northolt, Van
Egmond, and Paulsch (1979a), penicillic acid has only a low toxic effect.
This toxin has been isolated from corn and beans and is thought to
be a potential health hazard to humans.
RoqueJortine. This is a group of indole alkaloids (Fig. 6.14) produced by
Penicillium roqueforti and a few other penicillia. Roquefortine has been
detected in commercial samples of blue cheese at an average level of 0.8
JAg/g and as high as 6.8 JAg/g (Scott and Kennedy 1976).
From the amount of roquefortine found in blue cheese, Scott (1981)
stated that there is no potential acute human health hazard. The effect
of this toxin on humans is not known.
Sterigmatocystin. This mycotoxin (Fig. 6.14) is produced by Aspergillus versi
color and some fourteen other molds (Davis 1981; Terao 1983). This myco
toxin is structurally related to aflatoxin BJ and is mutagenic and carcino
genic. It attacks mainly the liver and kidneys, causing liver cancer in rats.
In mice, it affects the lungs and blood capillaries. It causes severe necrosis
of the kidney and the heart of monkeys. Sterigmatocystin naturally pres
ent in feed caused bloody diarrhea and death in dairy cattle (Vesonder
and Horn 1985).
In nine of thirty-nine samples of naturally moldy cheese, researchers
detected sterigmatocystin in the cheese surface at 500 to 600 JAg/kg (Nor-
tholt et al. 1980). In brown rice, sterigmatocystin was found at levels up
to 21.8 JAg/g (Takahashi et al. 1984). It has also been isolated from other
cereals and pecans (Davis 1981). So far, outbreaks in humans have not
been reported (Terao 1983). Perhaps this is due to its poor absorption
from the digestive tract.
Trichothecenes. The trichothecenes are chemically related toxins (sesquiter-
penoids) that are produced by various species of Fusarium, Cephalosporium,
Myrothecium, Stachybotrys, Trichoderma, Trichothecium, and Verticimonospor-
ium. There are more than 40 known trichothecenes. The ones found oc-
curring naturally include those in Table 6.24, deoxynivalenol (vomi-
toxin), fusarotoxin (T-2), neosolaniol, and nivalenol.
The trichothecenes were implicated in the human illness ATA in Rus-
sia. They also apparently are involved in cancer of the esophagus (Mara-
sas, van Rensburg, and Mirocha 1979). They have been involved in var-
ious symptoms and syndromes in animals such as feed refusal, vomiting,
growth depression, hyperestrogenism, intestinal tract inflammation,
bloody stools, lesions of various organs, skin lesions, tumors, and death
(Hoerr et al. 1982; Trenholm et al. 1984). These toxins inhibit the synthe-
sis of protein and DNA. Animals vary in their resistance to vomitoxin.
Without serious adverse effects, swine can ingest 2 mg/kg of feed, poultry
at least 5 mg/kg of feed, and cattle 6 mg/kg of feed (Trenholm et al. 1984).
The tricothecenes have been found in various grains and also in feeds
(Cote et al. 1984; Osborne and Willis 1984; Teich and Hamilton 1985).
Residues of ingested trichothecenes have been found in muscle tissue
(Robison et al. 1979), eggs, or milk of animals. This indicates that these
agents may be ingested by humans when consuming animal products.
Although deoxynivalenol in grain is not regulated in the United States,
the FDA has issued a "level of concern" of 2 jlg/g of whole grain, and 1
jlg/g of finished feed. It is regulated in Canada at these levels.
Zearalenone. Also called F-2 toxin, this mycotoxin (Fig. 6.14) is produced
by various Fusarium (Table 6.24). It is estrogenic, causing infertility in
animals, especially swine (Chang, Kurtz, and Mirocha 1979). No effect on
humans has been reported due to ingestion of the naturally produced
toxin. The toxin is found in various grains, but most commonly in corn
(Mirocha et al. 1979).
EFFECTS OF MYCOTOXINS. The ingestion of mycotoxins may cause
either short-term or long-term effects. Short-term or acute effects are
rapid and sometimes fatal. Long-term or chronic effects include genetic
and birth defects as well as cancer, which may occur after a number of
years of ingestion of mycotoxins_ Some effects are described in the previ-
ous section of individual types of mycotoxins. The biological effects of
mycotoxins on living systems were reviewed by Bullerman (1979) and
Hayes (1978). One study described a case of acute mycotoxicosis in which
the symptoms were a throbbing frontal headache, a feverish feeling, nau-
sea, vomiting, diplopia, weakness, bloody diarrhea, and tremors (Cole
et al. 1983). After some 30 hr the symptoms disappeared. Although not
involved in this case, other acute symptoms can include chills and skin
irritation.
The main chronic symptom is cancer. Of the mycotoxins that seem
to be carcinogenic, the toxins of primary concern are the aflatoxins.
Because foods may contain more than one type of toxigenic mold
and some of the molds can produce more than one toxin, the symptoms
of a mycotoxicosis may be a combination of multiple toxins, or there may
be synergism or antagonism among the toxins. Antagonistic reactions
between patulin and rubratoxin were described by Kangsadalampai, Sa-
lunkhe, and Sharma (1981).
PRODUCTION OF MYCOTOXINS. The ability of a fungus to produce
and accumulate toxins is dependent upon such factors as genetic poten-
tial, environmental conditions (substrate, moisture, temperature), light,
aeration (oxygen and carbon dioxide present), inhibitors, competitive
growth, and the time of contact between the mold and the substrate. Be-
ing secondary metabolites, to be produced, the mold must grow. How-
ever, growth is not synonymous with the presence of mycotoxin, since
some substrates suitable for growth are not suitable for toxin production.
After growing and producing the toxin, the mold might die and not be
recoverable from the food.
Molds can grow over a wide range of temperatures. Even cold storage
does not prevent growth or toxin production. The optimum temperature
for growth of A. flavus and A. parasiticus is 35 to 38C. However, maxi-
mum aflatoxin production occurs at lower temperatures (240 to 30C).
Aflatoxins are not produced below 8 to lOoC (Bullerman, Schroeder,
and Park 1984). The lowest temperature for growth of A.flavus was 15C
(Niles, Norman, and Pimbley 1985).
The production of ochratoxin by Penicillium cyclopium or P. viridicatum
can occur at 4 to 31C (Northolt, Van Egmond, and Paulsch 1979b).
However, the temperature range for ochratoxin production by Aspergillus
ochraceus was 12 to 37C. Reportedly, P. viridicatum grew at 5C but did
not produce ochratoxin (Damoglou, Downey, and Shannon 1984). It did
produce the toxin at lOe. More than fifty times as much ochratoxin was
produced at 25C than at 12C (Haggblom and Ghosh 1985).
According to Ciegler (1978), Fusarium tricinctum produces T-2 toxin
best near freezing temperatures, and Penicillium martensii produces peni-
cillic acid faster at 20 to 30C but accumulates more of the toxin at 4
to lOe.
In some studies, temperature cycling resulted in higher levels of toxin
production than if a constant temperature were used (Bullerman,
Schroeder, and Park 1984).
Even with other environmental conditions being optimum, the mini-
mum and optimum temperature for the production of mycotoxin is quite
variable and depends on the genus, species, and strain of mold, as well
as the mycotoxin that is elaborated.
Moisture satisfactory for growth and toxin production may be present
in foods prior to or during harvesting, before they are dried adequately.
Improper storage of dried foods can result in a moisture level adequate
for mold growth. During harvesting, damage to the protective covering of
foods increases the susceptibility of the food to invasion by fungi. High-
moisture corn is more readily damaged during harvesting than is low-
moisture corn. Most molds have a minimum a
w
for growth of between
0.70 and 0.80. The minimum aw for growth of A.flavus is 0.78 (Bullerman,
Schroeder, and Park 1984). The minimum a
w
for growth on peanuts var-
ied with the condition of this food (Diener and Davis 1970). The limiting
aw was 0.83 (broken immature kernels), 0.84 (sound mature kernels), or
0.86 (kernels from unshelled peanuts). In most cases, molds can grow in
a wider range of aw and temperature than needed for the production of
mycotoxins (Magan, Cayley, and Lacey 1984; Roland and Beuchat 1984a).
The effect of pH of the medium on aflatoxin production is related to
the type of substrate, the acids or bases used to alter the pH, and other
environmental factors. Jarvis (1971) observed that aflatoxin producers
did not grow well below pH 4.0. Maximum production of aflatoxin oc-
curs at pH 5.5 to 7.0 (Buchanan and Ayres 1975).
Substrates with a high concentration of carbohydrates favor aflatoxin
production (Diener and Davis 1969). Since plant products have a higher
level of carbohydrates than animal products, the majority of foods con-
taining aflatoxins are plant products (Table 6.24). Aflatoxins in animal
products usually are due to residues from the mycotoxins in feeds in-
gested by the animal.
Payne and HazIer (1983) reported that when used as the sole source
of nitrogen, proline or asparagine supported more toxin production
than did either tryptophan or methionine. The production of ochratoxin
increased as the protein concentration of the substrate increased (Hagg-
blom and Ghosh 1985).
Trace metals influence mycotoxin production. Both KH
2
P04 and
ZnS04 were found to be essential for growth and formation of aflatoxin
(Reddy, Viswanathan, and Venkitasubramanian 1979). Increasing the
zinc level from 0 to 10 t-tg/ml increased the amount of aflatoxin over 1,000
fold (Marsh, Simpson, and Trucksess 1975). The aflatoxin content offeed
samples correlated significantly with the zinc content (Jones, Hagler, and
Hamilton 1984). However, researchers reported that the addition of
ZnS0
4
to autoclaved soybean meal inhibited aflatoxin production (Hen-
sarling et al. 1983). The addition of sodium phytate relieved this inhibi-
tion. The production of patulin by Penicillium urticae required manganese
(Scott, Jones, and Gaucher 1984).
Various substances can inhibit mold growth and mycotoxin produc-
tion. The inhibitors include S02 (Roland and Beuchat 1984b), caffeine
(Buchanan and Lewis 1984), butylated hydroxyanisole (BHA) (Lin and
Fung 1983), potassium sorbate (Bullerman 1985), and ferulic acid (Bil-
grami, Sinha, and Singh 1981); but the most inhibitory substances seem
to be cinnamon and cloves, as well as eugenol extracted from cloves,
thymol extracted from thyme, and cinnamic acid from cinnamon (Buller-
man, Lieu, and Seier 1977; Hitokoto et al. 1980; Llewellyn, Burkett, and
Eadie 1981). Besides inhibitors, some chemicals tend to stimulate myco-
toxin production (Fanelli et al. 1983, 1984; Gareis et al. 1984; Tice and
Buchanan 1981).
Generally, molds are aerobic, so it might be expected that oxygen is
needed for growth and aflatoxin production. Reducing the oxygen con
centration or increasing the carbon dioxide or nitrogen concentration
reduces aflatoxin production (Clevstrom et al. 1983; Paster, Lister, and
Chet 1983). The inhibitory effect of CO
2
is enhanced as the temperature
and relative humidity are lowered.
The time needed for aflatoxins to appear varies with the environmen
tal factors. The toxins may appear in 24 hr, peak production may require
one or two weeks or longer and, with adverse conditions, it may not be
produced.
Competitive growth of fungi can result in inhibition of aflatoxin pro
duction. Scopulariopsis brevicaulis, Nocardia, and Aspergillus niger detoxify
aflatoxins. Rhizopus oryzae metabolizes aflatoxin (Jarvis 1971). A. chevalieri
and A. candidus reduced or prevented aflatoxin formation by A. parasiticus
(Boller and Schroeder 1974). A. niger inhibited aflatoxin production by
A. parasiticus (Misra, Sinha, and Singh 1981). The presence of T2 toxin
enhanced the production of aflatoxin by A. parasiticus (Fabbri et al. 1984).
Even in pure cultures of A. flavus or A. parasiticus, the aflatoxin level
falls after reaching a maximum. This seems to be true, even though some
strains appear to be nondegraders of the aflatoxin. Perhaps there is some
nonspecific chemical mechanism for degradation of the toxin.
METHODOLOGY. It would be an endless task to discuss all of the exist
ing methods for determining mycotoxins in food and feed. There are
certain steps involved in almost any analytical procedure. They are sam
piing and sample preparation, extraction of the toxin, removal of lipids,
cleanup, separation, and quantitation. Depending upon the product,
some steps may be excluded and additional steps such as removing spe
cific interfering substances, may need to be added.
Since mycotoxins are not distributed homogeneously, the first prob
lem is to obtain a representative sample. One report stated that a bin of
corn can be sampled adequately only while it is being filled or emptied
(Davis et al. 1980). Their reasons for errors included inadequate sample
size, biased sampling procedures, inadequate sample comminution, and
improper subsampling for analysis. The proportion of error due to sam
piing becomes greater as the aflatoxin concentration becomes lower
(Schuller, Horwitz, and Stoloff 1976). A random selection of samples is
more representative of the lot than selecting a sample from one location
(Waltking 1980).
Two types of tests have been developed for the assay of mycotoxin:
physicochemical and biological. Although some simple tests have been
suggested for screening foods and feeds for the presence of aflatoxins,
at least one research team believes that there is no reliable screening
technique (Calvert et al. 1983).
The simplest test that can be used for some commodities involves
scanning grain or seed with longwave ultraviolet light (365 nm) and
watching for a bright greenish yellow (BGY) fluorescence. For corn, there
were significant associations between kernel moisture, BGY fluores
cence, and aflatoxin (Lillehoj et al. 1983). Of the corn samples with no
BGY fluorescence, 98 percent had less than 20 ng/g (20 ppb) of aflatoxin,
the action level established by the FDA (Shotwell and Hesseltine 1981).
However, the BGY test is not intended to be a quantitative method. The
fluorescence is not due to the presence of aflatoxin, but rather to fungal
produced kojic acid. In cotton, Marsh and Simpson (1984) suggested that
the fluorescence was caused by interaction ofthe kojic acid and peroxidase
from the fiber. Bothast and Hesseltine (1975) reported the BGY test could
be used as a presumptive test for aflatoxin in wheat, oats, barley, corn, and
sorghum, but it was not satisfactory for peanuts, rice, or soybeans. Lee and
Cucullu (1978) questioned the value of the BGY test for cotton.
A fluorometriciodide method for screening corn for aflatoxin was de
scribed by Davis and Diener (1979). The system was altered to be faster
and more convenient by Davis, Guy, and Diener (1981).
Before extraction of the toxin, the sample may need some prepara
tion, such as milling or grinding, to reduce large particles. The toxins are
extracted with an organic solvent. They are soluble in methanol, chloro
form and acetone, but are only sparing soluble in water. The solvents
used for extraction depend upon the food or feed being analyzed (Whi
taker, Dickens, and Giesbrecht 1984). Further cleanup, such as removing
lipids and other interfering substances may be accomplished with col
umn chromatography systems. Then the aflatoxins can be separated by
thin layer chromatography (TLC) and detected with longwave UV light
(AOAC 1985; Shannon, Shotwell, and Kwolek 1983; Trucksess, Nesheim,
and Eppley 1984).
Confirmation of the presence of aflatoxin can be accomplished chem-
ically (Cauderay 1979; Van Egmond and Stubblefield 1981), or by mass
spectral methods (Haddon et al. 1977; Rosen, Rosen, and DiProssimo
1984). The extraction and TLC method is sensitive to 2 to 4 J!g/kg of
aflatoxin (Romer 1973).
Many variations of the physiochemical assay of aflatoxin have been
reported. In one system, a minicolumn or florisil tube is used for separa-
tion. A direct readout of aflatoxin concentration in the tube can be ob-
tained with a Velasco flu oro toxin meter, or the column can be observed
with long-wave UV light (Holaday 1981; Shotwell and Hesseltine 1981;
Velasco 1972)_
Systems using TLC have been used to detect ochratoxin A (Howell
1982; Letutour, Tantaoui-Elaraki, and Ihlal 1983), patulin (Ough and
Corison 1980), sterigmatocystin (Van Egmond et aL 1980), trichothecenes
(Trucksess, Nesheim, and Eppley 1984), and zearalenone (Howell 1982;
Swanson et aL 1984)_
Liquid chromatographic systems have been used to determine afla-
toxins in various foods (Hisada et aL 1984; Tarter, Hanchay, and Scott
1984; Yousef and Marth 1985)_ Systems using high-pressure liquid chro-
matography (HPLC) have been suggested to separate and determine the
various aflatoxins (Cohen and Lapointe 1981; Francis et aL 1982; Takeda
1984)_
Chromatographic systems (liquid, column gas, HPLC) have been de-
veloped to detect citrinin (Gimeno and Martins 1983), deoxynivalenol
(Bennett, Megalla, and Shotwell 1984; Chang et aL 1984), luteoskyrin
(Takeda et aL 1979a), ochratoxin A (Ehrlich and Lee 1984; Howell and
Taylor 1981), patulin (Gimeno and Martins 1983; Moller and Joffesson
1980), T-2 toxin (Cohen and Lapointe 1984), and zearalenone (Bennett,
Megalla, and Shotwell 1984; Chang and DeVries 1984)_
Systems using tandem mass spectrometry to detect and identify afla-
toxins have been developed (Grove, Plattner, and Peterson 1984; Platt-
ner, Bennett, and Stubblefield 1984)_ The inhibition of protein synthesis
of culture cells by T-2 toxin was suggested as a method to detect this
toxin (Thompson and Wannemacher 1984)_
Immunoassays for mycotoxins were reviewed by Chu (1984)_ A radio-
immunoassay (RIA) for T-2 toxin in corn and wheat was sensitive, accu-
rate, reproducible, and relatively simple (Lee and Chu 1981). The sensi-
tivity of the RIA was 10 ng/ml for T-2 toxin (Fontelo et aL 1983) or 5
ng/ml for zearalenone and zearalenol (Thouvenot and Morfin 1983). An
enzyme-linked immunoassay (ELISA) gave more consistent data, rela-
tively lower standard deviations, and lower coefficients of variation than
did RIA for determining aflatoxin BJ (EI-Nakib, Pestka, and Chu 1981).
An ELISA was reported to be simple, sensitive, and specific for aflatoxin
MJ in milk (Hu, Woychik, and Chu 1984), and for MJ at 10 to 50 ng/kg
(ppt) in various dairy products (Fremy and Chu 1984).
ELISA systems were effective for the analysis of ochratoxin (Lee and
Chu 1984) and T-2 toxin (Gendloff et aL 1984). Monoclonal antibodies
to certain toxins have been produced (Hunter et aL 1985; Woychik, Hins-
dill, and Chu 1984), which will aid in the immunoassay for specific toxins.
There are several systems for the biological assay of aflatoxin. In the
chicken embryo test, specific amounts of aflatoxin deposited in a fertile
egg will destroy the life of the embryo. Typical lesions develop in the em
bryo with subacute levels (less than 0.1 J.tg/kg) of aflatoxin Bj.
The effect of aflatoxin on ducklings can be used for assay purposes.
Aflatoxins inhibit cell cleavage in fertilized mollusk (Bankia setacea) eggs
without preventing nuclear division. The resultant cells are multinuclear.
Over 60 percent mortality of brine shrimp (Artemia salina) is obtained in
24 hr by 0.5 J.tg/ml (aflatoxin Bj). With 1 J.tg/ml, greater than 90 percent
mortality occurs. At a concentration of 1 J.tg/ml, aflatoxin Bj is lethal to
zebra fish larvae. With a concentration of 0.15 J.tg/ml, the water flea (Daph
nia) is killed in 40 hr. Schuller, Horwitz, and Stoloff (1976) stated that the
chick embryo bioassay is the most useful of these biological systems.
The reduction of bioluminescence of Photobacterium phosphoreum was
suggested as a possible assay system for some mycotoxins (Yates and Por
ter 1982). The cytotoxicity of trichothecenes on HEp2 and Chang cells
was more sensitive than the TLC system (Robb and Norval 1983). Swiss
mouse fibroblasts were adapted to a biological assay of triothecenes (Ab
bas, Shier, and Mirocha 1984). The sensitivity was 0.1 ng/ml for T-2 toxin
and 5 J.tg/ml for zearalenone.
The FDA requires that the presence of aflatoxin Bj in certain foods
be confirmed by negative ion chemical ionization mass spectrometry in
stead of the chicken embryo bioassay.
CONTROL OF MYCOTOXINS. The systems for controlling aflatoxins
in foods or feeds are essentially the same as for controlling any microbial
toxin. We might prevent contamination of the food by toxigenicA.flavus
or A. parasiticus and inhibit their growth and toxin production. We can
analyze food for aflatoxin and, if present, remove, destroy, or detoxify
the toxin.
Prevent Contamination. The ubiquitous nature of toxinproducing fungi
makes it difficult to grow crops without subjecting them to potential con
tamination. One solution might be the selection or development of plant
varieties that resist such contamination. Irrigation of crops may reduce
mycotoxin contamination by lowering the stress on plants caused by in
sufficient rainfall. No aflatoxin was detected in peanuts harvested from
plots treated with gypsum (Mixon, Bell, and Wilson 1984).
Prevent Fungal Growth. The fungi attack damaged seeds more readily than
they attack sound seeds. Control of insects and using care in harvesting
can reduce the number of damaged seeds. The removal of damaged seeds
and foreign material before storage will help control mold growth and
mycotoxin production.
The development of a low moisture content in the seeds, and storage
at low RH is perhaps the simplest and best method for controlling growth
and mycotoxin production. If there is improper ventilation during stor
age, localized areas can develop with sufficient moisture for fungal
growth. These areas can be caused by sweating, movement of moisture
through the food, and biological activity of seeds.
Cold storage will prevent growth of the aspergilli and production of
aflatoxin, but other toxigenic molds can produce mycotoxins at low tern
peratures (below lOC). Hence, cold storage alone is not beneficial from
an overall viewpoint.
The addition of certain chemicals will inhibit or reduce the produc
tion of aflatoxin. Davis and Diener (1967) found that soaking peanuts in
a solution of p-aminobenzoic acid reduced the production of aflatoxin
by 50 percent. Potassium sulfite and potassium fluoride also inhibited
aflatoxin production by the molds. Other effective chemicals are dis
cussed in the section on production of mycotoxins.
Destroy Organism. Spores of A. flavus do not survive a 45-sec treatment
with ultraviolet light (Bean and Rambo 1975). Gamma radiation at levels
of 0.25 to 1.0 Mrad inactivate fungi in stored products.
According to Doyle and Marth (1975), the conidia of A.flavus and A.
parasiticus strains have a D55 of 3 to 29 min and D60 of 8 to 59 sec at pH
7.0. Hence, it might be possible to heat certain foods prior to storage to
destroy the organism. However, the foods would then need to be stored
in a manner that would prevent recontamination.
Remove Toxins. Aflatoxins can be removed from food by segregating obvi-
ously contaminated grains or kernels, or by extraction of the food with
solvents. These procedures can be used only for certain foods or feeds.
The aflatoxin content of a batch of peanuts may be confined to a few
highly contaminated kernels. When peanuts are removed from the shell,
they are sorted to separate any moldy, discolored, shriveled, or damaged
raw peanuts. The aflatoxin content of peanut lots is reduced significantly
by this segregation procedure (Telford 1982). The difference in density
was used to separate contaminated from sound corn (Huff 1980), as well
as wheat (Huff and Hagler 1985).
The removal of mold growth from products such as cheese does not
remove all of the aflatoxin, since aflatoxins tend to diffuse away from
the mold mycelia. Hence, not only must the mold be removed, but also
any food material that may contain diffused aflatoxin.
Examination of seeds with UV light for greenish-yellow fluorescence,
and segregation of the fluorescent seeds, should aid in reducing the afla-
toxin content of remaining seeds. Schade and King (1984) reported that
almond kernels that fluoresce violet-purple under long-wave UV light
contain high levels of aflatoxin. By separation of these kernels, the afla
toxin content can be lowered.
For some types of foods, solvents have been used to extract the afla-
toxins_ Due to the distribution of toxin throughout the peanut, solvent
extraction of whole kernels is ineffective_ However, with crushed peanuts
or peanut meal, extraction can be used. Besides peanut meal, the extrac-
tion can be useful for other oilseed meals. Detroy, Lillehoj, and Ciegler
(1971) reviewed the systems, solvents, and results ofthe extraction proce
dures for removing aflatoxins. Activated charcoal avidly adsorbed afla-
toxin from a liquid medium (Decker and Corby 1980).
When animals eat feed containing aflatoxins, residues may be de
tected in various tissues. Researchers reported that feeding contaminated
pigs an aflatoxin-free feed resulted in a significant reduction of aflatoxin
in organs and tissues in one day, and no aflatoxin was found in any tis-
sues after four days (Furtado et al. 1982).
Inactivate Mycotoxins. Either chemical or physical treatments can be used
to inactivate or detoxify aflatoxin. Various methods of detoxification
have been reviewed by (Dollear 1969; Doyle et al. 1982).
Chemical treatments that have been reported for inactivation include
hydrogen peroxide, chlorine gas, sodium hypochlorite, benzoyl perox-
ide, sodium perborate, sodium hydroxide, ammonia, methylamine, chlo
rine dioxide, formaldehyde, nitrogen dioxide, ozone, and bisulfites.
Ammonia is effective in inactivating aflatoxins in cottonseed meal,
peanut meal, and corn (Bothast et al. 1982; Lee, Koltun, and Stanley
1984; Norred 1982). Ammoniation inactivates the aflatoxin, reduces the
carcinogenicity to rainbow trout, and does not reduce the nutritive value
of treated corn (Brekke et al. 1977). Although ammoniation reduced the
amount of ochratoxin A in barley, it did not influence the daily weight
gain or feed efficiency when fed to hogs (Madsen, Hald, and Mortensen
1983). The process is not acceptable for foods for human consumption
(Wood 1982).
Treatment of peanut meal with NaOH reduces the aflatoxin content.
A combination of calcium hydroxide and formaldehyde reacts with afla-
toxin in highly contaminated meal, so that it is acceptable for feed use
(Codifer, Mann, and Dollear 1976).
Sodium hypochlorite (NaOCI), or bleach, has been effective in inacti-
vation of aflatoxin (Draughon and Childs 1982). However, researchers
have found that the reaction of chlorine with aflatoxin BJ forms another
carcinogen (Castegnaro et al. 1981). Sodium bisulfite was more effective
than either NaOH or ammonia in inactivation of aflatoxins BJ and B2
(Moerck et al. 1980). According to Hagler, Hutchins, and Hamilton (1983)
bisulfite reacted with BJ to form BJS, but aflatoxin B2 or G2 were not
susceptible to the action of bisulfite. When vitamin C was present in
apple juice, patulin disappeared (Brackett and Marth 1979b).
Oil, as extracted from oilseeds (peanuts and cottonseed) may contain
small amounts of aflatoxins. The oil is treated with alkali and bleach to
purify it. This processing reduces the aflatoxin content from 812 p.g/kg
to less than 1 p.g/kg (Kensler and Natoli 1969).
The primary physical treatment used to reduce toxicity of aflatoxins
is heat. Roasting peanuts at IS00C for Y2 hr reduces aflatoxin B1 about
80 percent and B2 about 60 percent (Lee, Cuculla, and Goldblatt 1968).
Waltking (1971) roasted peanuts at 204C and found an average loss of
40 to SO percent aflatoxin B1 and G1 and 20 to 40 percent of B2 and G2.
Oil roasting of peanuts at 163C for 3 min reduces B1 and G1 by 6S per-
cent (Lee et al. 1969).
Roasting soybeans at 180C reduced levels of aflatoxin from 40 to 73
percent (Hamada and Megalla 1982). Normal cooking of rice destroyed
about 49 percent of the aflatoxin, while pressure cooking with-xcess
water reduced aflatoxins by 81 percent (Rehana, Basappa, and Murthy
1979). EI-Banna and Scott (1984) found that cooking fava beans or wheat
reduced ochratoxin A by only 16 to 20 percent. About 20 percent of
ochratoxin was lost by frying pork at IS00 to 160C (Josefsson and Moller
1980). Aflatoxins and at least some other mycotoxins are considered to
be heat stable.
Biological systems might be used to degrade aflatoxins (Mann and
Rehm 1976). Corynebacterium rubrum, Aspergillus niger, Trichoderma viride,
and Mucor ambiguus degrade aflatoxin B1 to aflatoxin Ro. Before such sys-
tems can be used in industry, much more research is needed, as well
as approval by the FDA. Aflatoxin Ro reportedly is less toxic than B1 to
ducklings, but is carcinogenic to trout.
Control by Regulation. The production of aflatoxin by toxigenic molds can-
not be controlled by a regulation of a government agency. However, it is
possible to regulate against the use of food or feed if it contains aflatoxin.
Aflatoxins are considered to be carcinogens. The Delaney clause to
Section 409 of the Federal Food, Drug and Cosmetic Act prohibits estab-
lishment of a tolerance for additives that "induce cancer when ingested
by man or animals."
Instead of tolerances, the FDA has used administrative guidelines, the
acceptable level depending upon the ability to detect aflatoxin. In 1964,
a control program was established requiring the analysis of shelled pea-
nuts by a certified laboratory. Peanuts with more than 40 p.g/kg (ppb)
could not be used in feed. In 1965, this was lowered to 30 p.g/kg, and in
1969, to 20 p.g/kg. Present methods enable the detection of aflatoxin at
levels of 2.S p.g/kg or less. Ideally, there should be no aflatoxin, but realis-
tically this would eliminate a large amount of food from an already over-
taxed food supply. In 1975, the FDA proposed a formal tolerance of IS
J1.g/kg for aflatoxin in peanuts and peanut products, but after more than
ten years, this lower level has not been adopted. Dichter and Weinstein
(1984) estimated that lowering the tolerance level to 15 J1.g/kg would be
cost effective, and that lower levels of 10 or 5 ppb would not be.
Besides peanuts and peanut products, FDA surveillance activity is di
rected at any food that might contain aflatoxin and action is taken
against any food or animal feed with 20 J1.g or more aflatoxin per kilo-
gram. There is an administrative guideline of 0.5 ppb for aflatoxin Ml in
fluid milk in interstate shipment. This lower level was set because milk
is consumed in large quantities by children. For laboratory animals in
toxicology studies, the FDA and Environmental Protection Agency spec-
ify that diets contain less than 5 ppb aflatoxin. Animal feed is included
in the guidelines because aflatoxins consumed by animals may be found
in the resultant food (milk, meat, eggs). However, during years when corn
has contained levels of aflatoxin above 20 ppb, the FDA has made excep-
tions in certain cases (Hamilton 1984; Labuza 1983).
In Australia, the permitted levels of aflatoxin are 15 ppb for peanuts
and 5 ppb for other foods (Pitt 1982). The only other restrictions are
various tolerance levels of deoxynivalenol (vomitoxin) in wheat in Can-
ada (Morrison 1982a, 1982b), and ochratoxin A in the kidney of hogs in
Denmark.
The continual surveillance and the elimination of aflatoxin-
contaminated products from the food or feed supply constitutes a system
of controlling these toxins.
VIRAL HEALTH HAZARDS
For most foodborne illnesses caused by bacteria, the organisms must
proliferate in the food for illness to occur. Viruses that cause human
illness cannot multiply in food. Thus, a virus that contaminates a food
either survives or becomes inactivated. This means that viruses tend to
be present only in very low concentration. This has made it difficult to
detect viruses in foods. The isolation and identification of viruses require
special techniques.
Bryan (1973) listed four viral diseases in which there is evidence of
foodborne transmission. These diseases are infectious hepatitis, polio-
myelitis, Bolivian hemorrhagic fever, and Russian spring-summer en
cephalitis. He also listed eight viral diseases that might be foodborne, but
proof is lacking. These include reovirus infections, serum hepatitis, and
nonbacterial gastroenteritis. Cukor and Blacklow (1984) stated that only
two viruses, rotavirus and Norwalk virus, are important etiological agents
of human gastroenteritis. Besides gastroenteritis, enteroviruses may in-
fect other systems and cause paralysis, encephalitis, menigitis, pleurody-
nia, myocarditis, or herpangia (Moore 1982; Phillips et aL 1980)_
There were thirty-three confirmed outbreaks of viral foodborne ill-
ness from 1977 to 1981 (CDC 1983b)_ This represents only a small part
of the total outbreaks of foodborne illness. However, many viral illnesses
are neither reported nor confirmed. According to Cukor and Blacklow
(1984), acute viral gastroenteritis is second in frequency only to the com-
mon cold. Estes and Graham (1979) stated that epidemic viral gastroen-
teritis is the leading cause of mortality among infants and children in
underdeveloped countries.
To cause an illness, the ingested viruses must be able to survive the
acidity of the stomach, digestive enzymes, and also bile in the duodenum.
The number of viruses needed for infection is not known. However,
swine became infected with ingestion of only 250 plaque-forming units
(PFU) (Cliver 1981)_ In many cases, little is known as to which host cells
are involved, how the viruses pass through the mucous membranes, or
other factors that influence the initiation or spread of the infection.
Viruses causing human diseases have been isolated from various do-
mestic animals. In some cases, the same virus was isolated from animals
and from people who had close contact with the animals. Human and
animal viruses have been recovered from raw and heated milk, dairy
products, meat, eggs, oysters, mussels, clams, and crabs (Goyal, Gerba,
and Melnick 1979; Larkin 1981; Wait et aL 1983). Raw or only partially
cooked foods are those primarily implicated as vehicles for virus trans-
mission. The usually long incubation periods and problems of isolating
viruses has made it difficult to implicate food in viral diseases.
The first viral illness reported to be transmitted by foods was polio-
myelitis. Raw milk was the predominant food vehicle for this illness.
Viruses also are found in sewage and polluted water. They can con-
taminate various inanimate objects, which can act as sources to contami-
nate other items, such as food.
Survival of Viruses
Mahl and Sadler (1975) studied the survival of various viruses on
hard-surfaced inanimate objects (glass, stainless steel, tile). Although inac-
tivation was observed, some viruses persisted for eight weeks at room
temperature (25C) and low RH (3 to 7 percent). At 37C and 55 percent
or 93 to 96 percent RH, survival time varied from one day to eight weeks,
depending upon the virus type.
The stability of viruses in foods is determined by the type of virus,
the moisture and pH of the food and the temperature of storage. Enteric
viruses survive longer than influenza viruses (Cliver, Kostenbader, and
Vallenas 1970). At low temperatures, poliovirus is extremely stable in
food with pH 7.0 or greater. As the temperature of storage is increased
from 4C, the inactivation also is increased. They found that enterovi-
ruses persisted in low-moisture food for over two weeks at room temper-
ature, and more than two months in the refrigerator.
Herrmann and Cliver (1973a) inoculated ground beef and Thuringer
sausage with a coxsackie virus_ When ground beef was held at 4C, about
85 percent of the viruses survived for eight days, but less than 1 percent
survived for fourteen days. Kantor and Potter (1975) reported that a
poliovirus and echovirus persisted in high numbers and were virtually
unaffected during the commercial production of salami and cervelat sau-
sages.
Larkin, Tierney, and Sullivan (1976) irrigated lettuce and radish crops
with sewage sludge and sewage effluent inoculated with poliovirus. This
virus persisted for up to thirty-six days on these crops. They suggested
that a cycle of infection could be established if contaminated effluent or
sludge were used on crops in the food chain. Enteric viruses survived at
least five months in food stored at - 20C.
Inactivation
Viruses can be removed from materials or be inactivated, by essen-
tially the same systems used to control bacteria. The action of sunlight,
water-treatment processes, physical and chemical inactivation by sus-
pended and dissolved materials, bacterial antagonism, as well as simple
dilution, serve to decrease the viral concentration in water. In a rather
extensive survey, viruses were not detected in chlorinated drinking water
(Clarke et al. 1975).
Just as viruses (bacteriophage) can affect bacteria, certain bacteria
may inhibit some viruses. Hydrogen-peroxide-generating bacteria are an-
tagonistic to poliovirus (Klebanoff and Belding 1974), culture filtrates of
Proteus mirabilis contain a substance inhibitory to Sindbis and vesicular
stomatitis viruses (Mahdy and Bansen 1974), and proteolytic bacteria in-
activate coxsackie virus type 9 (Herrmann and Cliver 1973b).
Viruses can be inhibited or inactivated by various chemical agents,
such as !3-propiolactone, guanidine hydrochloride, quaternary ammo-
nium, phenolic and iodophor compounds (Gaustad, McDuff, and
Hatcher 1974), ozone (Burleson, Murray, and Pollard 1975), glutaralde-
hyde (Saitanu and Lund 1975)_
Viruses are more resistant to gamma radiation than are other types
of microorganisms. Considering the high doses needed, the use of radia-
tion to inactivate viruses is not practical.
Thermal inactivation seems to be an acceptable and practical method
of controlling viruses in food. Many viruses are inactivated in 5 min when
heated to 65C. Therefore, it is sometimes assumed that properly cooked
or pasteurized foods are not a concern of public health unless the prod
uct is recontaminated. However, researchers have reported that the time
used to cook crabs may not be sufficient to inactivate viruses (DiGiro-
lamo et al. 1972). Filppi and Banwart (1974) inoculated ground beef with
poliovirus type 1 and heated the mixture at various temperatures. From
their data, it is evident that meat, when highly contaminated with a po-
tentially pathogenic virus, might be a source of infection, even if heated
to 70C. Other researchers found no surviving poliovirus 1 or coxsackie
virus B-2 in hamburgers broiled to either 71C or 76.7C (Sullivan et al.
1975).
Cliver (1971) reported that inoculated polio 1 virus was not com-
pletely inactivated in oysters by stewing, frying, baking, or steaming. Pas-
teurization treatments, such as in egg products or milk, cannot be relied
upon to destroy large numbers of heat-resistant viruses.
Methodology
The analysis of food for viruses requires techniques different from
those used for bacterial enumeration. Viruses are determined by inocula-
tion into host cells for enumeration, by observation with an electron mi-
croscope, or by immunological methods, either by determining viruses
directly or by determining antibody produced by a host in response to a
viral infection.
Since viruses, when present, are usually at a low level in food, a large
quantity must be analyzed. After making a suspension of the food sam-
ple, the larger food particles are removed by filtering through glass wool
or cheesecloth, or by slow centrifuging (Tierney"et al. 1973). After re-
moval of the food, the viruses are concentrated. This is accomplished by
membrane adsorption, adsorption to a precipitable salt, filtration, cen-
trifugation, electrophoresis, or dialysis (Guttman-Bass and Nasser 1984;
Sullivan et al. 1984).
To determine viruses in the concentrate, the plaque-forming units
that develop on a susceptible tissue culture are counted. Bacteria present
in food can interfere with the growth of cells in a tissue culture. There-
fore, if they are not removed, they must be inhibited. This is accom-
plished by adding antibiotics that inactivate the bacteria but not the tis-
sue culture or viruses.
Due to the cell specificity of viruses, the more types of tissue culture
cells that are used, the more likely that most types of viruses can be de-
tected and isolated.
In some cases, procedures using the electron microscope have been
used to detect viruses, viruslike particles, or virus-antibody complexes_
Serological systems for the detection and identification of viruses in-
clude fluorescent techniques, RIA, hemagglutination inhibition, immu-
noperoxidase, counterimmunoelectrophoresis, latex agglutination tests,
enzyme immunoassay, immunocytochemical and ELISA (Angarano, Lad-
dago, and Materi 1984; Herrmann, Hendry, and Collins 1979; Sambourg
et aL 1985; Smith 1985)_
Diseases
Although several human illnesses are caused by viruses, only those
that may be considered a foodborne illness are discussed_ These illnesses
were reviewed by Gerba, Rose, and Singh (1984)_
HEPATITIS. There are three types of viral hepatitis: hepatitis A, hepati-
tis B, and hepatitis non-A, non-B. Hepatitis A, sometimes called infec-
tious hepatitis, is caused by a picornavirus which is transmitted by the
fecal-oral route. The virus can be grown in cell culture (Deinhart and
Gust 1982). The illness is not chronic, and no chronic carriers are known.
Hepatitis B (serum hepatitis) is transmitted through blood or blood prod-
ucts or close contact with a hepatitis B-positive person. Hepatitis B may
result in a persistent or chronic infection. This virus has not been grown
in cell culture (Deinhart and Gust 1982). In 1983, the total number of
cases of hepatitis reported in the United States was 56,469. Of these cases,
38 percent were hepatitis A, 43 percent hepatitis B, 6 percent hepati-
tis non-A, non-B, and 13 percent unspecified (CDC 1985b). This was the
first year that the incidence of hepatitis B was greater than that of he pat i-
tis k
Since hepatitis A virus is spread by the fecal-oral route, waterborne
and foodborne outbreaks can occur. People with the infection who han-
dle food are a primary source for spreading the virus. Even then, only
about 6 percent of the cases are foodborne or waterborne (CDC 1985b).
The eating of raw shellfish (oysters, mussels, or clams) is an important
source of the hepatitis virus. This is due to the potential contamination
of water with raw sewage and the collection and concentration of the
virus by the shellfish. However, since infected food handlers are a pri-
mary source, various foods have been implicated as vehicles of the virus.
These foods include dairy products, meats, salads, bakery goods, and
fruits, which were contaminated during preparation and received no fur-
ther cooking prior to serving.
A series of outbreaks of hepatitis A due to eating raw clams was de-
scribed by CDC (l982d). Some 6 to 72 hr after consumption, gastrointes-
tinal distress (diarrhea, abdominal cramps, nausea, vomiting), which
lasted from one to three days, was experienced. From twentyone to thirty
seven days after ingestion of the clams, symptoms of hepatitis A were ev
ident. These symptoms include bile in urine (dark urine) and jaundice. In
an outbreak resulting from a family reunion of forty five people, four
teen cases of clinical hepatitis were identified from nineteen to fortyfour
days. The illness may be rather mild, allowing cases to be ambulatory. In
other cases, liver injury occurs, resulting in cirrhosis. A severe infection
can cause death.
During hepatitis, the level of serum glutamic-oxalacetic transaminase
(SGOT) or serum glutamic pyruvic transaminase (SGPT) is at least twice
as high as the normal laboratory standard, and there is a total level of
serum bilirubin over 2 mgllOO ml. Also, immunoglobin M hepatitis A
antibody (IgM anti HAV) is produced (CDC 1981b). This antibody usually
appears in the serum before or shortly after the onset of symptoms and
persists for two to three months. Detection of the antibody can identify
early and subclinical cases. A team of researchers developed an ELISA
system to detect IgM anti HAV in serum (Locarnini et al. 1979).
The hepatitis A virus is present in the feces some two weeks prior to
symptoms.
The disease can be attacked from four areas. First, people who have
been exposed or might be exposed to the virus can be given immune
serum globulin. This has been an effective deterrent for the secondary
spread of the illness. Second, the beds from which seafoods, such as shell
fish, are gathered should be clean and not polluted with raw sewage.
Harvested shellfish should not be eaten raw. However, there are some
people who insist on consuming these animals in the uncooked state.
The third means of control is to inactivate the virus in a food product
by heating, radiation, drying, or chemicals. Unfortunately, little is known
about the exact treatment to inactivate hepatitis virus A. Clams added to
a pot of boiling water, heated until they opened and then eaten, were
incriminated in an outbreak of hepatitis. This virus evidently is not inac-
tivated by limited heating. However, adequate cooking should inactivate
the virus.
Inasmuch as most of the foodborne outbreaks seem to be caused by
a food handler contaminating a food with no further cooking, the fourth
area of control is the most important. The infected food handler must
be controlled. This is not easily accomplished, since the virus may be
excreted in stools during the incubation period, from seven to ten days
prior to the onset of symptoms and by asymptomatic persons. However,
those clinically ill persons should not be allowed to work with food until
they are clearly convalescent. Employees can be screened by the analysis
of the SGOT or SGPT levels of their serum. If the levels are more than
twice the normal values, then the employee should not work with food.
Better personal hygiene of food handlers, including more frequent hand
washing and less handling of food and ingredients, would help prevent
the spread of not only infectious hepatitis, but also other illnesses.
ROTA VIRUS. Human rotavirus is a major cause of gastroenteritis in
infants and young children throughout the world (Albert, Bishop, and
Shann 1983; AlNakib et al. 1980; Coiro et al. 1983; Mata et al. 1983; Oishi
et al. 1979; Paniker, Mathew, and Mathan 1982; Rodriguez et al. 1980).
The illness is most prevalent and severe in children from six months to
two years of age. After the second year of life, rotavirus infections requir
ing hospitalization decline significantly. By age five, most children have
acquired serum antibodies to rotavirus.
The incubation period is two to four days, and the symptoms include
diarrhea, vomiting, fever, and abdominal pain. The duration of the ill
ness is two to ten days. Due to loss of fluids and severe dehydration,
death can occur, especially in children less than one year old. In temper
ate climates, the incidence of the illness is highest during the winter and
is virtually absent in the summer. This is the opposite of salmonellosis,
which is more prevalent during the summer. Some possible reasons for
wintertime prevalence have been suggested (Brandt et al. 1982). In tropi
cal areas, the incidence of rotavirus tends to be influenced by rainy or
dry seasons.
The transmission of rotavirus appears to be by the fecal oral route;
however, the respiratory route also has been suggested (Foster et al. 1980;
Oishi et al. 1979).
Adults that contact infected infants often become infected with rota-
virus as evidenced by serological tests. Most adults have only mild symp-
toms or are asymptomatic; however, a few may have a severe illness.
NORWALK VIRUS. This name was given to the causative agent in-
volved in an outbreak of nonbacterial gastroenteritis in Norwalk, Ohio,
in 1968. Particles 27 nm in size were observed in stool filtrates with the
aid of electron microscopy. Similar particles have been observed in other
outbreaks. Other characteristics of the viruses were described by Cukor
and Blacklow (1984). According to Dolin (1978), six agents (Norwalk,
Hawaii, Montgomery County, W, Ditchling, Cockle) are all similar, but
there are multiple antigenic types. At the present time, Norwalk virus is
the most prominent. None of these agents has been grown on cell cul-
tures.
Foodborne, waterborne, and persontoperson (both primary and sec-
ondary) modes of transmission occur (Goodman et al. 1982; Kaplan et al.
1982). The illness appears during all seasons and affects all age groups.
The incubation period is about 30 to 36 hr, and the duration of the illness
is 24 to 48 hr. The illness is rather mild, with symptoms including diar
rhea, nausea, vomiting, cramps, headache, and fever. Foodborne out
breaks have involved green salad (Griffin et al. 1982; Lieb et al. 1985), raw
oysters (Eyles, Davey, and Huntley 1981; Gunn et al 1982), cake frosting
(Kuritsky et al. 1984) and chicken sandwiches (pether and Caul 1983).
Control
The control of viral infections involves essentially the same effort as
other foodborne illnesses. There is a need for harvesting shellfish only
from satisfactory waters and practicing good personal hygiene.
The viruses do not multiply in food and tend to become inactivated
during storage. They can be inactivated with chemical and physical
agents in a manner similar to bacteria.
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7
Indicator Organisms
Indicator organisms have been used since 1892, the year Schardinger
tested water for what is now called Escherichia coli, instead of testing for
Salmonella typhi. It is difficult to detect S. typhi in a water supply. The pres
ence of E coli indicates that there might have been contamination from
sewage and that Salmonella or other intestinal pathogens might be
present.
Since the original studies, indicator organisms have been used to de
termine an objectionable microbial condition of food, such as fecal con
tamination, the presence of potential pathogens or potential spoilage of
foods, as well as the sanitary conditions of food processing, production,
or storage. Tompkin (1983) stated that the reason for using indicators is
to monitor raw materials, processing conditions, and products at various
stages of processing and distribution to forewarn of potential microbio-
logical problems.
According to Stadhouders, Hup, and Hassing (1982), the organisms
used to assess foods for the possible presence of certain organisms capa-
ble of causing foodborne illness should be called index organisms. How-
ever, these researchers stated that for many foods, a distinction between
indicator and index organisms is not possible. Mossel (1983) called these
indicator organisms "marker organisms," but in this text, they will be
called indicator organisms.
Certain criteria are used to determine the value of an indicator or-
ganism. If such an organism is to indicate possible fecal contamination
or potential pathogens, it must be associated exclusively with feces or
closely allied with the pathogens.
The indicator should not be present as a natural contaminant of the
material being analyzed. If it is usually present on a food product, its
detection would not necessarily indicate the presence of either fecal rna
terial or pathogens. Hence, for these two criteria, knowing the sources
of the indicator organisms is important.
The organism should be easy to grow and differentiate. There should
be a relatively simple, accurate, rapid, and standard test to enumerate the
indicator. If the procedure to determine the indicator is more difficult or
less accurate than that for pathogens, then we might as well analyze for
the pathogens.
371
If the indicator multiplies very rapidly at storage temperatures of the
food, the actual degree of contamination may be obscured. If it dies very
rapidly, its value as an index of contamination is lowered. The indicator
organisms should withstand processing treatments of the food in a man
ner similar to pathogens. If the indicator is less stable, it may disappear
before the pathogen. If it is more stable, it may persist long after the
pathogen is destroyed.
Many organisms or groups of organisms have been suggested as indio
cator organisms. Fecal microorganisms include the enteric bacteria, vi
ruses, and protozoa. In general, viruses and protozoa are more difficult
to enumerate than are bacteria. Bacteria such as coliforms, E. coli, Enter
obacteriaceae, enterococci, pseudomonads, clostridia, staphylococci, and
the aerobic plate count have been suggested as indicator organisms. How
well do these bacteria or groups of bacteria meet the general criteria
needed for a good indicator? We need to know the usual source, the
association with enteric pathogens, methods for enumeration, growth
conditions, survival during storage and processing, and the significance
of these organisms in foods or a particular food.
COLIFORMS
As a result of the successful use of coliforms as indicators in water,
they have been employed as indicators of possible fecal contamination
of foods.
Whenever organisms are given a group terminology, there is a ques
tion as to which organisms are included, or at least a definition is needed.
Coliforms include all aerobic and facultatively anaerobic Gramnegative
nonsporeforming bacilli which ferment lactose with gas formation within
48 hr at 35C. For techniques that are used in the membrane filter
method for water analysis, an alternative definition is that coliforms in
clude all organisms that produce a colony on eosin methylene blue agar
with a goldengreen metallic sheen within 24 hr of incubation at 35 to
37C.
With the procedures of the U.S. Food and Drug Administration (FDA
1978), the definition of a presumptive coliform is an organism that pro
duces gas in lauryl sulfate tryptose (LST) broth in 48 hr at 35C. A con
firmed coliform is a presumptive coliform that produces gas in brilliant
green lactose bile (BGLB) 2 percent broth in 48 hr at 35C.
The coliform group includes Escherichia coli, Citrobacter jreundii, Enter
obacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae. There are a
few strains of other species that ferment lactose and might be included
in a coliform determination.
INDICATOR ORGANISMS 373
Sources
Coliforms are common inhabitants of the intestinal tract of people
and animals. However, bacteria in this group are of both fecal and nonfe-
cal origin.
The organisms are found in undisturbed soil and can be at high levels
in fecal-contaminated soil. Under certain conditions they tend to die in
the soil, but with proper nutrients and moisture they can increase in
number. Soil dust can disseminate coliforms into the atmosphere. Rain
carries the surface contamination from soil to streams, rivers, or lakes.
Coliforms are found on all types of plant material (foliage, roots, flow-
ers). Klebsiella predominated in samples obtained from forest environ-
ments and from fresh farm produce by Duncan and Razzell (1972). Most
of the fresh vegetables these reseachers examined had coliform counts
of 10
6
to 10
7
per gram_ All thirty samples of lettuce examined by Kii-
ferstein (1977) contained coliforms (from less than 10 to over lOs/g).
Coliforms are found on the shells of freshly laid eggs and can pene-
trate through the pores if the egg surface is damp. The organisms can be
found in raw milk through contamination of the teat canals by feed or
manure. Coliforms cause infection of the mast cells (mastitis) of cattle,
from which they can be shed into the milk. Milk can be contaminated
after milking by air, people, or equipment.
Coliforms are present on the feathers of live poultry and on the hide,
hooves, and hair of other animals. Upon slaughter, the organisms can
contaminate the meat from these sources or from intestinal leakage dur-
ing evisceration.
Shellfish grown in polluted areas concentrate the organisms so that
they are contaminated at levels higher than that of the water.
Coliforms are widespread and can be detected on many types of food
products, especially those of animal origin.
Methods for Detection
Many methods have been used to detect coliforms. The fermentation
oflactose on or in a medium is the primary requirement for an organism
to be considered a coliform.
The procedure suggested by FDA (1978) consists of using the MPN
method by inoculating tubes of LST broth. Growth from those tubes
showing gas production (due to fermentation of lactose) after incubation
at 35C for 48 hr is inoculated into tubes of BGLB (2 percent) broth_
These inoculated tubes are incubated at 35C for 48 hr and observed for
gas production (due to fermentation of lactose). The production of gas
is considered to be confirmed positive for coliforms.
Many of the reported coliform investigations are performed by plat-
ing samples with violet-red bile (VRB) agar. Higher counts are often ob-
tained using an agar, since anaerogenic strains will be counted_ These
strains are not positive in the MPN tube test. Not all of the organisms
that ferment lactose on VRB agar prove to be coliforms_ This may be
because of lactose-fermenting organisms other than coliforms that are
not inhibited by the selective agents (bile salts and dyes), because the
carryover of food material may reduce the inhibition of the selective
agents, or because the sampled food can furnish other carbohydrates be-
sides lactose for the organisms to ferment. These factors also can affect
the results of the MPN test. Processed food material often contains
stressed coliforms that require a noninhibition medium for repair_ Hart-
man, Hartman, and Lanz (1975) suggested using a VRB2 agar to increase
counts of stressed coliforms in foods_ A similar procedure enumerating
injured coliforms was suggested by Ray and Speck (1978)_ They plated
the sample with trypticase soy agar and overlayed the surface with VRB
after a 1-hr incubation at room temperature_ For acid-injured coliforms,
Reber and Marshall (1982) reported higher counts with the VRB2 system
than with VRB_ A petrifilm VRB system compared favorably with the
VRB agar plates and the MPN method for enumerating coliforms (Nel-
son, Fox, and Busta 1984)_
An enriched sulfate-aniline blue medium was reported by Wright
(1984) to yield higher coliform and E coli counts than did media contain-
ing bile salts_ To overcome the natural variations in bile salts media, a
chemically defined minerals modified glutamate medium (MMGM) was
developed_ Other researchers reported that MMGM was superior in sen-
sitivity and favorable in specificity to other media tested, including LST
and BGLB broths (Abbiss et aL 1981)_
The membrane filter (MF) technique is used to enumerate coliforms
in water. After filtering a standard volume of water, the filter pad is
placed directly on a differential Endo-type medium for incubation, or a
preliminary enrichment in LST broth can be used_ One advantage of
the MF system is that organisms can be concentrated from low-density
materiaL Inhibition of coliform growth and interference in the produc-
tion of the typical sheen due to growth of non coliform bacteria has been
observed (Burlingame et aL 1984; Clark 1980). Anaerobic incubation re-
portedly overcame part of this problem (Franzblau et aL 1984).
Besides water analysis, the MF technique can be used for certain food
products. This would be especially advantageous if a component of the
food is a carbohydrate that could be fermented by organisms other than
coliform organisms, since filtration will remove these carbohydrates.
The hydrophobic grid membrane filter system to enumerate col-
iforms from food has been adopted as official first action by AOAC (En tis
1983). Resuscitation of injured organisms is accomplished by placing the
filter with food sample onto tryptic soy agar supplemented with 0.15 per-
cent magnesium sulfate (Brodsky, Boleszczuk, and Entis 1982).
The impedimetric system has been adapted for use in determining
coliforms in water and food (Firstenberg-Eden et al. 1984; Strauss, Mala-
ney, and Tanner 1984). Although this system is more rapid than the MPN
system, Tenpenney, Tanner, and Malaney (1984) suggested that it is more
expensive and the results deviated considerably from those obtained
with the MPN system.
Growth and Survival
Coliforms include psychrotrophic types capable of multiplying at 3
0
to 100C, or refrigerator temperature. They grow well on most carbohy-
drates and proteins.
Members of the coliform group may persist in water, soil, or foods
for long time periods. Usually, the organisms are not able to withstand
the pasteurization of milk. Therefore, the presence of coliforms in pas-
teurized milk indicates either improper pasteurization or recontamina-
tion after the heat treatment.
The organisms are rather hardy under natural conditions and resist
drying. Coliforms do not withstand the rigors of freezing or frozen stor-
age very well. The ultraviolet light in sunlight is bactericidal to coliforms.
Significance of Coliforms
There are good and bad aspects of using coliforms as indicator organ-
isms. Since members of this group are found in the intestines and dis-
charges of people and animals, their presence could be due to fecal con-
tamination, and enteric pathogens could also be present. Unfortunately,
since some members have nonfecal origins, the presence of these mem-
bers in a food would not indicate fecal contamination or potential path-
ogens. One can rationalize this predicament by saying that using nonfe-
cal coliforms as indicators gives an extra margin of safety. Finstein (1973)
called the coliforms the most conservative indicator.
Since coliforms can multiply outside the animal body, their presence
in high numbers in a food product may not be indicative of original
contamination, but of improper handling, which allowed multiplication
of the organisms.
A good indicator should be present if an enteric pathogen is present.
In one study, data showed that 11.4 percent of protein feed supplement
samples with a coliform count ofless than one per gram contained salmo-
nellae (Loken et al. 1968). Of 607 samples with less than ten coliforms
per gram, ninety-two (15.2 percent) contained salmonellae. Only 42 per-
cent of the samples with over 100 coliforms per gram revealed salmonel-
lae_ Hence, there is some question as to the correlation of the presence
or absence of coliforms to salmonellae. After examining their data from
raw and ready-to-eat foods, researchers stated that the coliforms cannot
serve as an indicator of potential pathogens in these foods (Solberg et al.
1976).
Smith (1971) stated that persons with severe diarrhea often do not
excrete coliforms, but primarily excrete the enteric pathogen. He ques-
tioned the use of coliforms as an index of contamination.
Coliforms in milk products generally are considered to be of animal
origin and tend to indicate the hygienic standard of the product and its
keeping quality, rather than the presence of human pathogens. The more
carefully the milk is produced and handled, the lower the coliform count.
Hartley, Reinbold, and Vedamuthu (1968) stated that the coliform count
is of only limited value with fresh, high-quality milk, and of no value for
older or low-quality milk.
No statistically significant correlation has been found between
enteric viruses in clams or clam meat and the water from which the clams
were harvested (Wait et al. 1983). The number of coliforms in shellfish
fluctuated with rainfall (Hackney, Ray, and Speck 1980)_
The value of the coliform group as an indicator is both questionable
and acceptable_ In some products, the coliforms may indicate fecal con-
tamination from either human or animal sources or from soil, poor sani-
tation of equipment, faulty pasteurization techniques, or recontamina-
tion after pasteurization or cooking. Because of their ubiquity in nature,
the ability to multiply outside the animal body, and the nonfecal habitat
of some strains, the coliform group might not be a reliable indicator of
the presence of pathogens in food. However, strains of non-E. coli col-
iforms, in addition to strains of E. coli, are enterotoxigenic (see Chap-
ter 6).
FECAL COLIFORMS
To overcome the criticism of nonfecal coliforms as indicator organ-
isms, there have been suggestions to determine only the fecal coliforms.
In 1904, Eijkman discovered that intestinal coliforms produce gas in glu-
cose broth incubated at 46C, and nonfecal coliforms fail to grow.
The fecal coliforms can be defined as Gram-negative facultative rods
that ferment lactose at 44.5C. The fecal coliforms consist primarily of
E. coli, but a few Enterobacter and Klebsiella strains can produce gas in lac-
tose broth at 44.5C (Duncan and Razzell 1972).
Sources
The fecal coliforms are relatively specific for fecal material of warm-
blooded animals. Fecal coliforms were reported on fresh produce (Dun-
can and Razzell 1972). Also the effluent from a potato-processing plant
as well as four fruit and vegetable canneries contained fecal coliforms
(Menon 1985). Fecal coliforms can be found in animals and plants and
foods derived from them, as well as in contaminated soil and water.
Methods
The usual procedure is to inoculate growth from positive presump-
tive coliform tubes into tubes of EC medium (Hastback 1981) and incu-
bate these at 44.5C for 24 hr. The incubated broth is examined for gas
production, and the number of fecal coliforms estimated with an MPN
table.
The direct inoculation of a sample into EC broth does not reveal all
of the fecal coliforms. An enrichment in a presumptive broth is needed
to attain a level of cells that will produce gas when inoculated into EC
broth and incubated at 44.5C.
With this test, a small portion of fecal coliforms is excluded and a few
nonfecal coliforms are included in positive tubes_ The use of both LST
(48 hr) and EC (24 hr) broths requires a total of 72 hr for incubation.
However, Hastback (1981) found that incubation of LST for 48 hr, as
compared to 24 hr, in most cases increased the count less than 2 percent.
An A-I broth was described by Andrews and Presnell (1972) for the
determination of E. coli in raw seawater. The inoculated tubes of A-I
broth were incubated at 44.5C for 24 hr. This system has been modified
so that the inoculated tubes are incubated for 3 hr at 35C (to allow
repair of stressed cells), followed by 21 hr at 44.5C. Although this modi-
fied A-I procedure was found to be satisfactory for analyzing various
substances (Hunt et al. 1981; Standridge and Delfino 1981; Varga and
Doucet 1984) and was given official status by the AOAC in 1980 for de-
tecting fecal coliforms in seawater, one study stated that the reproducibil-
ity of recovery of fecal coliforms depends upon the food being analyzed
and recommended that the technique be used only as a screening proce-
dure (Andrews et al. 1981).
A membrane filter (MF) method can be used. After filtration, the MF
is saturated with M-FC broth and incubated at 44.5C for 24 hr. Blue
colonies, observed with low-power magnification, are counted as fecal
coliforms. Researchers developed media and systems for resuscitation of
stressed cells for detection of fecal coliforms with the MF method (Du-
four, Strickland, and Cabelli 1981; LeChevallier et al. 1984.) A hydro
phobic grid membrane filter system has been tested for fecal coliforms
and E. coli detection (En tis 1984). The method was recommended for
adoption by AOAC as official first action.
Besides the elevated temperature test, the IMViC tests can be used to
differentiate coliforms, according to the reactions as listed in Table 7.1.
There are some problems associated with the IMViC system. Both the
Klebsiella and Enterobacter are IMViC - - + +. Klebsiella can be found
in feces, industrial wastes, or nature, while Enterobacter usually is found
in nonfecal sources. Further, this biochemical testing requries the isola
tion of a sufficient number of colonies from an agar surface so that the
tests are meaningful. These time consuming and laborious procedures
are not adaptable on a routine basis in many laboratories.
Other systems for detecting fecal coliforms have been suggested,
tested, and reported. These include fluorescent antibody (Abshire and
Guthrie 1973), coliphage (Kenard and Valentine 1974), gas chromato
graphic system (Newman and O'Brien 1975), Millipore coli count sam-
pler (Richards 1979), and electrical impedance (Silverman and Munoz
1979).
Growth and Survival
Although the fecal coliforms are associated primarily with the intesti
nal contents and discharges of humans and warmblooded animals, they
can multiply in an acceptable environment outside the intestines. Bagley
and Seidler (1977) reported that 49 percent of the environmental K. pneu-
moniae grew in EC broth at 44.5C, and 16 percent were fecal coliform
positive.
The fecal coliforms are destroyed by pasteurization or normal cook
ing temperatures. The survival of frozen fecal coliforms deper.ds upon
the strain of organism and the substrate (Splittstoesser, Stewart, and Wil
kison 1982). When inoculated vegetable homogenates (four types) were
TABLE 7.1. USUAL IMVIC REACTIONS OF COLI FORMS
Reaction
Type M V C
Escherichia (Type I)
+ +
Escherichia (Type II)
+
Enterobacter
+ +
Klebsiella
+ +
Citrobacter
+ +
I = indole, M = methyl red, V = VogesProskauer, C = citrate utilization.
held at -lODe for 200 days, the survival of fecal coliforms (six strains)
ranged from 0.014 to 75 percent. The E. coli strain (0.014 percent survival
in broccoli) when rerun, survived storage at a level of 7.7 percent.
Significance of Fecal Coliforms
The fecal coliforms are more closely related to fecal contamination
than are the total coliforms. This should be considered an advantage;
however, critics state that testing for only fecal coliforms does not give a
margin of safety that is important when using indicator organisms.
The organisms can originate from improperly sanitized working sur
faces in a processing plant. In these cases, their presence would reflect
the quality of sanitation and not the direct pollution of the product.
No salmonellae were found in fresh or stored shellfish with a level of
fecal coliforms below 230/100 g (Hood, Ness, and Blake 1983). Higher
levels of fecal coliforms were limited in predicting the presence of salmo
nellae.
Researchers found that the presence of V. cholerae in shellfish was not
adequately indicated by fecal coliform standards for shellfishgrowing
waters and market shellfish (DePaola et al. 1983).
The absence of fecal coliforms in disinfected sewage effluents does
not assure the absence of viruses (Berg et al. 1978). The level of fecal
coliforms in oystergrowing waters does not reflect the level of virus con
tamination (Ellender et al. 1980).
ESCHERICHIA COLI
E. coli is the most prominent fecal coliform. Hence, we might expect
that there is little difference between E. coli and fecal coliforms. Since
the main habitat of E. coli is the intestinal tract of humans and warm
blooded animals, some microbiologists believe that only the presence of
E. coli is indicative of fecal contamination of food.
Sources
E. coli is a common inhabitant of the intestinal tract of humans and
animals. The number of this organism varies in different animals, but an
animal will excrete from 130 million to over 18 billion E. coli each day.
Hence, there is a close association between E. coli, fecal material, and,
possibly, enteric pathogens. It is difficult to completely prevent contami
nation of animal carcasses with E. coli during slaughter.
The organism can be found on plants and plant foods, although usu
ally not in great abundance. The inoculation of cells onto dry soil reo
suIted in a 99 percent reduction in 24 hr (Chandler and Craven 1978).
The survivors persisted for eight weeks. The survival of S. typhimurium
cells was similar to that of E. coli.
Marcus and Amling (1973) detected E. coli on 23 percent of the sam
pIes of pecans harvested from orchards in which animals had grazed, but
from only 4 percent of the samples obtained from ungrazed orchards.
Hall, Brown, and Lewis (1967) analyzed various foods for coliforms
and E. coli. E. coli was found in cheese and cheese products (75 percent
of the samples), fish, and seafood (30 percent), raw and frozen vegetables
(15.8 percent), prepared and convenience foods (50 percent), raw meats
(76.5 percent), and sandwiches (16.3 percent). Only thirty of seventyfour
raw beef patties contained ten or more E. coli per gram (Surkiewicz et
al. 1975).
Bettelheim et al. (1974) suggested there is a difference in the serologi
cal groups of E. coli isolated from humans and from animals. The strains
of E. coli isolated from meat generally resembled animal strains. Typically,
human serotypes found on meat are believed due to contamination duro
ing handling. According to the review of Mason and Richardson (1981),
the types or strains of E. coli in human feces are changing constantly.
Some strains persist for several weeks before being replaced, while other
strains are merely transients. Hinton, Linton, and Hedges (1985) reo
ported a similar finding for E. coli in the feces of calves.
Methods
The Bacteriological Analytic Manual (FDA 1978) test for E. coli consists
of inoculating growth from presumptive positive coliform LST broth
into EC broth and incubating at 44.5C. Growth from those EC broth
tubes showing gas production is streaked onto EMB agar and incubated
(35C for 18-24 hr). Typical E. coli colonies (nucleated with or without a
sheen) are tested using the IMViC reactions and Gram stain. The designa
tion in Table 7.1 is used to classify the E. coli.
In a review, Silliker (1982) stated that all the imprecision of the col
iform test is inherent in this MPN method. It is also long and laborious,
requiring up to eight days for completion.
A rapid method requires inoculating and incubating LST broth at
44.5C, and then streaking on EMB agar. Typical E. coli colonies on EMB
are confirmed with the IMViC tests.
Although incubation of the EC broth for 48 hr will yield slightly more
E. coli, the number of false positive cultures increases. Therefore, to ob
tain optimum specificity, the incubation period should be limited to 24
hr.
The MPN method (FDA 1978) and an MF system (Anderson and
Baird-Parker 1975) were compared for enumerating E. coli. One team of
researchers reported higher recovery of E. coli from beans, cheese, and
ground beef with the MF system (Sharpe et al. 1983), while another team
reported higher recovery from estuarine water and shellfish with the
MPN method (Motes, McPhearson, and DePaolo 1984). Apparently the
type of food influences the method of choice for the enumeration of not
only E. coli but also other types of microorganisms.
A rapid E. coli assay using 4-methylumbelliferone glucuronide (MUG)
was developed by Feng and Hartman (1982). Glucuronidase, an enzyme
produced by E. coli and some strains of salmonellae and shigellae, hydro-
lyzes MUG to a product that is fluorescent when viewed with long-wave
ultraviolet light. They found this system to be sensitive and rapid for the
detection and enumeration of E. coli. Further work has revealed the MUG
assay to be accurate, rapid, simple, and inexpensive to perform (Alvarez
1984; Koburger and Miller 1985; Petzel and Hartman 1985; Robison
1984). However, the assay may not be useful for all foods, or certain modi-
fications are needed for it to be valid (Koburger and Miller 1985; Petzel
and Hartman 1985).
Survival
The type of food influences the rate of death of E. coli in frozen stor-
age. In orange concentrate, the organism may survive only one day or as
long as one week. In a product such as frozen gravy, the death rate is
lower and E. coli may survive frozen storage for several months_ Some
cells of most strains tested survived at least 200 days in frozen vegetable
homogenates (Splittstoesser, Stewart, and Wilkison 1982)_
E. coli and salmonellae decline in numbers at a similar rate in many
refrigerated foods. The reduction in E. coli and salmonellae is similar
during spray-drying. The thermal resistances of E. coli and salmonellae
are similar. In filter-sterilized water, the survival of E. coli and S. typhimu-
rium are essentially the same over normal seasonal temperatures
(Mitchell and Starzyk 1975).
Significance
The source of E. coli is generally the intestinal tract and feces of warm-
blooded animals and people. Hence, it should be an excellent indicator
organism. On the other hand, if fecal coliforms lack a desired margin of
safety, it is an even greater problem with the use of E. coli alone as an
indicator.
The determination of E. coli includes any problems associated with
the enumeration of coliforms plus the added necessity to do additional
procedures to confirm and complete the tests for these specific organ-
isms. Thus, the tests for E. coli are not as acceptable as for those for coli-
forms, and the direct enumeration of pathogens might be considered.
According to Evison and James (1973), E. coli is a reliable index of
fecal pollution of water sources in temperate climates. Other research-
ers reported that with increasing contamination of coastal water with E.
coli, the proportion of positive salmonellae samples also increases (Gart-
ner et al. 1975).
In many food products, the survival of E. coli is similar to that of sal-
monellae. However, many pathogens may persist after E. coli is destroyed.
On the other hand, the presence of E. coli does not mean that enteric
pathogens also are present. Lovell and Barkate (1969) found that more
than 92 percent of the samples of crayfish contained E. coli, but only 3
percent contained either coagulase-positive staphylococci or salmonel-
lae. Conversely, salmonellae have been found in food samples with no E.
coli. The presence or absence of E. coli in oysters was not indicative of the
presence of enteric viruses (Fugate, Cliver, and Hatch 1975).
The finding of high coliform or E. coli counts in a food has been the
basis for seizure by regulatory agencies on the basis that the food "con-
sists in whole or in part of a filthy, decomposed or putrid animal or vege-
table substance."
ENTEROBACTERIACEAE
Those enteric bacteria in food that do not ferment lactose may be
more important in food than those that do ferment lactose (coliforms).
Therefore, it has been suggested that the entire family Enterobacteria-
ceae be determined and used as an indicator group.
If the use of coliforms as indicators is criticized because of nonfecal
origins, some members of this family deserve to be criticized. Some
Enterobacteriaceae occur as saprophytes in nature, some are parasites
for plants, causing blights and soft rots, many are found in the intestinal
tract of animals, and others are potential pathogens of humans and ani-
mals. Thus, the direct relationship of the organisms of this entire family
to fecal contamination or potential pathogens is questionable.
Researchers found Enterobacteriaceae on poultry carcasses at various
stages of chilling (Cox et al. 1975). Salmonella and Shigella were not found.
As pointed out, this does not mean that these organisms were not pres-
ent. The most prevalent isolate was Escherichia, accounting for 87 to 96
percent of the detected Enterobacteriaceae. Thus, the use of E. coli or
fecal coliforms as indicators would reveal essentially the same informa-
tion as the Enterobacteriaceae. There are many foods in which the col
iform count and Enterobacteriaceae count are almost the same.
The Enterobacteriaceae most frequently detected on various foods
were E. coli, Enterobacter agglomerans, Serratia liquefaciens, Klebsiella pneumo-
niae, Enterobacter cloacae, Serratia marcescens, and Erwinia herbicola (Cox et
al. 1979; Mercuri et al. 1978; Oblinger, Kennedy, and Langston 1982;
Stiles and Ng 1981). The Enterobacteriaceae are of little value as indica
tors of contamination or growth of enteric pathogens on refrigerated
raw meats (Shaw and Roberts 1982). There was no relationship between
Enterobacteriaceae counts and Salmonella in broilers (Mercuri et al. 1978).
The methods suggested to enumerate the organisms were evaluated
by Drion and Mossel (1977). They stated that testing two samples of 1 g
of food for Enterobacteriaceae and allowing no positives gives the same
degree of safety as testing 25 to 60g samples for salmonellae and allow
ing no positives.
FECAL STREPTOCOCCI ENTEROCOCCI
Because coliforms-fecal coliforms and E. coli-are not very resistant
to heating or freezing, other organisms have been suggested as indica
tors. The fecal streptococci and enterococci are potential indicator
organisms.
The enterococci are in Lancefield's group D of the genus Streptococcus.
Since references are made to the group D streptococci, the enterococcus
group, and the fecal streptococci, some distinction should be made be
tween these categories.
S. faecalis and S. faecium, along with two subspecies of S. faecalis (liquifa-
ciens and zymogenes) comprise the enterococcus group. These organisms,
plus S. bovis and S. equinus are the group D streptococci. Buchanan and
Gibbons (1974) included S. suis and S. avium as members of group D. The
fecal streptococci include all of these group D streptococci, and S. mitis
and S. salivarius.
S. avium has been listed in group Q, but it also possesses the group
D antigen, is prevalent in chicken feces, and is also present in animal
and human feces. In most cases, the growth of S. faecalis, S. faecium, and
the group Q streptococci are not distinguishable. Thus, the group Q
streptococci (S. avium) may be enumerated and reported as enterococci.
S. avium fits most of the characteristics of the enterococcus group, except
it neither tolerates 0.1 percent methylene blue, nor hydrolyzes arginine.
The definitions and the organisms that are included are not necessar
ily those listed in some of the literature. The streptococci have been stud
ied for many years, but the status and interrelationships of the species
have been, and still are, controversial. For example, reports such as that
by Schleifer and Kilpper-Balz (1984) have recommended the transfer of
S. faecalis and S. faecium to the genus Enterococcus.
Although these terms sometimes are used interchangeably, in this
text, both fecal streptococci and group D streptococci include the enter-
ococcus group_ Conversely, the enterococcus group does not include all
of the organisms in either fecal streptococci or group D streptococci.
The use of enterococci or fecal streptococci as indices of fecal con-
tamination, or as a means of determining the sanitary history of food
products, their alleged association with foodborne illness, and their oc-
currence in human pathological conditions attests to their importance in
public health.
Sources
Since enterococci are part of the larger group fecal streptococci, the
sources of enterococci are also sources of fecal streptococci. However, if
fecal streptococci are present, there could be organisms not included in
the enterococcus group.
The primary source of the enterococci and fecal streptococci is the
intestines of humans and warm-blooded animals. Levels in human feces
may range from 10
4
to 10
9
per gram. The organisms are prevalent on
the hide, hair, feathers, and hooves of animals and, upon slaughter, the
processing equipment, the hands of the workers, and the food products
become contaminated.
Animals excreting these organisms can cause widespread distribution
of the enterococci. They have been reported in water, soil, vegetation,
and insects (Mundt 1982). Due to this distribution, the organisms are
found in a wide variety of foods at relatively low levels, and in some foods
at rather high levels.
Methods
In order to be an acceptable indicator, relatively simple and standard
methods should be available. No method for enterococci or fecal strepto-
cocci is listed in FDA (1978). Procedures for the KF streptococcal count
and the GTC streptococcal count are described in APHA (1984). In a
comparison of several media, M-Enterococcus agar yielded the best over-
all results (Pagel and Hardy 1980).
Since few media, if any, are specific for a particular organism or
group of organisms, and because the species of Streptococcus may indicate
the source of the organism, further biochemical testing of the organisms
is warranted.
The tests include the finding of Gram-positive cocci that are catalase
negative and reduce 0.1 percent methylene blue. Also, lactose, sorbitol,
glycerol, and arabinose fermentation, starch hydrolysis, gelatin hydroly
sis, hemolysis and reduction of potassium tellurite and TTC can be deter
mined for differentiation of the species. Facklam and Moody (1970) sug
gested using the bileesculin test to segregate group D from other
streptococci. Lee (1972) reported the use of a tyrosine decarboxylase me
dium. The enterococci are tyrosine decarboxylase positive.
Mundt (1973) suggested that the reaction in litmus milk and fermenta
tion of melezitose and melibiose might be employed to distinguish be
tween contamination representing recent pollution of human origin and
the presence of S. faecalis as a member of plants with no sanitary signifi
cance.
Growth
The enterococci are able to grow at both 10 and 45C. They can
grow in broth with 6.5 percent NaCI and in media at pH 9.6. They can
tolerate 40 percent bile.
The nutritional requirements for enterococci and some other fecal
streptococci is such that they may fail to grow on simple culture media.
However, most food products contain the nutrients needed for growth
of these organisms.
Survival
The fecal streptococci survive outside the intestinal tract better than
do the coliforms. S.faecalis remains viable longer than E. coli in acid foods.
In frozen foods, the fecal streptococci remain viable for long periods.
The enterococci survive spraydrying and storage in egg powder better
than coliforms and E. coli. Cool, moist conditions prolong the survival of
S. faecalis in soil (Kibbey, Hagedorn, and McCoy 1978).
The enterococci are thermoduric, withstanding heat that would de
stroy most nonsporeforming bacteria. The organisms can withstand 60
to 65C for 30 min. S. faecium is the most heatresistant species.
Enterococci are sensitive to chlorination. They are destroyed in 15
sec with 100 ppm chlorine at pH 8.4 and in 2 min with 10 ppm chlorine
at pH 5.8 (Shannon, Clark, and Reinbold 1965).
Significance
The chief source of fecal streptococci is the intestinal tract of humans
and animals. The occurrence of these bacteria in water or food implies
either direct or indirect fecal contamination.
The occurrence of fecal streptococci in water generally suggests fecal
pollution. Their absence indicates little or no warmblooded animal con
tamination. Fecal streptococci rarely multiply in polluted water; however,
this may not be true for food products.
The presence of S. bovis or S. equinus indicates animal pollution rather
than human contamination. Therefore, increased information is ob
tained if the species of fecal streptococci are identified.
Enterococci survive environmental conditions that destroy other mi
croorganisms of sanitary significance. Due to their resistance to freezing,
low pH, and moderate heat treatment, the enterococci have been sug
gested as an indicator in some types of food products.
The finding of enterococci or fecal streptococci in a food is not proof
that enteric pathogens are present. Once the enterococci or fecal strepto
cocci contaminate a food processing plant, they can become established
and multiply on surfaces contaminated with organic material. From these
sources, they can contaminate food products. Thus, the organisms are
able to multiply in environments far removed from the original source
of fecal contamination.
In this case, the presence of the organisms could be an indication of
poor handling or sanitary practices. However, complete elimination of
these organisms is extremely difficult, even under good sanitary condi
tions.
It has been stated that the enterococci cannot replace E. coli as an
index for pollution. Also, the presence of the fecal streptococci, per se,
gives little information, and either total counts or coliform counts are
needed in conjunction with the fecal streptococcus or enterococcus
count.
The use of these organisms in food products should be related to
specific product practices, handling procedures, and the microenviron
ments of the food. In some products, their presence has some meaning
as indicator organisms; in other products, their presence is meaningless.
OTHER INDICATORS
The staphylococci, or S. aureus, have been proposed as indicators. The
presence of large numbers of S. aureus is an indication of a potential
health hazard due to staphylococcal enterotoxin, as well as questionable
sanitation, especially of processes involving handling of foods by hu
mans.
Clostridia have been suggested, but they are not very specific for hu
man feces. Since they are sporeformers, they may persist in foods when
most enteric organisms are gone. However, two clostridia, C. perfringens
and C. botulinum, are important in foodborne illnesses.
Spore counts of thermophiles have been used as an index of washing
efficiency and cleanliness of certain vegetables.
Machinery mold (Geotrichum candidum) has been used as an indicator
of sanitation of foodprocessing operations. It readily grows on food ad
hering to equipment surfaces and contaminates food that moves through
the contamination. One study found little correlation between the aero
bic plate count and incidence of the mold in frozen blanched vegetables
(Splittstoesser et al. 1980).
The use of Pseudomonas aeruginosa as an indicator has been suggested.
The presence of the organism in the intestinal tract of people, as well
as its physiological characteristics, makes it a possible indicator of fecal
contamination.
Bifidobacteria are present in the feces of humans and hogs (Resnick
and Levin 1981) and only occasionally in the feces of cattle and sheep
(Mara and Oragui 1983). Carrillo, Estrada, and Hazen (1985) reported
that bifidobacteria show promise as a better indicator of recent fecal con
tamination in tropical freshwater than either E. coli or fecal coliforms.
The presence of certain viruses (phage) can be used as indicator sys
terns. The correlation between coliforms and coliphages in natural water
systems were highly significant (Wentsel, O'Neill, and Kitchens 1982).
Stetler (1984) found that enteroviruses in water were better correlated
with coliphages than with the b.acterial indicator sytsems. Other research
ers reported that bacterial indicators failed to predict the viral quality of
marine water (Goyal et al. 1984). The presence of phage can be demon
strated in 6 to 16 hr, which is much more rapid than the present methods
for bacterial indicators.
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8
Food Spoilage
As consumers, we depend upon our senses of sight, smell, taste, and
touch to evaluate food quality. When based upon past experiences, these
organoleptic evaluations are used to determine if food is spoiled. Since
people have different past experiences and abilities, there are conflicting
opinions concerning the point at which a food is no longer acceptable
for consumption.
The deterioration of food can be caused by rodents, insects, tissue
enzymes, nonenzymatic chemical changes, physical effects, and the ac
tion of microorganisms. Weare concerned primarily with the deteriora
tion of food by microorganisms and microbial enzymes. Tissue enzymes
may aid in microbial deterioration of food by altering certain food com
ponents so they are more readily available for microbial use. The effect
of tissue enzymes on deterioration is quite evident in overripe fruits.
The microbial deterioration of a food usually is manifested by alter-
ations in the appearance, texture, odor, or flavor, or by slime formation.
The appearance includes color changes, formation of pockets of gas or
swelling, and microbial growth, especially that of molds. As some food
products deteriorate, they tend to become soft or mushy. Degradation of
food results in the formation of compounds that have odors and flavors
different from those of the fresh food.
MICROBIAL FUNCTIONS
Like people, the microorganisms need food for energy, cell growth,
maintenance, and reproduction. In the scheme of nature, one of theim-
portant roles of microorganisms is to degrade organic material that is no
longer needed, so the elements can be reused. Unfortunately, the micro-
organisms attack our food resources with equal vigor.
The biochemical changes that occur in a food may be desirable or
undesirable. Microorganisms change a food into another product, such
as cabbage to sauerkraut, grape juice to wine, or alcohol to vinegar. Un-
der controlled conditions, these reactions are desirable, but if we want
grape juice, then the formation of alcohol and the production of wine
are undesirable. The useful aspects of microorganisms in foods are dis-
cussed in the next chapter. The basic reactions, whether desirable or un
desirable, are essentially the same.
The system by which undesirable changes occur in a food is deter-
mined by the composition of the food (carbohydrate, fat, protein), the
types and numbers of microorganisms associated with the food, the fac-
tors (intrinsic or extrinsic) that influence the growth of microorganisms,
and changes in these factors that occur during processing or during dete-
rioration.
The microorganisms present on food include those associated with
the raw material, those acquired during harvesting, handling, and proc-
essing, and those surviving a preservation treatment and storage. The
microorganisms may act singly or in groups to break down complex or-
ganic compounds. The active enzyme systems of the dominant microor-
ganisms, as well as the natural enzymes of the food, will determine the
course of degradation of a food.
FOOD COMPOSITION
Foods are composed primarily of water, carbohydrate, fat, and pro-
tein, as listed in Table 8.1. High-carbohydrate foods include fruits, vege-
tables, grains, honey, baked goods, and candy. Fats are prominent in
TABLE 8.1. APPROXIMATE RATIOS OF CARBOHYDRATES, FATS, AND PROTEINS
IN VARIOUS FOODS
Ratio of Components"
Food Carbohydrate Fat Protein
Fruit 88 7 5
Fruit juice 94 2 4
Jellies, jams 98 1 1
Vegetables 74 3 23
Cereals 85 3 12
Honey 99 0 1
Baked goods 75 15 10
Candy
83 11 6
Milk
42 29 29
Butter
1 98 1
Seafoods 12 8 80
Fish 0 18 82
Beef carcass, total edible
0 64 36
Pork carcass, total edible
0 84 16
Lamb carcass, total edible
0 58 42
Sausage products 6 62 32
Chicken carcass, total edible 0 21 79
Eggs
4 45 51
"Calculated from data of Watt and Merrill (1963).
FOOD SPOILAGE 395
foods such as butter, eggs, and red meats. Protein foods are considered
to be animal products. High ratios of protein are in seafoods and poultry.
The ratio of protein of vegetables and cereals is comparable to that of
pork carcasses. In milk, there is less difference in the ratio of carbohy
drates, proteins, and fats than in any other food listed in Table 8.1.
Carbohydrates are utilized more readily than are proteins or fats. The
fermentation of carbohydrates produces acids and lowers the pH. The
acidic condition tends to inhibit proteolytic organisms. The term protein
spaTing has been used to describe the metabolism of organisms in a food
containing carbohydrates and proteins. Since very few foods are all car
bohydrate, all fat, or all protein, more than one type of degradation may
occur.
DEGRADATION OF COMPONENTS
Carbohydrates
The general formula of carbohydrates is Cx (H
2
0)y. The carbohydrates
are divided into monosaccharides, disaccharides, and polysaccharides.
The monosaccharides are polyhydroxy aldehydes (aldoses), or polyhy.
droxy ketones (ketoses). Some carbohydrates as well as some polyhydric
alcohols are listed in Table 8.2.
There are some free monosaccharides, but most naturally occurring
carbohydrates are disaccharides and polysaccharides. For utilization, bac-
teria first break down these complex carbohydrates into their constituent
monosaccharides.
POLYSACCHARIDES. Plant cells and tissues have a fibrous substance
embedded in an amorphous support matrix. The fibrous material resists
tension, and the matrix resists compression. In plants, the primary fi-
brous substance is cellulose.
Plants have a rather wide range of matrix compounds. In higher plants,
the matrix polysaccharides are classified into a group of acidic polysaccha-
rides called pectins and a heterogeneous group of neutral polysaccharides
called hemicelluloses. When stiffness is needed, a non polysaccharide, lig-
nin, is incorporated. In algae (seaweeds), such things as carrageenans and
agar are the matrix materials.
Pectin. Pectolytic enzymes are produced by a wide range of microorgan-
isms, especially those causing soft rot. The pectic enzymes include pec-
tin esterase (EC 3.1.1.11), polygalacturonases (EC 3.2.1.15; EC 3.2.1.67; EC
3.2.1.82), pectate transeliminases (EC 4.2.99.3), and lyases (EC 4.2.2.2; EC
TABLE 8.2. COMPOUNDS DEGRADED AS CARBOHYDRATES
Monosaccharides
Hexoses
Aldoses
Glucose
Mannose
Galactose
Ketoses
Fructose
Sorbose
Polyhydric alcohols
Mannitol
Glycerol
Adonitol
Dulcitol
Sorbitol
Disaccharides
Maltose (glucose + glucose)
Lactose (glucose + galactose)
Sucrose (glucose + fructose)
Cellobiose (glucose + glu
cose)
Polysaccharides
Dextrins
Starch
Glycogen
Inulin
Cellulose
Hemicellulose
Galactan
Xylan
Complexed polysaccha
rides
Glucosides
Salicin
Amygdalin
Esculin
Tannins
Pectins
Gums
Mucilages
4.2.2.9; EC 4.2.2.10). The random splitting of glycosidic bonds results in
softening and liquefaction.
The pectic enzymes are found in plant tissues as well as in many types
of microorganisms. The plant enzymes act during the ripening of fruit
to cause a softening effect. This is desirable to an acceptable level, but
continued action by these enzymes causes excessive softness and a mushy
product. When a microorganism is isolated from a softened fruit, the
softening could have been caused by either the enzymes from the or-
ganism or the plant tissue. The degradation of pectin was reviewed by
Chesson (1980). Perhaps this breakdown is the primary spoilage defect
of plant tissue.
Cellulose. Only a few species of bacteria can decompose cellulose, but sev-
eral filamentous fungi are capable of doing so. Cellulose is either hydro-
lyzed directly to glucose or to intermediates such as cellobiose.
The presence of readily metabolized substances such as glucose re-
presses the production of the inducible cellulases. Hence, the breakdown
of cellulose is of secondary importance in the deterioration of plant
tissue.
Starch. This polysaccharide is an important component of many plant
products. Several bacteria and molds possess an extracellular enzyme,
diastase or amylase, which hydrolyze starch. Starch is converted either
directly to glucose or through intermediates such as maltose.

pectin esterase

OH t OH I OH OH OH
.... - ____ .L. ___ pectin lyase

OH OH OH OH l OH t OH
polygalacturonase I
pectate lyase
{)
OH COOH
COOH COOH
o .vt-0,'-"._
y+-O"-.J
" OH
OH OH
OH
OH
polygalacturonase

OH OH
-
0.
COOHO
.
.
, OH + OH o
OH H
OH
OH
pectate lyase
COOCH, COOCH,
COOCH, COOCH,
,,0
0
4>0" -
OH OH
OH
OH
pectin lyase
Figure 8.1. Breakdown of pectin.
Courtesy of Pilnik and Rombouts (1978).
397
MONOSACCHARIDES. Glucose is the main carbohydrate used as a
carbon and energy source. The breakdown of these sugars can proceed
by several pathways. Some of the reactions and intermediates of glycoly.
sis are summarized in Figure 8.2. There are other systems for the metabo
lism of glucose such as the pentosePphosphoketolase and Entner
Doudoroff pathways.
Pyruvic acid (CH3COCOOH) is a key compound in the metabolism
of glucose. Depending upon the metabolic pathways available to the or
ganism and the environmental conditions, the pyruvate can be involved
in many reactions, either fermentative or oxidative. Some of the meta
bolic products resulting from carbohydrates are listed in Table 8.3. The
products include organic acids, alcohols, CO
2
, H2 and H
2
0.
In aerobic respiration, pyruvate is converted into CO
2
and H
2
0 by
means of the tricarboxylic acid (TCA) cycle, Krebs cycle, or citric acid
cycle. To enter this system, the pyruvate is converted to acetate activated
with coenzyme A. Only the aerobic and some facultatively anaerobic mi
croorganisms possess an intact TCA cycle.
The pyruvic acid can be decarboxylated to form acetaldehyde
(CH3CHO) and CO
2
. The acetaldehyde can remain or be reduced to
ethyl alcohol (CH3CH
2
0H), oxidized to acetic acid (CH
3
COOH), or con
densed to form acetoin (CH3COCHOHCH
3
) or acetylmethylcarbinol
(AMC). The AMC can be oxidized to diacetyl (CH
3
COCOCH
3
), which has
a butter flavor, or reduced to 2,3butanediol (CH
3
CHOHCHOHCH
3
).
Pyruvate can be aminated to form alanine.
Lipids
The principle lipids in foods are fats. Fats are esters of glycerol and
fatty acids and are called glycerides. The generalized structure of triglyc
erides is
o
II
H - C- 0- C- R
2 I 1
H - C- 0- C- R
I 2
H - C- 0- C- R
2 II 3
o
Lactose
Maltose I
Galactose
0 I
Mg" CD hexokinase glucose-1,6-diP CD p.
ADP glucose-6-phosphate === glucose-I-phosphate --'- starch or glycogen
. Mg" hos ho lase
phosphohexOlsomerase II (i) phosphoglucomutase p p ry
M ,.
Fructose n fructose-6-phosphate
ATP Mannose ------
ATP Fructose
f:\ phosphofructokmase I
Mg" \..V
ADP Fructose-I-P
fructose-I,6-diphosphate
Aldolase 10
0) ==-
dihydroxyacetone triosephosphate glyceraldehyde-
phosphate 3-phosphate
NAD' P,
glyceraldehyde-3- f8\
phosphate dehydrogenase v
H+ +NADH
1,3-dlphosphoglyceric acid

Mg'+ CD phosphoglycerate kinase
ATP
3-phosphoglyceric acid
phosphoglycerate mutase \ j @
2-phosphoglyceric acid
enolase l (,;\
Mg2+ I - H,O \.!21
phosphoenolpyruvic acid
@ pyruvate kinase
ATP'-1
15 .
Pyruvic Acid lactIC lactic acid
Pyruvate
decarboxylase
acetaldehyde

@ Alcohol dehydrogenase
NAD+
ethanol
dehydrogenase
NADH
+ H+
NAD+
Anaerobic Glycolysis
Figure 8.2. A glycolytic pathway for the degradation of various
sugars.
Courtesy of Aurand and Woods (1973).
TABLE 8.3. SOME PRODUCTS OF CARBOHYDRATE METABOLISM
Organisms
Leucorwstoc mesenteroides
Leucorwstoc cremoris
Saccharomyces (yeast)
Clostridium botulinum
Propionibacterium
Escherichia coli
Bacillus cereus
Products
Lactic acid
Ethyl alcohol
CO
2
Acetic acid
Acetoin
Diacetyl
CO
2
Ethyl alcohol
CO.
Acetic acid
Butyric acid
Propionic acid
Isobutyric acid
Isovaleric acid
Propyl alcohol
Isobutyl alcohol
Butyl alcohol
Isoamyl alcohol
Propionic acid
Acetic acid
Isovaleric acid
Formic acid
Succinic acid
Lactic acid
CO2
Lactic acid
Acetic acid
Formic acid
CO2
H.
Acetoin
Glycerol
2,3Butanediol
Lactic acid
Succinic acid
Formic acid
Acetic acid
CO2
The Rgroups denote the carbon chain of the fatty acids. Some com
mon fatty acids are listed in Table 4.9. The triglycerides may have the
same fatty acid in all three positions, or two or three different fatty acids
may be present.
Many foods contain fat that is susceptible to hydrolysis, oxidation,
and other chemical processes that result in the formation of various com
pounds. Both desirable and undesirable flavor changes in foods are asso-
ciated with these compounds.
The enzymes that hydrolyze triglycerides are called lipases. The desig
FOOD SPOILAGE 401
nation of true lipase is given to enzymes that attack only water-insoluble
substrates_
The oxidative deterioration of fats involves the reactions of unsatu-
rated fatty acids with oxygen to give hydro peroxides_ The hydroperox-
ides are not flavor compounds, but readily decompose to carbonyl com-
pounds resulting in off-flavor or odor. The carbonyl compounds are
mixtures of saturated and unsaturated aldehydes and ketones.
Deteriorated fat is called rancid. The simple release of free fatty acids
is called hydrolytic rancidity, and oxidative deterioration is oxidative ran-
cidity.
Hydrolytic rancidity is important in fats. Short-chain water-soluble
fatty acids (butyric, caproic, and caprylic) cause obnoxious rancid flavors
in milk.
Except for the release of volatile fatty acids, the main flavor changes
are not due to hydrolysis, but to oxidation. The oxidation of the fat is
more often due to tissue enzymes or autoxidation than to microbial activ-
ity. Autoxidation is accelerated by heat and light and is catalyzed by
heavy metals and their salts, organometalic compounds, hemoglobin,
hematin compounds, and photochemical pigments. Certain species of
Aspergillus and Penicillium produce oxidative enzymes that catalyze the
oxidation of free fatty acids to methyl ketones.
A pure fat is not attacked by microorganisms, since there must be
a nutrient-containing aqueous phase in which the organism can grow.
However, most of our fatty foods (butter, cream, margarine), contain an
aqueous phase associated with the fat.
There are many lipolytic microorganisms. Some of the genera that
contain lipolytic species or strains are listed in Table 8.4.
TABLE 8.4. SOME GENERA CONTAINING LIPOLYTIC SPECIES OR STRAINS
Bacteria
Acinetobacter
Aeromonas
Alcaligenes
Bacillus
Chromobacterium
Corynebacterium
Enterobacter
Flavobacterium
Lactobacillus
Micrococcus
Pseudomonas
Serratia
Staphylococcus
Streptomyces
Fungi
Absidia
Alternaria
Aspergillus
Candida
Cladosporium
Endomyces
Fusarium
Geotrichum
Mucor
Neurospora
Penicillium
Rhizopus
Torulopsis
The lipolytic characteristics of microorganisms depend upon the fat
in the substrate. Tom and Crisan (1975) showed that, although many or
ganisms decomposed tributyrin or tween 80, only a few were able to de
compose fish oil added to a medium.
With the breakdown of fats, various products are formed, including
glycerol, free fatty acids, aldehydes, ketones, and alcohols.
Proteins
Proteins are composed of amino acids combined by peptide linkages.
These linkages are broken by the addition of water catalyzed by enzymes.
The proteins are degraded through proteoses, peptones, polypeptides,
and dipeptides to amino acids. The enzymes that hydrolyze the peptide
linkages of proteins are called proteases. These proteases convert the
proteins into diffusible polypeptides and dipeptides, which can enter the
bacterial cell. The polypeptides and dipeptides are without offensive
odor.
The native proteins are resistant to attack by microorganisms. There
are lowmolecularweight compounds, such as dipeptides and free amino
acids in fresh meat, fish, and poultry tissue. These substances are readily
used by the microbial flora. Spoilage of the proteintype foods may be
evident before any significant amount of protein is degraded. In ad
vanced spoilage, some protein is hydrolyzed by proteolytic enzymes,
whether produced by the tissues or the microorganisms.
AMINO ACIDS. The degradation of amino acids is of primary impor
tance in the spoilage of protein foods. The products that are formed
depend upon (1) the type of microorganism; (2) the types of amino acids;
(3) temperature; (4) the amount of available oxygen; and (5) the types of
inhibitors that might be present.
There are many amino acids and hence, many products of amino acid
dissimilation. Some products that are formed anaerobically are foul
smelling. This form of degradation is called putrefaction. Aerobic degra
dation, with oxidation of the metabolic products, is called decay.
The two main reactions of microorganisms on amino acids are decar
boxylation or deamination. Many of the reactions involving amino acids
are shown in Figure 8.3. Other reactions of amino acids include the fol
lowing:
Oxidation reduction
+ 3NH" + CO2
R CH2CHNH2COOH
amino acid
oxidative deamination
+2H
reductive deamination
hydrolytic deamination
hydrolytic deamination
and decarboxylation
deamination and desaturation
decarboxylation
Figure 8.3. Reactions involving amino acids.
FOOD SPOILAGE 403
R CH
2
CO COOH + NH3
0' -keto acid
R CH2CH2COOH + NH3
organic acid
R CH
2
CH OH COOH + NH3
hydroxy acid
R CH2CHOH + CO
2
+ NH3
alcohol
R CH-CHCOOH + NH3
unsaturated
organic acid
R C H C ~ N H
+ CO
2
amine
The overall reaction results in two organic acids (one with one less
carbon atom), ammonia, and carbon dioxide.
Anaerobic degradation-release of hydrogen
5COOHCH2-CH2-CHNH2-COOH + 6H20 -> 6CH"COOH +
2CH"CH2CH2COOH + 5C02 + 5NH3 + H2
The overall reaction involving, in this case, glutamic acid, yields acetic
acid, butyric acid, carbon dioxide, ammonia, and hydrogen.
Transamination
Although the amino acid is not degraded in this reaction, transamina-
tion does alter the composition of the substrate, which will determine
the final degradation product.
COOH-CH2-CH2-CHNH2-COOH + COOH-CH2-CO-COOH ->
COOH-CH2-CHT CO-COOH + COOH-CH2-CHNH2-COOH
In this example, glutamic acid reacts with oxaloacetic acid to form (X-
keto glutaric acid and aspartic acid.
Mutual oxidation and reduction
Some amino acids (alanine, leucine, phenylalanine, and valine) can
serve as hydrogen donators and are oxidized, while others (arginine, gly-
cine, hydroxyproline, ornithine, and proline) can serve as hydrogen ac-
ceptors and are reduced_
Tryptophan can enter reactions resulting in many products_ One
product, indole, is used in the identification of certain microorganisms_
Another product is skatole. Small amounts of skatole are present in the
perfume of the jasmine and orange blossoms. It is one of the two most
important nitrogenous substances found in natural perfumes. In larger
concentrations, skatole is a foul-smelling compound. It is found promi-
nently in fecal material.
When sulfur-containing amino acids such as cystine or methionine
are degraded, hydrogen sulfide as well as mercaptans, organic disulfides,
and other sulfur-containing (thio) compounds may be formed.
The decarboxylation of the diamino acids, lysine and ornithine, yield
cadaverine and putrescine accordingly:
NH2-(CH2).-CHNH2-COOH -> NH2-(CH2hNH2 + CO2
NH2-(CH2h-CHNH2-COOH -> NH2-(CH2).-NH2 + CO2
Arginine is degraded through ornithine to putrescine.
By the dissimilation of amino acids, many diverse products, such as
carbon dioxide, hydrogen, ammonia, hydrogen sulfide, organic acids, al-
cohols, amines, diamines, mercaptans, and organic disulfides may be
formed. The production of ammonia and amines tends to cause an in-
crease in the pH. Trimethylamine is more basic than is ammonia. An
increase in the pH of a protein food indicates protein degradation, just
as a decrease in pH results from the fermentation of carbohydrates.
OTHER DETERIORATION
Besides the changes caused by the degradation of carbohydrates, fats,
and proteins, microorganisms can create unacceptable foods by chang-
ing the appearance of the food, either by mold growth or by altering
the color of a pigment. Also, microorganisms can metabolize sugar and
produce dextrans or levans that feel like slime on the surface of foods
such as meat, poultry, or fish, or a condition called ropiness, in such
foods as milk or bread.
Appearance
When microbial growth can be observed on a food, it usually is con-
sidered to be unacceptable. Pigmented bacteria can, at times, be ob-
served on a food. Mold mycelia indicate potential spoilage; however,
FOOD SPOILAGE 405
mold is grown on certain types of cheeses, such as Roquefort and Cam
embert, to develop the distinctive flavors.
Various pigments (chlorophyll, carotene, myoglobin) are present in
foods. The degradation of chlorophyll and carotene is related to the lipo
lytic breakdown of fats. A lipase in peas effectively degrades chlorophyll
to pheophytin, and the degradation of both chlorophyll and pheophytin
is related to an increase in fat peroxide. Carotene is fat soluble, and its
destruction is related to fat decomposition.
The myoglobin pigment of meat muscle can be altered by oxidation
reduction reactions. Myoglobin is dark red to purple and is present in
the interior part of meat. When the muscle tissue is cut and exposed to
oxygen, the myoglobin is oxygenated to oxymyoglobin, which is bright
red and is the usual pigment associated with meat. In myoglobin and
oxymyoglobin, the iron is in ferrous form. When it becomes oxidized to
the ferric form, metmyoglobin is formed. This pigment is dark brown.
Although these pigment changes do not necessarily make the food
inedible, a consumer may consider the product unacceptable.
Slime Formation
Several bacteria produce microbial polysaccharides (dextrans, levans,
or amyloses) from various disaccharides present in food. These polysac
charides form unpleasant slime in and on food, causing the food to be
both unpalatable and unacceptable to the consumer. An example of this
is the slimy or ropy texture of fruit concentrate or milk when infected
with Leuconostoc mesenteroides, Bacillus subtilis, or E. coli. Sucrose and malt
ose are readily used by E. coli, L. mesenteroides, or B. subtilis to produce
amyloses and dextrans. B. megaterium and B. subtilis produce levans, which
are longchain polysaccharides.
Dextran is a glucose polymer. Sometimes it is encountered as a slime
in large globular masses during the processing of cane or beet sugar. It
increases the viscosity of the sugar solution and retards filtration and
crystallization. These globular masses are due primarily to activities of
Leuconostoc mesenteroides and L. dextranicum, although Streptococcus salivarius
and S. bovis are able to synthesize from sucrose and raffinose a dextran
type insoluble carbohydrate.
Slime on refrigerated fresh meat and poultry that appears soon after
off-odor is caused by the growth of Pseudomonas.
MICROBIAL DEFECTS IN SPECIFIED FOODS
Presumably the enzymes produced by microorganisms, even if the mi
croorganisms are not multiplying, could, over an extended storage time,
decompose the food and cause spoilage. However, spoilage is associated
with large numbers of microorganisms. Therefore, the organisms that
cause spoilage are those that can multiply and become dominant. The
factors affecting growth and dominance are discussed in Chapter 4.
These factors are considered in regard to the spoilage of specific types
of foods.
Fruits
A large and varied population of microorganisms, including the
spores of many types of fungi, contaminate the surface of fruits during
the growing season. Relatively few of the fungi are capable of attacking
the fruit before harvest. The ripening process increases the susceptibility
of the fruit to invasion. Fruits harvested from the ground have a more
varied microbial flora than those picked from the tree. Falling from the
tree causes bruising, and contact with the grass and soil provides an
added source of inoculum.
Fruits have a low pH that inhibits most bacteria. The acidtolerant
bacteria are mainly Grampositive lactobacilli and leuconostocs. Usually
fruits are spoiled by yeasts and molds that are acid tolerant.
The microbial defects of fruit and fruit products are listed in Table
8.S. Penicillium species are important spoilage organisms of most fruits.
P. digitatum and P. italicum cause soft rot in citrus fruits, and P. expansum
causes blue mold rot of deciduous fruits. Blue mold rot consists of soft,
watery, tan to light brown areas that are readily gouged out of the fruit
flesh. The tissue has a moldy or musty odor and flavor. There are typical
bluish green spores on the fruit.
Black rots in apples, citrus fruits, bananas, and pineapples are due to
species of Alternaria as well as other molds. The tissue becomes soft and
watery. For brown rot, the fruit has decayed areas, turning dark brown
to black at the center. The mold spore masses are yellowish gray.
Anthracnose is a defect with scattered black or darkbrown sunken
spots covering firm decayed tissue. Under moist conditions, there are
pink spore masses on the spots.
Gray mold rot is evidenced by light-brown, fairly firm, watery decay,
covered extensively with delicate dirty-white mycelium. Grayish brown
velvety spots may be seen in advanced spoilage.
Sassa and Onuma (1983) isolated phytotoxic metabolites of Macro-
phoma which they called fruit rot toxins. These chemicals induce necrosis
of pinhole-injured apples.
The loss of berries results from dehydration, discoloration, and over-
ripe, mushy, damaged, moldy, or spoiled fruit. Berries are spoiled primar-
ily by Botrytis cinerea and Mucor mucedo. However, certain fungi are prev-
TABLE 8.5. SOME MICROBIAL DEFECTS OF FRUITS AND THEIR PRODUCTS
Food
Fresh fruit, general
Apples
Bananas
Berries, general
Strawberries
Blueberries
Cherries
Citrus fruits
Dates
Figs
Olives
Peaches
Canned fruit
Apricots
Banana puree
Grapefruit sections
Mango juice
Fruit juice
Jelly, jam, preserves
Wine
Defect
Blue mold rot
Black mold rot
Souring, bitter flavor
Soft rot
Green mold rot
Anthracnose
Gray mold rot
Fungal
Fermentation
Moldy core
Brown firm rot
Storage rote
Black rot
Crown rot
Fungal rot
Fermentation
Soft rot
Black rot
Soft rot
Brown rot
Green mold
Blue mold
Black rot
Sour rot
Stemend rot
Fermentation
Souring
Softening (stemend
shrivel)
Sloughing of skin
Brown rot
Decay
Butyric acid
Soft rot
Softening
Gas
Gas (C0
2
)
Gas
Souring, O ~
Acetification, vinegar
Moldy, surface
Cloudy
Cloudy, alcohol
Buttermilk flavor
Fungal
Fermentation
Acetification
Mousy odor, cloudy, slimy
Cloudy, gassy
Acetaldehyde
Flowers
Organisms
Penicillium expansum
Aspergillus niger
Streptococcus faecalis
Byssochlamys fulva, Penicillium
Penicillium digitatum
Colletotrichum
Botrytis cinerea
Gloeosporium
Torulopsis, Candida, Pichia
Alternaria tenuis
Trichoderma harzianum
Colletotrichum, Gloeosporium,
Botryodiplodia, Erwinia
Alternaria
Colletotrichum, Fusarium, Verti
cillium
Botrytis cinerea, Mucor mucedo,
Epicoccum, Colletotrichum,
Rhizopus, Alternaria
Kloeckera
Rhizopus
Alternaria
Phomopsis
Monilia, Sclerotinia
P. digitatum
P. italicum
Alternaria
Geotrichum candidum
Phomopsis, Diploida
Saccharomyces, Candida, Hansen
iaspora, Torulopsis
Gluconobacter
Rhodatorula, Saccharomyces,
Hansenula
Klebsiella, Enterobacter, Escher
ichia, Aeromonas liquefaciens
Monilinia fructicola, Sclerotinia
Rhizopus stolonifer
Clostridium
B. fulva, B. nivea
R. stolanifer, R. arrhizus
Bacillus licheniformis
Lactobacillus brevis
Leuconostoc oenos
Lactobacillus, yeast
Acetobacter
Penicillium
Nonfermenting yeasts
Fermenting yeasts
Lactobacillus, Leuconostoc
Xeromyces bisporus
Osmophilic yeasts
Acetobacter, Gluconobacter
Lactic acid bacteria
Yeasts (Saccharomyces, Pichia,
Candida)
Saccharomyces ovifonnis, S. beti
cus
Candida vini
407
alent on specific berries at various times during production, harvesting,
and storage.
The species of several genera of yeasts (Saccharomyces, Hanseniaspora,
Hansenula, Pichia, Torulopsis, Candida, Debaryomyces, and Kloeckera) can
cause a fermentation defect in fruits.
The lactobacilli are important in the spoilage of fruit juice. Some
strains are quite acid tolerant and can metabolize citric and malic acid.
This reduces the acidity, resulting in a bland, rather flat flavor and a loss
in astringency. The production of diacetyl by these organisms results in
a buttermilk flavor in the fruit juice.
Leuconostoc mesenteroides produces dextrans, resulting in a slimy, un
pleasant texture of fruit juices. Yeasts contaminate and ferment fruit
juice, especially apple juice. If the sugar concentration is 10 to 30 per
cent, the osmophilic yeasts, Saccharomyces rouxii and S. mellis, can ferment
the sugars to alcohol. Acetobacter can convert the alcohol to acetic acid,
giving the fruit juice a vinegar flavor.
The most common defect of wine is acetification. This is caused by
Gluconobacter and Acetobacter. All alcoholic beverages containing less than
15 percent w/v ethyl alcohol are subject to this type of spoilage.
Species of Leuconostoc (especially L. mesenteroides), Streptococcus, as well
as some Acetobacter, can produce dextrans in sweet wines. In this case, a
ropy texture develops which becomes slimy and viscous.
The mature spores of Byssochlamys Julva and B. nivea are thermoduric
and may survive the heat treatment during fruit canning. These fungi
can grow at rather low redox potentials, and their pectic enzymes cause
spoilage, especially of canned berries. In some cases, such as Rhizopus
spoilage of canned apricots, the mold infects the fresh fruit and produces
heat-resistant enzymes. During storage ofthe processed fruit, the residual
enzymes cause an unacceptable softening of the product. Ethiraj and
Suresh (1985) isolated L. oenos from a blown can of mango juice.
Vegetables
Because vegetables are harvested from or near the soil, they are sub-
jected to a heterogeneous flora of soil as well as to airborne microorgan-
isms.
In general, the pH of vegetables is near neutrality, so that bacteria, as
well as fungi, cause deterioration. Because tomatoes have a lower pH, the
spoilage is similar to that of fruit, although bacterial spoilage also occurs.
Microbial defects of vegetables are listed in Table 8.6. Black rot of sweet
potato is shown in Figure 8.4.
Celery plants are infected by strains of Sclerotinia sclerotiorum, which
produces a pink rot at the base of the plant. Carrots, cabbage, and other
TABLE 8.6. SOME MICROBIAL DEFECTS OF VEGETABLES AND THEIR PRODUCTS
Food
Fresh vegetables
Beans, snap
Cabbage
Carrots
Celery
Onions
Potatoes
Tomatoes
Canned vegetables
Corn, green beans, peas
Tomato
Fermented vegetables
Brine
Pickles
Sauerkraut
Vegetable juice
Defect
Soft rot, mushy
Soft black rot
Black mold rot
Blue mold rot
Anthracnose
Blight
Leaf spot
Gray mold
Soft rot
Fungal rot
Decay, wet rot
Fungal rot
Pink rot
Neck rot
Rot
Brown rot
Black mold
Ring rot
Dry rot
Ferment
Fungal rot
Bacterial spot
Soft rot
Flat-sour
Sulfide stinker
Putrid swell
Hard swell
Flat-sour
Butyric fermentation
Film forming
Soft
Black
Soft, mushy, slimy
Reduced acidity
Pink
Sour
Organism
Erwinia carotovora, Pseudomonas
fluorescens
Alternaria, Rhizopus nigricans
Aspergillus niger
Penicillium
Colletotrichum
Xanthomonas
Alternaria
B. cinerea
E. carotovora, Rhizopus stolonifer
Fusarium
Sclerotinia sclerotiorum, Rhizoctonia
carotae
Mucor
S. sclerotiorum
Botrytis allii
Pseudomonas cepacia
P. aeruginosa
Aspergillus niger
Corynebacterium
Fusarium
Candida, Pichia, Hanseniaspora,
Kloeckera
Alternaria, Aspergillus, Botrytis, Col-
letotrichum, Monilia, Penicillium,
Rhizopus
Xanthomonas
Byssochlarnys julva
Bacillus stearothermophilus
Desulfotomaculum nigrificans
Clostridium sporogenes
Clostridium thermosaccharolyticum
Bacillus coagulans
Clostridium pasteurianum, C. butyri-
cum
Yeasts (Candida, Debaryomyces,
Hansenula, Kloeckera, Pichia,
Rhodotorula, Saccharomyces)
Bacillus
Bacillus
B. subtilis
Yeasts
Rhodotorula
Lactobacillus, Acetobacter
409
Figure 8.4. Black rot of sweet potato.
Courtesy of USDA.
vegetables are soft rot-spoiled by bacteria, such as Erwinia carotovora, as
well as the fungi Rhizopus, Aspergillus, and Alternaria.
Canned tomato juice is subject to flat sour spoilage if not properly
handled either during preparation or final heat treatment. Flat sour
spoilage is attributed to the presence of and growth of Bacillus coagulans,
which either survives normal heat processes or recontaminates the
product.
FOOD SPOILAGE 411
Other Carbohydrate Foods
The microbial defects of these foods are listed in Table 8.7. Cereals,
honey, molasses, syrup, biscuits, and candy ordinarily have water activi-
ties too low to support the growth of bacteria. However, due to storage
at high relative humidity or production of water by metabolism, bacteria
can be responsible for the spoilage of these products.
Fungi are the primary agents causing deterioration of damp grain.
The alterations of grain caused by fungi include discoloration, decreased
germination, loss in weight, formation of mycotoxins, mustiness, an in-
crease in temperature, and a loss of nutrients.
Most raw sugars have an aw between 0.65 and 0.75. Only osmophilic
yeasts and some xerophilic fungi can grow on these raw sugars.
Soft-centered fondants and chocolates can be fermented by yeasts,
which produce ethyl alcohol and CO
2
. The gas buildup may cause the
outer chocolate coating to burst. Foods such as molasses, honey, syrups
TABLE 8.7. SOME MICROBIAL DEFECTS OF MISCELLANEOUS CARBOHYDRATE
FOODS
Food
Beer
Bread
Candy (chocolate cream)
Cereals, grains
Wheat
Corn
Chocolate sauce
Coconut
Dough "refrigerated"
Honey
Molasses
Peanuts
Soft drinks
Sugar solutions
Defect
Ropy
Gas, slime
Off-flavor
Turbid
Fruity
Haze, diacetyl
Ropy, slime
Black mold
Blue mold
Pink mold
Sour
Red
Fermentation
Moldy
Discoloration
Pink
Blue-eye
Cloudy
Rancidity
Gas, slime, sour
Fermented, yeasty
Gas, for thy
Moldy
Turbidity
Slime
Ferment, yeasty
Organism
Gluconobacter
Streptococcus lactis
Lactobacillus, Pediococcus, Brettano-
myces, Candida, Pichia
Candida, Brettanomyces
Candida
Lactic acid bacteria, pediococci
Bacillus subtilis
Rhizopus nigricans
Penicillium
Neurospora
Lactic acid or coliform bacteria
Serratia marcescens
Yeasts
Aspergillus, Penicillium
Rhizopus nigricans
Erwinia rhapontici
Penicillium martensii
Xeromyces bisporus
Micrococcus luteus, Bacillus subtilis
Lactobacillus, Leuconostoc, Strepto-
coccus
Torulopsis, osmophilic yeasts
Clostridium, osmophilic yeasts
Fusarium, Penicillium, Aspergillus
Yeasts
Leuconostoc mesenteroides
Osmophilic yeasts
(maple, corn, chocolate, soft drink), and candy are usually spoiled by os-
mophilic or osmotolerant yeasts, such as Saccharomyces rouxii, S_ bailii, and
S. bisporus.
Lactobacilli are hops tolerant. These organisms produce lactic acid,
dextran hazes, and diacetyl in beer. A diacetyl flavor generally is not de-
sirable in the light lager beers produced in the United States. Several
yeasts, such as Brettanomyces, Candida, and Pichia can infect finished beer
and, with secondary fermentations, cause defects (dextran hazes, films,
off-flavors, and off-odors) in the beer.
The decarboxylation of hydroxycinnamic acids normally present in
barley, by certain enterobacteria, such as Hafnia, result in a phenolic off-
flavor in beer (Lindsay and Priest 1975)_ The genus name Pectinatus was
suggested for an anaerobic bacterium that can grow in hopped beer, and
cause turbidity with H
2
S production (Lee et al. 1980).
Animal Products
Since fresh animal products are perishable, they are chilled and
stored in ice or a refrigerator (0 to 4C). This means that psychrotrophs
become dominant and are the primary cause of spoilage. If these fresh
products are mishandled and allowed to remain at room temperature
(20C), a more diverse flora may be present, since not only will many
psychrotrophs grow, but also there will be some mesophilic strains.
The organisms most often involved with spoilage of refrigerated fresh
meat, poultry, fish, and eggs are species of the genus Pseudomonas. Pseudo-
monas putrefaciens has been designated as being important in spoilage of
meat and poultry. This organism does not conform to the given defini-
tion of Pseudomonas, so it was assigned the name Alteromonas putrefaciens.
Since the DNA base composition is somewhat higher than the range for
Alteromonas, the exact nomenclature should be delayed (Krieg and Holt
1984).
RED MEAT. In general, the muscle tissue of live, healthy animals is
sterile. The organisms that are present in the carcass are concentrated in
the lymph nodes. Researchers isolated organisms from 58 percent of 360
nodes they tested (Moo et al. 1980). Contaminated nodes are thought to
be a source of bacteria for deep spoilage of postrigor meat. After slaugh-
ter, the defense mechanisms are slowed or halted. This enables bacteria
to multiply and spread throughout the tissues.
The most common indications of spoilage are (1) off-odor and slime,
usually due to the growth of aerobic bacteria on the cut surfaces of meat;
(2) fungal growth, which is favored at water activities too low for bacterial
growth; (3) bone taint, or deep spoilage, due to anaerobic or facultative
FOOD SPOILAGE 413
microorganisms; and (4) discoloration, primarily due to alterations of
myoglobin, the muscle pigment.
There are some differences in the spoilage patterns of fresh and
cured meat. There is only limited information on cooked meat spoilage.
Generally, cooked meat is spoiled by the few types of organisms that can
survive the cooking or those that gain access to the cooked product.
Fresh, chilled meat spoilage is evidenced by off.odor and slime due to
Pseudomonas, Acinetobacter, and Alcaligenes. Cured meats become sour due
to the activity of Micrococcus, Lactobacillus, and Microbacterium species. Mi
crobial defects of meat and meat products are listed in Table 8.8.
Fresh Meat. The rate of spoilage depends upon the numbers and types of
organisms initially present, the conditions of storage (temperature and
gaseous atmosphere), and characteristics (pH, aw) of the meat.
In general, the whole carcass presents a surface of fat and connective
tissue affording little opportunity for bacterial growth. The surfaces of
meat cuts can support the growth of large numbers of bacteria, and
ground meat offers not only ample and desirable surfaces, but a thor
ough inoculation of the meat during grinding.
To retard microbial growth and biochemical changes, fresh meat is
stored at temperatures near OOC. However, at times, the meat is, or may
be, subjected to higher temperatures. The temperature of the animal is
approximately 37C. After slaughter, the temperature tends to increase
and then change to that of the room in which the meat is held. During
subsequent processing of the carcass, mishandling may occur and the
temperature may rise. Furthermore, if a consumer purchases the meat
and simply places it in a car trunk on a hot sunny day, the meat's temper
ature may be raised to a dangerously high level.
At room temperature (20C), the dominant organisms on spoilage
of sheep meat were Escherichia and Acinetobacterlike organisms (Rao and
Sreenivasamurthy 1985). They stated that Enterobacter, Pseudomonas, and
Staphylococcus also could form a major part of this flora.
To reduce the biochemical and microbial changes, fresh meat is
stored at temperatures near Oe. At low temperatures, spoilage is evi
denced on the surface. In storage conditions in which a moist meat sur
face in maintained, spoilage is due to Gramnegative bacteria, primarily
species of Pseudomonas. Species of Aeromonas, Alcaligenes. Acinetobacter, Mor
axella, Flavobacterium, Enterobacter, Microbacterium, Proteus, and Streptococcus
occasionally are found in the surface flora of spoiled meat.
The defects of the meat are off.odor, slime formation, and discolora-
tion. The flavor would be unacceptable to most people. In one study,
researchers could not detect proteolysis prior to the appearance of spoil-
age odor and slime (Dainty et al. 1975).
TABLE B.B. MICROBIAL DEFECTS OF RED MEATS AND THEIR PRODUCTS
Product
Fresh, refrigerated
(0-5C)
Vaccum packaged
Cured meat
Bacon
Vacuum packaged
Brines
Ham
Sausages
Fermented sausage
Canned
Commercially sterile
Semipreserved
Defect
Offodor, slime, discolora
tion
Lipolysis, pungent odor
Moldy
Whiskers
Black spot
White spot
Acid, sweet, rancid
Cheesy, sour, rancid
Discoloration
Slight souring
Putrefaction
Cabbage odor
Tainted
Turbid
Surface slime
Gassy or puffy
Green discoloration
Bone and meat "sours"
Surface growth (dry
cured)
Slime on surface
Gas production (vacuum
packed)
Greenish discoloration
Slime
Spots
Gas, putrefaction
Souring, discoloration
Putrefaction, gas
Organism
Pseudornonas, Aerornonas, Alcali
genes, Acinetobacter, Microbacter
iurn, Momxella, Proteus,
Flavobacteriurn, Alterornonas, Sac
charornyces
Pseudornonas, yeasts
Penicilliurn
Tharnnidiurn
Cladosporiurn
Sporot,-ichurn
Lactobacillus, Microbacteriurn, /<.'n-
terobacter, Hafnia
Micrococcus
Molds
Lactobacillus, Micrococcus, Vibrio,
Alcaligenes, Corynebacteriurn
Clostridiurn sporogenes
Proteus inconstans
Vibrio
Debaryornyces, Kloeckera
Micrococcus, Microbacteriurn, Yeasts
Clostridiurn
Lactobacillus, Streptococcus, Leucon
ostoc
Clostridiurn
Molds
Micrococcus, yeasts
Lactobacillus
Lactobacillus viridescens, Leuco-
nostoc
Yeasts
Molds
S poreformers (Bacillus, Clostri-
diurn)
Streptococcus
Bacillus, Clostridiurn
The important biochemical changes occur in the meat juices that con-
tain free amino acids, nucleotides, and peptides. These nutrients are suf-
ficient for microbial growth, and the metabolism of these compounds
leads to the formation of H
2
S, NH3 indole, and various amines and di-
amines.
The pigment stability in meat involves factors such as oxygen penetra-
tion, bacterial growth, lipolysis, and the presence of flavin compounds.
Exposure to light can enhance the destruction of oxymyoglobin on the
FOOD SPOILAGE 415
surface of beef. The H
2
S produced during breakdown of sulfur-
containing amino acids reacts with myoglobin at low oxygen levels, form-
ing the green sulfmyoglobin_
Water activity can playa role in spoilage_ Fresh meat will lose mois-
ture if stored below 99 percent RH_ Even at an RH of 99 percent, the
growth of Pseudomonas is reduced_ At an aw of 0_96 or less, most of the
usual microorganisms causing spoilage of fresh meat are inhibited_ When
the surface of meat is lower than a
w
0.96, the slower-growing fungi be-
come evident. In this situation, Thamnidium chaetocladioides and T. elegans
(cause of "whiskers"), Cladosporium herbarum ("black spot"), and Sporotri-
chum carnis ("white spot") growth can produce moldy meat. Gill, Lowry,
and Dimenna (1981) reported that besides C. herbarum, black spot is pro-
duced by C. cladosporioides, Penicillium hirsutum, and Aureobasidium pullu-
lans. White-spot colonies yielded Chrysosporium pannorum or auremonium.
Besides. T. elegans, whiskers colonies yielded Mucor racemosus (Lowry and
Gill 1984).
Storage atmosphere can influence meat spoilage. When meat is vac-
uum packaged, the dominant flora tends to be Lactobacillus instead of
pseudo monads. Other microorganisms include Leuconostoc, Brochothrix,
Enterobacter, Hafnia, and Altermonas. Odors of stored, vacuum-packaged
meat have been described as acidic, sour, sweet, cheesy, stale, or rancid.
The spoilage of meat under both aerobic and anaerobic conditions
was reviewed by Gill (1983).
Cured Meat. Meat that is treated with salts such as NaC1, NaN0
2
, and
NaNOs differ from untreated fresh meat. The most apparent differences
are in the color and flavor of fresh and cured meat. However, the intrin-
sic factors of cured meat, such as pH, a
w
, and the presence of inhibitors,
play an important role in the microbial ecology of these products. Be-
sides ham, bacon, and corned beef, there are cured comminuted meats
wieners, bolognas).
The aw of cured meat (0.88 to 0.95) is lower than that of fresh meat
(0.98-1.00) and the specific inhibitory effects of the added salts may ac-
count for the differences in the spoilage patterns of fresh and cured
meats.
For bacon, the important types of organisms are Micrococcus, yeast,
Vibrio, Acinetobacter, Alcaligenes, Arthrobacter, and Corynebacterium (Gardner
1971). It is generally believed that bacon in aerobic conditions is spoiled
by micrococci. However, a significant portion of the flora on the lean
portion of the bacon is Gram-negative rods. As the pH of bacon decreases
from 6.0 to 5.5, the yeasts in the flora increase during spoilage. Many
molds, such as Alternaria, Aspergillus, Mucor, and Rhizopus can cause sur-
face discolorations.
According to Gardner (1971), chemical changes do not occur in bacon
until the total microbial count reaches lO8/g or higher. The off.odors of
spoiled bacon were described as cheesy, putrid, or sour by Dempster
(1972), or as smoked fish and rancid cheesy by Ingram and Dainty (1971).
The rancid cheesy flavor was thought to be due to lipolysis of fat by mi
crococci.
The predominant microflora of vacuumpackaged bacon is deter-
mined by the salt content. Species of Micrococcus give way to lactic acid
bacteria in bacon with low salt (5 to 7 percent), while they remain domi
nant in bacon with higher salt ( 8 to 12 percent).
For ham, the important type of spoilage is "souring." This includes
various spoilage problems from mild degradation to extreme putrefac-
tion. Before the modern short-curing methods, clostridia caused bone
sours prior to the meat being chilled or cured.
The short-cure commercial method of curing has greatly reduced the
incidence of ham souring. However, gassy or puffy hams can result if the
fresh meat is held too long at a moderate temperature prior to cure. This
allows clostridia to grow. Proper slaughter and bleeding of hogs, prompt
chilling of the meat, prompt handling, good sanitation, and use of pump
and cover brines that have low microbial counts have aided in improving
the microbial quality of ham.
Modern commercial ham has a milder cure (with less salt), milder
smoking, a more moist surface, and vacuum packaging, than dry-cured
hams. Slicing during packaging creates sources of contamination and
more cut surfaces on which microorganisms can grow. Although the dry-
salt-cured hams suffer from mold growth on the surface, the commercial
hams allow the growth of micrococci, microbacteria, various lactic acid
bacteria, and yeasts.
Comminuted and cured sausage products are subjected to various
types of spoilage. Generally, these products are composed of mixtures of
pork, beef, salt, sugar, sodium nitrite, and spices. Hence, the flora of
these products is somewhat different from that of fresh meat. Bell and
Gill (1982) found that the flora of freshly cooked luncheon meat con-
sisted of about equal numbers of Bacillus and Micrococcus. These research
ers reported that little microbial growth could be detected when this
meat was held at lOoC for forty-two days. However, at 25C, the Bacillus
grew at the surface and reduced the nitrite level and lowered the pH
from 6.8 to 6.2, as well as produced amylase enzymes. By the fourteenth
day, a Streptococcus replaced Bacillus and continued lowering the pH to
5.2. A low pH, vacuum packaging and low-temperature storage help ex-
tend the shelf life of this type of meat product.
A surface slime of micrococci and yeasts can occur when sufficient
moisture is present. As the surface dries, inhibiting the bacteria, mold
growth can cause spoilage. When products such as luncheon meats are
FOOD SPOILAGE 417
vacuum packaged, aerobic growth is inhibited and lactic acid bacteria
become dominant. Facultatively anaerobic yeasts may grow. The lactic
acid bacteria produce CO2 and can cause a swelling of the package, while
the yeasts produce a surface slime. At 25C, the shelf life of chubpacked
luncheon meat was inversely proportional to the availability of oxygen
(Bell and DeLacy 1982). At 25C, the principal spoilage organisms of
chub packed luncheon meat were Bacillus licheniformis and Streptococcus
faecium. B. licheniformis caused surface softening, discoloration, gas pro
duction, and product liquefaction, while the S. jaecium produced lactic
acid which caused souring of the meat product.
For bologna products, a casing is used. Moisture accumulates at the
meatcasing interface, allowing the growth of bacteria. Micrococci can
cause a slime layer during growth at this interface.
During storage of these meats, microorganisms of the genera (Micro-
coccus, Lactobacillus, and Leuconostoc can grow and cause souring of the
product. The production of organic acids and reducing compounds by
the bacteria can cause a fading of the pink color at the outer surface of
the product, resulting in a ring of discoloration.
Green discolorations can occur in cured sausages. The greening may
appear as rings, cores, or on the surface. Lactobacillus viridescens can grow
in products with reduced oxygen tension and pH and is the organism
primarily involved in greening of these sausages. Other Lactobacillus and
Leuconostoc species sometimes are present. The bacteria produce perox
ides that react with the cured meat pigment, causing the green discolora-
tion. The first evidence is the appearance of small greenish spots on the
damp surfaces of the cured sausage. These spots tend to spread and cover
the sausage surface if favorable conditions exist for a sufficient time. In
some cases, a slimy appearance may be noted.
Fermented Sausages. Some comminuted sausages, such as cervelat, Thur-
inger, and Lebanon bologna, are not only cured but also are fermented
by lactic acid bacteria to produce a tangy flavor. Dependence upon the
natural contaminants to ferment the product may cause spoilage, so a
starter culture of homo fermentative lactic acid bacteria is used. The
lower pH of these fermented sausages helps control some types of bacte-
ria, but they are susceptible to spoilage similar to cured sausage products.
A surface slime due to yeasts, and discoloration due to mold, may occur.
Canned Meat. Canned meat products are subject to the same type of spoil-
age as other low-acid foods, if heat-resistance spores which survive the
process can germinate and grow. In semipreserved or pasteurized cured
products, such as canned ham, the curing salts and refrigeration are used
to prevent spore germination and outgrowth. If not adequately proc-
essed, thermoduric cells, such as Streptococcus jaecium, may survive and
cause souring. This organism may cause rapid discoloration after the
product is removed from the can. If the spores of clostridia are able to
germinate, and the resultant cells grow, gas may be formed along with
extensive putrefaction. If held at a high temperature (55C), Bacillus spe
cies (B. stearothermophilus or B. coagulans) may cause flat sour spoilage of
canned meats and of other canned foods.
POULTRY. The spoilage of uneviscerated and eviscerated poultry is
somewhat different from the spoilage of canned meat. However, because
commercial poultry in the United States is eviscerated, only this type of
poultry is considered in this section. Uneviscerated poultry is discussed
by Barnes and Shrimpton (1957).
Immediately after processing, any of several hundred species of mi
croorganisms might be found. However, as the poultry is chilled and held
in cold storage, psychrotrophic microorganisms predominate and cause
deterioration.
The main defects are off-odor, which appears at a bacterial load be
tween 10
6
and 108/cm2, and slime formation, which occurs soon after off-
odor is noted. As the number of bacteria increases, the flavor score
decreases, species of Pseudomonas are the principal spoilage organisms.
Besides Pseudomonas, other organisms, similar to those in fresh red meat
spoilage, are found on spoiled poultry (Table 8.9). These include Aeromo
nas, Moraxella, Alcaligenes, Flavobacterium, and Micrococcus. When antibiotics
were used on poultry, yeasts became the important spoilage micro organ
TABLE 8.9. MICROBIAL DEFECTS OF POULTRY AND POULTRY PRODUCTS
Product
Poultry meat
Shell eggs
Liquid whole egg
Liquid albumen
Defect
Off odor, slime
Black rot
White rot (colorless)
Sour
Green white
Musty
Moldy
Red rot
Custard rot
Yellow and green rot
Fishy
Offodor, sour
Offodor
Microorganism
Pseudomonas, Acinetobacter, Moraxella,
Alcaligenes, Aeromonas, Alteromonas
Proteus, Aeromonas
Citrobacter, Alcaligenes
Pseudomonas
P. jluorescens
Pseudomonas
Many types of molds
Serratia marcescens
Citrobacter, Proteus, Enterobacter
Alcaligenes, Flavobacterium, Cytophaga
Pseudomorws, Flavobacterium, Chromo
bacterium
Proteus, Alcaligenes, Escherichia, Flavo
bacterium, Pseudomonas, Bacillus
Pseudomonas, Acinetobacter, Enterobac
ter
FOOD SPOILAGE 419
isms. However, yeasts are not important on normally processed and
eviscerated poultry.
Daud, McMeekin, and Thomas (1979) studied the flora of chicken
meat held at 2C. Before storage, the flora included species of Micrococ
cus, Flavobacterium, Cytophaga, Acinetobacter, Moraxella, and Pseudomonas, as
well as enterics. During storage, the off.odor producers (principally
Pseudomonas II) became dominant, accounting for 62 percent of the popu
lation after twelve days. At this time, species of Pseudomonas accounted
for 98 percent of the bacterial population. Nonpigmented strains of
pseudomonads produce more intense odors than do pigmented strains.
Off.odors of poultry have been listed as sulfidelike, fruity, fishy, and like
evaporated milk.
The chemicals produced by pseudomonads grown in chicken meat
include acetone, 2butanone, 2pentanone, toluene, dimethyl benzenes,
ethyl methyl disulfide, 2-heptanone, and dimethyl trisulfide (Pittard et al.
1982). Some of the more important volatile sulfur-containing compounds
are dimethyl disulfide, dimethyl sulfide, methanethiol and propylene sul-
fide (Bowman et al. 1983).
Packaging poultry in oxygen-impermeable film or vacuum packaging
it causes inhibition of psuedomonads so that the predominant flora at
spoilage consists of lactic and enteric types of bacteria.
EGGS. It is generally accepted that, when an egg is laid, its contents are
free from bacteria. But there are exceptions because of ovarian infec-
tions.
The egg contents are protected by the shell and associated mem-
branes and chemical inhibitors in the egg albumen. This means that mi-
croorganisms must penetrate these barriers and then be able to grow
inside the egg to cause spoilage. Penetration of eggs is aided by moisture
on the shell. If the eggs are not properly stored or washed, penetration
may be quite rapid, and spoilage can occur.
For egg products, the shell and membranes are removed. During
processing, the liquid egg is subject to contamination from organisms
on the egg shell, equipment, humans, added ingredients, and the final
container.
Bacterial analysis of shell eggs during storage has revealed molds (Pen-
icillium, Aspergillus, Cladosporium, Rhizopus, and Mucor), yeasts (Rhodotorula),
and bacteria (Pseudomonas, Micrococcus, Bacillus, Proteus, Alcaligenes, Flavo-
bacterium, Citrobacter, Escherichia, and Enterobacter).
As with other protein foods, species of Pseudomonas are the main spoil-
age organisms of shell eggs and egg products.
When bacteria grow within the egg, they decompose the contents and
form by-products. This results in characteristic odors, appearance, or
colors from which the various rots acquire their name (Table 8.9).
The United States Department of Agriculture (USDA 1977) described
a loss egg as "an egg that is inedible, smashed or broken so that the con-
tents are leaking, frozen, contaminated, or containing bloody whites,
large blood spots, largely unsightly meat spots, or other foreign m t e ~
riaL" Inedible eggs are due to nonmicrobial defects as well as microbial
defects. An egg with a blood ring, embryo, or stuck yolk is inedible.
Besides these loss types, shell eggs will absorb odors from the storage
atmosphere. Most vegetables and fruits will impart flavors and odors to
shell eggs. If stored with apples, the eggs will be bitter and have a card-
board flavor and odor. If stored near gasoline or kerosene, the shell eggs
will taste and smell like these compounds.
Eggs that become inedible due to microbial growth are listed by
USDA (1977) as black rots, white rots, sour eggs, eggs with green whites,
mixed rots, musty eggs, and moldy eggs.
Other designations for rotten eggs are fluorescent green, red, custard,
colorless, green and yellow, mixed, and rusty red rots.
Black Rots. When viewed with a candling light, black rot eggs are virtually
opaque. When broken out, the egg content has a muddy (dark brown)
appearance and a repulsive putrid odor, and H2S is evident. In many
eggs, an internal gas pressure develops. The bacteria associated with this
type of spoilage are species of Proteus and Aeromonas. Rots of this type are
more likely to occur at room temperature (20C) than in cold storage
(4C or less).
White Rot. Threadlike shadows may be seen in the thin white, and in later
stages the yolk appears severely blemished when the shell egg is viewed
with the candling light. When opened, the egg yolk shows a crusted ap
pearance and frequently has a fruity odor.
This type of inedible egg sometimes is referred to as a colorless rot.
Various organisms have been associated with this rot, including Citro-
bacter, Salmonella, and Alcaligenes.
Sour Eggs. These eggs are difficult to detect by ordinary candling, but
they usually show a weak white and murky shadow around an off-center,
swollen yolk. These eggs also are called fluorescent and are quite readily
detected by observing with UV light. A green sheen is produced by spe-
cies of Pseudomonas.
Since a green fluorescence is observed, these inedible eggs also have
been termed fluorescent green rots.
Green Whites. This defect is caused mainly by Pseudomonas jluorescens.
Green whites of broken-out eggs fluoresce when observed with UV light.
FOOD SPOILAGE 421
Eggs with green whites mayor may not have a sour odor, since the green
fluorescence can be observed long before any odor can be detected.
Musty Eggs. These frequently appear clear and free from foreign material
when candled. The musty odor may be caused by odors in the atmo
sphere being absorbed by the egg contents. Also, some microorganisms
occasionally invade shell eggs and produce a musty odor.
Moldy Eggs. Mold growth is visible as spots on the shell, in checked areas
of the shell, or inside the egg.
The mold contamination of eggs seems to be due to the reuse of
moldy packing materials. Several molds, such as Penicillium, Alternaria,
and Rhizopus can grow on eggs. However, molds are not a prominent
cause of egg spoilage.
Red Rot. These eggs are distinguished by a red discoloration of the albu
men and the surface of the yolk. An ammoniacal to putrid odor may
occur. Serratia marcescens has been considered as the cause of red rot.
Custard Rot. In this rot, the yolk is encrusted with custardlike material
and occasionally flecked with olive green pigment. The albumen be
comes thin with an orange tint. There may be a slightly putrid to putrid
odor. Citrobacter and Proteus vulgaris have been associated with this type
of spoilage.
Mixed Rot. These addled eggs occur when the vitelline membrane of the
yolk breaks and the yolk mixes with the white, resulting in a murkiness
throughout the interior of the egg when viewed with a candling light.
With no offodor, these are referred to as odorless mixed rots.
Other Rots. Alcaligenes has been accused of causing both yellow rots and
green rots. These rots are similar in odor and in the appearance of albu
men. However, the yolk is dark yellow in the former and dark green to
black in the latter case.
Rust red rot is associated with growth of P. vulgaris.
Egg Products. The spoilage of egg products is evidenced by off-odors de-
scribed as sour, fecal, or fishy. Organic acids (lactic, succinic) are recog-
nized as indices of decomposition of egg products.
SEAFOODS. Fish and other seafoods are subject to contamination of
microorganisms in their marine environment, as well as those acquired
during catching, handling, and processing. However, as with other foods,
even though a mixed flora exists, certain types of organisms become
dominant during storage of the product. The dominant types are those
that can survive and multiply on or in the food at the temperature that
prevails. The defects of seafoods are listed in Table 8.10.
TABLE 8.10. SOME MICROBIAL DEFECTS OF SEAFOODS
Product
Fish
Fresh
Salted
Crayfish
Oysters
Shrimp
Squid
Defect
Off-odor
Fruity
Ammoniacal
H
2
S odor
Pink
Dun (red growth)
Cheesy, putrefactive
Sweet to foul odor
Pink
Off.odor
Yellow discoloration
Red discoloration
Microorganism
Pseudomonas, Alterumunas, Acinetubacter,
Vibrio, Aeromonas, Moraxella, Proteus
Pseudomonas
pseudomonas, Alteromonas
Pseudomonas, Alteromonas
Halobacterium, Halococcus
Hemispora stellata, Sporendonema epizoum
Red halophilic bacteria
Pseudomonas, Lactobacillus, Coryneforms
Yeasts (Rhodotorula)
Pseudomonas
P. putida
Serratia marcescens
Fish. The spoilage pattern depends somewhat on the species and type of
fish, the initial microbial flora, the area of catch, the method of catch,
processing method, and method of storage.
The initial microbial flora is dependent upon the contamination of
the water and bottom sediment from the area of catch. Fish caught on a
line have lower counts than fish that are trawled by dragging a net along
the bottom. The trawl net drags through the bottom sediment, which usu-
ally has high counts of microorganisms.
The temperature of the water affects the fish microflora. Fish from
warm seas tend to have mesophilic strains; in cold seas, the organisms
are primarily psychrotrophs.
The methods for handling the fish after catching influence the con-
tamination. Fish do not have a sphincter muscle, so when any pressure
is exerted, intestinal discharge occurs. This pressure can happen when
the net is hauled aboard, when the fish are piled on the deck, or when
whole fish are stored in ice.
The caught fish may be left whole, or they may be beheaded or gutted.
A gutted fish may spoil somewhat differently than a whole fish, due to
the intestinal flora.
The fish can pick up added contamination on shipboard if the hold-
ing areas are not maintained in a sanitary manner. The fish must be han-
dled rapidly on the ship, and chilled. Usually the fish are mixed with ice
or with chilled seawater for cooling; however, some ships have freezing
facilities.
During processing, the organisms in the surface slime layer or the
skin can be spread throughout the processing equipment, the workers,
and onto the flesh of the fillet. Hence, the normal potentially sterile flesh
FOOD SPOILAGE 423
can be inoculated with millions of bacteria. With this contamination, the
fish may be spoiled by the time they reach the consumer. The microbial
flora on the fish leaving the processing plant may be different from that
on the fish entering the plant.
Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Clostridium, coryneforms,
Cytophaga, Flavobacterium, Micrococcus, Proteus, Psuedomonas, and Vibrio have
been isolated from fresh and spoiled fish. Members of the family Enter
obacteriaceae rarely are found on fish but may be detected on fish har
vested in polluted areas of both fresh and saltwater.
During storage of the fish at oDe, the number of organisms increases
after a one to twoday lag period. During spoilage, the Pseudomonas and
Acinetobacter become increasingly dominant, with Flavobacterium showing
a transient increase. By ten to twelve days, Pseudomonas may comprise 90
percent of the population. The species of Pseudomonas tend to change as
spoilage progresses. Regardless of which species are involved, organisms
of the genus Pseudomonas are the most active spoilage organisms in fish
stored at ODe. The primary factor influencing the spoilage rate is temper
ature. As the temperature is increased from oDe the growth rate of pseu
domonads increases. However, as the temperature approaches 20
o
C, the
mesophilic flora becomes evident.
Lipids in fish have a high content of polyunsaturated fatty acids.
Hence, oxidative rancidity is more evident in fish than in most other
animal tissue.
In marine fish, the trimethylamine oxide (TMAO) is reduced by bacte
rial and enzymatic action to trimethylamine (TMA), a spoilage product.
The odor of trimethylamine at low levels is referred to as a stale fishy
odor. Piperidine, aamino valerie acid, and aamino valeric aldehyde also
have a fishy odor.
During spoilage, volatile bases, amines, and organic acids are formed
by decarboxylation or by deamination of amino acids and organic bases.
Hydrogen sulfide, mercaptans, and disulfides add to spoilage odors.
Spoilage odors have been designated as fishy, stale, musty, rancid, sour,
ammoniacal, yeasty, fruity, sweet, acid, and putrid.
Vacuum packaging of fish changes the spoilage flora in a manner
similar to that of fresh meat. Lactobacillus and Microbacterium grow and
cause souring, whereas the aerobic psuedomonads are inhibited. How
ever, for vacuum packaged sand flatheat fillets, McMeekin, Hulse, and
Bremner (1982) reported Alteromonas species dominated the spoilage
flora. Reducing the pH increased the number of Enterobacteriaceae.
Packaging of swordfish steaks in 100 percent CO
2
resulted in lactobacilli
becoming dominant (Lannelongue et al. 1982).
Shewan (1971) stated that bacterial spoilage can be prevented by stor
ing fish below - lODe, so, from a practical viewpoint, the bacteriology of
spoilage is somewhat academic. This is also true for spoilage of meat and
poultry products. It is unfortunate that so much of our meat, poultry,
and fish is maintained in the socalled fresh condition, rather than being
frozen.
Crayfish. The spoilage bacteria of freshwater crayfish was studied by Cox
and Lovell (1973). They reported that spoilage of the tail occurred when
the total aerobic count reached 109/g. In the fresh meat, Micrococcus, Staph
ylococcus, and Alcaligenes were the major genera, while in meat spoilage, at
OOC, Pseudomonas, and at 5C, Achromobacter (Acinetobacter, Moraxella) pre
dominated. These genera comprised the major bacterial flora of the
spoiled meat at either temperature (0 or 5C).
Clams. The microbiology of soft shell clams was reported by Cox (1965).
The clams were judged to be unacceptable on the basis of black discolora
tion or a slimy appearance. The predominating spoilage bacteria are
Gramnegative rods that produce sweet esterlike, musty, oniony, or
cesspoollike odors on agar media. He suggested that the spoilage flora
consisted mainly of Pseudomonas types.
Crabs. During processing of red crab, the aerobic plate count (35C) in
creased from 170 CFU/g after cooking to 270,000 CFU/g in the finished
product (Wentz et al. 1985). Loaharanu and Lopez (1970) studied the
spoilage of pasteurized crab cake mix. When stored at 18 or 30C Bacil
lus and Micrococcus species dominated, while at 2C, the dominant genus
was Alcaligenes.
Oysters. Frozen or fresh oysters spoil by either fermentative mechanisms
(characterized by a sour odor) or by putrefactive mechanisms. Canned
oyster decomposition is putrefactive. Oysters that are well cooked and
then refrigerated spoil due to fat rancidity, rather than to microbial de-
composition. Red or pink coloration of oysters may be due to S. marces
cens or to pink yeasts, such as Rhodotorula, as well as to phytoplankton or
eggs of the oyster crab. Various discolorations can occur in shellfish
(Boon 1977).
Shrimp. These animals are caught at sea by towing a trawl net along the
bottom. After one to three hours of trawling, the net is hauled aboard
and emptied. Since the bottom sediment is more contaminated with bac-
teria than the upper water, one would expect harvested shrimp to have
rather high bacterial counts. Novak (1973) reported counts of 31,000 to
1,200,000 bacteria per gram of whole shimp. He further reported that,
although the head was 40 percent of the shrimp weight, it contained 75
percent of the bacteria. Therefore, heading the shrimp lowered the mi-
crobial count.
FOOD SPOILAGE 425
Bieler, Matthews, and Koburger (1973) reported that the heading of
shrimp exposed tissues, allowed the gut to empty, and triggered enzy-
matic changes_ Hence, they found that shrimp stored in ice maintained
better quality with the head on than when headless_
The predominant organisms on iced raw shrimp are Moraxella, Pseudo-
monas, Acinetobacter, Arthrobacter, Flavobacterium and Cytophaga (Lee and
Pfeifer 1977). They believed that the presence of Arthrobacter and Acineto-
bacter may indicate inadequate cleaning. The presence of Moraxella, Flavo-
bacterium, and Cytophaga indicates the degree of secondary contamination,
and Pseudomonas indicates the potential shelf life of processed shrimp.
When shrimp was stored at OOC for eight days, Pseudomonas species pre
dominated (Matches 1982). Storage at 5.6 or 11C resulted in the dom-
inance of Moraxella, while at 16 or 22C, Proteus, which produced indole,
became dominant. Researchers reported that 42 of 1,647 isolates from
putrid shrimp produced indole (Smith et al. 1984). The indole levels of
severely decomposed shrimp were less than the 25 p.gllOOg of shrimp
suggested as the defect action level by the FDA (Chang et al. 1983).
DAIRY PRODUCTS. Milk, when obtained from the cow, might not be
sterile. During and after milking, the milk is subjected to organisms from
various sources, although contaminated equipment used to handle, trans-
port, store, and process the milk seems to be the main source of organ-
isms. The genera found in cold-stored milk include Pseudomonas, Acineto-
bacter, Aerobacter, Alcaligenes, and Flavobacterium as well as members of the
family Enterobacteriaceae. With extended storage of milk products at
refrigerated temperatures, psychrophilic or psychrotrophic organisms
are a cause of spoilage.
Spoilage of milk usually is caused by psychrotrophs that recontami-
nate the milk after pasteurization. Also involved are thermoduric psy
chrotrophs as well as heat-stable proteases produced before pasteurization.
The defects that can occur in milk due to microbial growth are (1) off.
flavors; (2) lipolysis with development of rancidity; (3) gas production; (4)
fermentation to lactic acid with souring; (5) coagulation of milk proteins;
(6) viscous or ropy texture; and (7) discoloration. Some of the defects of
milk and dairy products are listed in Table 8.11.
As with other animal products, species of Pseudomonas are prominent
in producing defects in milk. They are associated with a bitter flavor and
rancidity as well as a fruity odor. The fruity odor is due to a mixture of
ethyl butyrate and ethyl hexanoate.
Certain lactic acid bacteria are involved with defects in fresh milk.
Although the production of lactic acid and the resulting souring are de-
sirable in fermented milk products, they are undesirable in fresh milk.
Besides this type of defect, a strain of Streptococcus lac tis designated as
TABLE 8.11. SOME MICROBIAL DEFECTS IN DAIRY PRODUCTS
Product Defect
Milk
Pasteurized, Rancid
refrigerated
Ropy or slimy
Sour (acid, gas)
Discoloration
Bitter, fruity
Canned Swelling, gas
Cream Foamy
Butter Surface taint
Cheese Gassy, butyric acid
Gassy, floating, or split
curd
Moldy
Soft Black mold (eat's fur)
Surface growth
Cottage Slimy curd, putrid odor
Discoloration
Slimy, gelatinous
Fruity
Cheddar Sweet, yeasty, fruity
Swiss Gassy, sweet
Offodor
Yogurt Yeasty
Microorganism
Pseudomonas, Alcaligenes, Staphylococcus
Coliforms, Pseudomonas, Alcaligenes, Mi
crococcus, Bacillus subtilis
Lactic acid bacteria
Chromobacterium
Pseudomonas, Flavobacterium, Alcaligenes,
Proteus, Acinetobacter
Clostridium sporogenes
Candida, Torulopsis
Pseudomonas, Alteromonas
Clostridium tyrobutyricum
Leuconostoc, S. lactis subsp. diacetylactis
Penicillium, Scopulariopsis, Mucor, other
molds
Mucor
Torulopsis, Debaryomyces
Pseudomonas
Flavobacterium, yeasts, molds
Pseudomonas, Alcaligenes, Flavobacterium,
coli forms
Yeasts
Yeasts
Yeasts (Torulopsis)
C. sporogenes
Yeasts (Torulopsis)
variety maltigenes metabolizes leucine and produces 3-methylbutanol,
which gives a malty flavor defect in dairy products. At the time the aroma
first becomes detectable, counts range from 10
7
to 10
8
per milliliter. Ropy
milk has been ascribed to several organisms, such as Alcaligenes, Klebsiella,
Pseudomonas, and Flavobacterium. Streptococcus lactis longi and Streptococcus
cremoris longi were isolated from ropy milk (Macura and Townsley 1984).
Butter. This is primarily fat with some moisture, carbohydrate, and pro
tein. The main defect is due to rancidity either by autoxidation of fatty
acids or by lipolysis due to microbial enzymes. The presence of short
chain acids (butyric and caproic) results in a rancid flavor. Molds are able
to grow on the surface of butter. Putrid, proteolytic, and fruity flavors in
butters are caused by psychrotrophic bacteria.
Surface taint and yeasty butter are other defects of this product
caused by microorganisms. Butter is made from cream. If the cream has
a defect, such as rancidity, it can affect the quality of the butter.
FOOD SPOILAGE 427
Cheese. Defects that can occur in milk may appear in other dairy products
made from the defective milk.
In cottage cheese, the flavor defects of milk may be accompanied by
a gelatinous or tapioca curd formation. Lipolytic and proteolytic organ
isms cause defects in cottage cheese.
P.fragi and A. putrefaciens have been associated with a slimy curd defect
on the surface of cottage cheese. A fruity, putrid, or rancid odor and a
fruity or bitter flavor may accompany this defect.
Surface discolorations may occur due to the growth of the pigmented
Flavobacterium. E. coli can cause barny or unclean flavors and, if the cot-
tage cheese is held at room temperature, the organism can cause a gassy
defect.
The yeast Rhodotorula produces pink spots on the surface of the prod-
uct. These pink spots eventually become a pink slime. Torulopsis also pro-
duces a slime, but it is yellow. Geotrichum produces off-white, tan, or yel-
low surface discolorations.
Molds multiply rather slowly. Cottage cheese may be spoiled by other
organisms before mold growth is evident. However, there are occasions
when growth of Penicillium and Mucor appears on the surface of the
product.
A pink discoloration of Romano and other Italian cheese varieties
was investigated by Shannon, Olson, and Vonelbe (1969). The pink dis-
coloration occurred as a uniform band of color near the cheese surface,
or as discoloration throughout the entire cheese. According to them, spe-
cies of Lactobacillus (L. helveticus and L. bulgaricus) can cause this discolora-
tion. Pink discolorations due to strains of Propionibacterium, certain reduc-
ing Bacillus species and reducing micrococci have been suggested as
causing pink to red discolorations in various cheeses.
Early gas defects of semihard cheeses usually are caused by coliforms,
while late gas defects are caused by fermentation by Clostridium tyrobutyr-
icum.
The growth of molds such as Penicillium, Mucor, and Aspergillus can
appear on the surface of cheese. Some of the molds growing on cheese
can produce mycotoxins (see Chapter 6).
Fats and Oils
Rancidity, acidity, soapiness, and off-flavor defects can occur in fatty
foods, such as butter, margarine, lard, and vegetable oils. The develop-
ment of rancidity can be due to autoxidative deterioration, lipolysis by
microbial lipases, or lip oxidation due to lipoxidase enzymes.
There are several lipolytic microorganisms (Table S.4). Microorgan-
isms associated with the liberation of free fatty acids in butter and mar
garine include Cladosporium, Candida, Pseudomonas, Micrococcus, Penicillium,
and Geotrichum. The hydrolysis of olive oil, coconut oil, and butterfat can
be caused by species of Micrococcus, Pseudomonas, Serratia, and Staphylo
coccus.
The defect characterized by rancidity and acidity is due to the libera
tion of free fatty acids, especially butyric (C
4
), caproic (C
6
), caprylic (C
s
),
and capric (CIO), and the production of their corresponding methyl ke
tones. When lauric and myristic acids are liberated from triglycerides in
coconut oil and butterfat, a soapy flavor defect can occur.
The spoilage of mayonnaise and salad dressings was investigated by
Kurtzman, Rogers, and Hesseltine (l971). Saccharomyces bailii was the prin
cipal cause of mayonnaise spoilage, although Lactobacillus jructivorans also
was involved. Species of Bacillus were isolated from several spoiled sam
pIes. The yeast Zygosaccharomyces was involved in spoilage of French salad
dressing. Rather low numbers of yeast and bacteria were present in the
spoiled product. They reported only five yeasts per gram in one spoiled
mayonnaise sample. The highest yeast count they reported was 165,500
per gram.
Canned Food
Canned foods are discussed in the section on heat preservation, but
some recognition of them should be made here.
Bacterial spoilage of canned food may be due to underprocessing,
elevated storage temperature, or leakage of the container due to im
proper closures, rough handling, or defective cans.
Underprocessed cans of foods usually are spoiled by heatresistant
sporeformers (Bacillus, Desulfotomaculum, and Clostridium), which survive
the process. Leakage can result in spoilage by nonsporeformers, which
could not have survived the heat processing, but recontaminate the food
after the heat treatment. Heatresistant thermophilic organisms may sur
vive the processing and, if the food is not cooled after heating, or if the
cans are stored at elevated temperatures, these thermophiles can grow.
There are four prominent thermophilic organisms causing spoilage of
canned food. B. stearothermophilus causes flat sour spoilage of canned non
acid foods. It is facultatively anaerobic and can grow at 70C. B. coagulans
is less heat resistant but is more acid tolerant than B. stearothermophilus.
It causes flat sour spoilage of canned tomatoes.
Clostridium thermosaccharolyticum forms large volumes of gas and result-
ant hard swells or blown cans. The spores are very heat resistant. Blown
cans can be caused by various species of clostridia. Desulfotomaculum nigri
FOOD SPOILAGE 429
cans produces hydrogen sulfide. This gas reacts with metallic ions from
the container or food, forming sulfides, some of which are black.
Molds and yeasts are not significantly important in the spoilage of
canned foods, except for mold spores such as B. fulva, which can survive
fruit processing treatments.
The movement of bacteria from contaminated cooling water into the
processed canned food may result in various spoilage problems. Bean
and Everton (1971) discussed a problem of pigmented bacteria surviving
the chlorination treatment given the water used to cool heat-processed
cans of product. No gas was noted (no swelling of the cans), but a canned
pudding developed cheesy to sour flavors and there was separation of
the product.
Bacillus subtilis and Enterococcus faecium were isolated from a can of
coagulated evaporated milk by McKellar and Nichols-Nelson (1984). Nei-
ther organism alone caused the defect. They concluded that B. subtilis sup-
plied low-molecular-weight nitrogenous compounds for the growth of E.
faecium, which produced acid, causing the coagulation.
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Bean, P. G., and Everton,]. R. 1969. Observations on the taxonomy of chromogenic bac-
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Bell, R. G., and DeLacy, K. M. 1982. The role of oxygen in the microbial spoilage of
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FOOD SPOILAGE 431
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9
Useful Microorganisms
Microorganisms are used in many facets of the food industry. Desired
alterations of food by microorganisms are referred to as fermentations,
regardless of the type of metabolism. By definition, fermentation is the
anaerobic breakdown of an organic substance by an enzyme system, in
which the final hydrogen acceptor is an organic compound. Hence, the
aerobic oxidation of alcohol to acetic acid in vinegar production is not
a true fermentation. Hence, for our purposes these alterations of foods
are called food conversions. Since the enzyme systems ofthe microorgan
isms catalyze the changes in foods, for some reactions it is advantageous
to use purified enzymes separated from the microbial cells.
Food conversions may reduce the food volume as well as aid in preser
vation. An example is the changing of milk to cheese. These changes can
eliminate undesirable or toxic factors as well as enhance the nutrient
value of foods. It is possible to convert substances presently considered
wastes into useful products or foods.
Besides changing the form of food, microorganisms can be used to
produce chemicals for food additives, such as acids, flavors, vitamins,
amino acids, or protein. Systems using microorganisms or enzymes have
been developed to assay for vitamins, amino acids, and other constitu-
ents of foods. These systems have certain advantages over normally used
chemical systems for analysis.
FOOD CONVERSIONS
The conversion of one food to another may be called controlled deg-
radation. Various types of food conversions are listed in Table 9.1. This
table does not list all of the many food conversions. Other sources, such
as Pederson (1979) and Reed (1982) should be consulted for more com-
plete descriptions of food conversions than is possible in this text. In
many countries, food fermentation is used as a system of preservation,
whereas in the United States these foods are produced for their special
flavors or other characteristics.
433
TABLE 9.1. SOME MICROBIAL FOOD CONVERSIONS
Organisms
Lactic acid bacteria, species of
Leuconostoc, Lactobacillus, Pedi-
coccus and/or Streptococcus
Propionibacterium
Breuibacterium linens
Penicillium roqueforti
P. camemberti
Lactic acid bacteria with yeasts
Saccharomyces, Aspergillus oryzae
A. jlavus or Mucor rouxii, yeasts
Yeasts
Yeasts with Acetobacter or Glucon
obacter
Halophilic bacteria
Bacillus subtilis
Actinomucor elegans, Mucor
Rhizopus oligosporus
R. oligosporus
Neurospora sitophila
434
Substrates
Cabbage
Cucumbers
Olives
Vanilla beans
Red meat
Milk products
Cream
Milk
Milk
Milk
Unripened cheese
Unripened cheese
Unripened cheese
Unripened cheese
Taro
Flour (dough)
Ginger plant
Beans
Rice, black gram
Soybeans, wheat
Kaffir corn
Malt
Fruit
Wines
Molasses
Grain mash
Flour (dough)
Fruit peel
Sugar, fruit, potatoes,
honey, malt, grain
alcohol
Fish
Soybeans
Soybean curd
Soybeans
Coconut press cake
Peanut press cake
Products
Sauerkraut
Pickles
Olives (green, ripe)
Vanilla
Sausages, (salami, Thuringer,
Lebanon bologna, cervelat, sum
mer, pepperoni)
Sour cream, cultured butter,
ghee
Cultured milk, acidophilus, yo
gurt
Cheese-unripened (cottage,
pot, cream)
Cheese-ripened (Cheddar,
American, Edam, Cheshire)
Cheese (Swiss, Emmenthaler,
Gruyere)
Cheese (limburger, brick, Trap
pist)
Cheese (Roquefort, blue, Stilton,
Gorgonzola)
Camembert cheese
Poi
Sour dough bread, pancakes
Ginger beer
Vermicelli
Idli
Shogu (soy sauce), miso
Kaffir corn beer
Beer, ale, stout, lager, bock, por
ter, Pilsner
Wine, vermouth
Brandy
Rum
Whiskey
Bread
Citron
Vinegar
Nuocmamngapi
Natto
Sufu (Chinese cheese, bean
cake)
Tempeh, tempe
Tempeh bongkrek
Ontjom
USEFUL MICROORGANISMS 435
Food Cultures
The food conversions or biotransformations are listed in Table 9.1
according to the microorganisms involved. The addition of previously
fermented product or the selection of microflora naturally present have
been used in traditionally fermented foods. However, to assure that
proper reactions occur and to obtain larger yields of high quality prod
uct, cultures of selected and special organisms are added at the begin
ning of the food conversion. The culture may consist of one strain of
one organism, or more likely, multiple or mixed strains of one or several
organisms.
Microorganisms tend to be energy efficient, and they also have feed
back mechanisms to prevent the accumulation of a product that is useless
to them but may be useful to us. However, mutants of the organism may
be found that do accumulate these products. This selection of exception
ally good organisms may require the screening of hundreds or thousands
of organisms.
To improve the organisms in the culture, the manipulation of genetic
information in the cells may be used. By genetic engineering, certain
traits can be added to or subtracted from the cells. These manipulated
cells may produce an improved product with a greater yield.
Since much work is needed to acquire an exceptional culture, it is
important that the culture be properly maintained so that it does not
lose its desirable traits. Quite often, the organisms contain desirable pI as
mids that can be lost after several transfers. Hence, after growth, the cuI
tures are frozen or freeze dried for storage. Frozen cultures are stored at
- 20 to - 40C. How the culture is obtained and maintained depends
upon the food substrate as well as on the company policy. There are
companies that produce and sell commercial cultures for use by other
companies. People in the dairy, meat, and baking industries tend to use
commercial cultures, while beer and wine processors usually develop and
maintain their own cultures.
Although some foods may be pasteurized or treated with chemicals,
usually they are not sterile when subjected to fermentation. Hence, the
inoculated culture must be sufficiently large and active so that it can
outgrow the residual organisms. When an inoculum of 5 percent or more
of the weight or volume of the food is needed, the amount of culture is
increased by inoculation and incubation of increasing amounts of sub
strate.
In some cases, such as in the continuous production of alcohol, the
immobilization of microbial cells may be advantageous. The immobilized
cells are actually complex enzyme systems. The immobilization of cells
was reviewed by Fukui and Tanaka (1982). The process is discussed fur
ther in this chapter for some specific products as well as in the section
concerning enzymes.
Lactic Acid Bacteria
The organisms referred to as lactic acid bacteria include certain spe
cies in the genera Streptococcus, Pediococcus, Leuconostoc, and Lactobacillus.
The first two genera are homofermentative, the leuconostocs are hetero
fermentative, and the lactobacilli include both homofermentative and
heterofermentative types. The homofermenters convert carbohydrates
primarily to lactic acid, while the heterofermenters produce lactic acid
and substances such as acetic acid, ethyl alcohol, and carbon dioxide.
The homofermenters use the glycolytic (hexose diphosphate) pathway to
degrade carbohydrates, while the heterofermenters use the phosphoketo
lase as well as alternative pathways. Some recent studies indicate that
some homofermenters have the potential for heterofermentation. The
genetic determinant for lactose metabolism by various streptococci is
found in a plasmid, which can be lost or transferred (Fujita, Okamoto,
and Irie 1984; Orberg and San dine 1985; Scherwitz, Baldwin, and McKay
1983).
Many of the lactic streptococci cultures used in fermentations are sus
ceptible to lytic bacteriophage. Lysis by the phage is believed to be the
primary cause of slow acid production during the fermentation of dairy
products (Teuber and Lembke 1983).
The lactic acid bacteria usually grow in a sequence in a food product.
Generally, the leuconostocs or streptococci begin the fermentation and
are followed by pediococci and lactobacilli. The production of lactic acid
from sugar is used in vegetable, fruit, and dairy product fermentations.
There are many similarities in the lactic acid fermentation, but because
of the various substrates, there are some differences.
SAUERKRAUT. This means "acid cabbage." According to definition,
sauerkraut is the clean, sound product of characteristic flavor, obtained
by full fermentation, chiefly lactic, of properly prepared and shredded
cabbage in the presence of not less than 2 percent nor more than 3 per
cent salt. Upon completion of the fermentation, it contains not less than
1'h percent of acid, expressed as lactic acid. Sauerkraut that has been
rebrined in the process of canning or repacking contains not less than 1
percent of acid, expressed as lactic acid.
Sound heads of cabbage are prepared by washing and removing the
outer leaves and any defective leaves (Fig. 9.1). The core is removed and
the leaves are shredded. Shredding the leaves gives a larger total surface
area and allows the extraction of juice. About 2.25 percent salt is added
Wash ___ > Remove outer leaves ___ > Core
----> Trim--> Shred-->
Salt ---> Ferment in covered vat (21-30 d) ----'> Package-Process
Figure 9.1. Flow sheet for sauerkraut.
uniformly to layers of shredded cabbage. The combination of salt and
packing down of the cabbage expels the juice from the cabbage, and a
brine results. The juice contains sugars and other nutrients from the cab
bage. Depending upon the variety of cabbage and other factors, the sugar
content is about 3 to 6 percent. The amount of sugar influences the fer-
mentation and the final acidity.
When the vat or tank is essentially full, a plastic sheet is used as a
cover to keep out dirt and air. The environmental conditions, numbers,
and kinds of microorganisms, cleanliness of cabbage and vat, salt concen-
tration and distribution, temperature, and covering influence the fer-
mentation (Pederson 1979).
Many types of microorganisms are associated with raw cabbage. Most
of these are not involved in the fermentation. Cabbage contains sub-
stances that are inhibitory to Gram-negative bacteria (Fig. 9.2). With the
inhibitors, salt, and an anaerobic environment, the lactic acid bacteria
tend to dominate. The fermentation is started by Leuconostoc mesenteroides.
It converts the sugar to lactic acid, acetic acid, alcohol, CO
2
, and other
products that contribute to the flavor of sauerkraut. The CO
2
helps main-
tain anaerobic conditions in the fermenting cabbage.
As the acids accumulate, L. mesenteroides is inhibited, but the fermenta-
tion continues with Lactobacillus brevis, Pediococcus cerevisiae, and finally,
Lactobacillus plantarum. The sequence of organisms and acid production
during a typical sauerkraut fermentation are listed in Table 9.2.
The proper concentration of salt favors the growth of the lactic acid
bacteria in the correct sequence. Too little salt results in poor flavor and
soft kraut. Too much salt inhibits the lactic acid bacteria and may result
in an acid flavor, darkening, and growth of yeasts (Pederson 1979).
Although salt influences certain organisms, the inhibition of undesir-
able bacteria is due primarily to the acid produced by the fermentation
of sugars.
Keeping air from the fermentation is important in controlling molds
and yeasts. Air allows the growth of these microorganisms on the cabbage
surface and can result in softening, darkening, and the development of
undesirable flavors. The product is not microbiologically stable, so it is
important that it be refrigerated or heat processed after packaging.
7
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Figure 9.2. Bactericidal action of cabbage juice toward a Gram-nega-
tive bacterium obtained from a cabbage surface.
courtesy of Pederson (1979).
PICKLES. There are many pickle products produced from cucumbers.
Reportedly, three methods are available for pickle processing. Fresh-pack
pickle products are directly acidified cucumbers (acetic acid is preferred)
which are pasteurized. Various spices are added to give the characteristic
flavor of the particular product. Researchers found that a combination
of an acidity of 0.6 percent (acetic acid) and heating to an internal tem-
perature of 71 to 77C prevented spoilage of the product (Monroe et
al. 1969).
For refrigerated pickle products, an acid brine is added to the jars
containing washed, sorted cucumbers. Since they are not pasteurized,
these pickles must be refrigerated.
The oldest method of producing pickles is by fermentation. Fermen-
tation can be accomplished by the natural (traditional) method or by in-
oculation with pure cultures.
Natural Fermentation. Cucumbers from the field are washed and placed in
a salt brine of about 25 salometer and allowed to ferment. The salt in-
hibits the growth of undesirable microorganisms and allows the salt-
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tolerant lactic acid bacteria to ferment the sugars to lactic acid. Sugars
and other nutrients diffuse from the cucumbers into the brine and are
used by the microorganisms. The salt diffuses into the cucumbers during
the holding period in the brine.
The formation of sufficient lactic acid is an important factor in the
quality and preservation of the fermented pickle. The rate of acid pro
duction and the total acid produced depend on the variety and size of
cucumber, initial salt concentration, the temperature, and the natural
microflora of the cucumbers. During fermentation, the pH is lowered to
about 3.5. During storage, salt is gradually added to a salometer level of
45 to 60. This level of salt, with the low pH, halts enzymatic and bacte-
rial activities and helps preserve the pickles. An important aspect of pres-
ervation is the removal of all fermentable carbohydrates from the pickles.
The natural microflora of the cucumbers is quite variable and in
cludes bacteria, yeasts, and molds. The salt and a lowered redox potential
favor the growth of facultatively anaerobic organisms. The vats are cov-
ered with plastic, and UV rays from sunlight or UV lamps are used to
prevent surface growth of film-forming yeasts.
The initial microflora may contain molds primarily associated with
parts of the flower still attached to the cucumbers. Molds do not survive
due to the salt and to the low redox potential. However, the pectinolytic
enzymes from molds have been involved with softening ofbrined cucum-
bers.
There is a miscellaneous group of yeasts at the beginning of the fer-
mentation. Yeasts can affect the fermentation by utilizing sugars that
would otherwise be metabolized to lactic acid by the lactic acid bacteria.
Also, the yeasts can utilize the produced lactic acid, raise the pH, and
allow other potential spoilage types of microorganisms to grow. The
yeasts produce large amounts of gas. This is associated with bloater or
hollow defect (Fig. 9.3). However, there are other causes for this defect.
The sugars that diffuse from the cucumbers are fermented sequen-
tially by Leuconostoc mesenteroides, Pediococcus cerevisiae, Lactobacillus brevis,
and Lactobacillus plantarum. Depending on the condition of fermentation,
about 0.6 to 1.2 percent lactic acid is formed in about seven to fourteen
days. As the pH is lowered to 3.2, the metabolism of L. plantarum is in-
hibited; and, in one study, about 0.25 percent sugar remained after lactic
acid formation had ceased (Etchells et al. 1975).
Controlled Fermentation. The cucumbers are washed and sanitized with a
chlorine solution that removes most of the undesirable microorganisms.
After brining (20 to 25 salometer), the cover brine is acidified with
acetic acid and buffered with sodium acetate or sodium hydroxide. The
brine is purged with N2 to remove dissolved CO
2
. A pure culture of Lacto-
bacillus is added for the fermentation. With controlled fermentation, the
Figure 9.3. Severe bloater damage of pickles.
need to add more salt during storage is reduced. This is important be
cause people are seeking lowsalt diets and because the Environmental
Protection Agency is phasing out the dumping of brine into streams.
The exposure of cucumbers to 100 percent oxygen prior to brining
resulted in certain improvements during the early stages of brining
(Fleming, Pharr, and Thompson 1980). However, the resultant pickles
had serious bloater damage (Daeschel and Fleming 1981).
Defects. The two main defects of fermented pickles are bloaters and
softening.
Bloaters are those fermented and cured pickles that float on the brine
or are hollow or have large air spaces in the interior. Bloater formation
is due to the accumulation of gas inside the cucumber during fermenta
tion (Fig. 9.3). This can be caused by several factors. The growth of un de
sirable bacteria and yeasts can produce gases resulting in bloater forma
tion. The respiration of cucumber tissue plus the fermentation by
homofermentative P. cerevisiae or L. plantarum produces sufficient CO2 to
cause bloater formation (Fleming et al. 1973; Etchells et al. 1975). The
degradation of malic acid to lactic acid (malolactic reaction) was a major
source of CO
2
when L. plantarum fermented cucumber juice (McFeeters,
Fleming, and Thompson 1982). When a strain of L. plantarum that did
not degrade malic acid was used, bloating did not occur (McFeeters,
Fleming, and Daeschel 1984). Piercing of the fruit prior to brining con
troIs this defect. Controlled fermentation, increasing the depth of the
vat, and purging with N2 reduce the incidence of bloaters (Fleming et al.
1977).
Bloaters are not a complete loss, since they can be used in cut pickle
and relish products. However, their value is reduced by about 50 percent.
Softening of the saltstock pickles is attributed to pectinolytic en
zymes that degrade the cucumber tissue. One source of these enzymes is
molds that enter the vat with the cucumbers, especially with portions
of flowers that may remain attached to the cucumbers. Pectindegrading
enzymes are naturally present in the cucumber (fruit and seeds). The
purging of brines at high rates of airflow results in increased softening
(Costilow, Gates, and Lacy 1980). Gates and Costilow (1981) suggested
that softening is caused by microorganisms growing in or on the cucum
bers.
The control of softening involves the removal of any flowers attached
to the cucumbers, by blanching at about 81C, by adding CaCI 2, and
by acidification (Hudson and Buescher 1986; McFeeters, Fleming, and
Thompson 1985; Potts and Fleming 1982).
OLIVES. Olives are brined and fermented in a manner similar to cu-
cumbers. Before brining, the olives are soaked in a 1.25 to 2.0 percent
lye solution. This is necessary to hydrolyze oleuropein, a bitter factor in
the olive. After treatment, the lye is removed by rinsing the olives in
fresh water. Some nutrients are lost by this washing treatment. Therefore,
excessive, unneeded washing is not desirable. If too much carbohydrate
is lost, there will not be enough to develop sufficient acidity. To overcome
this problem, reduced washing, addition oflactic acid to neutralize resid-
uallye, or adding sugar to the brined olive for acid production has been
suggested. The lye also may affect the microbial flora. If so, it is necessary
to add cultures of desirable organisms.
After washing, the olives are brined. According to Pederson (1979),
the brine concentration varies from 5 to 15 percent salt, depending upon
the variety and size of the olives. As water diffuses out of the olives, and
salt penetrates, the brine concentration is reduced. Additional salt is
added to maintain the concentration. The vats, barrels, or other con-
tainers are covered to maintain a low redox level.
The entire fermentation process may take from two weeks to several
months. The same organisms active in the fermentation of cucumbers
also are involved in olive fermentations. A level of at least 0.6 to 0.7 per
cent acid is needed for proper preservation and flavor of the product.
Depending on the variety and type of treatment given the olives, the
acidity varies from 0.18 to 1.27 percent (Samish, Cohen, and Ludin 1968).
Various factors, such as the origin, maturity, and variety of olives,
treatment prior to brining, brine strength, sugar content, acidity, avail
able desired microflora, and temperature influence the fermentation.
A defect known as sloughing spoilage includes severe softening, skin
rupture, and flesh sloughing. The defect is caused by Gramnegative pec
tinolytic bacteria (Patel and Vaughn 1973) and occurs during the washing
to remove the lye prior to brining. They described the bacteria involved
as strains of Xanthomonas and various coliforms.
As with cucumber fermentations, yeasts can grow in or on the brine
during fermentation of olives. Pink yeasts (Rhodotorula) and fermenting,
pectinolytic yeasts (Saccharomyces and Hansenula) can cause softening of
olives (Vaughn et al. 1969; Vaughn et al. 1972).
RED MEAT. Fermentation of sugars to lactic acid is utilized in the pro
duction of certain semidry and dry sausages. Various formulations of
chopped meat are mixed with spices, sugar, salt, sodium nitrate, sodium
nitrite, or a combination of these.
The mixture is held at a temperature that allows the desirable bacte
ria to produce lactic acid from the sugar. Although it is possible to use
naturally occurring microorganisms, adding starter cultures of Pediococcus
and Lactobacillus is preferred (Bacus 1984; Smith and Palumbo 1983). The
culture may also contain one or more species of Micrococcus, although
one team of researchers found that what were assumed to be micrococci
in fermented meat were actually coagulasenegative staphylococci
(Seager et al. 1986). Concentrated cultures are added at the rate of 40 g
per 227 kg of meat. The use of starter cultures results in better color,
aroma, flavor, and texture of the fermented product. Also, the processing
time is reduced with a more rapid drop in pH and the yield is increased.
Both frozen and dried cultures are available. The addition of starter cui
tures to the curing pickle of bacon helps prevent the formation of nitros
amines in fried bacon.
The fermented sausages are smoked and dried, during which the de
sirable characteristic flavors are developed.
POULTRY MEAT. Chicken and turkey meat, either alone or combined
with beef, have been used in the production of dry fermented sausages.
The fermentation is similar to that with red meat. Acton and Dick (1975)
reported that fermented and dried turkey sausage, on a fat free basis,
had a lactic acid content of 3.1 to 3.2 percent and a pH of 4.6.
DAIRY PRODUCTS. The production of lactic acid from the lactose in
milk and the formation of flavor compounds are important in the manu
facture of fermented dairy products (Fig. 9.6).
The main lactic acid formers are the homofermentative streptococci,
S. lac tis, and S. cremoris (Table 9.3). Strains of these organisms vary in the
rate of acid production. Also, the rate is influenced by the temperature,
pH, antibiotics, bacteriophage, stimulants, inhibitory compounds, milk
composition, available nutrients, the condition of the culture, strain com
patibility, and strain dominance.
Some strains of S. cremoris can degrade citric acid with the formation
of diacetyl, an important flavor component of fermented milks. S. lactis
subsp. diacetylactis produces not only considerable lactic acid, but also
degrades citrate to diacetyl. Other flavor components include volatile
acids, dimethyl sulfoxide, methyl ketones, lactones, acetaldehyde, and
various esters.
The organisms that constitute the aroma and flavor producers are
strains of Leuconostoc species (L. cremoris, L. dextranicum, and L. mesente
roides). S. lactis subsp. diacetylactis is included as a flavor producer. Some
strains of L. dextranicum and L. mesenteroides produce lactic acid in milk,
but rather slowly and in low amounts. L. cremoris produces diacetyl only
in acidic substrates and is optimal at pH 4.3.
S. lactic subsp. diacetylactis degrades citric acid faster and produces
more carbon dioxide than the leuconostocs. The excess gas gives fer
TABLE 9.3. CULTURES AND CONDITIONS USED IN THE PRODUCTION OF SOME
FERMENTED DAIRY PRODUCTS
Product
Cultured buttermilk or
cultured sour cream
Butter
Acidophilus milk
Yogurt
Cottage cheese
Cheddar cheese
Swiss cheese
Cultures
Streptococcus lactis, Streptococcus
cremm'is, S. lactis subs. diacetylac-
tis
Lactobacillus acidophilus
Streptococcus thermophilus, Lactoba
cillus bulgaricus
S. lactis, S. cremoris
S. lac tis, S. cremoris
S. thermophilus, L. bulgaricus, Pro-
pionibacterium
Tempera-
Time (hr) ture (0C)
18 22
16-18 37-40
3 43-45
18 22
or
5 35
men ted milk drinks a desirable effervescence but is undesirable in cheese
manufacture, since it may crack the cheese.
Besides streptococci and leuconostocs, certain species of lactobacilli
are used in milk fermentations. Lactobacilli that have been found in fer
mented milk products include L. bulgaricus, L. acidophilus, L. lac tis, L. casei,
and L. helveticus. Some strains of lactobacilli produce diacetyl as well as
lactic acid.
After pasteurization or heat treatment of milk, cultures containing
the desired organisms are used to inoculate the milk for fermentation. A
sufficient amount of culture must be present for the quantity of milk
being fermented. Concentrated cultures that can be used to inoculate a
vat of milk are available commercially. The propagation of cultures must
be done under conditions that minimize contamination with undesirable
bacteria or with phage that can infect the desirable bacteria (Fig. 9.4).
An important problem in the dairy industry is the susceptibility of
lactic acid bacteria to phage. The rapid reproduction of phage can reo
duce or eliminate the streptococci in a fermenting milk so that an unac
ceptable product is produced. Phage resistance can be transferred be
tween strains of streptococci (Sing and Klaenhammer 1986).
Both acid formers and flavor producers are important in the fermen
tation and hence, in the culture. If insufficient acid is produced, a prod
uct with poor keeping quality results. Without aroma and flavor pro
ducers, the fermented product is flat or acid and may have a metallic
flavor.
The consumption of fermented milk rather than fresh milk has been
suggested for people with an intolerance for lactose. There is less lactose
in fermented milk, and the presence of viable lactic culture aids in the
digestion of residual lactose.
The culture also produces chemicals besides acids that may inhibit
spoilage or illness-producing organisms (Speck 1981).
Cultured Buttermilk. This can be made from whole milk, reconstituted non
fat dry milk, partially skim milk, or skim milk. From 0.5 to 2 percent fat
tends to improve the consistency and flavor of cultured buttermilk (Fig.
9.5). The milk is heated to 85C for 30 min or 88 to 91C for 2.5 to 5
min. Heating destroys many bacteria, inactivates natural bacterial inhibi-
tors, and helps prevent "wheying-off' of the product.
After heating, the milk is cooled to about 22C and inoculated with
a culture. Since good cultured buttermilk contains lactic acid and flavor
compounds, the culture must contain a lactic acid producer (S. lactis or
S. cremoris) and a flavor producer (a Leuconostoc, S. lactis subsp. diacetylactis,
or both). The inoculated milk is incubated at 21 to 22C until the titrata
ble acidity (as lactic acid) reaches about 0.85 percent with a pH of 4.4 to
4.5. This requires about fourteen to sixteen hours. A desirable cultured
Using Frozen Culture
Special media for lactic cultures
!
Disperse in 378.5,1136, or 1893 liters (100,
300, or 500 gal) (depending upon the cul-
ture can size) of liquid skim milk or water
to get 11.5% solids at 52-57 C
!
Heat to 85C for 40 min. Cool to 22C. In-
oculate 1 can of frozen culture concen-
trate. Incubate at 22-25C to a pH of of 4.9
approx. 16-18 hr
Bulk Starter
Using Lyophilized Culture
Antibiotic-free skim milk or non-fat milk
powder suspension
9% WIV
!
Dispense 750 ml milk into 241-liter bottles.
Cap and heat at 85C for 60 min. Cool and
use up to 10 days. Add lyophilized culture
to milk medium at 22C. Incubate for 16-
18 hr. May be subcultured 3 times before
starting again with original lyophilized CUl-
ture
Mother Culture
(Acidity> 0.7% lactic acid)
Maintain daily transfer by inoculating 1 %
mother culture in milk medium and incu-
bating at 22C for 16-18 hr. Heat 37.85 li-
ters (10 gaL) milk in a 37.85 liter (10 gaL)
can to 85C for 30 min. Cool to 22C and
inoculate 0.5% mother culture. Mix for 2
min and incubate at 22C for 16-18 hr
Semi-bulk or Intermediate
Starter
(Acidity -0.80% lactic acid)
Repeat the above process using 378.5-3785
liters (100-1000 gaL) of milk and 0.5-1.0%
semi-bulk starter
Store at 5C up to 48 hr
Figure 9.4. Flow diagram for production of bulk starter for certain dairy products.
Courtesy of Chandan (1982).
milk should contain at least 2 mg diacetyl per liter. Hence, it is desirable
to add citrate (about 0.25 percent) to the milk so that an acceptable level
of flavor compounds is produced during fermentation. When the cul-
tured buttermilk reaches the desirable stage, the fermentation is halted
by cooling the product to 5C for storge. A continuous fermentation
process for buttermilk was discussed by Lelieveld (1984).
Bakery Products
Cultured Buttermilk
Standardize milk to
10.0% milk solids-nat-fat.
0.5% milk fat
Heat Treat 85C for 30 min
or 88-91 C for 2.5-5 min
Homogenize 13.7 kPa
(2000 psi)
Ripening Tank 22C to pH
4.5 for 14-16 hr
Butter Flakes
1 % culture or
frozen concentrate
Figure 9.5. Flow sheet for the manufacture of cultured buttermilk.
courtesy of Chandan (1982).
Acetaldehyde, produced by S. lactis subsp. diacetylactis and some other
lactic acid formers, causes a defect in cultured milk that is called "green"
or "yogurtlike" flavor. L. cremoris reduces the acetaldehyde to alcohol and
prevents this defect. The chemical changes in milk during fermentation
and during storage of the cultured buttermilk were reported by Marsili
(1981). The formation of metabolites in milk by lactic cultures is shown
in Figure 9.6.
Rather than fermenting milk to make cultured buttermilk, an acidi-
dihydroxyacetone
phosphate
Figure 9.6. Formation of lactic acid and other metabolites in milk by lactic cultures.
448
fied buttermilk can be produced. Reportedly, the acidified, flavored
product is consistently good, has a long shelf life, and can be made in
less time, with less labor.
Cultured Sour Cream. This product is pasteurized and homogenized cream
fermented in a manner similar to cultured buttermilk. It contains not
less than 0.20 percent lactic acid and 18 percent butterfat.
Cultures in Butter. A lactic culture is added to cream and fermentation
proceeds at 18
0
to 20C. An acidity of 0.5 to 0.6 percent may be attained
prior to churning. This butter is sold as the unsalted product. If cultured
acid cream is used to manufacture salted butter, the product tends to
deteriorate with a fishy flavor. Salted butter has better shelf life if the pH
is near neutral.
For salted butter, a distillate from a culture can be added to give the
desirable flavor.
Yogurt. In some countries, goat, ewe, mare, or cow milk is used to produce
yogurt. In the United States either whole or skim milk from cows is used.
The milk is standardized to 10.5 to 12.5 percent solids, heated to about
95C for 30 min and, after homogenizing, cooled to about 43C (Fig.
9.7). The frozen culture or bulk starter is a mixture of Streptococcus thermo
philus and Lactobacillus bulgaricus in a 1:1 ratio. The combined action of
these two organisms is needed to obtain the desired acid and flavor of
the product. Yogurt is processed as either stirred or set, depending on
whether the fermentation occurs before or after packaging.
The flavor depends to some extent on the production of acetalde
hyde. Together, the cultures will produce about 25 mg per liter, but either
organism alone produces about 8 mg per liter or less.
In. a survey of 152 yogurt samples, Arnott, Duitschaever, and Bullock
(1974) found that only 15.1 percent had a desired 1:1 ratio of S. thermophi
lus and L. bulgaricus. According to Moon and Reinbold (1976), S. thermophi
lus tends to outgrow L. bulgaricus. Symbiotic growth is inferred, but they
found commensalism and competition between these organisms. This as
sociated growth was reviewed by RadkeMitchell and Sandine (1984). To
halt the fermentation, the product is cooled to 50 to 100C.
A continuous process for yogurt production using S. thermophilus and
L. bulgaricus was described by Driesser, Ubbels, and Stadhouders (1977),
MacBean, Hall, and Linklater (1979), and Prevost, Divies, and Rousseau
(1985). The overall yogurt process was reviewed by Tamime and Deeth
(1980).
Cottage Cheese. This is a soft, unripened cheese. Cottage cheese manufac
ture consists of coagulating the casein of skim milk by acid, cutting the
coagulum into cubes, heating to reduce the moisture in the curd, washing
Set Type
Standardize yogurt mix
Milk fat 1-2%
MSNF 12.S%
Low Fat Milk
Cream
Skim Milk
Nonfat Dry Milk
Cool to 43C Frozen culture or
bulk starter
Stirred Type
Standardize yogurt mix
Milk fat 1-2%
MSNF 10.S%
Stabilizer 0.7%
Cool to 43C
Mix in holding vat Culture vat. hold to
43C pH 4.S at 43C
Package in containers
Incubate containers at
43C to pH 4.S
Cool and store plain yogurt
at SoC up to 3-4 weeks
Cool to 2SoC
Package in containers
Cool and store plain yogurt
at SoC up to 3-4 weeks
Figure 9.7. Flowsheet outline for the manufacture of set and stirred plain low-fat
yogurts.
450
to remove residual whey, and cooling the curd. A cream dressing may be
added for texture and flavor. The product should not have more than SO
percent moisture.
The skim milk is pasteurized (52.SoC for 30 min or 71.7C for 15 sec).
Overheating can result in a soft coagulum and a lower yield. The inocu
lum should be able to produce lactic acid rapidly. S. lac tis, S. cremoris, or
a combination of these organisms is the culture of choice. S. lac tis subsp.
diacetylactis is not satisfactory since it produces large amounts of carbon
dioxide. This gas can result in a floating curd defect. The culture is added
at a level of 5 percent of the amount of milk.
The incubation temperature may vary from 20 to 33C. For an S
hr setting time, a temperature of 30 to 33C is used, while for a 12hr
incubation, from 20 to 24C is used.
Rather than using a lactic culture, the skim milk can be acidified by
direct addition of lactic or other acid. The coagulation of the curd can
be accomplished with acid, rennet, various commercial enzyme prepara
tions, or a combination of acid and enzymes.
The curd is ready to cut when it is firm but not hard and brittle. The
size of the cubes determines to some extent the size of the particles in
the finished cheese. Cutting the curd is done when the pH is between 4.5
and 4.S. In this pH range, the curd will expel moisture most readily when
stirred and heated, since pH range includes the isoelectric point of ca
seln.
Heating of the curd halts the fermentation and aids in expelling wa
ter. When the curd has attained the proper firmness, the whey is drained
and the curd is washed with cool tap water. A final washing is made with
ice cold water. When the curd is firm and dry, it is salted for flavoring.
The curd may be creamed to enhance the flavor. The flavor is ob
tained by adding starter distillate or cultured skim milk to the creaming
mixture. As high a level of this mixture as possible is desirable, but a
normal amount is 44 g of 14 percent fat dressing per 100 g of curd.
Skim milk concentrated by ultrafiltration is being used in the produc
tion of various cheeses. For cottage cheese, Kosikowski, Masters, and Mis
try (1985) added skim milk retentate (the portion retained on the filter)
to pasteurized milk to various protein levels. They reported that opti
mum flavor, body, texture, and appearance were obtained from
retentatesupplemented skim milk at 1.7:1 total protein concentration.
The main considerations in producing a highquality cottage cheese
include controlling the properties of milk, using proper cultures, cutting
the curd at the correct level of acidity and firmness, and cooking the curd
to the desired firmness and solids content.
The cottage cheese can be contaminated by the wash water or by poor
sanitation during creaming and packaging. With poor sanitation, the
shelf life may be only three to four days rather than three to four weeks.
Surface spoilage (slimy defect) can be caused by Pseudomonas and Alcali
genes. Yeasts and molds indicate poor sanitation in the processing plant
and can cause spoilage of cottage cheese with pH less than 5.0.
Cheddar Cheese. This cheese was first made in the village of Cheddar in
Somersetshire, England. So much of this cheese is made in the United
States that it is often called American cheese, or American Cheddar
cheese. Cheddar is a firm, ripened cheese, ranging in color from nearly
white to yellow. orange, depending upon the amount and type of coloring
added.
In making Cheddar cheese, the lactic acid culture is added to a vat of
pasteurized whole milk. The culture may be a pure culture of S. lactis or
S. cremoris, but preferably a mixture of these organisms. L. cremoris may
be added for flavor and its presence increases the rate of acid formation
by lactic acid. A mixture of strains with different bacteriophage sensitivit
ies is desirable to prevent potential lysis of the culture and failure of the
starter to produce acid.
Kosikowski (1985) listed nine general steps used to produce cheese:
prepare the milk; form the curd; cut the curd; cook; separate the curd
and whey; salt; apply microorganisms for ripening; press; ripen the
young cheese. These steps vary for different types of cheese. The process
for Cheddar cheese is listed in Table 9.4.
Accelerated ripening of Cheddar cheese has been attempted by in
creasing the level of starter organisms, addition of lactase (JDgalacto
sidase) to hydrolyze lactose prior to fermentation, by adding certain lipo
lytic or proteolytic enzymes, adding trace materials, or increasing the
temperature of the aging process (Arbige et al. 1986; Aston et al. 1985;
EI Soda 1986; Law and Wigmore 1983; Ridha, Crawford, and Tamime
1984; Sood and Kosikowski 1979). The flavor of aged Cheddar cheese is
regarded as a blend of fatty and organic acids, amino acids, carbonyl
compounds, esters, alcohols, and sulfur compounds. Researchers re-
ported the volatile flavor compounds to include ketones, aldehydes, alco-
hols, acids, esters, lactones, terpenes, alkanes, alkenes, alkylbenzenes, and
chlorinated compounds (Liebich et al. 1970). However, McGugon, Em-
mons, and Larmond (1979) believed that the nonvolatile, water-extract-
able compounds, such as salts, amino acids, and peptides, make a signifi-
cant contribution to the flavor of Cheddar cheese.
Ultrafiltered or reverse osmosis whole milk retentates have been used
successfully in the production of Cheddar cheese (Bynum and Barbano
1985; Green 1985; Kealey and Kosikowski 1985). Although milk can be
concentrated to 10:1 volume concentration ratio, the optimum concen-
tration is 1.7:1 to 1.8:1 (Kosikowski 1986).
The defects in Cheddar cheese include bitter, fruity or acid flavors,
TABLE 9.4. TRADITIONAL PROCESSES OF CHEDDAR CHEESE MANUFACTURE
Process Step
Temperature ad
justment
Starter addition
Settings
Cutting
Cooking
Draining
Cheddaring
Milling
Salting
Pressing
Curing
Purpose
To bring temperature to optimum
for starter organisms
To develop about 0.01 to 0.02%
acidity to assist rennet coagulation
To coagulate the milk
To promote whey removal
To remove whey and firm curd
To remove whey
To mat the curd and to develop the
typical body of Cheddar curd
To prepare curd for pressing
To stop further acid production
To remove additional whey and ar
rive at final desired moisture con
tent.
To ripen the curd to give the char
acteristic body and flavor of
Cheddar cheese
SOURCE: Adapted from Harper and Seiberling (1976).
Remarks
Milk added to vat and adjusted to
30-32C
Lactic starter used at about 1 % for
Y2 to 1 hr
Rennet, or suitable substitute,
added at a rate of about
18 gil,OOO kg of milk to coagulate
in 30 min.
Curd is cut into 0.5- to lcm cubes
Temperature raised to 38 to 40C
in about 45 min and held about
45 min until firm.
Whey may be pumped through a
separator to recover fat and then
be stored for further processing
This is the characterizing step in
Cheddar cheese manufacture;
curd is cut into slabs, turned
every 15 min, and piled every 30
min to give three to four high
piles of matted curd. The curd is
matted until the acidity reaches
0.5% (pH about 5.2)
The curd is cut into pieces about
2.5 X 5 cm
Salt added to give 1.5 to 1.8 % in
finished curd. Salt is mixed and
allowed to become absorbed into
curd for 20 to 30 min
Cheese is hooped into forms or
round or square hoops; usually 9
to 18 kg of finished cheese (9.7 to
19.5 kg of curd)
Cheese is pressed at about 1.2 kgl
cm
2
for 1 hr, then the cheese is
removed from the press and
dressed by placing a cloth around
the curd to ensure a smooth
surface. The curd is then pressed
for an additional 14 to 16 hr
Cheese is placed in a 7 to 16C
curing room at 80 % humidity for
ripening. For a fully cured
cheese, the curing requires 6
months to 2 years.
453
or lack of flavor. Bittertasting peptides are a result of proteolysis of the
casein and the inability of the culture to degrade the pep tides to amino
acids. Fast acid producers have a tendency to yield bitter flavored cheese.
Fruitflavored cheese tends to contain high levels of ethyl butylate, ethyl
hexanoate, and ethyl alcohol. Carbon dioxide produced by micoorgan
isms causes a slitopen defect in Cheddar cheese. This is the result of too
many S. lactis subsp. diacetylactis in the starter culture or high levels of
citrateutilizing lactobacilli during ripening of the cheese.
Lactic Acid Bacteria and Other Bacteria
In some fermented products, not only is the production of lactic acid
important; so are other compounds involved with flavor or other charac
teristics. Both dairy and vegetable products are manufactured with a
combination of lactic acid bacteria and other microorganisms.
OTHER DAIRY PRODUCTS. As stated previously, lactic acid produc
tion is part of the process for all fermented dairy products. In some cases,
acidulation with lactic acid can be used.
Swiss Cheese. This cheese is manufactured by using lactic acid bacteria and
propionic acid bacteria. Swiss cheese is a hard cheese characterized by a
sweet, nutty flavor, and by gas holes, or eyes, distributed throughout the
cheese.
The organisms used in Swiss cheese manufacture are the lactic acid
producers (S. thermophilus and L. bulgaricus) and a species of Propionibacter.
ium, which produces propionic acid. Propionibacterium freudenreichii
subsp. shermani is usually given credit as the species in Swiss cheese. How
ever, Propionibacterium freudenreichii subsp. freudenreichii and globosum, as
well as P acidipropionici and P. jensenii, may be found in Swiss cheese.
The propionibacter are responsible for the characteristic flavor and eye
formation in Swiss cheese.
After coagulation and separation of the Whey, the curds are put into
a cheese hoop and pressed lightly. The cheese is turned several times and
is pressed more firmly each time. During the first two or three weeks of
ripening, the cheese is salted and turned everyone to three days.
Next, the cheese is moved to a prewarming cellar (17 to 20C) for
ten to fourteen days, and then to a fermentation room for six to eight
weeks. The fermentation room is 22 to 23C, with a relative humidity
of 80 to 90 percent. During this time, the cheese is turned and washed
with saltwater two to three times per week (Fig. 9.8).
The final process consists of curing at 7 to lOoC and high relative
humidity. After four months, the cheese is mild and has the typical sweet
Figure 9.B. Swiss cheese in curing room.
Courtesy of Switzerland Cheese Association.
nutty flavor. Stronger flavors are developed by leaving the cheese in the
curing room for eight to twelve months.
The propionibacter produce CO
2
, which is not able to escape, so it
concentrates in various places in the cheese. The pressure produced by
this gas results in the eyes in Swiss cheese (Fig. 9.9). The turning, salting,
washing, temperature, and humidity are essential to obtain uniformity
ofthese holes. Undesirable organisms, such as butyric acid bacteria, pro
duce offflavors as well as gases such as hydrogen, which results in poor
eye formation.
Further information on the production of Swiss cheese can be ob
Figure 9.9. Typical eye formation
in high-quality Swiss cheese.
Courtesy of Switzerland Cheese Asso-
ciation.
tained from Auclair and Accolas (1983), Biede and Hammond (1979),
Gilles, Turner, and Martley (1983), and Lawrence, Heap, and Gilles
(1984).
Limburger. This is a semisoft surface-ripened cheese with a distinct odor
and flavor. It is regarded as one of the most delicate and difficult cheeses
to make. After the milk is coagulated, the cnrd is packed into rectangular
forms, and residual whey is allowed to drain. When the cheese is firm
enough to retain its shape, it is removed from the form and salted and
turned frequently.
At the beginning of ripening, fungi predominate. They reduce the
acidity so that bacteria can grow. After the fungi, micrococci may be pres-
ent along with Brevibacterium linens. This organism (B. linens) is common
in the slime of surface-ripened cheese. It is thought that proteolytic en-
zymes secreted by the organism diffuse into the cheese and cause the
characteristic softening and flavor.
The flavor of Limburger cheese is probably due to sulfur compounds
such as dimethyl disulfide, methanethiol, 2,3,4-trithiapentane, S-methyl-
thioacetate, and dimethyl polysulfides as well as to phenol and indole
(Cuer et al. 1979; Parliment, Kolor, and Rizzo 1982).
Although yeasts are listed as the initiators, these have been described
as Geotrichum candidum, Oidium lac tis, or Oospora. At the present time, Oi-
dium lactis is Geotrichum candidum, which is a mold. Some Oospora species
are now included in the yeast genus Trichosporon.
Lactic Acid Bacteria with Yeasts
Some fermented milks, such as kumiss and kefir, utilize a simultane-
ous production of lactic acid and alcohoL The alcohol is a product of
yeast metabolism_ The alcoholic content of kefir is about 0_3 to LO per-
cent, while that of kumiss is from 1 to 2 percent.
The lactic acid bacteria are Streptococcus lac tis and Lactobacillus bulgari-
cus_ The yeast is a lactose fermenter (perhaps Kluyveromyces lactis)_
Other products in which lactic acid bacteria and yeasts are involved
include sour dough, idli, and ogi_
SOURDOUGH. This type of dough was so prominent among pros-
pectors of the Old West that they were named sourdoughs_ San Francisco
is still noted for sourdough bread_
The sourdough process uses a lactic acid bacterium for the souring
and a yeast for leavening_ Sugihara, Kline, and Miller (1971) screened
200 yeast isolates from sourdough_ They reported two types of yeast, Sac-
charomyces exiguus and Saccharomyces inusitatus_ The lactic acid bacteria iso-
lated from sourdough were difficult to grow, and their characteristics did
not fit any known species (Kline and Sugihara 1971)_ They suggested the
name Lactobacillus sanfrancisco for this organism_
Lactic Acid Bacteria with Molds
Molds are involved in the aging and curing of several cheeses (Table
9_1)_ Two of the more familiar cheeses are Roquefort and Camembert.
ROQUEFORT CHEESE. Roquefort cheese is made from sheep's milk
and is ripened in caves near Roquefort, France_ A similar cheese that is
common in the United States is made with cow's milk and is called blue
cheese_
One of the characteristics of this cheese is the greenish-blue marbling
of its soft, creamy-white interior_ This marbled appearance is due to the
growth of Penicillium roqueforti, a blue-green mold, throughout the cheese_
So that oxygen is available to favor interior growth, the cheese is pierced
with needles_ P roqueforti is able to grow at a lower redox potential than
many other molds_
The characteristic sharp, biting flavor of blue cheese is generally at-
tributed to hydrolysis of the cheese fat to fatty acids, and then the conver-
sion of these fatty acids to methyl ketones_ The development of blue
cheese flavor was described by Coghill (1979), Jolly and Kosikowski
(1975), and Kinsella and Hwang (1976)_
CAMEMBERT CHEESE. This is a soft, surfaceripened cheese (Fig.
9.10). The coagulated curd is placed into hoops and allowed to settle for
about two days. The cheese is then removed from the hoop, salted, and
inoculated with Penicillium camemberti. The cheese is cured at about 12C
Curing requires at least sixty days. The mold plus bacteria and yeasts
ripens the cheese from the outside toward the center- During curing, a
grayishwhite feltlike growth of mold is followed by a secondary growth
that produces a sliminess, and the surface becomes reddish to russet col
ored. The cheese has a mild to pungent flavor, probably due to carbonyl
compounds (Karahadian, Josephson, and Lindsay 1985).
Milk concentrated by reverse osmosis or ultrafiltration can be used
readily in the production of Camembert cheese (Honer and Horwich
1983; Rash and Kosikowski 1982).
Yeasts
The metabolism of sugar by yeast yields alcohol and carbon dioxide.
The production of alcohol is important in various beverages (beer, wine,
liquor), the CO
2
is used in the baking industry, and the yeast cells are a
source of protein, lipid, and various useful chemicals and enzymes (Table
9.5). Although yeasts are the main organisms utilized in the production
of these foods, other organisms may playa role.
ALCOHOL. Alcohol is produced during the metabolism of various
sugars by yeasts as well as other microorganisms. Industrial alcohol,
Figure 9.10. Camembert
cheese showing white sur
face mold during curing.
Courtesy of The Borden Co.
TABLE 9.5. THE USE OF YEAST IN INDUSTRY
Fenventation
Conditions
Time of fermentation
Temp. of fermentation
pH
Final EtOH cone. (%)
Cells," start
end
Gas production rate'
Bread Dough
1-3 hr
30-35C
5.2-4.7
2-3
275
300
18-35
"For primary fermentation.
hExpressed in million cells/g or 1m!'
'Expressed as mM EtOH/hr/g yeast solids.
SOl)RCE: Ponte and Reed (1982).
Product
Lager" Ale
8-10 days
10C
5.2-4.2
4
6-10
30-70
16-25
2-6 days
20C
5.2-4.0
4
6-10
30-70
16-25
Wine
5-10 days
15-27C
3.5
11-13
5-10
50-150
25-32
which usually is produced chemically by the oxidation of ethylene, is
used for many purposes. However, this discussion is limited to the alco
holic beverages beer and wine. Various strains of species of Saccharomyces
are involved in the production of these beverages. Since the strains that
are presently used cannot utilize starch or cellulose, these substances
must be converted to glucose with enzymes or other microorganisms.
However, yeasts are being isolated or developed through genetic engi
neering and tested for their ability and efficiency in converting starch or
cellulose directly to alcohol (Amin et al. 1985; Calleja et al. 1982; Hawke
et al. 1983; Laluce and Mattoon 1984; Russell et al. 1986). Perhaps in
the future, new types of organisms will be used in food fermentations
producing alcohol.
Beer. Beer is produced in several steps (Fig. 9.11). The first step is to make
malt (Hudson 1986). Barley is cleaned, graded, washed, and steeped. It is
then allowed to germinate for about five days. After germination, heat is
used to stop the sprouting process and to dry the grain. The rootlets
are screened from the resultant barley malt. Malting is used to develop
enzymes such as amylases. The amylases degrade the starch in the grain
to fermentable carbohydrates.
An adjunct, such as ground rice or corn, is mixed with the barley
malt, wetted with clear, filtered water, and cooked. This mash is then
strained. The filtered, clear, amber liquid, called wort, is obtained and
pumped into the brew kettle. Here, hops, or hops extracts, the seasoning
of beer, are added to the wort and the mixture is boiled to obtain the
correct delicate hop flavor (Buckee, Malcolm, and Peppard 1982; Clarke
1986; Sharpe and Laws 1981; Verzele 1986). By this time the amylases
have degraded the starch. The temperature attained during wort boiling
halts the action of these enzymes.
F.ed
Figure 9.11. Processes of brewing.
Courtesy of Helbert (1982).
;-HOPS
t t ~
Br.w
K.ttle
Clar i ficat ion
CO
2
Saturation
Ruh Storage
AgIng
Packaging
The hopped wort is strained, cooled, and then pumped to fermenta
tion tanks. Here, yeast is added. The fermentation changes the sugars in
the wort into alcohol and carbon dioxide.
For aging and natural carbonation, the beer is pumped into tanks
that may contain substances such as beechwood chips to provide surfaces
for the yeast. Some freshly yeasted wort is added, and the beer is allowed
to become naturally carbonated by a slow and quiet fermentation.
During this secondary fermentation, chillproofing of beer can be ac
complished by adding a proteolytic enzyme, such as papain. This hydro
lyzes residual protein that would otherwise precipitate and cloud the
beer when chilled. The addition of acetolactic decarboxylase helps reo
move acetolactic acid, the diacetyl precursor, and also aids in the matura
tion of beer (Godtfredsen et al. 1984).
The so called light (lite) beers are made by including an enzyme, glu
coamylase, before or during fermentation. This enzyme hydrolyzes most
of the unfermentable dextrins in wort to glucose, which can be fer
mented by the yeast to alcohol. Hence, the beer contains a lower calorie
level.
The beer is then filtered to remove yeast cells. The beer is either pas
teurized (60C for 15 to 20 min) after packaging, or it is packaged asepti
cally after bulk pasteurization or microfiltration. Bulk beer in kegs is not
pasteurized, so it must be refrigerated.
Some yeasts settle to the bottom of the fermenting vat and are called
bottom yeasts. These are strains of Saccharomyces uvarum and are used to
produce lager beer. Other yeasts, strains of S. cerevisiae, tend to collect at
the surface and are called top yeasts. The product of this fermentation
is called ale. Each brewery has certain strains of these yeast species for its
particular products. Not all strains will produce an acceptable beverage.
Hence, when a good strain is obtained, it is handled very carefully.
Through genetic alteration, new strains of yeast for brewing are being
developed (Hammond and Eckersley 1984; Hinchliffe 1985; Hopwood
1981; Panchal et al. 1984).
Besides alcohol and carbon dioxide, there are various metabolic
products that can affect the flavor of the beer. These compounds include
ethyl acetate and other esters, fusel alcohols (pentanol, isopentanol, and
isobutanol), diacetyl, 2,3'pentanedione, sulfur compounds (sulfites, sul-
fides, mercaptans, mercaptals, thioaldehydes, and thioketones), and leak-
age of amino acids and nucleotides from the yeast cells. The amount of
these compounds depends upon the conditions of fermentation, wort
composition, and yeast strain. Top-fermented ale usually contains more
fusel alcohols than bottom-fermented lagers. The effect of the chemical
composition on the flavor of beer was discussed by Meilgaard (1982).
There are factors in beer that limit the types of organisms that can
grow. These factors include a low pH and redox potential, the isohumu-
lones of hops that inhibit Gram-positive bacteria and the alcohol pro-
duced by the yeast. The contaminants are usually acetic acid bacteria,
lactic acid bacteria, coliforms, and wild yeasts.
The acetic acid bacteria (Acetobacter and Gluconobacter) can oxidize
ethyl alcohol to acetic acid. In the anaerobic environment of active fer
mentation, this is not possible.
Some lactic acid bacteria (Lactobacillus and Pediococcus) have a toler
ance to the isohumulones of hops. They are microaerophilic and tolerate
acids. They produce lactic acid, diacetyl, off.flavors, turbidity, and ropi
ness in beer (Dolezil and Kirsop 1980; McMurrough and Palmer 1979).
The coliforms (Klebsiella and Escherichia) grow only when the pH is
above 4.3. They impart various odors and flavors to the wort. If the wort
is stored for future fermentation, coliforms can become a problem.
In addition to the so-called wild yeasts, the various strains of brewer's
yeasts can cause contamination. Bottom yeasts may become contami-
nated with top yeasts, and vice versa_ This will result in an alteration of
the desired product.
A bacterium, Zymomonas anaerobia, has been found in beer and cider.
It produces acetaldehydes and H
2
S. The beer has a very unpleasant odor
and flavor.
N-nitrosodimethylamine is a carcinogen and has been reported in
beer (Scanlan et al. 1980; Spiegelhalder, Eisenbrand, and Preussmann
1979). The possible source of nitrosamines in beer as well as reduction
or elimination were discussed by Wainwright (1986a, 1986b).
Wine. Wine is considered to be the oldest fermented alcoholic beverage.
The term wine is applied to the product made by alcoholic fermentation
by yeasts of grapes or grape juice, with an aging process. However, the
products of fermentation of berries, fruit, and such things as honey, palm
juice, rhubarb, and dandelion also are called wines. These are designated
by the substance from which they were made (blueberry wine, dandelion
wine).
The quality of the finished wine depends upon the grapes, fermenta-
tion techniques, aging, and blending. Anyone can make wine, but only
the experts can produce high-quality wine.
Some of the factors influencing the quality of the grape are climate,
soil conditions (temperature, fertility, type, and drainage), and variety of
grape (Amerine, Berg, and Cruess 1972). A year with a good climate is
known as a vintage year. If the climate is too cold, the grapes do not
mature as well, producing less sugar and more acid.
The grapes are crushed and pressed to release the juice, which is
called the must. The sugar content is determined with a hydrometer. The
sugar content can be adjusted by adding sugar, mixing high and low
musts together, or by adding water. Multiplying the hydrometer reading
in degrees Brix by 0.55 to 0.56 generally gives an estimate of the percent-
age alcohol in the finished product. Jones and Ough (1985) reported
some yields of 0.60 or higher.
The must contains many types of microorganisms. To control these
contaminants, the must is treated with sulfur dioxide (S02), or potassium
metabisulfite. The yeast primarily involved in the fermentation has been
called Saccharomyces ellipsoideus, S. vini, or S. cerevisiae var ellipsoideus. How
ever, Van der Walt (1970) classified the yeast as Saccharomyces cerevisiae.
There are many strains of this yeast species. One study listed other Saccha-
romyces (S. chevalieri, S. bayanus, S. italicus, and S. uvarum) as so called wine
yeasts (Rosini et al 1982). Besides the various strains of Saccharomyces,
Kunkee and Amerine (1970) listed about 160 species of yeasts that have
been found on grapes, in musts, or in wine. These also may influence the
overall fermentation of the must, especially if they are resistant to S02
treatment.
The added S02 not only inhibits unwanted organisms, but also stabi
lizes wine color. Due to health problems caused by the consumption of
sulfites, there is a desire to eliminate this additive. Some strains of S.
cerevisiae can stabilize wine color, apparently by producing sulfite. One
strain produced 30 to 80 mg/L (ppm) (Suzzi, Romano, and Zambonelli
1985). Even if other yeasts are not inhibited, as the fermentation pro
ceeds the alcohol tolerant wine yeasts soon become dominant.
Thornton (1983) listed twelve desirable characteristics of a wine yeast:
1. Efficient conversion of grape sugar to alcohol
2. Rapid initiation of fermentation (48 hr)
3. S02 Tolerance
4. Ability to cause even fermentations
5. Ability to ferment at low temperatures
6. Ability to ferment to dryness (alcohol tolerant)
7. Good flocculation after fermentation to aid in removal
8. Production of a desirable bouquet
9. Low foaming
10. Low H2S or mercaptan fermentation
11. For sensory quality of the wine, a relatively high glycerol produc
tion
12. Production of a relatively low amount of higher alcohols.
Although all of these characteristics are probably not possible to attain in
anyone yeast strain, improvements can be made through hybridization,
mutagenesis, cell fusion, transformation, and genetic engineering
(Eschenbruch et al. 1982; Romano et al. 1985; Thornton 1983, 1985).
Killer wine yeasts that will inhibit (kill) unwanted yeasts, are being devel
oped (Hara, Iimura, and Otsuka 1980; Seki, Choi, and Ryu 1985; Shimizu
et al. 1985).
For red wine, the must is allowed to ferment with the skin and pulp of
the red grapes. The fermentation is allowed to proceed until the correct
amount of color is extracted from the skin. During this early stage, aera-
tion is used to promote the growth of the yeast.
After this initial fermentation, the must is drawn off in a process
called racking. The must is put into closed tanks (Fig. 9.12) to provide
anaerobic conditions for the main fermentation. The produced CO
2
tends to purge the must of oxygen and inhibits aerobic organisms, such
as Acetobacter. The fermentation of white table wines is usually at 10 to
15C, while red wine is at 25 to 30 C. It may require from one to four
weeks to complete the fermentation, which is evidenced by the cessation
of bubbling due to CO
2
production.
The wine is pumped into barrels, vats, or tanks for aging. The aging
time varies with the type of wine. During aging, the bouquet and aroma
of the wine are developed. Various compounds, such as esters, are
formed. Most wines are blended to maintain flavor consistency. This is
usually done during the aging period.
After aging, the wine may be pasteurized at 60C for 30 min and
Figure 9.12. Stainless steel fermenting tanks. Note their location outdoors.
courtesy of Wine Institute, Amerine, Berg, and Cruess (1972).
bottled or, in some cases, packed in kegs for shipment, and bottled else
where. When properly aged wine is bottled, it will continue to improve
for several years.
There are special procedures for certain products, such as dessert
wine and champagne. Dessert wines are sweet and have an alcohol con
tent of about 20 percent. To attain these conditions, the fermentation is
halted while there is sufficient sugar remaining by adding brandy. The
higher alcoholic content of brandy increases the alcohol in the resultant
wine.
For champagne and other sparkling wines, the wine is put through a
secondary fermentation. New must, sugar, and yeast are added to the
stock wine, which is then bottled or put into a closed tank. The CO
2
that
is produced remains in the wine, which causes the effervescence.
Although acids in grape juice contribute to the organoleptic quality
of wine, retard microbial spoilage, and stabilize color, too much acid is
undesirable. Amelioration (adding a sugar solution) not only dilutes the
acid but also dilutes other components, including flavor compounds.
A fermentation called the malolactic fermentation, can be used to
reduce the acidity. Malic and tartaric acids account for about 90 percent
of the acidity in grapes. Primarily three genera of lactic acid bacteria
(Lactobacillus, Leuconostoc, Pediococcus) are involved in the reaction to con
vert malic acid to lactic acid and CO
2
This fermentation usually follows
the alcoholic fermentation. The general equation of the reaction is
HOOCCHTCHOH-COOH -> CHyCHOHCOOH + CO2
Malic Acid Lactic Acid
The reaction goes through an intermediate, pyruvic acid, which is
then reduced to lactic acid. The monocarboxylic lactic acid is not as acid
as the dicarboxylic malic acid. Hence, the acidity of the wine is reduced.
Not all lactic acid bacteria can tolerate the alcohol and low pH in
order to convert malic acid to lactic acid. Leuconostoc oenos is usually the
organism involved. It grew in wine with 14.2 percent alcohol and 11.2
ppm S02 at pH 3.2 or above (Davis et aL 1986). The organism did not
grow or alter malic acid in wine with 11.9 percent alcohol and 72 ppm
S02 at pH 4.0 or lower. It is evident that all conditions that may affect
growth of an organism need to be considered. The use of immobilized
cells of L. oenos for the malolactic fermentation was considered by Mc-
Cord and Ryu (1985).
The yeast Schizosaccharomyces pombe utilized all of the malic acid when
the must pH was over 3.0 (Yang 1973). Even at pH 2.5, about 70 percent
of the malic acid was metabolized. With S. pombe, ethyl alcohol and car
bon dioxide are the end products; however, the production of undesir
able flavors and aromas has limited its usage. It may be possible to alter
the genetic material in a desirable yeast to produce the enzyme systems
to convert malic to lactic acid (Snow 1985; Williams et al. 1984).
Certain defects can occur in wine. The lower the sugar content, the
less likely that spoilage is to occur. Thus, dry wines are more stable than
other types of wine. Spoilage is evident by flavor or odor changes as well
as haze or gas formation. Alterations may be due to molds (Daly, Lee,
and Fleet 1984), lactic acid bacteria (Edinger and Splittstoesser 1986),
acetic acid bacteria (Drysdale and Fleet 1985; Joyeux, Lafon-Lafourcade,
and Ribereau-Gayon 1984) and chemical reactions (Simpson, Bennett,
and Miller 1983; Somers and Ziemelis 1985).
To prevent microbial spoilage of the finished wine, it is important to
deactivate any residual microorganisms before or after bottling. This can
be accomplished by pasteurization, addition of inhibitors such as S02, or
filtration. The delicate flavors of some wines are harmed by heating or
by adding S02. For these wines, filtration is the preferred method of reo
moving microorganisms (Reeves 1983; Scott, Anders, and Hums 1981).
Baked Products. The leavening or raising of dough or batters is due to
the incorporation of air into the product. This may be accomplished by
whipping, such as egg whites in angel cakes, by adding chemical agents
that react to produce gas, or by fermentation of sugars by microorgan-
isms to produce carbon dioxide.
Although many microorganisms ferment sugars with the release of
carbon dioxide, Saccharomyces cerevisiae, or baker's yeast, is best adapted
for leavening of bakery products. Yeast fermentation has certain advan
tages over chemicals, since it can contribute a characteristic flavor and
aroma to the product and gas evolution can continue over a longer time.
The products of yeast fermentation also affect the texture of the dough.
Baker's yeast is used in the manufacture of bread, rolls, sweet dough,
pretzels, and crackers. Although some yeasts can hydrolyze starch present
in the flour, baker's yeast (S. cerevisiae) cannot. Enzymes (amylases) natu-
rally present in the flour, or added enzymes (diastatic malt or amylase)
hydrolyze the starch, making it available to the action of the yeast en-
zymes.
Baker's yeast is available as dried or compressed yeast. Commercial
dried yeast has about 8 percent moisture and compressed yeast about 70
percent moisture. Trivedi, Cooper, and Bruinsma (1984) described the
process of protoplast fusion used to combine the desired characteristics
of two yeast strains into a hybrid yeast strain. The resultant commercial
product of this new strain has been called quick-rising, highly active, and
instant-active dried yeast.
Generally, baker's yeast is contaminated to some extent with bacteria,
molds, and other yeasts. Proteolytic and lactic acid bacteria found in
USEF UL MICROORGANISMS 467
yeast are potentially important in altering the pH and structure of
cracker dough (Fields, Hoseney, and VarrianoMarston 1982).
A typical sponge dough process is shown in Figure 9.13. It involves
blending a portion of the flour and water with yeast and yeast food and
allowing this to ferment 3 to 5 hr. The remainder of flour and water plus
salt, fat, milk product, and such things as dough improver, crumb soft
ener, mold inhibitor, and enrichment (vitamins and minerals) are mixed
with the fermented sponge (Dubois 1981; Fowler and Priestly 1980; Mann
1986).
In continuous breadmaking processes, the yeast is grown and fer
mentation occurs in a preferment or brew, which contains little or no
flour. The fermentation products (alcohol, acids, aldehydes, carbon diox
ide) in the preferment contribute to the flavor of the finished product.
The leavening action is accomplished during proofing (holding the
mixed dough for about 45 min in pans before baking).
The heat to which the fermented dough is exposed during baking
kills the yeast cells, drives off the alcohol, and sets the flour protein sur
rounding the entrapped carbon dioxide.
Yeasts with Acetic Acid Bacteria
This combination of organisms is used in the production of vinegar
and in the fermentation of cacao beans and citron.

INGREDIATOR MIXER FERMENTATION ROOM TROUGH MIXER
HOIST
-TO PROOF
MOULDER PRooFER ROUNDER
Figure 9.13. Schematic diagram of sponge dough process.
From Seiling (1969).
DIVIDER TROUGH
HOIST
VINEGAR. Vinegar is produced by an alcoholic fermentation followed
by oxidation of the alcohol to acetic acid. The vinegar must contain at
least 4 g of acetic acid per 100 ml. The strength of vinegar is referred to
in grains, with 10 grains equal to 1 percent. Hence, 4 g/lOO ml is 4 percent
or 40-grain vinegar.
Vinegar can be produced from any food product that can be fer-
mented by yeast to ethyl alcohol. Among the materials used are fruits,
fruit juices, tubers, cereal grains, molasses, honey, coconut, beets, malt,
and refiners' syrup. Vinegars are classified according to the raw material
from which they were made. In the United States, the term vinegar implies
that it was made from apples. The names cider vinegar and apple vinegar
also are used for this product. Wine vinegar is made from grapes. Dis-
tilled, grain or spirit vinegar is made from an alcoholic solution that has
been distilled_
It is essential that alcoholic fermentation be complete before vinegar
formation, since the acetic acid interferes with further alcohol produc-
tion. The overall chemical changes can be represented by the reaction
C2H"OH + O2 ---> CHsCOOH + H20
Acetobacter
Several side reactions occur which alter the final composition of the vin-
egar.
There are various systems for converting alcohol to vinegar_ All of
the methods provide a means of bringing together the alcohol, air, and
Acetobacter_ The air supplies the oxygen needed by the strictly aerobic Ace-
tobacter, and for oxidizing the alcohol through acetaldehyde to acetic acid.
The efficiency of aeration determines the rate of conversion of alcohol
to acetic acid. There are three basic methods: (1) the slow, open-vat
method; (2) the trickle, generator method; and (3) the submerged or bub-
ble method_
Open-Vat. In the open-vat method, the substrate is placed into vats or bar-
rels and inoculated with fresh vinegar or with mother of vinegar (a thick
gelatinous skin formed during acetification). Since aeration is inefficient,
the conversion may require several months. The system developed in Or-
leans, France, was a slow process adapted from the older methods and is
known as the Orleans process.
Generator. In the quick process, the vinegar is manufactured in a genera-
tor in which the substrate is trickled over a packing material (beechwood
shavings, coke, charcoal, ceramics, corn cobs) on which the Acetobacter
is inoculated. The packing material allows a large surface area for the
components to come together. The heat caused by the reaction results in
air moving from the bottom through the generator and out of the top.
When excess heat is produced, cooling of the generator system is needed.
One pass of the substrate through the generator is not sufficient to con
vert all the alcohol to acetic acid, so the product is recirculated to com
plete the conversion. Not only is the generator method quicker than the
vat method, but also higher grain vinegars (over 100 grain) may be ob
tained. The amount of acetic acid in the vinegar depends on the alcohol
content of the substrate. Theoretically, 1.0 percent alcohol should yield
1.3 percent acetic acid. However, actual yields vary from 0.8 percent to
1.0 percent, due to inefficiencies and residual alcohol in the vinegar.
Bubble. The submerged culture, or bubble method, was developed from
the methods used for antibiotic production. In this system, air is pumped
into the vat containing the stirred substrate (alcohol) and Acetobacter. Re
cent research has attempted to increase the productivity of this system
(Ghommidh, Cutayar, and Navarro 1986; Okuhara 1985).
Besides acetic acid and water, vinegar contains residual ethanol,
traces of ethyl acetate, fuse! alcohols and their acids, and traces of other
compounds.
Biologically produced acetic acid in vinegar can be distinguished
from that produced chemically from petroleum or coal by measuring the
14C content with a scintillation counter (Kaneko, Ohmori, and Masai
1973). The basis is that fossil fuels have a lower 14C content than recently
produced carbon.
Yeasts with Molds
In the production of sake, molds are used to hydrolyze the starch of
rice to carbohydrates fermentable by yeasts to alcohol. Although many
microorganisms may be present and affect the reactions and product,
the principal organisms are Aspergillus oryzae and a strain of Saccharomyces
cerevisiae. Kodama (1970) discussed the manufacture of sake and the var-
ious organisms that might be involved.
Molds
The molds are used in many fermentations, especially in Oriental
countries. The most readily recognized mold-produced product is soy
sauce.
SOY SAUCE. This is a dark-brown liquid that is an important flavoring
material for the normally bland rice diet of the Far East. Also, it is used
on vegetables, meats, poultry, and fish. Of all the fermented foods of
Asian countries, soy sauce has received the greatest acceptance in the
United States. The soysauce production process is shown in Figure 9.14.
Soaked, steamed soybeans are mixed with roasted wheat in a ratio of
about two to one. This is inoculated with a mold, koji. The koji is cooked
rice that is inoculated with a species of Aspergillus and incubated for three
to five days. The mold species that have been used are A. oryzae, A. soyae,
Wheat

Roas ted

Starter
A sperg'//us oryzae
[Mixed strains)
Soybeans

Soaked

Crush lightly Mixed Cooked
----- -----
Incubation room

Mold mixture
IKoji)
Salt solution ------....,...
117-19%) Mash
IMoromi)

lactic acid fermentation
I Pecliococcus soyae)
Yeast fermentation
1 Sacchorom yces roux", Toru/opsis sp.)
+
Aged
+
___ Pressed -----....
Cake ----- liquid
+ +
Animal feed
Figure 9.14. Flow sheet for production of soy sauce.
Courtesy of Wang and Hesseltine (1982).
Pasteurized
+
Soy sauce
or A. japonicus. This mixture is stored until the mold covers the surface
of the soybean-wheat mixture_ Brine (1 kg saltl4 liters of water) is added
at the rate of about 2 liters/kg of soybeans. This blend, or moromi, is
fermented for three to six months, first at 15e, then at 25e. The mold
enzymes hydrolyze the carbohydrates and proteins. Bacteria appear in
the early stages of the fermentation and lower the pH from 6.7 to 5.0.
These bacteria are in the genera Pediococcus or Lactobacillus.
In the later stages, the osmophilic yeast Saccharomyces rouxii ferments
part of the sugars (from the hydrolysis of carbohydrates) to produce alco-
hol (about 2.5 percent). Besides Saccharomyces, other yeasts that may be
involved include Hansenula and Torulopsis.
In the final phase of the fermentation, the pH is lowered to about 4.5
to 4.8. At this time, the fermented mash is filtered, and the liquid ob-
tained (soy sauce) is pasteurized at 80 to 85e and bottled for use.
There are several variations in the procedure and several varieties of
soy sauce in Asian countries (Fukushima 1985; Pederson 1979). The fla-
vors in soy sauce were discussed by Nunomura, Sasaki, and Yokotsuka
(1976a, 1976b, 1984).
A system using enzymatically hydrolyzed soybeans fermented by im-
mobilized whole cells of bacterial and yeast cells has been described
(Osaki et al. 1985).
TEMPEH. Tempeh is a fermented food characteristic of Indonesia.
The product made from soybeans is called tempeh kedalee, while that
made from coconut press cake is called tempeh bongkrek.
There are several methods for making tempeh. Briefly, for the soy-
bean product, the beans are soaked and boiled and the hulls removed.
The beans may be cooked, and the bean mash made into cakes, inocu-
lated with tempeh mold, wrapped in banana leaves, and fermented.
There are several species of Rhizopus that can ferment the beans to a
desirable product, but R. oligosporus is considered to be the tempeh mold.
At a temperature of 30 to 40
0
e, the tempeh is ready to eat in 24 to 48
hr.
Tempeh has been described as having a bland flavor, is quite nutri-
tious, and has simple, low-cost processing techniques.
Miscellaneous Fermentations
There are certain fermentations in which individual or many micro
organisms may be involved. Among these is the fermentation of glucose
in egg white.
EGG WHITE. Egg white contains about 0.5 percent glucose, a reducing
sugar. In commercially dried egg white (3 to 9 percent moisture), glucose
will combine with amino acids to form brown insoluble products, due to
the Maillard reaction.
The simple solution to this problem is to remove the glucose. There
are several systems that can be used for this purpose. Many microorgan
isms ferment or oxidize glucose so that it is not reactive with the amino
acids. In a natural fermentation, the liquid albumen is allowed to remain
at room temperature and the contaminants acquired during the breaking
and separation of the egg white will eliminate the glucose. With this sys
tem, there may be undesirable organisms that degrade the protein, or
those that are health hazards, such as the salmonellae, which also grow
in the fermenting product.
Seeding of the albumen with specific organisms was used, so that
there would be some semblance of a controlled fermentation. The orga
nisms included coliforms (mainly Escherichia coli or Aerobacter [Klebsiella]),
yeasts (species of Saccharomyces and Candida), and streptococci. Although
these fermentations were acceptable, due to the lack of microbiologists
in many egg products operations, control tended to suffer.
An enzyme isolated from Aspergillus niger is called glucose oxidase
since, in its presence, glucose is oxidized to gluconic acid. The advan
tages of using this system are that the microbial load is not increased as
in fermentation, and the glucose is not lost but remains in the product
as gluconic acid. This means the product yield is greater. This enzyme
system is discussed further in the section on enzymes.
ENZYME SYSTEMS
Enzymes are organic catalysts that allow reactions to occur under rela
tively mild conditions. Hence, they are well suited for use in the food
industry. There are cases in which enzymes can be used more effectively
than live microbial cells. When a series of reactions or several types of
reactions are desired, such as in wine production, the intact microorgan
ism is the system of choice, but if only one reaction is needed, separated
enzymes may be beneficial. Also, some reactions or new products or
processes may be possible only through enzyme activity.
Industrial enzymes are obtained from plant and animal tissues and
from microorganisms. Since microorganisms can be grown in large
amounts in controlled conditions, they offer an unlimited source of
many enzyme systems. Of the thousands of enzymes, only a relative few
have been developed for commercial use.
Microorganisms synthesize all enzymes intracellularly. They secrete
certain hydrolases (carbohydrases and proteases) into the surrounding
substrate. These enzymes hydrolyze large molecules into smaller ones,
which can then be brought into the cells for further breakdown. All types
of enzymes remain inside the cell to conduct the normal cellular activi-
ties_ It is evident that the extracellular hydrolases are easier to obtain than
are the intracellular enzymes, since obtaining the latter requires break-
age of the cell wall and membranes_
On a comparative basis, enzymes are rather expensive_ If they are
added directly to a food, they are difficult or impossible to recover, so
they are used only once. Since enzymes are catalysts, they should be reus-
able. To accomplish this, processes have been developed for immobiliz-
ing enzymes on inert carriers_ Immobilization may be accomplished by
adsorption, chemical attachment (covalent bonding), microencapsula-
tion (enclosing within a material), covalent cross-linking, entrapment, or
copolymerization (Fig. 9.15). Various carriers or supports have been sug-
gested for the immobilization process (Baing 1982; Fadda et al. 1984;
Imai et al. 1986; Weetall and Pitcher 1986; Wongkhalaung et al. 1985).
Since various enzymes and substrates differ in their properties, no one
system is useful for all enzyme systems. Also, the process in which the
enzyme is used will influence the type of carrier that is used. Each of the
immobilization methods has certain advantages and disadvantages. The
advantages are that the enzymes can be removed from the reaction and
they can be reused. In some cases, an immobilized enzyme can be used
at higher temperatures than can the corresponding free enzyme (it has
a higher temperature of inactivation). Since the rate of chemical reac-
tions is increased as the temperature is raised, this is an advantage to the
processor. Some problems with immobilized enzymes include the reduc-
tion of enzyme activity upon immobilization, loss of activity after re-
peated usage, mechanical degradation of the enzyme or its support after
usage, and microbial growth, with the enzyme acting as the substrate.
Enzyme activity (enzyme decay) declines simply because of time.
Besides stability and reuse, other advantages of immobilization in-
clude using a continuous process rather than a batch operation, better
con trol of the process, halting the reaction by removing the enzyme, and
less product inhibition. One can conceivably use immobilized enzymes
in a series so that several reactions can be obtained during the treatment.
The concept of enzyme immobilization has been applied to microbial
cells by attaching or entrapping in various substances (Bisping and Rehm
1986; Fukui and Tanaka 1982;Johansen and Flink 1986)_ The advantages
of cell immobilization over that of enzymes include the formation of mul-
tistep and cooperative enzyme systems and the elimination of the need
to extract and purify enzymes from the cells. Also, the biological activities
of immobilized cells are maintained much longer than those of immobi-
lized enzymes. Some disadvantages are that the cells may contain un-
wanted enzymes, and there are permeability barriers of the cells and en-
trapment supports to the reactants.
Immobilized cells can be used advantageously in continuous pro-
A
EOE EE
E E
E E
E
A1
A2
B
B1
B2
c
-E E
ry
E E
E---\ A
E E-
Figure 9.15. Methods of enzyme immobilization. (a) Bonding: (1) co
valent bonding; (2) adsorption. (b) Inclusion; (1) entrapment; (2) mi-
croencapsulation. (c) Cross-linking.
From Baing (1982).
cesses. These cells can be removed from the product more readily than
can the free cells, and they can be reused. Compared to free cells, the
immobilized cells have a higher rate of product formation, and the yield
is greater. This is due to an increased cell density of immobilized cells.
With higher productivity, smaller reactors can be used, with a reduction
in overall cost of the product.
Some microbial enzymes that are useful in the food industry are listed
in Table 9.6.
Amylases
These enzymes hydrolyze starch. Most starches are composed of amy-
lose and amylopectin. Amylose is an unbranched polysaccharide with
glucose units joined by 1,4-a-glucosidic linkages. Amylopectin is a
branched polysaccharide with the glucose units as in amylose and 1,6-a-
glucosidic linkages at the points of branching. The products of hydrolysis
of starch depend upon the specific amylase that is present.
Commercial amylases are not pure and contain small amounts of mal-
tase and other carbohydrases. In general, amylases can hydrolyze gelati-
nized starch more readily than raw starch.
The dextrins and oligosaccharides released from starch by the amy-
lases are important in the color and texture of crust and the shelf life of
bread. These enzymes can replace the diastatic malt in doughs when the
flavor of malt is not needed. Amylases help prevent the staling of bread.
They are also used in the manufacture of syrup for use in confections,
as well as in combination with glucose isomerase to produce high-fruc-
tose corn syrups.
ALPHA AMYLASE (3.2.1.1). These enzymes are obtained from Asper-
gillus oryzae, Aspergillus niger, Rhizopus oryzae, Bacillus subtilis, and B. licheni-
[ormis. They rapidly fragment starch to dextrins. They do not hydrolyze
the 1,6-a-glucosidic linkages of amylopectin.
The enzyme facilitates filtration by converting gelatinous starch to
soluble dextrins. It clarifies fruit juices which are turbid due to starch. It
is useful in the baking, brewing, and alcohol industries, converting starch
to fermentable components. By its action on cocoa starch, it helps ensure
stability of chocolate syrup.
The thermostabilities and effective pH ranges vary among the fungal
and bacterial amylases (Wasserman 1984). Hence, all of the characteris-
tics of amylases or any other enzymes must be considered when an en-
zyme is selected for a particular application.
Beta Amylase (3.2.1.2)
These enzymes hydrolyze starch from the nonreducing ends of the
glucosidic chains and release maltose. They do not attack the 1,6 link-
ages. In combination with alpha amylases, they are used in brewing, fer-
mentation, and baking industries.
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GLUCOAMYLASE (3.2.1.3). This is a glucose-liberating amylase. Glu-
coamylase is used to convert partially hydrolyzed corn starch (treated
with acid and alpha amylase) to corn syrup containing more than 90 per-
cent glucose on a solids basis (Walton and Eastman 1973). An immobi-
lized two-enzyme (glucoamylase and alpha amylase) system for this pur-
pose was described by Hausser, Goldberg, and Mertens (1983).
The enzymes can be obtained from organisms such as Aspergillus niger,
A. oryzae, Rhizopus delemar, R. oryzae, and R. niveus.
Catalase (1.11.1.6)
Catalase is produced by many organisms. Commercially, it is obtained
from a strain of Aspergillus niger or Micrococcus lysodeikticus. This enzyme
catalyzes the reaction in which hydrogen peroxide is decomposed to wa-
ter and oxygen. When hydrogen peroxide is added to milk for cold steri-
lization, catalase is used to remove the excess hydrogen peroxide_ Cata-
lase is present in commercial glucose oxidase preparations to release O2
from H
2
0
2
for the oxidation of glucose to gluconic acid, such as in the
process for liquid egg white.
Cellulase (3.2.1.4)
Cellulase catalyzes the hydrolysis of the 1,4 linkages of cellulose to
form dextrins and usable sugars. Several fungi, including the thermo-
philic fungi, are sources of cellulase. The fungi include species or strains
of Myrothecium verrucaria, Stachybotrys atm, Trichoderma viride, Trichoderma
reesii, Aspergillus niger, Chaetomium thermophile, Sporotrichum, Talaromyces,
Thermoascus, and Humicola.
Cellulase is used to remove the cellulose cloud and clarify citrus
juices, to improve the body of beer, to aid in extraction of flavoring com-
pounds (such as essential oils), to increase the digestibility and nutritive
value of plant products, and to treat cellulosic wastes for potential use
as feed, food, or as fermentable sugars (to alcohol). Some problems in-
volved in cellulose conversions were discussed by Saddler (1986).
Beta Galactosidase (3.2.1.23)
The common name for this enzyme is lactase. It catalyzes the hydroly-
sis of lactose to glucose and galactose. This enzyme can remove the lac-
tose from milk, making it acceptable to people with lactose intolerance.
Lactose is less soluble and less sweet than the hydrolysis products glucose
and galactose. Hence, hydrolysis of lactose improves the solubility of
dried milk and the consistency of concentrated milk, ice cream bases,
and frozen milk. Lactose can crystallize in frozen products, which results
in graininess or sandiness in these foods. Frozen desserts made with the
hydrolyzed product requires the addition of fewer sweeteners, resulting
in a lower-calorie product.
The high lactose content of cheese whey has made this by-product
difficult to use. With hydrolysis of the lactose, a sweet whey is obtained
that can be used in other products or as a substrate to produce yeast.
The enzymes from different sources vary in their physical properties.
They have many microbial sources, including Kluyveromyces marxianus,
Aspergillus niger, and A. oryzae.
Glucose Isomerase (5.3.1.5)
Glucose is obtained by the enzymatic (amylases) hydrolysis of starch,
such as corn starch. Glucose isomerase catalyzes the reversible isomeriza-
tion of glucose to fructose. This reaction is important because the relative
sweetness of fructose is two and one-half times that of glucose. With corn-
starch as the starting material, high-fructose corn syrup is obtained. This
syrup contains about 42 percent fructose on a dry basis. With further
processing, higher-level fructose products can be produced (55 percent
or 90 percent).
The enzyme is produced by various bacteria, including species of
Streptomyces, Bacillus, Actinoplanes, Arthrobacter, and Microbacterium.
Glucose Oxidase (1.1.3.4)
This enzyme catalyzes the oxidation of glucose to gluconic acid. The
excess hydrogen peroxide is then catalyzed to water and oxygen by the
presence of catalase. Hence, the enzyme system is called the glucose
oxidase-catalase system.
Glucose oxidase is obtained from Aspergillus niger, although other or-
ganisms, such as Aspergillus oryzae, Penicillium glaucum, and P. notatum also
produce this enzyme. Researchers reported that the best source of the
enzyme was a strain of Penicillium purpurogenum (Nakamatsu et al. 1975).
This enzyme can be used to remove glucose from egg white, whole
eggs, or pork prior to dehydration. This improves the quality and shelf
life of the dried product.
Since it is specific for glucose, the enzyme can be used as an analytical
tool to estimate the amount of glucose in foods. This is especially useful
in the estimation of glucose formed by the hydrolysis of lactose or starch
in the food industry. Also, the assay is used clinically for determining
glucose in blood and urine (Jackson and Conrad 1985). The enzyme can
also be used in immunohistochemical procedures (Rathlev et al 1981).
Since the enzyme uses oxygen to react with glucose, it can be em
ployed as an oxygen scavenger for food systems that deteriorate in an
oxygen atmosphere. The removal of oxygen by this system may increase
the shelf life or aid in retaining the quality of foods. An increase in shelf
life of fish treated with glucose oxidase was reported by Field et aL (1986).
Invertase (3.2.1.26)
Invertase catalyzes the hydrolysis of sucrose to fructose and glucose.
The resultant invert sugar is sweeter than sucrose.
Although several microorganisms possess the ability to hydrolyze su
crose, the yeasts have been used as a source of invertase. The yeasts in
clude S. cerevisiae, S. rouxii, K. marxianus, and Candida utilis.
Invertase can be used whenever it is desirable to have sucrose hydro
lyzed. It is used in confections to convert solid sucrose mixtures to a fluid
consistency after covering with a substance such as chocolate, to produce
softcentered confections.
Limonoate Dehydrogenases
Limonin is a bitter component in citrus products. If this bitterness
could be controlled, there might be an increased demand for citrus prod
ucts. Immobilized cells of Arthrobacter globiformis or Corynebacterium jas
cians, which produce the enzyme, were used to reduce the bitterness of
citrus juices (Hasegawa, Patel, and Snyder 1982; Hasegawa et aL 1985).
Lipases (3.1.1.3)
Lipase acts on fats, hydrolyzing them to monoglycerides, diglycerides,
or glycerine, and free fatty acids. Many microorganisms produce lipase
enzymes. The commercial sources include A. niger and A. oryzae as well as
Candida lipolytica, C. cylindracea, Penicillium roqueforti, Mucor, Rhizopus,
Pseudomonas, and Torulopsis ernobii.
Lipases isolated from microorganisms have been used to develop fla
vor in cheese or cheeselike foods and for treating milk fat for use in ice
cream, margarine, and butter (Kilara 1985a). It can be used to improve
the flavor of bakery and other products. It can improve the whipping
properties of egg white by removing any contaminating egg yolk fat.
Since the reactions are reversible, it is thought that fats can be taken
apart and other fatty acids replaced, thereby producing special modified
fats for specific uses.
Pectic Enzymes
These enzymes hydrolyze pectin and pectic substances to lower
molecularweight compounds. Various names have been used for these
enzymes. The usual enzyme system is pectinase, which generally is a mix
ture of polygalacturonase (3.2.1.15) and pectic methylesterase (3.1.1.11).
Pectic lyase (4.2.2.3) and pectin transeliminase (4.2.99) were suggested as
clarifying agents for fruit juices (Ishii and Yokotsuka 1973).
Pectic methylesterase causes demethylation, while the polygalacturo
nase catalyzes the hydrolysis of a14galacturonide bonds of pectin. The
release of methyl groups exposes carboxyl groups, which, in the presence
of calcium or other multivalent ions, form insoluble salts that can be
removed.
The commercial sources are Aspergillus niger and Rhizopus oryzae; how
ever, several other fungi, such as Aspergillus soyae, A. japonicus, A. wentii,
Penicillium glaucum, and P. expansum, may be used.
Pectin remaining in fruit juices tends to hold other substances in sus
pension as a colloidal system. By hydrolyzing the pectin, the protective
colloidal action is destroyed, so suspended substances will settle out and
can then be removed from the juice by filtration.
Not only do pectic enzymes act as clarifying agents, they also improve
pressability and color extraction, prevent gelation of fruit juice concen
trates, speed filtration, and increase the yield of freerun juice. Pectinase
is used to remove the gelatinous coating from coffee beans for fermenta
tion and processing.
Proteases
This is a group of enzymes that catalyze the hydrolysis of peptide
bonds in proteins and yield pep tides of lower molecular weight. Most
proteases split specific peptide linkages. Hence, it is necessary to select
the appropriate protease or combination of enzymes so that the desired
reaction is obtained.
Proteases can be obtained from any proteolytic organism. The com
mercial sources of proteases include Aspergillus oryzae, A. saitori, Bacillus
subtilis, B. licheniformis, and Streptomyces griseus.
The applications of proteases include the chill proofing of beer, meat
tenderization, the production of protein hydrolysates, mellowing of the
dough in bakery products, the production of fish protein solubles, and
the improvement of protein recovery from oilseed meals.
In baked products, the use of proteases in the sponge dough reduces
mixing requirements. The treated doughs are more extensible and can
be molded more easily than untreated dough. Proteases are of value in
controlling the pliability, eliminating buckiness, and assuring proper
machinability of the dough, as well as increassing loaf volume.
Proteases are added to beer or ale during the finishing operation to
prevent an undesirable haze when these beverages are cooled. The hy
drolysis of highmolecularweight proteins prevents the formation of the
haze. The use of proteases in foods has been discussed by Kilara (l985b)
and Loff1er (1986).
Milk-Clotting Enzymes
These are proteolytic enzymes that have an important function in the
manufacture of cheese from milk. During lactic acid fermentation of
milk, rennet (rennin) is added to the vat to aid in the clotting process of
casein to form a curd (McMahon and Brown 1984). Rennet is obtained
from the fourth stomach (abomasum) of suckling calves. It is a mixture
of enzymes, primarily chymosin, which has a high ratio of milk clotting
activity to proteolytic activity. Because fewer calves are being slaughtered,
rennet substitutes have been investigated. Problems of using rennet sub
stitutes can usually be traced to the extent and type of proteolytic activity
relative to their ability to clot milk.
Although many microorganisms produce enzymes that clot milk, only
three fungi have been used to any large extent for commercial enzyme
production (Endothia parasitica, Mucor miehei, and M. pusillus).
CHEMICALS
Some organisms do not participate in food fermentations directly but
are used to produce chemicals that are added to foods. Such chemicals
as vitamins and amino acids upgrade the nutrient value of food, and
gums and dextrins improve the physical characteristics of foods. Demain
(1971) listed alcohols, amino acids, antibiotics, antioxidants, coloring
agents, enzymes, nucleotides, organic acids, plant growth regulators, pol
yols, polysaccharides, protein, sugars, and vitamins as fermentation
products used in the food or feed industries.
Amino Acids
Amino acids can be produced by chemical synthesis, but both optical
isomers are obtained. In contrast, the synthesis by microorganisms reo
sults in the Lisomer which is the form used by biological systems. The
microbial sources of certain amino acids are shown in Table 9.7.
Lysine and methionine are important as additives to plant proteins
TABLE 9.7. SOME MICROBIAL SOURCES OF AMINO ACIDS
Amino Acids
Alanine
Aspartic acid
Glutamic acid
Lysine
Methionine
Ornithine
Phenylalanine
Threonine
Tyrosine
Tryptophan
Valine
Microbial Sources
Corynebacterium, Brevibacterium
Escherichia, Serratia, Bacillus, Pseudomonas
Corynebacterium, Brevibacterium, Micrococcus, Microbacterium, Bacil
Ius, Pseudomonas, Streptomyces, Aeromonas, Flavobacterium
Micrococcus, Bacillus, Escherichia, Torulopsis, Saccharomyces, Proteus,
Erwinia, Candida, Corynebacterium
Pseudomonas, Rhodotorula, Micrococcus, Candida
Micrococcus
Escherichia, Micrococcus
Escherichia
Micrococcus
Claviceps, Serratia, Micrococcus, Rhizopus, Hansenuia, Candida,
Escherichia
Enterobacter, Escherichia, Klebsiella, Micrococcus, Brevibacterium
to increase the nutrient value. Glutamic acid is used in the production
of monosodium glutamate, which is a flavor enhancer in many foods.
Compared to free cells, immobilized cells produce increased amounts of
amino acids (Umemura et al. 1984; Wada et al. 1980).
Other Organic Acids
Various organic acids are added to foods. The main supply of citric
acid was lemons, but now it is obtained by fermentation of sugars with
Aspergillus niger. Acetic and lactic acids are discussed in regard to vinegar
production and vegetable and dairy fermentations. In lactic acid produc-
tion, Lactobacillus delbrueckii is used because the reaction can be accom-
plished at 50C. This high temperature suppresses many other organisms
(psychrotrophs and mesophiles). Some microbial sources of organic acids
are listed in Table 9.8.
TABLE 9.8. SOME MICROBIAL SOURCES OF ORGANIC ACIDS
Acid
Acetic
Citric
Gluconic
Lactic
Propionic
Pyruvic
Succinic
Microorganism
Acetobacter
Aspergillus, Penicillium, Trichoderma
Penicillium
Lactobacillus delbrueckii
Propionibacterium
Pseudomonas
Brevibacterium
Microbial Gums
Gums are used in the food industry in aqueous systems when there
are problems involving suspension, thickening, emulsion stabilization,
and rheological modifications. One of the sources of gums is microorgan
isms. Several microbial gums have been produced and tested. One that
is available commercially is xanthan gum. It is an optional emulsifying
and stabilizing ingredient in French dressing and a stabilizing ingredient
in certain cheeses and cheese products. It has applications in canned
foods, dry mixes, frozen foods,juice drinks, relish, sauces, gravies, syrups,
bakery fillings, and bakery flavor emulsions.
In the production of xanthan gum, Xanthomonas campestris is grown in
a well aerated medium containing glucose, a nitrogen source, potassium
phosphate, and trace elements. The gum is produced as an exocellular
polysaccharide coat surrounding the cell wall.
Vitamins
Vitamins are added to various foods for enrichment. Only a few vita
mins are produced by fermentation. Riboflavin, produced chemically, is
used in food, while that produced by fermentation is used in feeds. Some
microbial sources of vitamins are listed in Table 9.9.
Flavoring Compounds
Autolyzed yeast extract is used as a flavoring agent in many foods
(soups, bouillons, sauces, gravies, entrees and side dishes, snack foods,
canned meat products). An enzymatic system for autolysis of the yeast
cells was described by Knorr et al. (1979).
MICROBIAL PROTEINS
Due to the potential increase in population and the limited supply
of land for agriculture, alternative sources of food have been investi
TABLE 9.9. SOME MICROBIAL SOURCES OF VITAMINS
Vitamin
Ascorbic acid
/3-Carotene
Bl2
Riboflavin
Microorganism
Pseudomonas
Rhodotorula
Bacillus, Propionibacterium, Streptomyces, Pseudo-
monas, Escherichia, Nocardia, Rhizobium
Aspergillus niger, Eremothecium ashbyii, Ashbya gossy-
pii, Candida, Streptomyces
gated. One of these sources is microbial cells. Various names, such as
novel protein, unconventional protein, minifoods, petroprotein, and
single-cell protein (SCP), have been used for these products. Actually, the
products are microbial cells or microbial proteins. Microbial protein
(MP) is used in animal feed more than directly as human food.
Either live or dead microorganisms are present in nearly all of the
food that we eat. In some products, such as yogurt, the presence of cer
tain microorganisms is used as a sales gimmick. Thus, the use of microor
ganisms for food should not be objectionable if the microorganisms are
not pathogenic and produce no harmful toxins.
Advantages
Microorganisms have many advantages over other types of food
sources. Microorganisms do not require much space; they do not com-
pete for the arable land that is available; they can be genetically manipu-
lated to produce certain types of products; they have a rapid rate of
growth and, since growing conditions can be easily controlled, there are
no environmental problems as in normal agriculture, such as freezing,
floods, or drought. Besides these advantages, microorganisms can be
used to digest and convert wastes into useful products. This also reduces
the problems of waste disposal.
Substrates
Various substrates ranging from CO
2
with sunlight for algae to com-
plex substances for bacteria, yeasts, and molds have been proposed for
the production of microbial protein. These substances are derived from
petroleum, natural gas, industrial products, agricultural crops, and var-
ious wastes.
According to Calam and Russell (1973), to be economical, the mini-
mum capacity of a processing plant should be 50,000 tons of SCP per
year. This means that an adequate supply of substrate is needed for mi-
crobial growth and SCP production. Petroleum and natural gas products,
such as kerosene, fuel oil, gas oil, n-alkanes, methanol, and ethanol were
considered desirable because they were readily available in an appar-
ently unlimited supply. However, there are problems in utilizing fuel oil,
gas oil, and n-alkanes. Besides the real or imagined shortages and the
increasing prices of petroleum, these substances are not miscible with
water. Hence the mixture must be agitated constantly to keep the cells
in contact with the substrate. A high volume of oxygen is needed, and
ammonium salts or nitrates must be added as a nitrogen source for the
microbial cells. Only about onehalf of the hydrocarbon is converted to
cells, the rest being oxidized to carbon dioxide and water.
Other problems with petroleum usage include the need to remove
the excess heat due to the oxidation of the hydrocarbons and also the
separation of the cells from substrates, such as fuel oil or gas oil. There
are potentially carcinogenic polycyclic hydrocarbons in oil-based fer-
mentations.
Methanol is regarded as a very promising substrate. This alcohol can
be produced from natural gas that would normally be burned at remote
oil wells. Also, it can be produced from coal or wood. Methanol is rela-
tively cheap, there is an ample supply, it is essentially pure (99.5 to 99.8
percent), it is miscible with water, less oxygen is required, less heat re-
moval is needed, it is easily stored and handled, contamination is mini-
mized due to restrictive use by only a few microorganisms, and the cells
are readily separated from this substrate (Cooney and Makiguchi 1977;
Dijkhuizen, Hansen, and Harder 1985;Jara, Allais, and Baratti 1983; King
1982).
Ethanol can be obtained from ethylene, a petroleum product, or by
fermentation of carbohydrates. The use of ethanol as a substrate is simi-
lar to methanol (Laskin 1977).
Methane, derived from fossil fuels or by degradation of waste prod-
ucts, can be used as a substrate. The process was discussed by Wilkinson
(1971). Another gas that has been considered as a substrate is carbon
monoxide (Meyer 1980).
Some agricultural crops that are primarily carbohydrate could be
used as substrates to produce SCP, and this protein could then be used
to upgrade the nutritional value of the food crop.
The disposal of our wastes is a problem that is increasing every year.
If our wastes could be used to produce a usable product such as SCP, we
might be solving two problems, waste disposal and a food source.
For the United States, organic agricultural wastes have been estimated
at over a billion metric tons annually (Bellamy 1974). The annual world
production of straw is estimated at a billion tons. Generally only one-
third to one-half of the agricultural crop is harvested and the remainder
is usually left in the field. This residue is often a harbor for pests and
plant pathogens. For some vegetables, such as peas, the entire plant is
hauled to the processing plant and, after the peas are removed mechani-
cally, the pea vines accumulate and may become a disposal problem.
Crop residues, such as corn stalks and cobs, straw, pea and bean vines,
and other vegetable-processing wastes contain from 30 to 60 percent cel-
lulose, 10 to 30 percent hemicelluloses, 5 to 20 percent ash, 4 to 18 per-
cent lignin, and relatively small amounts of protein and fat.
Urban and industrial organic wastes accumulate at about 200 million
metric tons annually. Some of these wastes are not biodegradable and
others may contain toxic chemicals that make them unsuitable as sub-
strates.
The suitability and economics of these wastes depend upon their
availability (the amount and location), composition, biodegradability,
treatment(s) needed, presence of toxic materials (including pesticides),
and various costs (product gathering, transportation, and storage).
Some of the wastes that have been considered as potential substrates
are animal wastes (Kargi et al. 1980), sewage, sugar cane bagasse (Molina
et al. 1984), straw (Pavlostathis and Gossett 1985; Taniguchi et al. 1982).
Various wastes from food-processing plants have been investigated as po-
tential substrates for SCP production (Calleja et al. 1986; Clementi, Mor-
esi, and Rossi 1985; Rale 1984; Shay and Wegner 1986). Some wastes are
seasonal and diluted with water. Hence, there are costs to concentrate
the dilute wastes and to accumulate enough to use over the entire yearly
operation of a processor.
Some wastes, especially cellulosic wastes, need to be treated with acid
or alkali so that they can be used by microorganisms. A source of nitro-
gen (ammonium salts or nitrates) and minerals such as calcium, iron,
phosphorus and magnesium needs to be added for acceptable growth
of microorganisms. Since these different types of wastes vary in their
composition, it is difficult to establish a plant and procedure to utilize
all of them. Only a few accumulate in a volume capable of supplying an
SCP facility, and even then, the gathering and transportation costs must
be considered.
Microorganisms
All types of microorganisms (bacteria, yeasts, molds, higher fungi, al-
gae, and protozoa) have been suggested as potential sources of protein.
For economic as well as other reasons, the organisms should be able to
grow on inexpensive, simple media (preferably waste products); it is
helpful if they can grow in a continuous culture; they should be easily
separated at harvest; pure cultures with known genetic factors and means
of altering these factors should be available; there should be no toxic or
allergenic factors in the culture; the product should be palatable or read-
ily disguisable, contain high-quality protein, and be easily packaged and
stored; and the remaining effluent should be of a quality that is easily
disposed of, with little or no further treatment.
There are certain advantages and disadvantages for using each type
of microorganism in this process. Bacteria have the fastest growth rate,
but being the smallest, they are more difficult to harvest. Molds are large
and are the easiest to harvest, but they have a slower growth rate than
either bacteria or yeasts. The yeasts are easier to harvest than are the
bacteria.
The type and species of microorganism used in the process depend
upon the substrate, the conditions for growth, the equipment, and other
factors.
YEASTS. As a food for humans, the yeasts have been more apparent
than have the other microorganisms. Yeasts have been used as a food
source since workers in Germany developed the process during World
War I. The first successful commercial process of yeast production was
from molasses and ammonia. Since this beginning, several types of sub
strates have been used to grow yeasts for feed and food. Yeast extracts
are used in soups, sauces, relishes, and some processed meats.
Although some yeasts can multiply by a sexual process of spore fu
sion, under most circumstances they reproduce by budding. The time
required for a mother cell to produce a daughter (the doubling time) for
most yeasts with favorable growing conditions varies from one to three
hours. Cooney, Levine, and Snedecor (1975) cited references showing
that for three species of yeast on a methanol substrate, the doubling time
varied from three to nine hours.
Various yeasts have been considered as potential food sources, includ
ing species of Candida, Saccharornyces, Rhodotorula, Torulopsis, Hansenula,
Kluyverornyces, Debaryornyces, Pichia, and Kloeckera.
Yeasts can be used for producing vitamins, enzymes, highquality pro
tein, and other physiological essentials. Since yeasts can be bred for selec
tive purposes, it is possible to obtain special yeasts that are richer than
ordinary yeasts in certain nutritional components, such as highprotein
yeasts.
In considering the nutritive value of yeast, the proximate analysis of
the yeasts is usually published. The protein content can vary from 30 to
65 percent, depending upon the yeast and the substrate. The amino acid
content of these proteins varies considerably. In general, the amino acid
analysis of the yeast protein compares favorably with animal protein, ex
cept that the methionine content is low. According to the review of Reed
(1981), the protein efficiency ratio (PER) of baker's yeast (2.02) can be
increased to 2.77 by the addition of 0.5 percent methionine. Martini,
Miller, and Martini (1979) calculated the PER of various yeasts and reo
ported that they varied from 1.2 to 2.2. The replacement of 8 percent
wheat flour with torula yeast flour increased the PER for rats fed the
resultant bread from 1.31 to 2.28 (Lin, Chastain, and Strength 1986). Ac
cording to Shacklady (1972), the biological value (BV) of dried whole egg
is 90, while that of yeast protein varied from 46 to 61. When 0.3 percent
methionine was added to the yeast protein, the BV increased to values
of 91 to 96, indicating that the supplemented yeast compares favorably
to plain dried whole egg, or dried skim milk (BV of 87). The relatively
high lysine content of yeast will improve the nutrient value if added to
grains such as corn, wheat, or rice. It would not be as useful to add yeast
to soybeans, since they too are low in methionine. Macmillan and Phaff
(1973) listed Rhodotorula gracilis as a source of methionine.
One problem with SCP in general is the high nucleic acid content (6
to 16 percent) of microbial cells. The end product of purine metabolism
is uric acid. Although we can tolerate low levels, when the daily nucleic
acid intake is over 2 g, the uric acid, being insoluble at body pH levels,
can cause conditions such as gout or kidney stones. Methods for reducing
or removing the nucleic acids from yeast protein have been reported by
Gierhart and Potter (1978), Sarwar et al. (1985), and Shetty and Kinsella
(1979), and reviewed by Waslien and Steinkraus (1980).
In a review, Harrison (1970) reported that ingestion oflive yeast cells
is not desirable, because they can remove vitamins and other nutrients
from the digestive tract. Whole dead cells are not easily digested because
of the cell wall. With this inability to digest the cells, the protein is not
usable. To make the protein available, the cells can be disintegrated me
chanically or by enzymes.
DeGroot et al. (1975) fed yeasts to rats over a twoyear period and
found no adverse effect on mortality, rate of body weight gain, hematol
ogy, kidney function, fertility, histological changes, or incidence of tu
mors. Yeast SCP fed to rats yielded no toxicological effects and no abnor
mal reproductive performance in either male or female rats (Ashraf et
al. 1981). Other researchers found an increased resistance to respiratory
disease among monkeys fed brewer's yeast (Sinai et al. 1974); they
thought that the yeast stimulated phagocytosis.
Chickens fed 5 to 10 percent yeast diets were significantly lighter than
those on a control diet (Shannon, McN ab, and Anderson 1976). This was
thought to be due to less feed intake when yeast was incorporated.
Yeast, per se, has not replaced meat proteins in the human diet, pri
marily on the basis of flavor and other quality attributes. Dried yeast has
very few functional properties, but yeast protein has several. The refined
proteins can be spun or otherwise processed to function in various ways
and can be added to foods in the same manner as vegetable proteins
(Hayakawa and Nomura 1978; Huang and Rha 1978).
BACTERIA. The use of bacterial cells or protein as food lags behind
yeasts. However, they have been consumed in fermented foods such as
yogurt.
Generally, bacteria multiply faster than do yeasts. Most bacterial SCP
has a higher level of protein and a higher BV than yeast SCPo Although
bacteria, being smaller, are more difficult to recover than are yeasts,
there are methods using flocculation, filtration, and centrifugation that
allow recovery, but at added expense.
The type of bacteria that is used depends upon the substrate. Species
of Pseudomonas are grown on substrates of methanol, kerosene, fuel oil,
or gas oil (Lipinsky and Litchfield 1974). Abbott, Laskin, and McCoy
(1973, 1974) described the growth of Acinetobacter calcoaceticus on ethanol.
They stated that the composition of an organism is strongly dependent
upon its growth rate and environment. They obtained a higher protein
content of the cells when the growth rate was increased. The fermenta
tion of rice straw with a species of Cellulomonas and Alcaligenes was dis
cussed by Han (1975) and Han and Callihan (1974). It was suggested that
the microbial protein could be used for nonruminant feeding and the
residue could be fed to ruminants.
Rhodopseudomonas gelatinosa is a photosynthetic bacterium utilizing
CO
2
and sunlight. Thermomonospora and Thermoactinomyces are thermo
philic bacteria that produce protein on cellulose substrates. Methylococcus
and Hyphomicrobium species utilize methane. Hydrogenomonas can assimi
late hydrogen and carbon dioxide for SCP production. These were dis
cussed by Bellamy (1974) and Litchfield (1977, 1980). A system using im
mobilized E. coli to produce protein has also been described (Inloes et al.
1983).
The dry weight of bacterial cells is 47 to 86 percent protein. The nitro
gen digestibility and weight gain of rats were improved when cells of
Pseudomonas were homogenized before feeding (Yang, Yang, and Thayer
1977). At levels of 10 to 20 percent of the diet, methanolgrown bacterial
cells resulted in reduced growth rates and other adverse effects on young
chicks (D'Mello and Acamovic 1976).
Besides the problem of harvesting, the nucleic acid content of bacte
rial cells appears to be higher than that of yeast cells. Nucleic acid con
tent up to 20 percent was suggested (Shacklady 1972). This can be reo
duced with known processing systems (Yang, Thayer, and Yang 1979).
When fed to humans, bacterial cells have caused gastrointestinal prob-
lems and other adverse reactions (Litchfield 1980).
MOLDS. Molds are complex, multicellular, aerobic organisms. They
grow over a wide range of pH, temperature, and substrates. They contain
the vitamin B complex and from 13 to 60 percent crude protein. The
methionine content of mold protein is rather low, as is true for yeast
protein. The tryptophan and cystine contents are also low. For other
amino acids, mold protein compares favorably with milk and fish meal
proteins.
Molds have a slower growth rate with a lower nucleic acid content
and are easier to separate from the substrate than either yeasts or bacte-
ria_ The mold filaments can be physically aligned into a fibrous texture
resembling meat or poultry or even the flaky texture of fish_ A fungal
protein, mycoprotein, made from Fusarium graminearum, was to be market
tested in the United Kingdom in 1981 (Anon. 1981). Reportedly, it is
nutritious and safe to eat. However, there was a concern that it might be
deficient in iron and zinc (Litchfield 1983).
The cellulolytic fungus Trichoderma viride and other Trichoderma have
been grown on feedlot wastes, cereal straws, and other cellulosic wastes
(Griffin, Sloneker, and Inglett 1974; Han and Anderson 1975; Peitersen
1975a). Other molds tested as potential sources of SCP include an acid-
tolerant Scytalidium acidophilum grown in peat extract (Martin and White
1985), and a thermo tolerant Phanerochaete chrysosporium grown on vinasse
(Cardoso and Nicoli 1981a, 1981b).
MUSHROOMS. Higher fungi, or mushrooms, have been known as a
source of food since the beginning of human history. Their efficiency of
converting carbohydrates to protein is approximately 65 percent. As with
other fungal protein, mushrooms have a low content of methionine and
tryptophan. The nucleic acid content of mushrooms is lower than that
of microorganisms (Fabregas and Herrero 1985). The use of mushrooms
was reviewed by Chang (1980).
MIXED CULTURES. In nature, mixed cultures are present during the
degradation of cellulosic and other wastes. In these wild, mixed cultures,
the organisms consume each other, so that a large amount of protein
does not accumulate. However, in a controlled mixed culture, two organ-
isms may grow better and produce more protein.
Mixed cultures of Trichoderma viride and a yeast (Candida utilis or Sac-
charomyces cerevisiae) were used with alkali-treated barley straw (Peitersen
1975b). The overall rate of protein production was increased by use of
the mixed culture.
ALGAE. The algae are the simplest plants that contain chlorophyll.
Their use as a potential source for food should be readily apparent, since
they begin the food chain for sea life (Ryther and Goldman 1975).
The algae vary in size from microscopic forms to the giant seaweed
or kelp that may be 40 or more feet long. The familiar habitat of algae
is any body of sunlit water, ponds, streams, lakes, or oceans. There are
also terrestrial algae.
Since algae contain chlorophyll, they can utilize solar energy by pho-
tosynthesis, like the higher plants, to produce food from CO2, H20, and
inorganic salts. They have many of the advantages listed for the other
microorganisms. Generally, the algae have a slower growth rate than
other microorganisms. The algae require light for growth, so either artifi
cial light is needed, or growth is limited to areas with warm, sunny eli
mates. When grown outdoors in ponds or on sewage, as has been sug
gested, there is the problem of contamination with undesirable bacteria.
Spiruiina maxima is grown successfully in the naturally alkaline water of
Lake Texcoco in Mexico.
For centuries, algae have been harvested and consumed by people in
the Lake Chad area of Africa, with no apparent ill effects. The red algae
have been harvested for their agar and carrageenan. The brown algae,
Laminaria and Macrocystis, are sources of algin. Agar is used for solidifying
media, as well as in gels for dessert. Carrageenan and algin are added to
food products to improve the texture. A red algae, Porphyra, has been
used in Japan. It has been estimated that algae can produce from 30 to
80 tons of protein per hectare per year compared to conventional crops
producing 0.4 to 2.5 tons per hectare per year (Grobbelaar 1979).
The protein concentration of dried algae varies from 5 to 64 percent.
Cells of Chiorella contain about 60 percent protein (Endo, Nakajima, and
Chino 1974). The different levels are due to differences in algae and in
growth conditions. The PER of algal protein ranges from 1.25 to 2.6 and
the BV from 54 to 72. The analysis of the proteins of algae shows that
they are low in the sulfurcontaining amino acids (cystine and methio
nine).
There have been toxic effects noted due to certain algae. The toxic
effect of "red tide" on the killing of fish is well known. Some species of
bluegreen algae have caused the death of animals. Shilo (1967) reviewed
algal toxins, but no toxins were discussed for the micro algae Chiorella,
Scenedesmus, or Spiruiina. Humans can tolerate some algal protein, but if
the daily intake of algae exceeds 100 g per day, there may be gastrointesti
nal distress.
For good digestibility, the cells must be broken. For human accept
ance, the protein will need to be purified and processed into other foods.
Due to the many difficulties in growing, processing, and use in hu
man foods, other microorganisms show more promise as potential
sources of protein than do algae.
The Potential of Microbial Protein
Many people are optimistic about the future prospects of microbial
protein. With improved processing techniques, the potential will, no
doubt, also increase. The main use of SCP is in animal feed, but some
has been used in human food and this use should increase in the future.
The two main concerns are the safety and cost. As with any situation
or product, there is always a risk factor. Feeding trials with rats and other
animals indicate that the product is safe. However, for human consump
tion, it is necessary to reduce the nucleic acid content of the product.
Gastrointestinal distrubances have occurred in humans fed certain SCPo
Until problems such as these are solved, only low levels of SCP can be
used as human food.
The cost of producing SCP is such that it may not be able to compete
with other plant proteins, but it can compete with many animal proteins.
The nutritive value of SCP is generally lower than animal proteins, but
the SCP is improved by processing the cells, isolating the protein, and
supplementing it with the limiting amino acids.
If wastes, which cost money for disposal, can be used to produce SCP,
we might consider the savings in the disposal cost in the overall econom
ics of the SCPo Since SCP can be spun into fibers like plant proteins, it
could be used to make synthetic meats or added as extenders of meat
products. By means of genetic engineering and biotechnology, better
yields at lower costs may be achieved, resulting in advantages for the
production and use of SCPO
With our present and future needs for protein, the use of micro organ
isms as a source of feed or food cannot be overlooked.
ASSAY
Microorganisms can be used to determine or assay the amount of
vitamins and amino acids in food products.
The use of living cells for analytical purposes gives a high degree of
sensitivity as well as biological specificity, because of the particular reo
sponses of the metabolic processes of the cell. The reagents are the test
organism and the medium used for growth. The reaction is the metabolic
response, or lack of response, by the cell. By using serial dilutions of
the sample in the growth medium, inoculating with the test organism,
incubating, and determining the response of the cell, we can determine
the amount of substance being assayed. Automated systems for vitamin
assay were described by Einarsson and Snygg (1986) and Guilarte (1983)
and were reviewed by Gregory (1983). Enzymatic assays of amino acids
have been developed (Roy 1979; Tonogai et al. 1983; Tuffnell and Payne
1985).
Some of the microorganisms and vitamins assayed are listed in Table
9.10. The test microorganisms and corresponding amino acids are listed
in Table 9.11. Special strains, or mutants of the microorganisms, are used
for these assays.
TABLE 9.10. ASSAY OF VITAMINS
Vitamin
Biotin
Choline
B" (pyridoxine)
Nicotinic acid
Thiamin
Pantothenic acid
Folic acid
Riboflavin
Inositol
Microorganism
Lactobacillus plantarum
Sacchammyces cerevisiae
Neurospora crassa (mutant)
Saccharomwes uvarum
Neurospor"a sitophila (mutant)
Lactobacillus leichmannii
E-scherichia coli (mutant)
Euglena gracilis
Ochromonas malhamensis
Lactobacillus viridescens
Saccharomyces uvarum
Pediococcus acidilactici
Lactobacillus casei
Lactobacillus casei
Lactobacillus helveticus
Neumspora crassa (mutant)
Sacchammyces uvarum
Besides vitamins and amino acids, microorganisms can be used to
assay materials for antibiotics, some types of pesticides, and bacteriolytic
enzymes such as lysozyme (Bitton, Koopman, and Wang 1984; Kingdon
1985; Mueller, Reed, and Barkate 1979; Rajkowski, Peeler, and Messer
1986).
TABLE 9.11. ASSAY OF AMINO ACIDS
Amino Acid
Arginine
Aspartic acid
Cystine
Glutamic acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Microorganism
Leuconostoc mesentemides
Microorganisms or their enzymes have been adapted to analyze foods
for various chemicals in general (Schwimmer 1981) and specifically, such
as acetaldehyde in beer (De1cour et al. 1982), cyanide in cassava (Rao and
Hahn 1984), cholesterol in egg yolk (Shen, Chen, and Sheppard 1982),
starch in meat products (Skrede 1983), and ethanol in beer (Waites and
Bamforth 1984). In many cases the cells or enzymes are part of a bio
chemical electrode system (Hopkins 1985; Schwimmer 1981). These have
been used to determine glucose (Ikeda et al. 1985; Wingard et al. 1984),
monoamines in meat (Karube et al. 1980), ethanol in wine (Mason 1983),
aspartame (Renneberg, Riedel, and Scheller 1985), and hypoxanthine
and inosine in fish (Watanabe et al. 1984). Enzymes are also used in the
enzyme linked immunosorbent assay (ELISA) as described in Chapter 6.
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10
Control of Microorganisms
The control of microorganisms is one of the major concerns of food mi-
crobiologists_ This control is needed to retard or prevent spoilage and to
reduce or eliminate health hazards associated with foods_ The control of
contaminants also aids in obtaining better results when specific microor-
ganisms or enzymes are used in food processing_
Four basic systems are used to aid in the control of microorganisms
in foods_ These are (1) prevent contamination (asepsis); (2) remove con-
taminants; (3) inhibit growth; and (4) destroy contaminants_
In most food products, two or more of these systems are used to con-
trol the microbial leveL Preventing contamination is practiced for all
foods, but since contamination will still occur, other safeguards are
needed_
Besides controlling the microorganisms, foods must be protected
from reactions that are catalyzed by inherent enzymes, from chemical
degradation such as fat oxidation, loss of nutrients, and from the destruc-
tion by pests, such as insects and rodents_ There are various chemicals
and procedures that will control microorganisms and pests but cannot
be used for foods_ This is because the food must be safe for consumption,
be of acceptable organoleptic quality, and have good nutritional value_
Although the control of microorganisms in food is usually relegated
to the food processor, everyone involved in the production, processing,
handling, warehousing, retailing, preparation, and serving should be in-
volved in the control process as well as in maintaining a safe and nutri-
tious food supply_
The need for this overall effort is evident from the fact that only a
few outbreaks offoodborne illness are caused by problems at the process-
ing leveL Most of the outbreaks are caused by mishandling and contami-
nation of foods at foodservice establishments or in the home_
In sume cases, we assume that a food will be handled and used in a
particular manner; but this assumption may not always be correct. For
example, we assume that people will cook meat before eating it. How-
ever, a series of outbreaks of salmonellosis revealed that some people
thought that eating raw hamburger would make them healthy, strong,
and vigorous_ Because of such misuse of foods by some people, it is neces-
sary to take precautions beyond the normal processing requirements_ A
505
food microbiologist must be aware of all aspects of a food, from produc
tion to consumption.
A primary function of food processing is the preservation of foods.
This preservation ranges from short periods of a few days or weeks to
long terms of a year or more. Preservation by the removal, inhibition, or
destruction of microorganisms is easier and the results are more satisfac
tory when the original microbial index of the food is low. Keeping the
contamination low by sanitary means is very important-
CONTROL BY ASEPSIS
If all of the microorganisms in a food were either useful or inert, we
would have no concern about them. Unfortunately, the spoilage and
health hazard types are quite prevalent- Generally, the higher the micro
bialload, the greater the possibility that undesirable types are included.
The exceptions are foods in which specific microorganisms are grown to
produce desirable products and a high level of the useful microorganism
is needed.
It is much easier to inhibit or destroy low numbers of microorganisms
than high numbers. A food with a low microbial load will generally have
a longer shelf life than a food with a high microbial leveL The shelf life
is the time after packaging during which a food maintains its best quality
if it is stored under proper conditions of humidity and temperature.
Because microorganisms are ubiquitous, it is impossible to keep them
from contaminating food. However, we can reduce contamination by
controlling the potential sources of microorganisms.
The control of contamination is referred to as sanitation. Sanitation
may be defined as a modification of the environment in such a way that
maximum health, comfort, safety, and wellbeing are ensured for people.
The sanitation involved in the food industry is only a small part of overall
sanitation.
The control of the microbial quality of food must begin with the pro
duction and harvesting of food. Then it must carryover to the processor
and on to the ultimate consumer-
Production and Harvesting
The raw materials used by the food processor affect the quality of the
finished product- To have adequate supplies of high quality raw materials
with acceptable microbial loads, the food processor must work closely
with the producer or become the producer. In some cases, a food proces
sor who is also the producer seems to be the best method. As a result, we
CONTROL OF MICROORGANISMS 507
now have companies that own or control food-producing and -processing
systems_ By owning a system, the company can integrate all of the pro-
cesses to end up with a uniform, high-quality product_
With many foods, the processor must rely on the producer to deliver
an acceptable raw material. Some ingredients are obtained from foreign
countries where the level of sanitation is different from that in the
United States_ Hence, processors sometimes have to work with raw mate-
rials that are produced in areas beyond their control. Sometimes a visit to
the foreign supply areas will help in obtaining more acceptable products.
When the raw materials are obtained from local areas, the processor
can usually visit the sources of supply, make evaluations, and suggest pro-
cedures that will improve the quality. Sometimes the processor obtains
help from local, state, and federal regulatory agencies that inspect the
producing areas.
MILK. Many years ago, the occurrence of epidemics of various diseases
caused by the consumption of tainted milk caused health authorities to
decide that milk production should be regulated. To help control the
spread of tuberculosis, cows were tested and reactors eliminated. From
this beginning, many regulations and codes of operations have been de-
vised to control the production and handling of milk at the farm. No
other commodity is produced with as much regulation as grade A milk.
As a result, tuberculosis and brucellosis in milk are controlled. At pres-
ent, the main infectious problem at the farm is mastitis.
Besides the health of the dairy cow, standards have been developed
for the housing and milking areas so that they can be maintained in a
satisfactory manner. There are requirements for the equipment used to
obtain, handle, and store the milk, and for the equipment to be properly
cleaned, sanitized, and maintained (Guterbock, Blackmer, and Duffy
1984; Stone, Myhr, and Davie 1983). Grade A milk must be cooled to
lOoe or less within two hours after milking to maintain the quality. The
requirements are more stringent for the production of grade A milk than
for the manufacturing of grade milk. This latter milk is used in various
dairy products other than liquid milk.
Insect and rodent control are needed at the farm level as well as at
the processing level. Only approved insecticides can be used in dairy
barns or around other food products. Removing potential breeding
places helps control insects and rodents.
ANIMAL PRODUCTION. Healthy, disease-free animals should be
maintained in a relatively clean, disease-free environment. Modern hus-
bandry practices have tended to increase rather than decrease infections
in domestic animals. The practice of crowding animals into feedlots,
broiler houses, laying houses, and holding pens increases the potential
for spreading diseases, including salmonellosis. Sanitation of all animal
quarters on the farm may help, but will not prevent, infection.
With meattype chickens (broilers), flocks that are Salmonellafree tend
to result in carcasses that are Salmonellafree. When salmonellae are iso
lated from birds prior to slaughter, the carcasses tend to contain these
organisms.
The transportation of animals to market or from one farm to another
causes stress, and animals held in dirty pens until slaughter shed salmo
nellae at a higher rate than on the farm. This indicates that stress induces
shedding of organisms already present in the intestinal tract, or it makes
the animals very susceptible to Salmonella infection, or both. One solution
may be to hold animals at the farm and then move them into clean, un
crowded conveyances to the slaughterhouse for immediate slaughter.
One source of contamination of carcasses during slaughter is of fecal
origin. Withdrawal of feed and water 8 to 10 hr before slaughter reduces
the fecal content of chickens and might reduce the potential contamina
tion of the carcasses.
SHELL EGGS. Regardless of whether laying hens are housed on the
floor or in cages, it is desirable to keep the egg shells clean. The incidence
of spoilage is much greater in dirty eggs than in clean eggs, whether the
shell is washed or not. When eggs are washed, the temperature of the
wash water should be 20C higher than the temperature of the egg and
an approved germicide should be added to the cleaning solution. The
addition of a germicide tends to reduce spoilage of the washed eggs dur-
ing storage. Only odorless, nontoxic cleaner-sanitizers should be used for
washing and sanitizing egg shells.
FRUITS AND VEGETABLES. The production of quality plant prod-
ucts starts with quality plant varieties and seeds. The disease resistance
and the amount and quality of food produced are important consider-
ations for the selection of plant varieties.
The World Health Organization (WHO 1969) recommended several
hygienic practices for fruits and vegetables. The environmental sanita-
tion during growing included sanitary disposal of human and animal
wastes, sanitary quality of irrigation water, and control of animals, pests,
and diseases. The sanitary harvesting aspects include the equipment and
containers, which should be cleaned and maintained and not be a source
of microorganisms. Unfit produce should be segregated and disposed of
in a satisfactory manner. The product should be protected from contami-
nation by animals, insects, birds, chemicals, or microorganisms.
The transportation of produce should be done in conveyances that
can be cleaned and not be a source of contamination. All handling
should be done with care and, if needed, refrigeration or ice of sanitary
quality should be used.
The contamination of plant products by soils is important to the food
processor, since soils contain bacterial spores, including those of Clostrid
ium botulinum. Bacterial spores are resistant to various sanitizing agents,
as well as foodpreservation treatments. Hence, it would be a definite
advantage to the processor if spores could be kept out of the raw mate
rials.
SEAFOODS. Seafoods are contaminated by the environment in which
they grow, as well as during harvesting and transportation to the process
ing plant.
The main problem of contamination during growth involves the bi
valve mollusks, such as oysters, clams, and mussels. They grow in estuar
ies or protected coastal waters. These are also the most convenient places
for the disposal of sewage and other wastes of coastal communities. The
mollusks obtain nourishment by pumping water through a complex sys
tern of gills that filter and concentrate food, such as bacteria and marine
organisms. With the dumping of sewage, the bacteria may include poten
tial pathogens. With oysters and clams, the entire animal, including the
intestinal tract and its contents, is consumed, frequently raw or after suo
perficial heating. If the bivalves have consumed enteric pathogens, they
can serve as vehicles for organisms that cause foodborne illness.
Due to a series of illnesses culminating in a major typhoid fever epi
demic in 1924 from the consumption of contaminated oysters, a volun
tary cooperative program for the certification of interstate shellfish ship
pers was established in 1925 by the U.S. Public Health Service. The
program is now known as the National Shellfish Sanitation Program
(NSSP). The coastal states adopt laws, make sanitary and microbial sur
veys of shellfishgrowing areas, delineate and patrol restricted areas, pre
vent illegal harvesting, inspect shellfishprocessing plants, and conduct
other activities needed to ensure that shellfish are grown, harvested, and
processed in a sanitary manner.
The FDA administers the NSSP at the federal level and is responsible
for evaluation of state and foreign programs, standards development,
research, and training.
The survey of the growing areas has three parts: (1) the area is suffi
ciently removed from major sources of contamination so that shellfish
are not subject to fecal contamination; (2) the area is free from pollution
by potentially harmful industrial wastes; and (3) the median coliform
level does not exceed 701100 ml of water and not more than lO percent
of the samples exceed 2301100 ml for a fivetube MPN, or 3331100 ml for
a three-tube MPN_ In 1974, the FDA proposed that the coliform standard
be changed to fecal coliforms as follows: The median fecal coliform MPN
value shall not exceed 141100 ml of sample, and not more than 10 percent
of the samples shall exceed 43 for a five-tube, or 49 for a three-tube, MPN
test (Hunt 1977).
Even with safe coliform levels, occasional outbreaks of foodborne ill-
ness have occurred after the consumption of contaminated shellfish re-
portedly harvested from approved areas. Vibrio cholerae was detected in
estuarine-type waters even when coli forms were not detected (Guthrie
and Scovill 1984; Hood and Ness 1982), and there was no strong linear
correlation between V. cholerae and E. coli or fecal coliforms (Hood et al.
1983). Also, there does not seem to be a relationship between safe water
and the presence of enteric viruses (Gerba et al. 1980; Vaughn and
Landry 1984). Although there are problems, Larkin and Hunt (1982)
stated that the shellfish control programs are working reasonably well
and that there is no guarantee that raw shellfish will be free of disease-
producing organisms or toxic materials. However, Madden, Buller, and
McDowell (1986) suggested using Clostridium perfringens as the indicator
organism for contaminated shellfish.
In one study, researchers found that fish, as caught, have low bacterial
loads (Huss et al. 1974). The hygienic standards on board the ship deter-
mined the level of contamination of the fish. To reduce the growth rate
of the bacteria, proper refrigeration, icing, or freezing must be used. The
fish should be processed as soon as possible after catching. Some ships
have processing systems on board. In these cases, seawater is used to wash
and process the fish. If not treated with chlorine, this water may serve as
a source of microorganisms. The sanitary conditions of the working areas
and equipment, and the health and hygiene of workers on the ship
should be the same as in processing plants on land.
Processing
If care is taken to produce and deliver a high-quality product to the
processing plant, it is the duty of the processor to maintain the quality
at a high level.
The U.S. Food, Drug and Cosmetic Act of 1968 states: "A food shall
be deemed to be adulterated if it contains any poisonous or deleterious
substance which may render it injurious to health; or if it consists in
whole or in part of any filthy, putrid, or decomposed substance; or it has
been prepared, packed, or held under insanitary conditions whereby it
may have become contaminated with filth or rendered injurious to
health." With these stipulations, the presence of any microorganisms or
chemical that may cause illness or that would indicate that the food may
contain harmful agents makes the food adulterated and subject to sei-
zure. The presence of filth, such as insects or rodent hairs or droppings,
is evidence of an adulterated product. However, there are tolerances for
these agents (see Chapter 13). Even if the food does not actually contain
filth or potentially harmful agents, if conditions exist so that these sub-
stances may be present, the food can be considered adulterated. Al-
though this act applies only to foods in interstate commerce, many states
have copied the federal regulations.
The buildings, environment, and processing facilities, as well as raw
materials, personnel, equipment (including cleaning and sanitizing), and
refuse disposal facilities, are important to the overall plant operations.
The level of sanitation maintained in the plant has a direct bearing
on the quality of the final product and should be a basic part of the
quality-control program of the processor.
Quite often, a sanitation program is considered to be a cost that is
passed on to the consumer, making the price of the food product less
competitive. More realistically, unsanitary conditions result in increased
costs, which increase the price of the food to the consumer. Additional
costs, such as those caused by loss of food due to a short shelf life and
spoilage of perishable products, medical bills of consumers ingesting
pathogenic organisms or poisons, legal fees, and settlement of court
causes due to illness or death of former consumers, are passed on to
present consumers. Regulatory agents may seize a product that violates
the regulations. As a result, the least that can happen to a food processor
is to have inventory tied up. The seizure may result in destruction of the
product, corporation or personal fines, prison terms, and the closing of
the processing plant. Hence, although sanitation may be a cost, the lack
of sanitation can create a greater cost. Sanitation is a necessity in order
for a food processor to stay in business and has resulted in advantages
to both the processor and the consumer.
Proper sanitation reduces or eliminates potential spoilage hazards
and creates a longer shelf life of perishable products. This reduces losses
due to spoilage, increases the efficiency of plant opeations, results in
easier maintenance of equipment, develops better employee relation-
ships, workmanship and safety, increases consumer acceptance and pub-
lic relations, improves the quality and safety of foods for the consumer,
and with better consumer acceptability, there is a potential increase in
sales. This results in faster turnover and less spoilage of perishable foods.
As a result of these benefits, the actual cost of the product may decrease.
The "may have" section of the U.S. Food, Drug, and Cosmetic Act of
1968 (Section 402 (a) (4 describes adulterated food. To help interpret
this description and to establish criteria to aid industry in complying with
this section of the act, the FDA has published regulations called current
good manufacturing practice (CGMP) for various foods. These regula
tions cover essentially all operations of a foodprocessing plant, includ
ing its outside appearance, equipment, processes, and personnel. The
USDA regulates the processing of meat, poultry, eggs, and egg products.
Each food processing operation has certain points at which failure to
prevent contamination can be detected by laboratory tests with maxi
mum assurance and efficiency. These are known as critical control
points. Bauman (1974) defined hazard analysis as the identification of
sensitive ingredients, critical process points, and relevant human factors,
as they affect product safety. Together these form the concept called haz
ard analysis critical control points (HACCP).
Foods have been placed into five categories in terms of health haz
ards, which are based on three hazard characteristics (NAS 1969). These
are described in Chapter 6 in regard to controlling Salmonella in foods.
These hazard characteristics are used as the basis for the HACCP inspec
tions.
According to Kauffman (1974), the HACCP inspection approach is
used to determine the points in the process that are critical to the safety
of the product, and these can then be used by the processor to identify
critical points. HACCP inspections, guided by the CGMPs, will tell the
FDA and industry what needs to be done to assure safe, highquality food
production and distribution.
The critical control points are determined for each type of food pro
cess and for each food processing plant. The critical control points have
been discussed for frozen foods (Peterson and Gunnerson 1974), canned
foods (Ito 1974; Warne, Capaul, and Moffitt 1985), and a foodservice
system (Cichy, Nicholas, and Zabik 1982).
With this concept of inspection, not only is visible evidence of filth
(insects, rodents) or unclean equipment and facilities important, but also
the microbial analysis for potential hazard or spoilage types is used at
critical control points to determine the problems involved in the process.
This concept increases the importance of the microbiologist.
Although the CGMP and HACCP concepts provide a basis for satisfac
tory operation for food processors, they do not state how to meet the
requirements in all cases, or why some of the CGMPs are needed. There
fore, it seems worthwhile to discuss some of the these factors.
PLANT AND GROUNDS. The plant should be designed so that mate
rials can be received, stored, processed, warehoused, and shipped in an
efficient and satisfactory manner.
Some operations, such as washing dirt off vegetables or slaughtering
animals, are not considered to be clean activities and must be separated
from areas where processed food is exposed. The segregation of facilities
should be such that no crosscontamination can occur between raw prod-
ucts and processed products.
AIR. Fresh air is needed in food-processing areas. Since air is a poten-
tial source of microorganisms, a system or systems are needed for micro-
bial control. The sources of microorganisms and the means of dispersal
throughout the plant must be considered in designing a method for con-
trol.
The ventilating system is the main place to control microorganisms
in the air. Filters have been developed to remove microorganisms, as well
as other materials from air. Viruses were effectively removed from air by
filters with an efficiency rating of 93 percent or higher (Roelants, Boon,
and Lhoest 1968). There are filters available with ratings of 99.9 percent
and an ultra filter of 99.999 percent. The filter should be rated by the
dioctyl-phthalate filter test using 0.3 iJ-m particles to challenge the filter.
However, in some cases, organisms such as Bacillus spores (Robertson and
Frieben 1984) or Pseudomonas cells (Leahy and Gabler 1984) have been
used to test filter systems.
By pumping in more filtered air than is removed by the outlets, a
positive air pressure is established in the room. With positive air pres-
sure, when a door is opened for personnel or product to move in or out
of the room, unfiltered air will not come in, since air is moving only out
of the room. The clean, fresh air should enter the cleanest areas of the
processing plant, move to the less clean areas, and leave the plant from
the areas that are "dirty." This procedure reduces the potential airborne
contamination of processed product from these less clean areas.
To eliminate turbulence factors, laminar airflow (unidirectional) has
been developed. Laminar airflow devices are effective in reducing and
controlling airborne contamination. Due to the relatively higher cost,
they have not been readily adopted for use in the food industry. They
should find usage in cabinets or special rooms where contamination of
products must be tightly controlled, such as in aseptic packaging.
Besides mechanical filters, the air may be treated by chemical germi-
cides, UV radiation, electronic air cleaners, thermal sterilization, centri-
fuging, or "scrubbing." When meticulous air cleaning is essential, electro-
static precipitation might be employed. The larger the particles, the
easier it is to remove them. Thus, molds and yeasts can be removed by
electrostatic precipitation more easily than bacteria or viruses. Due to
the high cost of installation, this system has been used seldom, if at all,
in the food-processing industry. Electrostatic precipitation is used to re-
move dust and other pollutants from effluent air.
WATER. The water may be from a municipal supply, in which case it
should be potable, that is, free from potential pathogens. However, mu
nicipal water is not tested for potential spoilage organisms, and reser
voirs and pipelines at the plant can serve as a place for multiplication.
Some pseudomonads grow in distilled water. Hence, it may be necessary
to treat the water by chlorination at the processing plant.
The chemicals in the water influence the type of cleaning agents that
are used in the processing plant. When water is an ingredient ofthe food,
chemicals causing odors and flavors may affect the characteristics of the
food product. In these cases, the water should be monitored and given
the necessary treatments that are indicated by the analysis and by the
food that is being processed. The treatment of water ranges from filtra
tion, flocculation, softening, demineralization, reverse osmosis, distilla
tion, to various combined processes to obtain pure water. A water soft
ener is not a means of removing organisms and, in some cases, the resins
in softeners may act as a source of inoculum of the soft water.
EQUIPMENT AND UTENSILS. The equipment should be designed
so that food does not become lodged on or in it to form pockets of micro
bial growth for continual contamination of foods moving over or
through it. The sanitary design of equipment results in easier cleaning
and sanitizing, thereby saving labor and materials.
The equipment surfaces in contact with food must be nontoxic, non
absorbent, nonporous, and noncorrosive. Although equipment is made
of several types of material, stainless steel is preferred for surfaces that
are in contact with food.
Stainless steel is not just one metal, but is a group of alloys of iron
with chromium, nickel, carbon, molybdenum, titanium, silicon, phospho
rus, manganese, and sulfur.
Although stainless steel is corrosion resistant, it is not corrosion
proof. Stainless steel is protected from corrosion by its self-repairing sur
face film of chromium oxide. When this film is broken down by cleaning,
it will reform when exposed to the air. If abrasive materials are used in
cleaning, the metal surface will be scratched, which allows corrosion to
occur. If harsh chemicals are used in cleaning, they can cause pitting of
the metal. Scratches and pitting make it difficult to clean, sanitize, and
maintain the equipment in a satisfactory manner. Scanning electron mi
croscopy revealed that there were flaws such as scratches, cracks, and pits
in samples of stainless steel prior to any stress or wear (Stone and Zottola
1985a).
Certain materials should not be used in equipment that contacts food.
Due to its porous nature and difficulty in maintenance, wood should not
be used. Copper and its alloys accelerate rancidity of fats, discolor food
products, and contaminate foods with copper salts. Cadmium is toxic.
Lead should not be used, except in small amounts when soldering is
needed. Aluminum is attacked by acids and alkalis and it has a tendency
to rub off and cause black marks. Iron can cause off.flavor in products,
such as milk, and it is impossible to clean because of rusting and pitting.
Zinc and antimony are not acceptable metals for surfaces in contact with
food.
As an alternative to stainless steel, glass (usually borosilicate glass
pipelines) or glass fiber-reinforced polyester resin are used in some
foodprocessing plants. A unique substitute for metal is the use of liquid
jets for cutting. This system is based on the principle of forcing a liquid
(water, glycerine, vegetable oil, alcohol) through a tiny nozzle at ex
tremely high pressure. These conditions accelerate the liquid to about
1,000 m/sec. This results in a more sanitary cutting method than using
knives, and it is easier to maintain in a sanitary manner.
There are some areas or conditions in which flexibility is needed, and
plastics or rubber products are used. Plastics may not be acceptable if
they contain free phenol, formaldehyde, or any other substance that can
transfer to or alter the food. Since additives used in the manufacture of
plastics can be transferred to foods, the suitability of each type of plastic
must be determined for each particular food.
Ease of assembly and disassembly is important if the equipment is to
be cleaned by hand methods. When possible, equipment should be de
signed for clean in place (CIP) or automatic methods. These systems may
cost more at the beginning but usually pay for themselves by requiring
less labor and more effective cleaning and sanitizing.
The design and installation of equipment for proper drainage are
important. This is needed in CIP systems, so that soil removed by clean
ing solutions, as well as the cleaning solutions, sanitizing solutions, and
rinse waters can be removed from the equipment.
Many years ago, 3A Sanitary Standards for dairy equipment were de
veloped (Atherton 1986). Since then, equipment standards have been
written for other food products. These standards can be found in publi
cations such as the Journal of Food Protection and Dairy and Food Sanitation.
Processing equipment does not have a normal microbial flora. It be
comes contaminated with food residues that can support high levels of
microorganisms. As the buildup of the microbial load progresses, it be
comes a source for contaminating food that contacts it. Hence, it is neces
sary that the equipment be cleaned and sanitized at appropriate intervals
to keep the microbial load at a reasonable level.
Cleaning. Equipment that is less than 100 percent clean is still dirty. It is
difficult, if not impossible, to sanitize equipment that is not completely
clean. Therefore, cleaning is a very important part of the operation.
Before cleaning, the food must be removed from the line and pro
tected from the possible contamination by the cleaning and sanitizing
agents, as well as the water and soil removed from the equipment.
In some processing plants, all of the equipment is cleaned and sani
tized manually. In other plants, some or all of the cleaning and sanitizing
may be semiautomated or fully automated. In any of these systems, the
human factor is the most important ingredient, since even with a fully
mechanized system, it is necessary to set up the operation and supply the
system with the cleaning and sanitizing agents.
The first part of any cleaning operation is to remove waste material
and to rinse loose soil from the surfaces with clean water. For fatty mate
rials, hot water may aid in this initial rinse, but cool water is desirable
for protein soils (Anderson et aL 1985; Dunsmore et aL 1981; Koopal
1985; Middlemiss et aL 1985).
Even if most of the soil can be removed by hand and by water rinsing,
the equipment is still not clean, since a residue remains that is difficult
to remove. An input of energy is needed to remove this residual soiL The
energy forms are thermal (hot water, steam), chemical (cleaning agents),
and mechanical (highpressure spray, manual scrubbing). Not only is soil
attached to the equipment surfaces by physical forces, but microorgan
isms are also attached and are difficult to remove (Powell and Slater
1982; Stanley 1983; Stone and Zottola 1985b).
The cleaning process consists of bringing the cleaning solution into
intimate contact with the soil on the equipment surface, removing the
soil from the equipment, dispersing the soil into the cleaning solution,
and preventing redisposition of the soil, as well as scale due to hard
water, onto the cleaned surface. Various cleaning agents are available
that perform these activities. Cleaning agents are designed to reduce the
amount of work needed to remove the adhering soiL
Cleaning Agents. A cleaner may consist of a single compound or be a com
plex mixture of various chemicals.
Only approved cleaners and sanitizers should be used in foodpro
cessing plants. Various government agencies compile lists of compounds
considered to be acceptable and safe for washing foodprocessing equip
ment, as well as the floors and walls of plants.
A cleaning agent should have the following characteristics:
L Quickly and completely soluble
2. Noncorrosive to metal surfaces
3. Nontoxic
4. Easily rinsed
5. Economical
6. Stable during storage
7. Not harsh on the hands if used for manual cleaning
8. Possess germidical action, if required.
The types of cleaners and their properties are listed in Table 10.I.
Substances that lower the surface tension of a liquid, or the interfacial
tension between two liquids, are known as surfaceactive agents, or sur
factants. Chelating agents are chemicals that can form a soluble ringlike
complex with a metallic cation. In this form, the metallic ions do not form
insoluble films on the equipment, but are removed from the equipment
by rinsing. The chief organic chelating agents, or sequestrants, are so
dium salts of EDT A and related compounds, the hydroxy carboxylic com
pounds (citric, gluconic, tartaric, and hydroxyacetic acids) and the amino
hydroxy compounds (such as tetraethanolamine). The important
inorganic sequestrants are the sodium polyphosphates (sodium pyro
phosphate, sodium tripolyphosphate, sodium hexametaphosphate). They
have a softening effect on hard water. The chelating agents can increase
the effectiveness of sanitizing agents.
Conventional cleaners are used and rinsed with hot water. For low
temperature cleaning of poultry and meat processing areas, proteolytic
enzymes are added to surfactants. The surfactants possess a penetrating
and emulsifying action on fats, while the enzymes soften and break up
protein soils.
SANITIZING. If the cleaning procedure removed all of the organic mao
terial, the equipment would not need to be sanitized. The sterilization
of all equipment would be ideal, but it would be neither practical nor
economical. Thus, we are satisified if the equipment is sanitized.
In some cases steam, hot water, or hot air is used for sanitizing. In
most processing plants, these agents are not practical, so chemical sanitiz
ers are used. Sanitizing does not mean that all living bacterial cells are
destroyed or removed. A sanitized surface does mean, however, that all
pathogenic or disease producing bacteria, as well as a large percentage
of nonpathogenic ones, are destroyed.
Some sanitizing agents are added to detergents (detergentsanitizers)
so that the bacteria are killed during cleaning. This single operation of
cleaning and sanitizing saves time and labor. When chlorinated cleaners
are used, the chlorine not only kills bacteria, but also aids in the removal
of soil and reduces redeposition.
The effectiveness of a sanitizer depends upon the concentration and
the time of exposure. The pH modifies the sanitizing action by affecting
both the bacteria and the chemical. An increase in temperature usually
increases the rate of sanitizing. The effectiveness of the sanitizer depends
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upon the types and numbers of microorganisms, as well as on their his-
tory_ The use of a high concentration of a sanitizing agent as a substitute
for thorough cleaning is inefficient and expensive, and, in some cases,
may cause corrosion of the metal. Chemicals that leave bacteriostatic or
bactericidal residues on surfaces may help with sanitation programs in
food-processing plants_
Thousands of chemicals have germicidal powers, but only those com-
pounds that are approved by a regulatory agent can be used as sanitizers_
The four most popular types of sanitizers are the chlorine compounds,
iodine or iodophors, quaternary ammonium compounds, and acid-an-
ionic surfactants_
Chlorine_ The most widely used sanitizers are the chlorine-containing
compounds_ Six factors determine the effectiveness of chlorine as a sani-
tizing agent. They are (1) type and concentration of chlorine compound
used; (2) pH; (3) temperature; (4) contact period; (5) types of organisms;
and (6) presence of organic material.
The types of chlorine compounds include the sodium and calcium
hypochlorites, the salts of isocyanuric acid, the hydantoin derivatives and
chlorinated trisodium phosphates, as well as gaseous chlorine_ In pow-
ered chlorinated cleaners the source of chlorine is usually chlorinated
trisodium phosphate, potassium dichlorocyanurate, dichlorodimethyl-
hydantoin, or chloramine T. The first two give the most active chlorine
in the cleaning solution_ Besides these compounds, mixtures of chlorine
and bromine, as well as chlorine dioxide, have been used as sanitizing
agents_ The most widely used agents in the food-processing area are the
hypochlorites_
The advantages and disadvantages of the use of hypochlorites as sani-
tizing agents are listed in Table 10_2_ The hypochlorites are effective at
relatively high dilutions and are active against a wide range of bacteria
and bacterial spores, as well as molds, yeasts, bacteriophages, and some
viruses_ The hypochlorites are considered to be more effective against
Gram-negative than against Gram-positive bacteria_ Viruses are more re-
sistant than bacteria to the action of chlorine_
In moderately strong concentrations, the hypochlorites can cause irri-
tation of the skin and may cause an objectionable odor. In some cases,
unacceptable flavors may develop in foods exposed to hypochlorites_
There is a reduction of efficiency of hypochlorites by organic com-
pounds and oxidants_ Due to their high corrosion potential, they should
not be used at high temperatures or be allowed to contact the surfaces
of equipment for excessively long periods_
Chlorine compounds are used in various aspects of food processing_
They are added to water used for washing and conveying raw food prod-
ucts, for cleaning and sanitizing food-handling equipment, and for cool-
TABLE 10.2. ADVANTAGES AND DISADVANTAGES OF HYPOCHLORITES
AS SANITIZERS
Advantages
Relatively inexpensive
Quick acting
Not affected by hard water salts
Harmless residue, does not form a film
Effective at high dilution
Active against a wide variety of microor
ganisms, including spores and phage
Relatively nontoxic at use dilutions
Nonstaining
Colorless
Easy to prepare and apply
Concentration easily determined
Can be used for water treatment
D isadvan tages
Unstable during storage
Inactivated by organic compounds
Corrosive if misused
Irritating to skin
Odor may be undesirable
Precipitate in iron waters
Effectiveness decreases with increasing pH
of solution
May remove carbon from rubber parts of
equipment
ing heatsterilized cans of food. Citrus packers in Florida dip or rinse
fresh fruit in 200 ppm for 2 min prior to shipment to prevent the spread
of citrus canker, which includes the presence of darkbrown and yellow
lesions on the fruit.
Inplant chlorination is accomplished by injecting gaseous chlorine
or liquid sodium hypochlorite into the water supply of a processing plant
by an automatic proportioner. Sufficient chlorine is added to satisfy the
demand of substances present in the water (the breakpoint), as shown in
Figure 10.1, and to give a free available residual chlorine of from 2 to 20
mg per liter. The free available chlorine is in the form of hypochlorous
acid (HOCl) and hypochlorous ions (OCl-). The combined chlorine reo
sidual is that chlorine reacted with amino nitrogen (chloramines). The
total available chlorine includes the combined and free chlorine.
Inplant chlorination provides a continuous bactericidal action of
chlorine on food preparation equipment during its operation. The use
of chlorinated water sprays at selected points reduces or prevents accu
mulation of microbial slimes and off.odors in food processing plants.
The total microbial load of the finished product tends to be lower in
food plants with inplant chlorination. Chlorination cannot substitute for
good sanitary practices and good plant operations.
Sodium hypochlorite reacts with water to form hypochlorous acid
and sodium hydroxide.
NaOCI + H20 <=' HOCI + NaOH
Thus, the solution is alkaline, and the hypochlorous acid ionizes to
form hydrogen ions and hypochlorous ions:
t
...J

:;)
o
(j)
w
a::
w
z
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J:
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chlorine demand ...... ,'
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____ J
CHLORINE
,
,
,
,
,
,
,
Figure 10.1. Chlorine demand characteristics of water.
Courtesy of Heid and Joslyn (1967).
HOCl +Z H+ + OCl-
,
"
,
The hypochlorous acid can dissociate to form hydrochloric acid and
nascent oxygen:
The HOCl, OCl- and 0 have germicidal properties. In an acid solu
tion, the hypochlorous acid tends to remain as HOCl, which is mote bac
tericidal than the hypochlorous ion. Hence, the bactericidal efficiency of
chlorine solutions is higher in acid than in alkaline solutions (Fig. 10.2)
and is increased with temperature increases.
Apparently there is more than one mechanism by which chlorine
compounds can affect living cells. Chlorine reacts with cell membrane
proteins, forming Nchloro compounds. This impairs the transportation
of nutrients (carbohydrates, amino acids) into the cells. Also, damage to
the cell membrane can result in the leakage of cellular components from
the cell. The HOCI can penetrate the cell and oxidize sulfhydryl groups
on key enzymes, interfering with cellular metabolism and resulting in
cellular death. It may react with DNA of living cells, and oxidize purine
and pyrimidine moieties (Wlodkowski and Rosenkrantz 1975).
The effect of chlorine on spores of Bacillus and Clostridium was investi
gated by Wyatt and Waites (1975). They found that chlorine disrupts the
spore coat by combining with and removing protein. This inactivated the
150
I
(f)
W
I-
::J
Z
100
:2:
uS
:2:
i=
(!)
50
z
::i
-.J

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/
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V
7 8 9 10
pH
Figure 10.2. Effect of pH on germicidal efficiency of hypo-
chlorite solution.
courtesy of Heid and Joslyn (1967).
11
germination mechanism, but if spores were able to germinate, most were
damaged to the extent that no outgrowth occurred. Degradation of B.
subtilis spore cortex by chlorine appeared to correlate with loss of colony
formation and may be the primary lethal effect (Gorman et al. 1984).
The heat resistance of the spores was reduced by a chlorine treatment.
Treatment of B. cereus spores with a solution of 0.25 percent NaOCI at
20C for 19.5 min resulted in a 99 percent loss in viability (Kulikovsky,
Pankratz, and Sadoff 1975). They found that the spores lost calcium ions,
dipicolinic acid, ribonucleic acid, and deoxyribonucleic acid. The pri-
mary effect was the degradation of the outer spore coats, which led to a
disruption of the normal permeability barriers. In general, Bacillus spores
are more resistant than Clostridium spores to the action of chlorine
(Foegeding 1983). However, Bacillus spores were more sensitive to chlo
rine dioxide than were Clostridium spores (Foegeding, Hemstapat, and
Giesbrecht 1986). Spores are about ten times as resistant to chlorine as
are vegetative cells.
Chlorine inactivation was due to degradation of RNA of polioviruses
(O'Brien and Newman 1979). The rate of inactivation of viruses is af-
fected by pH, temperature, chlorine concentration, viral aggregation,
and the type of virus (Engelbrecht et al. 1980; Harakeh and Butler 1984;
Keswick et al. 1985; Vaughn, Chen, and Thomas 1986).
For sanitizing equipment, regulatory agencies recommend a chlorine
concentration of 100 to 200 mg per liter, with a holding time of 2 min.
A 200mg-perliter hypochlorite solution circulated for 2 to 5 min is
usually satisfactory for sanitizing pipes cleaned in place. Enclosed vats
or tanks can be sanitized by fogging with a chlorinated (200 mg per liter)
solution. With open vessels or vats, the sanitizer can be pressuresprayed
onto the surfaces.
The use of chlorine has received a great deal of bad publicity because
of its reactions with organic compounds and the resultant mutagenic and
carcinogenic potentials. Due to these aspects, it has been suggested that,
in some cases, the use of chlorine may create a health hazard. The use of
chlorine dioxide, however, does not seem to produce these toxic com
pounds (Wei, Cook, and Kirk 1985).
Iodine. Iodine is well recognized for its germicidal properties against a
wide variety of microorganisms. However, its corrosiveness, toxicity, in
stability, low solubility in water, and other unacceptable characteristics
have limited the usefulness of iodine in food plants.
An iodophor is a combination of iodine and a solubilizing agent, usu
ally a surface-active agent (surfactant), which, when added to water, re-
leases free iodine at a low rate. The surfactant can be nonionic, cationic,
or anionic, but certain nonionic types are preferred. The advantages and
disadvantages of using iodophors as sanitizers are listed in Table 10.3.
As with hypochlorites, the iodophors show greater bactericidal activ-
ity at an acidic pH. Hence, phosphoric acid buffers are added to the
sanitizer to maintain the pH at 4.0 to 5.0.
In the iodophor combination, the germicidal properties of iodine are
maintained or enhanced, but the characteristic odor of iodine is elimi-
TABLE 10.3. ADVANTAGES AND DISADVANTAGES OF IODOPHORS
AS SANITIZERS
Advantages
Good stability
Long shelf life
Surface activity
Generally active against all microoorgan-
isms except bacterial spores and bacteri-
ophages
Destroy yeast cells at a greater rate than
do hypochlorites
Not affected by hard water salts
Relatively nontoxic
Not corrosive
Do not penetrate the skin
Good penetrating and spreading proper-
ties
Acid nature prevents mineral film forma-
tion
Built-in color indicator
Concentration easily determined
Less sensitive than hypochlorites to or-
ganic matter
Easily dispensed and controlled
Disadvantages
Effectiveness decreases with increase in
pH, such as carryover of alkaline deter-
gent solutions
Less effective than chlorine against bacte-
rial spores and bacteriophages
May cause off-flavors in dairy products
Should not be used at temperatures above
49C
May cause discoloration
More expensive than chlorine
Causes staining of some material such as
plastic hoses
nated. At the recommended dilution, the iodophor solution has a rich
amber color and, as it is used, the color fades. Hence, the iodophors have
a builtin indicator of effectiveness.
The iodophors have good stability with a long shelflife. A high degree
of surface activity gives them good penetrating and spreading power and
aids in quick draining. Residues or films of minerals are prevented, since
they are solubilized by the acid nature of the iodophors.
Iodophors cannot be used at temperatures above 49C due to subli
mation of the iodine, which results in loss of effectiveness.
Although the iodophors have good germicidal properties, they are
less effective than hypochlorites against bacterial spores and bacterio
phages.
There is less reaction of iodophors than hypochlorites with organic
matter. However, the germicidal power is reduced by starch.
The widespread use of iodophors to sanitize equipment on dairy
farms in Australia resulted in higherthannormallevels of iodine in milk
(Wheeler, Fleet, and Ashley 1982). This, in turn, caused thyroid problems
in some consumers of dairy products.
Quaternary Ammonium Compounds. The quaternary ammonium salts (quats)
have the general formula
The R stands for organic groups ranging from methyl (CH3) to long
chain aliphatic groups (CsH17 to C1sH,17) and phenyl groups. For bacteri-
cidal properties, at least one R is a plain or substituted long-chain alipha-
tic group. The X is an anion, such as a halide (Cl, Br), sulfate, or acetate.
The quats may be bacteriostatic in low concentration and bactericidal
in high concentration. Their lethal effect has been attributed to various
activities, including reactions with cell membranes, denaturation of es-
sential cell proteins, or enzyme inactivation. The effects on the cell mem-
brane may be due to membrane lysis, membrane enzyme inactivation, or
interference with the transport system. Quats may act on the cells by
increasing the permeability of the cell membrane, allowing water to enter
until the cell bursts.
The quaternary ammonium compounds have certain advantages and
disadvantages when used as sanitizing agents. These are listed in Table
10.4.
The quats are not as effective as the hypochlorites in destroying bacte-
TABLE 10.4. ADVANTAGES AND DISADVANTAGES OF QUATERNARY
AMMONIUM COMPOUNDS AS SANITIZERS
Advantages
Stable
Long shelf life
Stable to temperature changes
Effective in alkaline conditions
Noncorrosive
Odorless
Less affected by organic matter
Residual bacteriostatic effect
Nonirritating to the skin
Easily dispensed and controlled
Control off.odors
Nontoxic
Active against many microorganisms, espe
cially thermoduric types
Good penetration qualities
Combined with nonionics for detergent
sanitizing agents
Disadvantages
Incompatible with anionic agents in deter
gents
Expensive
Low activity in hard water
Less effective in activity against spores
and bacteriophage, as well as coliforms
and psychrotrophs
Need to rinse residual film from equip
ment
Problem with foam during mechanical ap
plication
rial spores of Gramnegative bacteria, including coliforms and psychro-
trophs. They are definitely inferior in acting against bacteriophages.
However, the addition of certain sequestering agents enhances the viruci-
dal activity. In general, the quats are very effective against Gram-positive
bacteria.
Generally, residues of quats are not allowed on equipment when food
is being processed. Therefore, it is necessary to rinse the equipment,
preferably with chlorinated water, prior to processing food.
Acid-Anionic Sur/actants. These are combinations of an organic or inor-
ganic acid (usually phosphoric) with a surfactant (usually an alkyl aryl sul-
fonate). The low pH as well as the activity of the surfactant provide a
bacteriostatic or bactericidal effect. The advantages and disadvantages of
this class of sanitizers are listed in Table 10.5.
RAW MATERIALS. To control the raw material, it may be necessary
for the processor to become the producer. For ingredients not directly
controlled, specifications are needed, if for no other reason than to pre-
vent the acceptance of rejects from another company. The specifications
must be written so that they are flexible, but rigid enough so that they
are useful. Some conditions for microbiological specifications of raw ma-
terials were described by Davies (1968) and Mossel (1969). The allowable
number of the significant types of microorganisms, as well as the process-
ing treatment that these ingredients are given, should be considered. The
presence of microorganisms that may be a public health hazard should
TABLE 10.5. ADVANTAGES AND DISADVANTAGES OF ACID-ANIONIC
SURFACTANTS AS SANITIZERS
Advantages
Stable
Long shelf life
Active against a wide spectrum of micro
organisms including thermodurics, con
trois phage and most yeast strains.
Residual antibacterial film
Noncorrosive and nonstaining to stainless
steel
Relatively low toxicity
Effective in hard water or with organics
Action enhanced at high temperatures
Disadvantages
Effective only at acid pH (optimum range
1.9-2.2; above 3.0, action decreases rap
idly)
Slow activity against sporeformers, not ef.
fective in destruction of most spores.
Foam problem in mechanical and clean
inplace applications
be of concern to the processor, especially if the processing treatments
do not destroy these potential pathogens. Spoilage organisms cannot be
overlooked. The presence of a significant number of resistant bacterial
spores in ingredients such as sugar, flour, or spices may cause spoilage
of canned foods.
Before worrying about microbial analysis, the raw materials should
be given a preliminary examination with normal human senses for evi
dence of filth or decomposition. The presence of rodent pellets or hairs,
insect infestation, field rot, or decay of fruit and vegetables, a diseased
condition of animals prior to slaughter, parasitic infestation of fish, or
decomposition of the material are causes for rejection. Since the quality
generally does not improve with processing, only clean and sound mate
rials should be brought into the processing plant.
After the raw material is inspected and passed, it should be processed
as soon as possible. When delays are unavoidable, facilities for storage
should be maintained to protect the raw material from contamination
and infestation as well as from deterioration. Some fresh raw materials
can be modified by storage to obtain a more uniform product. An exam
pie is the storage of potatoes for making potato chips. At low temper
atures, reducing sugars will accumulate and result in a dark potato chip.
By conditioning these potatoes for a short time at high temperatures, the
reducing sugars are returned to a low level.
Many foods have an outside peel, skin, or shell to protect the interior
portion of the food from microorganisms. However, the organisms on
the outside surface serve as a source of contamination when the protec
tive covering is removed during processing. Hence, the raw material
should be washed, when needed, to remove field dirt, other material, and
many of the microorganisms from the outside surface.
The washing of the raw food material should be done in an area sepa-
rated from the rest of the processing facilities_ The water should be free
of microorganisms that may cause foodborne illness or spoilage_
OPERATIONS. It is important that the equipment is maintained, kept
in good repair, and is properly cleaned and sanitized. When fruits and
vegetables are cut or peeled, or an animal is killed and the hide removed,
the natural defenses are broken down and the food is subject to contami-
nation and the beginning of a chain of events that lead to quality loss
and finally to destruction of the food.
Bacteria multiply rapidly. Therefore, the speed of handling becomes
important. The operations should be timed so there is no backup of
product, since long delays in processing can cause microbial problems.
Bacteria develop more rapidly as the temperature rises; hence, the tem-
perature should be kept as low as possible in keeping with processing
requirements.
When organisms attain a high level in a food, even if they are in-
hibited or killed, their enzymes may remain active and cause changes in
texture, flavor, and color.
Equipment such as trays or vats must not be used interchangeably for
raw and cooked products unless this equipment is completely cleaned
and sanitized before being used for cooked products.
Equipment is designed to reduce the handling of foods by workers.
This equipment causes less contamination than the people handling it if
it is properly cleaned and sanitized.
To reduce the temperature of poultry carcasses, continuous chillers
are used. With proper sanitation during processing, washing of the car-
caasses before chilling, maintaining low water temperatures, chlorina-
tion of the chill water with an acceptable input of fresh water and ice,
with resultant overflow of excess water, microbial contamination of the
carcasses is reduced.
PACKAGING. Food containers and packages are made from paper,
plastic, glass, and metal, as well as combinations of these materials. The
package should identify and advertise the contained product as well as
present an attractive appearace to give it sales appeal. The package usu-
ally gives the product a standardized shape. The main function of pack-
aging is to separate the food from the surrounding environment so that
the food is protected from recontamination by microorganisms and pests
and from physical and chemical damage from atmospheric oxygen, light,
storage odors, and moisture_
Packaging materials must meet certain standards. The package should
be easily filled and emptied. It must be strong and durable, since damage
to the package will result in contamination. The material used for pack
aging must not contaminate the food with foreign odors, flavors, or toxic
substances. Further, the food must not corrode or weaken the package.
The package must be able to withstand the processing treatment of the
food (heating, freezing, cold storage), and the package must be obtain-
able at a reasonable cost.
If not properly handled and protected, the package may be a source
of microorganisms. The food may acquire chemicals from the packaging
materials, such as plastics or waxes, either alone or coated onto paper.
Makinde, Gilbert, and Lachance (1976) listed chemicals such as mono-
mers (ethylene, vinyl, propylene, styrene) and catalysts used in preparing
plastics or various stabilizers, pigments, and antistatic, antifogging, bacte
ricidal, and antifungal agents incorporated into plastics. Polycyclic aro-
matic hydrocarbons or carcinogens such as 3,4benzopyrene may be asso-
ciated with waxes. Polyvinyl chloride plastics are made from vinyl
chloride, a human carcinogen.
With proper packaging, the greatest number of people can be sup-
plied with a clean, wholesome supply of food. Extremes of food packag-
ing are now available. The poisoning of packaged drug products with
cyanide and the threatened contamination of foods have been responsi-
ble for an increase in tamper-evident packaging. These types of packages
include film overwraps, blister-strip packs, bubble packs, shrink seals,
sealed tubes, breakable caps, bottle seals, pouches, sealed cartons, aerosol
sprays, and tape seals. This extra effort increases the cost of the product
and in some cases makes it difficult for the consumer to open the pack-
age for use.
At the other extreme is no packaging. There is a growing trend for
retail stores to sell bulk foods that are in a box, barrel, or case, or simply
sitting on a shelf. The consumer selects the type and amount of foods
and puts them into a container such as a plastic bag for purchase. It
is questionable whether these foods are handled in a sanitary manner.
Although these bulk foods are often presumed to be cheaper because of
reduced labor and packaging costs, they may not actually be priced any
lower per unit than packaged foods. At the same time, they offer a poten-
tial target for contamination by toxic substances. However, as long as
bulk foods are purchased, the retailer will continue to allot more space
for their sale, until a government agency decides that regulations are
needed.
Different packaging materials vary in their physical and chemical
properties. The permeability to moisture, oxygen, gases, light, and even
microorganisms varies with different materials. With the various proper-
ties of packaging materials, the characteristics of a package can be engi-
neered to fit almost any food product or processing and storage require-
menL
PERSONNEL. People are an important cause of microbial contamina-
tion offoods. The CGMPs stress (1) disease control; (2) cleanliness; (3) edu-
cation and training; and (4) supervision. The concern of the plant person-
nel for sanitation and personal hygiene must begin with plant
management. Management must set a good example, furnishing the
proper environment and establishing within the personnel a desire to
comply with the requirements necessary to maintain sanitation and per-
sonal hygiene.
All personnel should be involved in keeping the processing plant
neat and orderly. However, of special concern are those people who han-
dle ingredients, foods, or food-contact surfaces. In the handling of cream-
filled pastries, cooked meat and poultry products, milk products, and
salads, the hygiene of workers is a critical control point (Bryan 1974).
Disease Control. Even with the development of equipment for various pur-
poses, there is still direct handling or close contact of food by humans.
Although equipment is a major source of microorganisms, these are usu-
ally spoilage types normally associated with foods. Humans are a poten-
tial source of pathogens. In this respect, humans are more important
than equipment as a source of food contaminants.
Anyone who is sick, has a communicable disease, is a carrier of such
a disease, or has boils, sores, or infected wounds must not be allowed to
work in any area of a food plant where there is a possibility of contami-
nating the food or a food-contact surface.
According to California health authorities, education in food han-
dling and personal hygiene, along with inspection of food establishments
for sanitation, will prevent more foodborne disease than any program
involving the routine screening of food handlers for communicable dis-
eases. In general, the value of routine medical examinations of food han-
dlers is questioned for the following reasons: (1) It has not been estab-
lished that most outbreaks of foodborne illness are caused by food
handlers; (2) medical examinations and laboratory tests are very expen-
sive; and (3) because of the rapid turnover of food handlers, it is difficult
to assure that all employees are examined.
Cleanliness. Employees should maintain a clean appearance. The CGMPs
suggest the wearing of clean outer garments. Even though the employee
may dress in clean clothing at home, it is not sterile. Furthermore, by the
time of arrival at the plant, the clothing has been subjected to various
sources of contamination, which can include both food-spoilage and
disease-producing microorganisms. Hence, in some cases, it is required
that employees wear special protective outer clothing that is not worn
outside the processing plant. In the general guidelines for handling
ready-to-eat products, employees who work on raw and ready-to-eat prod-
ucts must change any garment that has contacted a raw product. Due to
human shedding of microorganisms, special clean-room clothing may be
needed in selected areas. This clothing is essentially that worn in operat-
ing rooms, including coveralls or smocks with hand, foot, and head cover-
ings. In many food plants, the employees wear company-furnished uni-
forms. Uniforms of different colors should be provided for different
departments. In this way, there will be less chance for cross-contamina-
tion of food by personnel.
The hands are used for many things. Therefore, it is essential that
before working with food, the hands are washed and, if necessary, sani
tized. Simply rinsing the hands is not sufficient. Washing with soap and
water does not remove all of the microorganisms; however, a thorough
washing with soap and water will remove most of the transient organ-
isms. Antimicrobial soaps will aid in removal of the resident flora. Sani-
tizing solutions for hand dips may contain chlorine, iodine, or any other
approved bactericidal agent.
Not only are hand washing and sanitizing needed before beginning
work, but also after each break, after leaving and returning to work, or
when the hands may be contaminated from handling contaminated
equipment or picking up debris from the floor.
The handwashing facilities should be provided with foot-operated
controls to obtain warm water. Tap handles have been found to be highly
contaminated. It does little good to wash one's hands and then promptly
contaminate them with enteric-type organisms from the tap handles.
Probably not surprising, McBride (1984) found that bar soap had sig-
nificantly higher levels of organisms than did liquid soaps. Stiles and
Sheena (1985) found that, of the products tested, only 4 percent chlorhex-
idine gluconate liquid detergent and iodophor containing 0.75 percent
available iodine were significantly better than nongermicidal soap for
reduction of transient bacteria. They also reported that a barrier cream
on the hands reduced the shedding of organisms from the fingertips
(Sheena and Stiles 1983).
In some cases, gloves are worn to control potential contamination.
These should be disposable rubber gloves and should be maintained in
a sound, clean, and sanitary condition.
Hair on the head, -face, and body is a source of microorganisms, as
well as loose hair that contaminates food. Hence, hair coverings, such as
hair nets, beard nets, headbands, caps, or other effective hair restraints
must be worn by employees.
Personnel should not have extraneous materials in the food-process-
ing area. Smoking, chewing of gum or tobacco, spitting, eating, or drink
ing cannot be tolerated in areas that will affect the food. Also, the food
must be protected from contaminants such as perspiration, cosmetics,
and medicants.
With the regulations of various government agencies relative to such
things as health and safety, no one except authorized plant employees or
government inspectors should be allowed in the foodhandling or pro
cessing areas.
People tend to have bad habits, forget what they are supposed to be
doing, and get careless. People forget to wash their hands, or merely rinse
them. Food is left at room temperature rather than being put into the
refrigerator or freezer. Some people believe that there is nothing wrong
with salvaging unfit food. It is relatively easy to observe examples of poor
sanitation as well as questionable practices when eating in a restaurant
or other foodservice unit.
In a processing plant, the cleanup crew must do a complete and thor
oughjob of cleaning and sanitizing the plant and equipment. If they are
not careful, they can contaminate the equipment more than they clean
it. The food processing crew must follow rules for acceptable operations.
Government inspectors should be clean and set an example for the
plant personnel. It is illogical for an unkempt person to try to explain
sanitation and personal hygiene to other people.
Training. Many foodprocessing jobs are seasonal and often offer rela
tively low salaries. Hence, the food industry tends to obtain young
people, part-time workers, and various others who cannot find work else-
where. Some of them are poorly trained people in the lower socioeco-
nomic group. With this combination of jobs and types of people, the
turnover rate is rather high and sanitary training is difficult.
The employees must be properly trained and retrained in the correct
use of equipment, sanitation, and personal hygiene. They must be in-
formed of the perishable character of the food product, sources of con-
tamination, and their role in the production of a top-quality product.
Since a majority of outbreaks of foodborne illness are due to contami-
nation or mishandling of food at foodservice establishments and in the
home, it seems logical that the general population needs training in the
care and handling of food products and in personal hygiene. A course
concerning these aspects given in high school might be beneficial.
Supervision. The supervision of the plant sanitation and the sanitary train-
ing of personnel should be under the direction of a well-trained sani-
tarian.
The sanitarian and other plant supervisors must be able to recognize
unsatisfactory employee habits or health problems_ If employees cannot
or will not follow the necessary rules, the supervisor must remove them
from critical areas, since the supervisor is ultimately responsible for com
pliance with the regulations that are applicable to the product being
processed.
Microbial Evaluation
A visual inspection of raw material, equipment, packages, and fin
ished product can detect gross filth, but microbial analyses are needed
to determine microbial contamination.
Samples for analysis should be obtained from the equipment sur
faces, hands, raw material, the material during processing (online sam
pIes), finished processed product, and other things such as air, water, or
packaging materials that might be involved in product contamination.
Routine tests will show daytoday variation and help reveal weaknesses
in raw materials, ingredients, equipment sanitation, or personal hygiene.
Not only the total number of organisms, but also pathogenic types,
such as Salmonella or Staphylococcus or indicators such as coliforms, Escher
ichia coli or enterococci, should be determined. In some processing plants
the sporeforming bacteria (Bacillus, Clostridium, and Desulfotomaculum) ,
yeasts, molds, psychrophiles (Pseudomonas), or other spoilage types might
be evaluated. In the citrus industry, a colorimetric test for the presence
of diacetyl and acetylmethylcarbinol is effective in quality control. These
are metabolic products of the degradation of citric acid by species of
Lactobacillus and Leuconostoc.
The total aerobic plate count of samples obtained from various stages
of production may be used as a general index of plant sanitation. How
ever, processing treatments such as heating will decrease the count, and
the addition of certain ingredients may increase the number of microor
ganisms. If sharp increases in numbers occur in a particular area, this
is evidence that something may be amiss and needs investigation and
corrective action.
A significant increase in the daytoday microbial level of equipment
surfaces indicates faulty cleaning or sanitation. Close observation of the
cleaning and sanitizing may reveal the problem so it can be resolved.
Microbial surveys of various foods reveal that products handled with
good sanitary conditions usually have significantly lower counts than
products produced by plants operating with poor sanitary conditions.
There are times when poor sanitary practices are not revealed by micro
bial numbers of the final product, but with sufficient sampling over sev
eral sampling periods and analysis of online samples, a correlation can
be obtained between plant sanitation and bacterial loads. When ana
lyzed, the coliforms, E. coli, and coagulase positive staphylococci counts
generally reveal the sanitation of the plant and personal hygiene of the
workers.
Foodservice Establishments
Of the reported outbreaks of foodborne disease in which the place
of mishandling is known, most are due to foodservice establishments. If
health authorities are truly interested in reducing the number of food
borne outbreaks, then foodservice establishments need more regulation
and observation than has been evident. It is unfortunate that people
must be forced to do an acceptable job of producing food. Too many
owners of foodservice businesses have no background in sanitation and
the proper handling of foods.
The same general requirement for housekeeping, equipment, clean
ing, sanitizing, and personnel problems and regulations that apply to
food processing plants also are required for foodservice establishments.
In many food processing plants, one or only a few foods are handled
and processed, whereas in a restaurant all types of foods in various stages
of processing are handled and utilized. Some raw foods are served with
only a little preparation, such as in salads. Other foods, such as meats,
are cooked. The common organisms (salmonellae and staphylococci) that
cause foodborne illness are destroyed by cooking meat to an internal
temperature of 74C (Bryan and McKinley 1974). Hamburgers or other
meats may not receive sufficient heat treatment during the cooking pro
cess to destroy pathogens. Cooked meats for sandwich use may be left at
room temperature rather than being refrigerated. The same knives or
other equipment used to handle raw meat may be used for cooked meat.
Hence, there may be crosscontamination between raw and cooked prod
ucts.
Foods that are prepared just prior to serving normally do not cause
health problems. It is food that is prepared a day or two before serving
and then not properly refrigerated, or food that, after preparation, is
held for serving during a two or threehour lunch or dinner period that
can cause problems of foodborne illness.
To prevent growth of microorganisms, warming tables should keep
hot foods at or above 60C, and cold tables should keep cold foods at or
below 7C. If foods are to be held for any period over two or three hours,
they should be refrigerated.
Various accessories (salt, pepper, catsup, sugar, steak sauce, syrup)
may be found on the table or served to several customers at different
tables. The customers can be a source of microbial contamination, in
cluding potential pathogens, of these supplies.
Certainly things have improved since the days when only a superficial
rinse was given to many utensils and glasses. However, further improve
ments are needed in the foodservice industry to eliminate contamina
tion and foodborne illness.
Due to the rapid turnover of workers in the foodservice industry, for
a national training program to work, it seemed advisable to train manag
ers (Baker 1982), who could then train their personnel. There are fewer
managers than workers, and managers tend to have less rapid turnover
than the workers. A few health departments have made manager training
mandatory (Wright and Feun 1986). However, the reports on voluntary
manager training programs has indicated little, if any, benefit of man
ager training (Cook and Casey 1979; Wright and Feun 1986). Perhaps
foodservice people are more interested in profit than in food service,
or perhaps the training program needs to be revised so that it is more
realistic.
The HACCP concept for food processing also can be used in food
service operations (Bryan 1981). The examination of raw materials, pro
cesses, practices, personnel, products, equipment, and premises is used to
determine things such as potentially hazardous foods, pathogenic food
borne organisms, and unacceptable employer practices, timetemper
ature combinations, procedures, and environmental conditions. Various
foodservice operations have been studied with this concept in mind, in
cluding Mexican and Chinese foods, caterers and hospitals (Bryan and
Bartleson 1985; Bryan and Lyon 1984; Bryan, Bartleson, and Christopher
1981; Bryan, Harvey, and Misup 1981; Bryan et al. 1982a, 1982b, 1982c;
Snyder 1986).
Salad bars, smorgasbords, buffets, and Sunday brunches are the bulk
food sections of the foodservice industry. Problems of salad bars, such
as overreuse offood, people's coughing and sneezing on the food, or stick
ing thejr fingers into the food for sampling were described by Dahl
(1986). Fortunately, most vegetables and fruits found in salad bars are
not involved in foodborne illnesses (Chapter 6). However, that is not true
for many of the other self-serve foods. Surprisingly, the number of out
breaks of foodborne illness involving these foods found in self-serve sys
terns has been relatively small (see Chapter 6). However, lesser known
foodborne illnesses may be associated with consumption of these foods.
Retail Stores
The modern food supermarket resembles a group offoodprocessing
operations as well as a foodservice unit mixed in with normal retail func
tions. Some stores have a meatcutting and packaging area, a produce
packing area, a candymaking area, a bakery, a delicatessen, an orange
juice squeezer, and a lunchroom with a salad bar.
With all of these activities going on, there are many chances for mis-
handling of food, with subsequent problems of foodborne illness. As dis-
cussed previously, the bulk food section is subject to contamination not
only by store personnel, but also by the general public. Many other foods
are not packaged in tamper-evident systems.
Wyatt (1979) pointed out the importance of sanitation in retail stores.
However, the sanitary condition of the meatcutting area was not as im-
portant as the original bacterial load on the carcass and temperature of
holding in affecting the microbial contamination and shelf life of steaks
or ground meat (Greer and Jeremiah 1980; Greer, Jeremiah, and Weiss
1983; Wyatt and Guy 1980). Obviously, if the original count is 100,000
per gram, the addition of another 10,000 bacteria will not have as great
an effect as the multiplication of the original bacteria. However, sanita-
tion should be practiced to aid in the control of pathogenic organisms.
To provide industry and governments (state and local) with a uniform
system for the operation and regulation of retail food stores, the Associa-
tion of Food and Drug Officials (AFDO) initiated and jointly developed
with the FDA a sanitation ordinance (FDA 1982). It includes such things
as general provisions, food (supplies, storage, preparation, display), per-
sonnel (health, cleanliness, clothing), equipment and utensils, cleaning
and sanitizing, sanitary facilities and controls (water, sewage, plumbing),
physical facilities, and compliance procedures.
Sanitation at Home
The practice of sanitation and personal hygiene in the home will go
far toward preventing problems in other food-handling establishments.
Many people seem to be unaware of their responsibility for proper home
storage, hygienic food preparation, and sanitary practices in the kitchen.
Efforts to improve food protection in the home must overcome family
customs and personal habits.
For a foodborne illness to occur, a potentially pathogenic organism
must be in an acceptable food environment for growth or production of
toxin. A sufficient amount of this contaminated food must be eaten by a
susceptible person for illness to occur. Thus, not all unsanitary acts will
result in illness. The person who prepares the food becomes complacent
and careless. Besides carelessness, other factors that may be involved in
foodborne outbreaks are apathy, ignorance, and poor judgment.
Food protection in the home encompasses essentially the same sys-
tems as in the food-processing plant. It begins with the selection of food
and continues with transportation, storage, preparation, serving, and
handling of leftovers and wastes.
At the store, we can make a judgment about certain foods by their
appearance. Damaged or opened packages should not be selected. If pro
duce has a shriveled appearance, discoloration, evidence of mold, or
other microbial growth, it should be avoided. In the frozen food counter,
do not select packages that are piled above the freezer. Canned foods
that show bulging or the leaking of contents should be pointed out to
the manager. Although, in general, the food in dented cans may be satis
factory, this type of can does show evidence of rough handling. This
could affect the can seams and result in spoilage or a health hazard.
Perishable food should be protected while being transported home.
Allowing food to remain in a hot trunk of a car parked in a sunny park.
ing lot for several hours will cause problems. Many people own cooling
chests that give some protection to perishable foods during the period
of transportation.
The home should have adequate space for dry storage as well as reo
frigerated storage. These areas should be kept neat and clean. The foods
that are brought home must be promptly stored in the proper area, espe-
cially those that require refrigeration or freezing.
The food pre parer should practice good personal hygiene. Thor-
oughly washing the hands is essential, and rewashing is needed when
food-preparation activities are interrupted for any reason. It is expected
that animals should be kept out of processing plants, but some people
allow their house pets to run around the kitchen and think little about
this. Rodent and insect control are as important at home as in the pro-
cessing plant.
The kitchen should be kept clean. This includes the floors, walls, cup-
board, stove, refrigerator, and other accessories. Pots and pans should be
cleaned thoroughly. Have you ever seen anyone sanitize the utensils at
home? This failure may account for some of the problems that occur in
the home. Although wiping cloths in processing plants and foodservice
establishments is considered to be unsatisfactory, it is unusual to find
a home in which dish towels are not used to wipe at least some of the
utensils or dishes.
Fresh meats, poultry, and fish should be cooked sufficiently to destroy
pathogenic types of microorganisms. Eating raw animal products is not
recommended. Cross contamination between raw meats and prepared
foods should be avoided.
With most meals, more food is prepared than is eaten. Hence, there
are leftovers. If these are to be used again, they should be placed
promptly into the correct storage conditions, and perishable foods
should be refrigerated. It is not considered acceptable to prepare food
ahead of time and allow it to remain at room temperature before serving.
Sooner or later, this practice will result in illness.
Various sites in several homes were surveyed for microorganisms
(Finch, Prince, and Hawksworth 1978; Scott, Bloomfield, and Barlow
1982). Surprisingly, except for washcloths, tea cloths, and sink and basin
drains, most sites in the home had relatively low levels of contamination.
CONTROL OF MICROORGANISMS BY REMOVAL
One means of controlling microbial levels in food is the removal of
microorganisms. The systems used commercially for this procedure in-
clude washing, centrifugation, and filtration.
Washing
Washing of fruits and many vegetables is used in the preparation of
these foods for canning, freezing, or fresh consumption. Washing re-
moves soil, microorganisms, and some residual pesticides (Munsch, Sim-
ard, and Girard 1982; Wright et al. 1979).
During processing, broiler carcasses go through a spray washer. This
washing process removes many of the microorganisms from the carcass
surface (Mulder and Bolder 1981; Notermans, Terbijhe, and VanSchot-
horst 1980).
Significant reductions in bacterial numbers on beef and lamb car
casses by using high-pressure water sprays have been reported. However,
spray washing was considered to be impractical (Sheridan 1982), and
there was no increase in storage life of the sprayed carcasses (Kelly,
Lynch, and McLoughlin 1982). Kriaa, Arthaud, and Fournaud (1985)
found that the microbial contamination of carcasses was not greatly af-
fected by spray washing. One problem seems to be that bacteria become
attached to meat surfaces and are then difficult to remove. The addition
of acid (acetic or lactic) or sodium hypochlorite to the spray water yielded
some increased benefits (Anderson et al. 1979; Skelley et al. 1985; Smul-
ders and Woolthuis 1985; Woolthuis and Smulders 1985).
The addition of KCI (0.1 m) to a water rinse resulted in a greater
removal of microorganisms from meat surfaces (Appl and Marshall
1984). Pedraja (1973) recommended that fish or seafoods be passed un-
der a spray of chlorinated (10 mg/L) water before and after processing
to control bacteria.
Washing may not be beneficial in all cases. Fellers and Pflug (1967)
reported that washing of pickling cucumbers "reduced the storage life to
half that of unwashed fruit." The polishing of fruit with saran brushes
can cause invisible injuries to the peel. This can result in increased decay
during storage.
If the washing of shell eggs is done improperly, there is increased
spoilage during storage.
Centrifugation
This operation involves the separation of a solid from a liquid or a
liquid from a liquid by means of centrifugal force. This process has been
used in the preparation of sugar, clarification of fruit juice, and the sepa
ration of cream from milk.
By increasing the centrifugal force of the milk separator, it is possible
to separate bacteria from the milk. In this centrifuge, the bacterial cells,
as well as other sediment, are thrown to the vertical wall of the bowl. The
outer edge of the bowl has two small holes (0.3 mm) through which about
1.5 percent of the milk containing from 90 to 97 percent of the bacteria
is removed. By using two centrifuges in series, 99 percent or more of the
bacteria in the original milk is removed.
Although originally described for milk, other applications have been
developed, including the separation of microorganisms from substrate
in the production of singlecell protein, the purification of molasses for
use in the production of baker's yeast (Saccharomyces cerevisiae), and the
separation of distiller's yeast from the substrate prior to distillation of
the alcohol.
When bacteria are removed by centrifugation, there is no damage to
the flavor or other quality factors such as occurs with heat. Also, the cells
are removed, not just destroyed.
Spores are more dense than bacterial cells and are readily removed
from liquid foods. The removal of over 99 percent of the heatresistant
spores and vegetative cells makes it easier to destroy the remaining
spores by heat treatment.
There may be problems with some applications. Centrifugation also
may remove some protein, calcium, and phosphorus from milk. To aid
in separation, some foods may need to be heated. To centrifuge viscous
molasses, it is diluted and heated.
The use of centrifugation is limited to liquid foods and then only
when the density of the bacteria is greater than that of the food compo
nents. Since not all of the microorganisms are removed, these liquid
foods cannot be considered sterile. However, by reducing the microbial
load, it is easier to achieve sterilization by heat treating the food.
Filtration
Filtration is the separation of solids from a liquid or gas by subjecting
the mixture to a porous material, the filter. The size of the filter pores
determines the particles that pass through and those that are retained.
Bacterial membrane filters (MF) have pores of 0.45 /-tm. However, if
all but the smallest viruses are to be retained, a pore size of 0.1 to 0.2 /-tm
is needed. Although one manufacturer indicated that the pore size varied
from 0.43 to 0.47 /-tm, according to Olson (1983) the range for pore diam
eter of this size MF is 0.20 to 1.0 /-tm. This variation is perhaps why a 0.20
/-tm size is recommended for sterilization.
There are some advantages in using membrane filters. The inert be
havior, high initial flow rate, particle retention due to the "sieve" effect,
no shedding of fibers, negligible loss of solution, and no effect on the
concentration, pH, color, or flavor of the filtrate are good features of a
membrane filter (Grubert 1974).
Before the membrane or other filter is used to remove bacteria from
substances, it must be sterilized. This usually is accomplished by means
of steam heat, ethylene oxide, or gamma radiation. After filtration, all
pipes, filling apparatus, and packages must be sterile or the product will
be recontaminated.
Filtration is used in the sterilization of media or media components
that are adversely affected by heat treatment. It is used in the microbial
analysis of liquids, such as potable water with low microbial counts (mem
brane filtration method).
Millipore (1970) listed several uses for membrane filters. These in-
clude (1) the filtration of beer or wine for cold pasteurization or for anal-
ysis of yeasts and bacteria; (2) filtering air to determine microbial con
tamination or to remove microorganisms and other particles from air
used in contact with food products, in fermentation vats, and in packag-
ing food products; (3) in the analysis of additives, pipelines, packaging
equipment, and foods, such as soft drinks, syrups, and liquid sugar for
microbial contamination; (4) remove yeasts and bacteria for the biologi-
cal sterilization of champagne; and (5) remove microorganisms or partic
ulate material from water, such as that used in blending of alcoholic bev-
erages, to achieve clarity.
Cold pasteurization or sterilization by filtration can be used for clear
fruit juices, vinegar, soft drinks, syrups, and vegetable oils. Since filtra-
tion to remove bacteria does not destroy or remove enzymes, if these
cause undesirable reactions in foods, heating must be used to inactivate
them. With ultrafiltration, enzymes can be removed. Systems such as re-
verse osmosis or ultrafiltration can be used to concentrate foods prior to
drying.
Filtration has some advantages, as well as disadvantages, as a means
to control microorganisms in foods. Among the advantages are that less
energy is needed to filter than to heat liquid foods. Not only are the
microorganisms removed, but particulate matter is also removed, and clar-
ity is thus improved. Problems include the prefiltering or clogging of the
filters, the residual enzymes, the necessity for sterilizing the filter system
prior to use, and the necessity for observing aseptic handling and pack
aging.
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by Retarding Growth
11
By altering the factors (discussed in Chapter 4) to retard or inhibit the
growth of microorganisms, the storage life of foods can be extended.
These factors included temperature, water activity, acidity, oxidation-
reduction potential, chemical inhibitors, and microbial interactions. Be-
sides these factors, microorganisms can be affected by electrical current
(Shimada and Shimahara 1981) as well as homologous phage (Greer
1986).
In some cases there is a rather fine line between inhibition and death.
Some chemicals are bacteriostatic at low concentrations and bactericidal
at higher levels. Low temperatures will reduce or inhibit microbial
growth, while high temperatures will kill the cells.
The retardation of growth means that the food product is not sterile
and, if mishandled or abused, the microorganisms may grow and cause
spoilage or be a health hazard.
LOW-TEMPERATURE STORAGE
Temperature is one of the most important environmental factors in
fluencing the growth and activity of microorganisms. Both microbial and
biochemical activity can be reduced by lowering the temperature of a
food and holding it in a refrigerator or freezer. The lower the temper
ature, the lower the rate of biochemical reactions or microbial activity.
Therefore, it might be assumed that freezing and freezer storage are best
for all foods. However, freezing damages food tissues, and, for some types
of foods, this damage is unacceptable. For some foods, low-temperature
refrigeration can cause injury. Thus, the temperature at which a food can
be stored depends upon the type of food as well as economic factors.
Removing heat and maintaining a controlled cold environment are ex
pensive. Although many types offoods or ingredients might benefit from
low-temperature storage, we are concerned mainly with perishable foods.
Refrigeration
The minimum growth temperature for most mesophilic organisms is
about lOoC. Since refrigeration generally refers to temperatures below
lOoC, these mesophiles do not grow and are not a problem with refriger
ated food. Some mesophilic microorganisms are psychrotrophic and can
grow on refrigerated foods. However, the optimum growth temperature
of these organisms is 25
0
to 30
0
C, and reduced growth occurs at temper
atures of lOoC or less.
The main organisms of concern on refrigerated foods are the psy-
chrophiles. These organisms can grow at temperatures below OC, and
some reportedly have an optimum growth temperature as low as lOOC.
Although psychrotrophic and psychrophilic microorganisms can
grow at low temperatures, they grow more slowly as the temperature is
reduced. The lower growth rate is very evident as the minimum growth
temperature of an organism is approached. Also, as the refrigerator tem-
perature is reduced from 100C, fewer strains can grow and cause spoil-
age. Hence, food will spoil about four times as fast at lOoC and twice as
fast at 5C as at OC.
Some microorganisms causing food borne illness are psychrotrophic,
but most will not grow or produce toxins below 4.4C. None of these
organisms grow below 1.7C. For safety, perishable foods should be held
below these temperatures. Under no circumstances should perishable
foods be left at room temperature longer than necessary, as dictated by
good handling procedures.
Quite often, other systems for preserving food are used in conjunc-
tion with refrigeration. Since psychrophiles are primarily aerobic, vac-
uum or carbon dioxide packaging with the exclusion of oxygen will delay
microbial spoilage. Salting, curing, smoking, or other chemical systems,
as well as mild heat treatments, may be used to inhibit or reduce the
microorganisms on refrigerated food.
COLD SHOCK. This is a form of direct chilling injury to the cells, re-
sulting from temperature reduction without freezing of the substrate.
Damage is evident immediately upon reaching the low temperature. Cold
shock is dependent upon the rate of cooling and the magnitude of tem-
perature reduction. Cells in the early exponential growth phase are the
most sensitive to cold shock.
Cold shock results in a loss of one or more membrane permeability
barriers. Hence, there is a leakage of amino acids and nucleotides from
the cells. According to Sato and Takahashi (1970), the DNA of cold-
shocked cells contains more single-strand breaks or nicks than either un-
shocked or recovered cells.
Injury to certain cells can occur simply by holding them at temper-
atures approaching OOC (Patterson and Jackson 1979a, 1979b).
RAPID CHILLING. It is necessary to remove the field heat from fruits
and vegetables, and the animal heat from milk, eggs, or meat, to prevent
CONTROL OF MICROORGANISMS BY RETARDING GROWTH 547
spoilage of these foods. There are many ways in which the temperature
of a food can be lowered rapidly. Liquid foods can be put through a plate
or other type of heat exchanger. Other foods are rapidly cooled by forced
air cooling, spray chilling, hydrocooling, icing, or in continuous chillers
in slush ice. Fluidized beds are used for cooling vegetables after blanch
ing and prior to freezing.
Food should be refrigerated in small containers, so it can be cooled
as thoroughly as possible within a short time. Bulk refrigeration can be
a contributing factor in foodborne illness.
When food is placed in storerooms, it should not be stacked tightly,
since allowance must be made for air circulation. This will aid in keeping
a uniform temperature and humidity in the warehouse, storeroom, reo
frigerator, or freezer.
LATENT-ZONE CHILLING. Refrigeration usually is considered to be
storage at temperatures from 0 to 5C. The freezing point of most foods
is below OC (Table 11.1). Hence, storage below OC but above the freez
ing point has several advantages (Smith 1976). At these colder temper
atures there is a longer shelflife than obtained with normal refrigeration.
The energy requirement is lower and there is less product alteration or
damage than when the food is frozen.
TYPES OF FOOD. Due to the different characteristics of foods, the
cooling and refrigeration requirements vary. The potential refrigerated
shelf life is influenced by the chemicals in a food.
Some foods, such as bread, should be stored at room temperature or
be frozen instead of refrigerated. Crackers and certain cookies tend to
lose their crispness in the refrigerator, due to moisture. The crystalliza
tion of sugar in marshmallows or marshmallow cremes occurs in refriger
ated storage. When foods are refrigerated, they should be packaged in a
proper manner to prevent moisture or aroma exchanges between the
various foods.
Meat. It is desirable to cool and hold meat below 3C and as close to
- 2C as possible to prevent growth of potential pathogens and to retard
growth of spoilage organisms and maintain the meat in a fresh condition
(Table 11.2). Below - 2C, meat will freeze.
From a microbiological viewpoint, it is desirable to remove the body
heat from carcasses as soon as possible after slaughter. The body temper-
ature varies from 38 to 40.5C, with an average of 38.9C, depending
on the animal. The rate at which a carcass is cooled depends not only on
the chilling room temperature, but also on the size, heat capacity, and
amount of fat covering on the carcass, and the velocity at which the air
is circulated in the room.
TABLE 11.1. APPROXIMATE FREEZING POINTS OF SELECTED FOODS
Food Temperature (0C) Food Temperature (0C)
Beef -1.6 to -2.2 Oranges -2.2
Pork -1.6 to -2.2 Peaches -1.4
Poultry -2.8 Pears -1.9
Fish -0.6 to -3.3 Pineapples -1.2 to -1.6
Shellfish -2.2 to -2.7 Asparagus -1.2
Shell eggs -2.2 to -2.8 Beans -1.1 to -1.3
Egg yolk -0.6 to -0.7 Beets -2.8
Egg white -0.4 to -0.5 Carrots -1.3
Milk -0.5 to -0.6 Celery -1.1
Cheeses -2.2 Corn, sweet -1.7
Brick -8.7 Cucumbers -0.8
Cottage -12.7 Lettuce -0.4
Ice cream -2.8 Melons -1.5 to -1.7
Apples -2.0 Peas, green -1.1
Apricots -2.2 to -2.3 Peppers, green -1.1
Avocados -2.7 Potatoes -1.7
Bananas -1.0 to -3.3 Pumpkins -1.1
Berries -0.9 to -1.7 Rhubarb -2.0
Cherries -2.2 to -4.2 Spinach -0.9
Grapes -2.5 to -3.8 Sweet potatoes -1.9
Lemons -2.2 Tomatoes -0.9
Limes -1.5 Turnips -0.8
Nuts -4.5 to -10.5 Fruit pies -3.0 to -4.0
Olives -1.9 Meat pies -1.4 to -1.9
TABLE 11.2. STORAGE CONDITIONS OF SELECTED ANIMAL PRODUCTS
Storage
Temp
Product
(0C)
Meat
Beef
Fresh -2.0 to 1.11
Pork
Fresh -2.0 to 1.11
Bacon 1.11 to 4.44
Sausage
Smoked 4.44 to 7.22
Lamb
Fresh - 2.0 to 1.11
Poultry
Fresh -2.0toO
Eggs
Shell -1.7 to 0.6
Dried 1.67
Cheese 1.67
Brick - 1.11 to 1.11
Cottage Oto1.11
Fish
Fresh 0.56 to 4.44
Smoked 4.44 to 10.0
SOURCE: Adapted from Farrall (1976).
548
Conditions
Humidity
(%)
88-92
85-90
85
85-90
85-90
85-90
Low
65-70
50
45
90-95
50-60
Approximate
Storage
Life
1-6 weeks
3-12 days
2-4 weeks
5-12 days
1 week
8-9 months
6-12 months
5-20 days
6-8 months
Light carcasses (lamb, pork, veal, and small beef) will cool in one-
third to one-half the time required for heavy beef carcasses_ Increasing
the air velocity will reduce the cooling time but may increase shrinkage
from moisture loss_ High humidity will help reduce shrinkage but may
stimulate microbial growth on the carcass surface_ Although for micro-
biological reasons the carcasses should be chilled as rapidly as possible,
doing so may result in less tender meat (James, Gigiel, and Hudson 1983).
Beef carcasses are generally aged to increase the tenderness of the
meat. Smith, Arango, and Carpenter (1971) suggested that chilling the
carcasses for the first 16 to 20 hr at 16C appears to be the most practical
system to use to develop tenderness. The use of ultraviolet light has made
possible higher room temperatures to obtain a more rapid tenderization
of the meat. The UV light, as well as the ozone produced by these lamps,
helps retard microbial growth. Processes to circumvent the aging room
include electrical stimulation of beef muscles and the hot boning of car-
casses. Raccach and Henrickson (1978) reported that electrical stimula-
tion of beef carcasses extended the shelf life of resultant ground beef by
three days over meat from control carcasses. The disassembly or break-
ing of carcasses before chilling (hot boning) has economic advantages.
Rapid chilling of this cut-up meat can be accomplished with solid carbon
dioxide (Gigiel, Swain, and James 1985). Reportedly, hot-boned cuts of
meat had higher microbial counts than did meat from chilled carcasses
(Kennedy, Oblinger, and West 1982).
The vacuum packaging of beef and lamb tends to minimize shrinkage
and discoloration as well as retard microbial growth. The low O2 and rela-
tively high CO2 developed in the packages inhibit pseudomonads, the
usual spoilage organisms of refrigerated meats. Low gas permeable bags
containing lamb carcasses were flushed with N2 or filled with CO2
(Grau, Eustace, and Bill 1985). The addition of CO
2
significantly reduced
the growth of Gram-negative oxidative (pseudomonads) and fermenta-
tive (enterobacteria), Brochothrix thermosphacta, and lactic acid bacteria.
Lower-grade carcasses are boned and the meat is used in sausage and
other processed products. The USDA requires that all fabrication areas
(beef breaking, pork cutting, or boning rooms) be maintained at lOoC or
lower to retard bacterial growth. Smith (1985) stated that maintaining the
boning rooms at 10C or less does not appear to be necessary, providing
the meat is processed with time limits which he determined for various
temperatures, for example, 8.4 hr at 15C.
Fresh meat is packaged for retail sale and put into a refrigerated dis-
play case. The salable life for this meat is about three days. In the home,
refrigeration (2.2 to 4.4C) of meat should be limited for maximum
quality. Ground meats, liver, and poultry should not be stored more than
two days, roasts and steaks for five days, and ham or bacon for seven days
(Table 11.3). For longer storage, the meat should be frozen.
Cured Meats. The psychrophilic flora is inhibited by the curing salts so
these products are more stable than fresh meats during refrigerated stor
age. They often receive a pasteurization treatment that also increases the
stability. Vacuum packaging aids in preventing discoloration, and the an
aerobic environment inhibits aerobic bacteria and molds (see Tables 11.2
and 11.3).
Poultry. The storage life of poultry carcasses is limited by surface micro
bial activity that is influenced by the initial bacterial load and holding
temperature.
After processing, it is important to remove body heat rapidly. This
may be accomplished by continuous immersion chilling, spray chilling,
TABLE 11.3. HOME STORAGE TIMES FOR MEAT AND POULTRY
(TO MAINTAIN QUALITY)
Refrigerator at
1.7 to 4.4C Freezer at
Product (days) -17.8C (months)
Meat, fresh
Roasts (beef and lamb) 3 to 5 8 to 12
Roasts (pork and veal) 3 to 5 4 to 8
Steaks (beef) 3 to 5 8 to 12
Chops (lamb and pork) 3 to 5 3 to 4
Ground and stew meats I to 2 2 to 3
Variety meats I to 2 3 to 4
Sausage (pork) I to 2 I to 2
Meat, processed
Bacon 7
Frankfurters 7 Y2
Ham (whole) 7 I to 2
Ham (half) 3 to 5 I to 2
Ham (slices) 3 I to 2
Luncheon meats
3 to 5 }
Freezing
Sausage (smoked)
~ to 21
not recom
Sausage (dry and semidry) mended
Meat, cooked
Cooked meats and meat dishes I to 2 2 to 3
Gravy and meat broth I to 2 2 to 3
Poultry, fresh
Chicken and turkey I to 2 12
Duck and goose I to 2 6
Giblets I to 2 3
Poultry, cooked
Pieces (covered with broth) I to 2 6
Pieces (not covered) I to 2 I
Cooked poultry dishes I to 2 6
Fried chicken I to 2 4
SOCRCE: USDA (1970).
or air chilling. Immersion chilling is the usual practice in the United
States. It is more efficient, effective, and economical than other systems.
For retail sale, the carcasses are packed on a fiberboard base and over-
wrapped or are packed in a plastic bag. They may be whole or cut up_ At
- 2.0C, the approximate storage life is one week (Table 11.2) and home
storage at 1.7 to 4.4C is only one to two days (Table 11.3). Poultry car-
casses are more subject to spoilage than beef, pork, or lamb carcasses.
The surface-to-volume ratio is greater for poultry carcasses, which may
c c o u ~ t for some of this difference. The method of slaughter and pro-
cessing may result in a greater contamination by psychrophilic types of
spoilage bacteria.
Shell Eggs. As soon as an egg is laid, its quality begins to decline. With our
present system of producing, distributing, and marketing shell eggs, the
main concern is with maintaining quality rather than with microbial
spoilage. Low temperature, shell treatments, thermostabilization, carton
overwraps, controlled humidity, and various other innovations have been
suggested or used to help maintain egg quality.
When shell eggs are stored at - 1.1 C, the rate of moisture loss is
decreased, the amount of carbon dioxide lost is reduced, the rate of
chemical change is slowed, and microbial growth is retarded. Soft-cooked
eggs could be stored at 10C for thirty days or at 25 to 35C for two to
three days (Imai 1981).
Egg Products. These are obtained by carefully breaking the egg shell and
removing the liquid. Most of the egg products are obtained by using egg-
breaking machines. The highly perishable nature of egg yolk and whole
eggs makes it necessary that this liquid be cooled to 4.4C or less within
1'h hr after mixing, and straining (to remove egg shell particles). Egg
whites can be held at a higher temperature than yolk or whole egg due
to the microbial inhibitors and the less available nutrients in egg white.
Liquid eggs can be held at 0 to 5C with relatively little change in
the microbial load up to 24 hr. However, at a temperature of 10C or
more, significant increases occur in the number of microorganisms.
Milk and Milk Products. When obtained, milk is at the body temperature
of the cow. This raw milk should be cooled and held near Oc. At 3.3C
or less, the coliform count tends to decrease, while in three days at 7.2C,
the psychrotrophs can show a tenfold increase. Gram-negative bacteria
are the organisms in raw milk held in cold storage. These include Pseudo-
monas, Acinetobacter, Aeromonas, Alcaligenes, and members of the family
Enterobacteriaceae. To obtain the maximum shelf life, pasteurized milk
should be held at 5C or less_
Buttermilk, yogurt, cottage cheese, and other soft cheeses are chilled
promptly after manufacture to near OOC. When held at OC, they have a
shelf life of a week or more. Unfortunately, due to the practice of stacking
of cartons in the display case, those packages at the top may reach 5C
or even 10C. This shortens the useful life of the product.
The refrigerator in the home may not be as cold as desired, and the
food is removed from the refrigerator during preparation of the meal.
Allowing these products to remain at room temperature for one hour
each day can reduce the storage life.
Some dairy products such as hard cheeses can have a refrigerated
storage life of several months or more. Mold growth can occur on the
surface of hard cheese. This growth is not desirable due to the formation
of off.flavors in the immediate area of mold growth. Also, the potential
production of mycotoxins by the molds must be considered.
Fishery Products. These products are very perishable. Fishery products
must be cooled promptly and held in this condition until either con
sumed or used in further processed items. The methods for cooling in
clude ice, chilled seawater, refrigerated seawater, cold air refrigeration,
and cryogenic gas or liquids.
There is a marked extension in the storage life of fish by small reduc
tions in the temperature in the range ofOC. A reduction in temperature
from 3 to OC doubles the storage life, and a reduction from 10 to
OC increases the storage life by a factor of 5 or 6. Fish spoils rapidly at
temperatures of 15 or 20C.
Atlantic mackerel were held satisfactorily in ice for nine days, after
which bacteria increased rapidly Uhaveri, Leu, and Constantinides 1982).
Molin and Stenstrom (1984) found that the shelf life of herring fillets was
one day at 15C or seven days at OOC. They found that the shelf life
increased to three days when it was packed in CO
2
at l5C or thirty three
days at OOC. Molin, Stenstrom, and Ternstrom (1983) reported that, at
2C, CO2 storage prolonged the shelf life of herring fillets by a factor of
3.5. The storage life of trout and herring at OC can be extended from
one to two weeks by vacuum packaging (Hansen 1972). This packaging
also retards the oxidative rancidity of fats and oils in the fish.
According to Cobb, Vanderzant, and Hyder (1974), shrimp boats reo
main at sea for four to twenty days. To maintain the quality, the shrimps
are packed in ice. As with other fishery products, icing can cause a cer
tain amount of leaching of nutrients from the shrimp. With ice storage,
the shrimp in the lower layers tend to have a higher bacterial load be-
cause the melting ice carry bacteria from the upper layers. The weight of
the shrimp and ice will tend to crush the shrimp at the bottom of the
pile. This crushing, plus the bacteria washing down from the upper lay-
ers, increases the rate of deterioration.
Fruits and Vegetables. Substantial losses occur in the shipping, distribution,
and retailing of fresh fruits and vegetables. These losses are due to bio-
chemical, enzymatic, and bacteriological activities that cause overripe-
ness and decay. Prolonging the storage life of fresh produce is advanta-
geous, not only to the food industry, but also to the consumer, because
it is the consumer who pays for the losses in the form of higher prices.
To help control these losses, it is essential that the field heat be re-
moved from the fresh produce as soon as possible after harvesting. Then
the produce should be maintained in a cool condition.
Two systems for removing field heat are hydrocooling and vacuum
cooling. Hydrocooling is applicable to both fruits and vegetables. In this
system, cold water sprayed over the fresh product reduces the temper-
ature of the produce at a very high rate. The warmed water is recirculated
through ice or a refrigeration system to cool it before respraying. In some
cases, hydrocooled products have developed more decay than untreated
produce. The addition of chlorine helps control the microorganisms in
the water as well as those on the surface of the produce.
Vacuum cooling is adaptable to products that have a large surface-to-
volume ratio. This system works in a manner similar to human perspir-
ing. The evaporation of water from the surface produces a cooling effect.
To prevent dehydration of the fresh produce, water is sprayed onto it.
Then the vacuum treatment evaporates this sprayed-on water and cools
the produce. The amount of cooling is roughly proportional to the water
loss. Products in which vacuum cooling is beneficial include lettuce, spin-
ach, and cabbage.
Besides cold storage, controlling the atmospheric gases (oxygen and
carbon dioxide) and the relative humidity (RH) are beneficial in main-
taining quality during storage of fruits and vegetables (Tables 11.4 and
11.5). Hypobaric storage (precisely controlling the pressure, temperature,
and humidity of storage) has been discussed for various fruits and vegeta-
bles (Jamieson 1980).
As is generally true of all foods, most fruits and vegetables keep their
fresh quality longer if stored at slightly above their temperature of freez-
ing. The freezing temperature of fruits varies from about - 3.9 to
-1.0
0
e and vegetables, from about - 3_7 to - 0.5e (Table 11.1).
Some fruits and vegetables are injured by low-temperature storage
(Table 11.6). When injured produce is returned to room temperature,
the respiration rate rises, then declines rapidly, due to the death of cells
in the tissues.
Tropical and subtropical fruits are especially subject to low temper-
ature injury. Even some fruits that are grown in temperate climates can
be affected.
Oranges are less susceptible to chilling injury than are other citrus
TABLE 11.4. RECOMMENDED STORAGE TEMPERATURES, RELATIVE HUMIDITIES,
AND APPROXIMATE STORAGE LIFE OF FRUITS AND NUTS
Storage Relative Approximate
Temperature Humidity Storage
Fruit or Nut (0C)
(%) Life
Apples -1.1 to -0.6 85 to 90
Apricots -0.6 to 0 85 to 90 1 to 2 weeks
Avocados 2.8 to 8.9 85 to 90
Bananas 11.7 to 15.6 85 to 90 1 to 3 weeks
Berries
Blackberries -0.6 to 0 85 to 90 7 to 10 days
Dewberries -0.6 to 0 85 to 90 7 to 10 days
Gooseberries -0.6 to 0 85 to 90 3 to 4 weeks
Loganberries -0.6 to 0 85 to 90 7 to 10 days
Raspberries
Black -0.6 to 0 85 to 90 7 to 10 days
Red -0.6 to 0 85 to 90 7 to 10 days
Strawberries -0.6 to 0 85 to 90 7 to 10 days
Cherries -0.6 to 0 85 to 90 10 to 14 days
Coconuts 0 to 1.7 80 to 85 1 to 2 weeks
Cranberries 2.2 to 4.4 85 to 90 1 to 3 months
Figs -0.6 to 0 85 to 90 10 days
Grapefruit 0 to 10 85 to 90
Grapes
Vinifera -1.1 to -0.6 88 to 92 3 to 6 months
American -0.6 to 0 80 to 85 3 to 8 weeks
Lemons 12.8 to 14.4 85 to 90 1 to 4 months
Limes 7.2 to 8.9 85 to 90 6 to 8 weeks
Mangoes 10.0 85 to 90 15 to 20 days
Nuts 0 to 2.2 65 to 70 8 to 12 months
Olives 7.2 to 10 85 to 90 4 to 6 weeks
Oranges -1.1 to 1.1 85 to 90 8 to 10 weeks
Papayas 7.2 85 to 90 15 to 20 days
Peaches -0.6 to 0 85 to 90 2 to 4 weeks
Pears -1.7 to -0.6 88 to 92 2 to 7 months
Persimmons -1.1 85 to 90 2 months
Pineapples
Mature green 10 to 15.6 85 to 90 3 to 4 weeks
Ripe 4.4 to 7.2 85 to 90 2 to 4 weeks
Plums, prune -0.6 to 0 85 to 90 3 to 8 weeks
Pomegranates -0.6 to 0 85 to 90 2 to 4 months
Quinces -0.6 to 0 85 to 90 2 to 3 months
SOURCE: Adapted from Desrosier and Desrosier (1977).
fruits. With the banning of ethylene dibromide fumigation, cold expo
sure (OOC for ten days or 2.2C for sixteen days) is the only quarantine
treatment for the Mediterranean fruit fly. This treatment causes peel pit-
ting in grapefruit. However, preconditioning or delayed cooling (holding
the fruit at 17C for six days or 16C for seven days prior to the cold
treatment) reduced the incidence of chilling injury (Chalutz, Waks, and
Schiffman-Nadel 1985; Hatton and Cubbedge 1981). Film wrapping or
TABLE 11.5. RECOMMENDED STORAGE TEMPERATURES, RELATIVE
HUMIDITIES, AND APPROXIMATE STORAGE LIFE OF VEGETABLES
Storage Relative Approximate
Tern perature" Humidity Storage
Vegetable
(0C)
(%) Life
Artichokes, Jerusalem 0 90 to 95 2 to 5 months
Asparagus 0 90 to 95 3 to 4 weeks
Beans
Green 7.2 85 to 90 8 to 10 days
Lima-unshelled 0 90 to 95 2 to 4 weeks
shelled 0 90 to 95 15 days
Beets
Topped 0 90 to 95 1 to 3 months
Not topped 0 90 to 95 10 to 14 days
Broccoli, Italian 0 90 to 95 7 to 10 days
Brussels sprouts 0 90 to 95 3 to 4 weeks
Cabbage 0 90 to 95 3 to 4 months
Carrots
Topped 0 90 to 95 4 to 5 months
Not topped 0 90 to 95 10 to 14 days
Cauliflower 0 90 to 95 2 to 3 weeks
Celery -0.6 to 0 90 to 95 2 to 4 months
Corn, green -0.6 to 0 90 to 95 4 to 8 days
Cucumbers 7.2 to 10 90 to 95 10 to 14 days
Eggplants 7.2 to 10 90 to 95 10 days
Endive 0 90 to 95 2 to 3 weeks
Garlic 0 70 to 75 6 to 8 months
Horseradish 0 90 to 95 10 to 12 months
Leeks, green 0 85 to 90 1 to 3 months
Lettuce 0 90 to 95 2 to 3 weeks
Melons (ripe)
Watermelon 2.2 to 40 80 to 85 I to 2 weeks
Muskmelon 4.4 to 10 80 to 85 10 to 14 days
Honeydew 4.4 to 10 80 to 85 2 to 4 weeks
Casaba, Persian 4.4 to 10 80 to 85 4 to 6 weeks
Mushrooms 0 85 to 90 5 days
Okra 10 85 to 95 2 weeks
Onions 0 70 to 75 6 to 8 months
Parsnips 0 90 to 95 2 to 4 months
Peas, green 0 85 to 90 I to 2 weeks
Peppers, green 7.2 85 to 90 8 to 10 days
Potatoes 3.3 to 4.4 85 to 90 6 to 9 months
Pumpkins 10 to 12.8 70 to 75 2 to 6 months
Rhubarb 0 90 to 95 2 to 3 weeks
Rutabagas 0 90 to 95 2 to 4 months
Spinach 0 90 to 95 10 to 14 days
Squashes
Summer 4.4 to 10 85 to 95 2 to 3 weeks
Winter 10 to 12.8 70 to 75 4 to 6 months
Sweet potatoes 12.8 to 15.6 80 to 85 4 to 6 months
Tomatoes
Ripe 4.4 to 10 85 to 90 7 to 10 days
Mature green 12.8 to 21.1 80 to 85 3 to 5 weeks
Turnips-topped 0 90 to 95 4 to 5 months
555
TABLE 11.6. COMMODITIES SUSCEPTIBLE TO COLD INJURY WHEN STORED AT
ONLY MODERATELY LOW TEMPERATURES
Fruit or Vegetable
Apples
Certain varieties
Avocados
Bananas
Green or ripe
Beans-snap
Cranberries
Cucumbers
Eggplants
Grapefruit
Lemons
Limes
Mangoes
Melons
Cantaloupe
Honeydew
Casaba
Crenshaw and Persian
Watermelons
Okra
o lives, fresh
Oranges, California
Papayas
Peppers, sweet
Pineapples
Mature-green
Potatoes
Chippewa and Sebago
Squash, winter
Sweet potatoes
Tomatoes
Mature-green
Ripe
Approximate
Lowest Safe
Temperature
(0C)
1.1 to 2.2
7.2
13.3
7.2 to 10
1.1
7.2
7.2
7.2
12.8 to 14.4
7.2
10
7.2
4.4 to 10
4.4 to 10
4.4 to 10
2.2
4.4
7.2
1.7 to 2.8
7.2
7.2
7.2
4.4
10 to 12.8
12.8
12.8
10
Character of ~ u r y When Stored
Between OOC and Safe Temperature
Internal browning, soggy breakdown
Internal browning
Dull color when ripened
Pitting increasing on removal, russet-
ing on removal
Low-temperature breakdown
Pitting, water-soaked spots, decay
Pitting or bronzing, increasing on re-
moval
Scald, pitting, watery breakdown, inter-
nal browning
Internal discoloration, pitting
Pitting
Internal discoloration
Pitting, surface decay
Pitting, objectionable flavor
Discoloration, water-soaked areas, pit-
ting decay
Internal browning
Rind disorders
Breakdown
Pitting, discoloration near calyx
Dull green when ripened
Mahogany browning
Decay
Decay, pitting, internal discoloration
Poor color when ripe; tendency to de-
cay rapidly
Breakdown
holding at 21C for three days reduced the chilling injury of lemons
when stored at 1C for twenty-one days (McDonald 1986). However, film
wrapping of mature green bell peppers did not reduce the incidence of
chilling injury (Miller and Risse 1986). The treatment that might be used
to alleviate chilling injury depends upon the fruit or vegetable. Systems
for controlling chilling ~ u r y have been discussed for avocados (Chaplin,
Wills, and Graham 1983), pineapples (Paull and Rohrbach 1985), and
muskmelons (Lipton and Aharoni 1979).
When cucumbers are stored at 7C or lower for ten days or more,
they develop pits, dark-colored watery patches, and tissue collapse. These
damaged tissues can readily become infected with microorganisms that
cause rotting. A temperature of lOoC and 95 percent RH are the best
conditions for storing and transporting cucumbers (Etchells et al. 1973).
Potatoes can heal injured areas of the skin if held at normal room
temperatures. This wound healing is very slow at lOoC. Sweet potatoes
need a curing treatment at about 29.5C and a humidity of 85 percent
to heal cuts and abrasions. After this treatment, 12.5C is recommended,
since chilling injury occurs at lower temperatures. The injury causes
darkening of the tissues and makes the potatoes susceptible to rotting.
Some vegetables deteriorate rapidly if not placed promptly into low-
temperature storage. Quick cooling and storage at 0.5 to 1.5C are
needed for corn, peas, and green lima beans_ Leafy vegetables, beets, and
carrots need quick cooling and storage at 0.5 to 4.5C (the temperature
depending on the vegetable) to maintain their quality.
Canned Food. Various biochemical reactions can occur in canned foods
during storage. These changes include loss in organoleptic quality, alter-
ations in chemical constituents, and losses in nutrients. These reactions
are generally reduced when the canned food is held in cold storage (lOC
or less), than at ambient-temperature storage. The extent of any benefit
depends upon the type of food.
Freezing
Foods are frozen to extend their storage life over that obtained in a
refrigerator. The food is preserved by using temperatures low enough
to stop or greatly reduce the deterioration caused by microorganisms,
enzymes, or chemicals such as oxygen. It is generally recognized that
freezing offers one of the best means of maintaining the fresh color, fla-
vor, and appearance of many foods.
GENERAL ASPECTS. Pure water at atmospheric pressure freezes at
OC_ As the temperature is lowered below OC, the vapor pressures of
water and ice are reduced so that the water activity below -lOoC is lower
than that necessary for the growth of most bacteria (Table 11.7).
The fluids in plant or animal tissues are aqueous solutions_ The freez-
ing points of these solutions may range from 0 to -lOoC (Table 11.1).
When food is subjected to a temperature below freezing and the extracel-
lular water begins to freeze, the solutes in this water tend to migrate to
the remaining liquid_ This increases the concentration of solutes and low-
TABLE 11.7. VAPOR PRESSURES AND WATER ACTIVITY OF WATER AND ICE
Temperature (0C)
o
-5
-10
-15
-20
-25
-30
-40
-50
Vapor Pressure
Liquid Water
(mm Hg)
4.579
3.163
2.149
1.436
0.943
0.607
0.383
0.142
0.048
Ice
(mmHg)
4.579
3.013
1.950
1.241
0.776
0.476
0.286
0.097
0.030
Water Activity
(VPkcNP ",a <or)
1.00
0.95
0.91
0.86
0.82
0.78
0.75
0.68
0.62
ers the freezing point of this liquid. The extent of solute concentration
is influenced by the product characteristics and the rate of freezing.
Some solid material is occluded within or among the ice crystals, the
amount depending upon the freezing rate.
As the solute concentration of the extracellular liquid increases, an
osmotic differential occurs and water tends to leave the plant, animal, or
microbial cells. Slow freezing causes a greater degree of solute concentra
tion and dehydration of the cells than does rapid freezing.
When a liquid is cooled slowly, solidification commences at a rela
tively small number of places, and the resultant crystals grow to large
sizes. Rapid cooling, on the other hand, results in the initiation of a large
number of smaller crystals. The rate of freezing, therefore, may alter the
physical structure of the tissue. Rapid freezing of food is desired.
The rupture of the cellular structure may result in a poor texture of
the thawed food. The exudation of juices, resulting in "drip," may occur
in frozen and thawed plant and animal tissue. It contains dissolved pro-
tein, other nitrogenous substances, vitamins, and minerals.
Although most of the water in a food is frozen at - 20DC, there is
usually a certain amount of bound water that does not freeze. This unfro-
zen water is considered to be unavailable for biological activity, so it
should not affect microbial deterioration of the frozen food.
SYSTEMS FOR FREEZING. Heat can be removed from foods by con-
vection, conduction, evaporation, or radiation. Commercially, freezing
systems use convection, conduction, or combinations of these. The prod-
uct may be frozen before packaging, after packaging, or even after casing.
Packaged foods freeze more slowly than unpackaged foods, given the
same conditions of freezing, due to the insulating effect of the packaging
material.
Systems developed for freezing food include the following:
Plate freezer (single, double, or pressure)
Still air (sharp freezer)
Air blast (room, tunnel)
Slush freezer
Fluidized bed
Liquid immersion (brine, dichlorodifluoromethane)
Liquefied gases (C0
2
, N
2
)
Freezant spray
The system selected for use depends on the type of food, speed of freez-
ing, and cost factors_ Although relatively slow, tunnel air-blast freezing
can be used for most foods_
Fluidized bed freezing is a means ofrapidly freezing small particulate
materiaL It is the best system for the production of individually quick-
frozen (IQF) foods_ Mattick, Rawnick, and Ellis (1982) described a fluid-
ized system in which the food travels through a tunnel on a stainless
steel wire-mesh belt. Blasts of cold air (- 34C) from underneath the belt
agitate, fluidize, and freeze each individual food particle_ Symons (1982)
described a fluidized bed with no belt. The holes in the trough carrying
the food were angled so that the air coming through moves the food
upward and forward. According to Symons, there is no moving part in
the fluidized bed; only the air moves_ Advantages are that the food is
frozen quite rapidly and individually, so an entire carton of food does
not need to be thawed when only a small portion is needed for use.
Liquid is a better conductor of heat than is air, so more rapid freezing
is accomplished by liquid immersion freezing than by air-blast freezing.
Since the liquid refrigerant is in direct contact with the food, it must
meet the FDA criteria for food additives. Only a few liquid refrigerants
are acceptable. Solutions ofNaCl or CaCh have been used for immersion
fluids. Another fluid used for immersion freezing is dichlordifluorometh-
ane (Freon 12), at - 29.8C. This freezant is under scrutiny by the Envi-
ronmental Protection Agency because it is suspected to react with and
damage the ozone layer in the atmosphere, which is needed to protect
the earth from the sun's UV radiation. Although the system is enclosed
so the fluorocarbon can be recycled, some losses may occur.
For very rapid freezing (cryogenic freezing), liquefied gases (C02 or
N2) are used, either as immersion liquids or sprays. There are certain
advantages for using liquid nitrogen freezing. A nitrogen spray system is
shown in Figure ILL Since the food is frozen in an inert atmosphere
of nitrogen, surface oxidation is prevented. The rapid freezing and low
temperature (- 196C) prevent microbial growth. There are problems
associated with this cryogenic freezing_ The sudden reduction in the tem-
perature of food by direct contact with liquid nitrogen can cause shatter-
Figure 11.1. Liquid nitrogen flash freezer.
courtesy of Farrall (1976).
ing of the food. Other materials, such as plastics, rubber, or glass, become
brittle and can crack or shatter due to the thermal shock caused by liquid
nitrogen.
STABILITY OF FROZEN FOODS. Some aspects that affect the storage
life of frozen foods are the treatments prior to freezing, the type of pack
aging, the temperature, fluctuations in the temperature, and conditions
for thawing of the frozen food. The frozen storage life of selected foods
is listed in Table 11.S.
The lower the temperature of storage, the longer the storage life of
the product. The deterioration of foods can occur at high freezer storage
temperatures. Many enzymes are not inactivated by these temperatures.
To help control enzymatic activity, some foods are given a heat treatment
(blanching) to destroy the enzymes prior to freezing.
Good packaging materials prevent the diffusion of oxygen into the
package to retard rancidity or oxidation. Also, the package will prevent
evaporation of moisture from the frozen food. The addition of ascorbate
or various antioxidants to certain frozen foods enhances the storage life
by retarding oxidative changes.
The freezing rate affects the storage life of frozen foods. Slow freezing
conditions may allow a food to remain at temperatures above OOC for
several hours. The quality lost due to slow freezing may be equivalent to
that lost during several months of storage at -lSoC.
TABLE 11.8. APPROXIMATE STORAGE LIFE (MONTHS) FOR QUALITY
MAINTENANCE OF SELECTED FOODS
Temperature C
Product -17.8 -12.2 -6.7
Orange juice (blanched) 27 10 4
Peaches 12 <2 0.2
Strawberries 12 2.4 0.3
Cauliflower 12 2.4 0.3
Green beans 11-12 3 1
Green peas 11-12 3 1
Spinach 6-7 <3 0.75
Raw chicken 27 15.5 <8
Fried chicken <3 <1 <0.6
Turkey pies or dinners <30 9.5 2.25
Beef (raw) 13-14 5 <2
Pork (raw) 10 <4 < 1.5
Lean fish (raw) 3 <2.25 < 1.5
Fat fish (raw)
2 1.5 0.8
If a malfunction occurs during storage, transportation, distribution,
or retailing, the frozen food can be subjected to defrosting temperatures.
In many cases, it is necessary to defrost the frozen food prior to use.
Thus, defrosting is of concern whether it is accidental or deliberate.
If a food is only partially defrosted, there is usually no harmful effect,
at least from a public health viewpoint. This is also true concerning a
product that is thawed but still cold. These foods usually can be refrozen
with no potential problems. If a food becomes thawed and warmed to
room temperature, it mayor may not be safe to refreeze, depending
upon the type of food and the time that it has been at room temperature.
For usage, fast thawing of frozen foods helps retard bacterial growth,
as well as loss of quality attributes. The rate of thawing usually is lower
than the rate of freezing. This is because of the lower temperature differ
ential during thawing than during freezing, and because the rate of heat
transfer is lower through the defrosted surface than through the frozen
surface during freezing.
Some foods can be cooked from the frozen state without intermediate
thawing. With other foods, thawing in the refrigerator usually is sug-
gested because the temperature of the defrosted food will remain low
enough to prevent the growth of potential pathogens. Thawing in warm
water will hasten the process, but if the warm water is in direct contact
with the food, nutrients from the food will be dissolved into the water
and lost. With this method of thawing a packaged frozen turkey, the outer
part of the turkey will be warm enough to support the growth of spoilage
and potentially pathogenic organisms while the inside is still frozen. A
microwave oven is useful for tempering frozen foods used in further
processed items.
ADVANTAGES AND DISADVANTAGES OF FREEZING. The advan-
tages of freezing as a method of preserving food are that it (1) neither
adds nor removes any components; (2) imparts no new flavor nor alters
the natural flavor; and (3) does not diminish the digestibility or cause a
significant loss of nutrient value.
Some disadvantages of freezing are as follows: (1) microorganisms
may be reduced in number but not destroyed; (2) spores are very resistant
and toxins are not destroyed; and (3) frozen foods not properly wrapped
dehydrate very rapidly, which causes marked deterioration in the flavor
and general appearance of the food.
SPECIFIC FOODS. Besides the general aspects of freezing foods, cer-
tain types of foods have some special problems, conditions, or character-
istics.
Red Meats. The storage life of frozen meat depends upon the initial qual-
ity, condition, and pH of the meat, the interval between slaughter and
freezing, the method of preparation, the temperature of holding before
freezing, package method and materials, method and temperature of
freezing, and the temperature and other conditions of frozen storage.
The cryogenic freeezing of meat was discussed by Sebranek (1982).
The quality of red meats can be maintained if the meat is packaged
in a wrapper impermeable to oxygen and moisture, rapidly frozen, and
then stored below -18C. Beef and lamb can be stored for one year or
more if properly protected from dehydration (Table 11.8). Pork, espe-
cially smoked or cured, has poorer storage life. However, it can be held
for six months or more if oxygen is excluded from the product and dehy-
dration is prevented. The fatty components of meats are susceptible to
changes in odor and flavor, with pork fat being more susceptible than
beef or lamb.
Fresh meats are seldom frozen for retail sale to consumers. Surveys
indicate that consumers prefer to buy fresh meat. However, most of this
fresh meat is then frozen in home freezers. Freezing in commercial freez-
ers would produce a more satisfactory product.
Meats preserved by freezing are stored at temperatures low enough
to prevent microbial growth. Freezing with subsequent thawing will kill
some microbial cells. Microorganisms tend to die slowly during storage,
but those that survive will grow in the thawed meat at a rate similar to
that exhibited by the same strain in raw meat (at the same temperature)
that had never been frozen. The rate of microbial growth in thawed meat
is dependent upon the temperature of the meat surface.
A condition called freezer burn may occur on the surface of frozen
meat. It appears as patches of lightcolored tissue and gives the meat a
bleached, unattractive appearance. The cause is similar to a freeze drying
system in which ice crystals are sublimated. The control of freezer burn
is aided by packaging in a moistureimpermeable film.
Poultry. Poultry meat that is frozen should be fresh, well finished, and
properly packaged. Although the quality is not improved, freezing
should maintain the quality as closely as possible to the value at the time
of freezing. Freezing can affect 'the appearance, the exudation of fluid
(drip) on thawing, and a progressive loss of flavor, tenderness, andjuici
ness.
Freezer burn can occur on frozen poultry in a manner similar to that
in red meat. With poor packaging, this dehydration can occur in one
month or less of storage.
The long bones in young frozen poultry (under fifteen weeks of age)
usually are discolored. The freezing and thawing processes hemolyze the
red blood cells, releasing hemoglobin. The hemoglobin is able to pass out
of the inner porous bone. This darkening is a normal condition found in
frozen poultry and in no way affects the food value or flavor of the meat.
It does affect the attitude of the consumer concerning frozen poultry,
however. Bone darkening was nearly absent when poultry was frozen in
liquid nitrogen at -196C (Cunningham 1974).
There was little change in the quality of broilers through five thaw
ings and refreezings (Baker et al. 1976). The microbial load increased
slightly after the five thawings and refreezings. The bacterial load on the
chickens before freezing was 3.8 (10
7
) CFU per chicken, and after five
thawings the loads were 2.1 (l08) and 3.0 (l08) CFU per bird for fast and
slow freezings, respectively. Thawed poultry does not spoil more rapidly
than poultry that has not been frozen.
Chickens are generally retailed in the refrigerated fresh form, but
turkeys are usually packaged in an evacuated, tightfitting plastic film in
the frozen condition. Turkey freezes at a faster rate in evacuated pack
ages than in unevacuated packages. Microwavedefrosted frozen chicken
breasts have been found to be more tender than those thawed in a home
refrigerator (Younathan, Farr, and Laird 1984).
Seafoods. The s t o ~ g e life of frozen fish is significantly longer than that of
iced or refrigerated fish. It is desirable to lower the temperature rapidly.
The frozen fish should be stored at - 20C and, for some fish, even
- 40C (Gates et al. 1985; Jiang, Ho, and Lee 1985).
The main deteriorative alterations in frozen seafoods are flavor
changes such as rancidity, toughening or loss of tenderness, formation of
drip, development of freezer burn, and gaping. Some of these changes
are due to protein denaturation and breakdown of fat. Changes of fish
during storage have been the subject of studies (Kelleher et al. 1982; Ron
sivalli and Baker 1981).
The storage life for seafoods is generally shorter than that for either
red meat or poultry (Table 11.8). The storage life of frozen salmon or
whitefish was improved by vacuum packaging in low oxygen permeable
film (Josephson, Lindsay, and Stuiber 1985; Yu, Sinnhuber, and Crawford
1973).
Egg Products. When egg yolk is frozen and thawed, it becomes a viscous,
rubbery mass. This irreversible increase in apparent viscosity is known
as gelation. Certain additives, such as sugar (sucrose), fructose, galactose,
or salt, will reduce gelation of the frozen and thawed yolk. Proteolytic
enzymes added to the liquid will partially inhibit gelation. If the egg yolk
is frozen rapidly, such as with liquid nitrogen, there will be less gelation
than when it is frozen by slower freezing systems. Rapid thawing also
reduces gelation. Rapid freezing, combined with rapid thawing, is more
effective in preventing gelation of frozen and thawed egg yolk than
either one alone.
When sugar or salt is added to yolk, the freezing point is lowered.
The temperature of the liquid must be reduced to -12C within 60 hr
after pasteurization. The eggs freeze from the outside of the carton
toward the middle, so it is a race between ice formation and microbial
growth. If the bacteria win, the product becomes sour. With pasteuriza
tion to destroy Salmonella, many of the spoilage bacteria are destroyed.
Hence, there should be less spoilage today than in the prepasteurization
era.
Dairy Products. These are frozen as a means of preservation, or in the
manufacture of ice cream or ice milk.
The freezing point of milk varies within relatively narrow limits. The
average freezing point is considered to be - 0.55C. The determination
of the freezing point provides a method of detecting added water. If wa
ter is added to milk, the freezing point is closer to OC. There is a linear
relationship between the hydrolysis of lactose to monosaccharides and
depression of the freezing point of milk (Jeon and Bassette 1982).
The deterioration in physical stability is one of the principal defects
encountered in frozen milk. During frozen storage, the proteins become
unstable and when the milk is thawed, the proteins settle out. Besides
the appearance, the milk has an undesirable chalky texture.
Fruits and Vegetables. Most of these products can be frozen and stored at
-17.8C for several months. The quality losses include color, texture
and flavor. The factors that influence the quality of the frozen product
include the initial quality, the type and variety of the fruit or vegetable,
processing operations, freezing temperature, packaging, and storage
temperature (Ahvenainen and Miilkki 1985; Kramer 1979).
Some varieties of certain fruits and vegetables withstand freezing and
frozen storage much better than other varieties. Freezing does not im
prove quality, so only high-quality products should be frozen.
Most vegetables must be heat treated (blanched) before freezing to
inactivate the enzymes, or these catalysts may cause flavor or color deteri-
oration during storage.
Baked Goods. Bakery products are especially adaptable to frozen storage.
Although products such as crackers and cookies generally are not frozen
commercially, cookies can be frozen at home to extend the storage life.
The stability of frozen dough was affected more by final freezing temper-
ature than freezing rate_ Freezing without fermentation produced better-
quality bread than freezing after fermentation. Damage to yeast cells is
a major problem in frozen doughs (Rsu, Roseney, and Seib 1979; Wolt
and D'Appolonia 1984a, 1984b).
The freezing point of most bakery products is between - 7 and
- 2C. The richer doughs freeze at the lower temperatures. Some ad-
juncts, such as jams or frostings, require even lower temperatures for
freezing.
Miscellaneous Foods. The freezer cases in the retail grocery stores contain
frozen dinners, pot pies, pizzas, and various mixtures of many types of
foods. Although there has been much concern about the possible health
hazards of certain precooked foods, they have had a good record for
noninvolvement in foodborne disease outbreaks.
MICROBIOLOGICAL ASPECTS. Although freezing is not a method
for sterilizing food, many of the associated microbial cells are killed or
damaged by freezing, frozen storage, and thawing. Various treatments
before freezing can affect the microorganisms associated with food.
Calcott and MacLeod (1974) listed reasons for the cryoinjury of cells.
These are (1) thermal shock; (2) effect of concentration of extracellular
solutes; (3) toxic action of concentrated intracellular solutes; (4) dehydra-
tion; (5) internal ice formation; and (6) attainment of a minimum cell
volume.
Freezing and thawing can damage the cell wall, membrane, ribosome,
and DNA. Cell wall damage results in loss of resistance of Gram-negative
cells to various agents such as bile salts, sodium lauryl sulfate, and lyso-
zyme (Ray, johnson, and Wanismail1984; Takano et al. 1979). Cell mem-
brane damage allows leakage of various compounds (proteins, phospho-
lipids, lipopolysaccharides) from the cell (Calcott et al. 1984; Souzu 1980).
The wall and membrane damage can be repaired (Calcott, Steenbergen,
and Petty 1979). Freezing of yeast induced a decrease of respiratory activo
ity (Mori, Suzuki, and Nei 1986). A loss of ribonucleic acid (RNA) from
the cell is associated with ribosome damage. The DNA may have both
single and doublestrand breaks (Grecz and ElZawahry 1984; Grecz et
al. 1980). According to ElZawahry and Grecz (1981) freezing caused sub
stantially greater cell injury to Y. enterocolitica than did radiation.
We are concerned with the lethal effects of freezing foods because we
would like to destroy as many cells as possible without destroying the
organoleptic or physical quality of the food. On the other hand, freezing
is used to preserve cultures for research as well as those organisms used
in food fermentations, as a source of enzymes, or as a source of single
cell protein.
There are many conflicting reports in the literature concerning the
effects of freezing on microbial cells. Part of the problem may be due to
the use of different types of microbial cells in various suspending media
which are cooled at various or unknown rates. The response of microor
ganisms is dependent upon many factors. Those that have been consid
ered include (1) the type, species, and strain of the microorganism; (2)
the age, population density, and nutritional status of the cells; (3) the
cooling rate; (4) the minimum temperature to which the cells are cooled;
(5) composition, pH, and type of suspending medium, including the pres
ence of protective agents; (6) the temperature and time of storage; (7) the
rate of warming or thawing; and (8) the methods used to determine cell
viability.
Bacterial spores are the most resistant microbial entity to freeze dam
age. Grampositive cells are more resistant to freezing and thawing than
are Gramnegative cells. Survival of microorganisms is greater in a super
cooled than in a frozen environment.
The cooling rate during freezing can affect the survival of micro organ
isms. At low cooling rates, the extracellular water begins to freeze and
form ice crystals. The solutes are concentrated into the remaining water.
This causes the water to emigrate from the cells due to the osmotic pres
sure gradient between the intracellular and extracellular solutions or the
vapor pressure difference between the supercooled intracellular water
and the extracellular ice. The slower the process of cooling and the
greater the permeability of the cells, the greater will be the loss of water
from the cells. The movement of water out of the cells concentrates the
solutes of the cells, lowers the intracellular vapor pressure, and dehy
drates the cells. The cell volume also decreases and solutes precipitate.
The maximum survival of Escherichia coli is at a cooling rate of about
6C/min, and the minimum at about 1000C/min (Calcott and MacLeod
1974). Calcott, Lee, and MacLeod (1976) determined the survival of Strep
tococcus faecalis, Salmonella typhimurium, Klebsiella aerogenes, Pseudomonas aero
uginosa, and Azotobacter chroococcum at various cooling rates. The cooling
rate for optimum survival varied from 7C/min for A. chroococcum to 11C/
min for P. aeruginosa. The minimum survival for these five organisms was
found to be at about lOOC/min. At higher cooling rates, the survival
increased.
Meyer, Sinclair, and Nagy (1975) studied the effect of cooling rate on
a mesophilic yeast and a psychrophilic yeast. The optimum cooling rate
for survival of both yeasts was between 4.5 and 6.5C/min. The level of
survival varied considerably even at the optimum cooling rate. The sur
vival of the psychrophilic yeast varied from 5 percent when cooled from
the exponential phase, to 71 percent when in the stationary phase. The
mesophilic Candida utilis survival for exponential phase cells was 47 per
cent, and for those in the stationary phase it was 71 percent.
Four Gramnegative bacteria studied by Calcott, Lee, and MacLeod
(1976) showed less survival when frozen in saline than in water. However,
the Grampositive S.faecalis did not show this reduction in survival. They
believed the difference might be due to the respiratory systems of the
bacteria. The Gramnegative bacteria respire aerobically with cyto
chrome systems integrated into the cytoplasmic membranes. The S. fae
calis is anaerobic and has no cytochrome system.
At very high cooling rates, small crystals of ice are formed. Due to
their high surface energies, the small ice crystals tend to enlarge during
warming. Mazur (1970) stated that a forty fold decrease in survival of
yeast cells occurred when the time for warming from -70 to OOC was
increased from 0.06 sec to 3.6 sec. The damage apparently can be very
rapid and is thought to be due to the growth of the intracellular ice crys
tals, rather than to the initial ice formation.
The enlarging intracellular ice crystals exert sufficient force to rup
ture plasma membranes. Cells killed by intracellular freezing suffer memo
brane damage and become leaky.
High thawing rates result in greater survival of fastfrozen cells than
do low thawing rates. Enhanced recovery of slowly frozen cells by slow
thawing is believed to be due to the ability of cells to reconstitute more
readily during slow thawing. Rapid thawing of the dehydrated cells
causes a rapid entrance of water, which may cause excessive internal pres
sure and resultant rupture of the membranes.
Cryoprotective Agents. There are many substances that protect the micro
bial cell during freezing and thawing. Some, such as glycerol and dimeth
ylsulfoxide, penetrate the cells, while others do not. Besides glycerol and
dimethylsulfoxide, protective agents include egg white, carbohydrates,
peptides, serum albumin, malic acid, milk, glutamic acid, yeast extract,
diethylene glycol, Tween 80, dextran, sodium glutamate, glucose, polyeth
ylene glycol, erythritol, polyvinyl-pyrrolidone, ionophores, acetamide,
and urea_ Also, mixtures of cryoprotectants have been used.
Glycerol reduces damage to the cell wall and membrane, while Tween
80 prevents only membrane damage (Calcott and MacLeod 1975). The
mechanism of action of glycerol is not fully understood. The presence of
glycerol decreases the mole fraction of solutes in the suspending fluid at
freezing temperatures and prevents the high concentration of solutes
that might affect the cell wall and membranes.
Microorganisms in Frozen Foods. Many factors influence microorganisms in
foods that are frozen. During freezing, the solutes and some microorgan-
isms become concentrated in the unfrozen portion. Until the temper
ature is reduced below the minimum temperature for growth, presum
ably some microorganisms can multiply. However, the solutes can
become concentrated to the level that microbial growth is inhibited. Mi-
crobial growth can occur in frozen foods stored at -7C and possibly
even at -12C.
The greatest reduction in the microbial load occurs during or shortly
after the freezing of foods. During frozen storage, the numbers are grad
ually reduced further.
Although food that is being thawed is susceptible to microbial growth,
there is generally a long lag phase before growth commences if the tem
perature is kept near OC.
Although salmonellae, staphylococci, and other potential pathogens
can survive freezing and frozen storage, the saprophytic flora tends to
inhibit their growth. During freezing and thawing of food, the temper-
ature favors the growth of the psychrophilic organisms. Hence, in nearly
all cases, if a frozen product is mishandled, spoilage is apparent before
the food becomes a health hazard.
There have been suggestions that freezing foods disrupts the cellular
structure of flesh foods and that bacteria can grow more readily on
thawed meat that was frozen than on unfrozen meat. Sulzbacher (1952)
found no indication that frozen meat was more perishable after thawing
than was fresh meat. Elliot and Straka (1964) reported that frozen and
thawed chicken decomposes at about the same rate as unfrozen chicken.
Thawed meats, like fresh meats, are perishable and should not be held
very long before cooking.
In some studies of the microorganisms associated with frozen foods,
the total number of aerobic bacteria was determined by incubation at
37C. At this temperature, mesophilic types are enumerated, but the psy
chrophiles are the important potential spoilage organisms in frozen
foods. Incubation should be conducted at 20C or less to obtain an esti
mate of psychrophilic types.
The microbial characteristics of frozen seafoods are similar to those
of red meat and poultry. During freezing of seafoods, from 60 to 90 per
cent of the bacterial population is inactivated. During frozen storage,
there is a gradual decline in the number of cells. Grampositive cells sur
vive frozen storage better than do Gramnegative cells. As with red meats
and poultry, defrosted fish spoils at about the same rate as unfrozen fish.
Poliovirus inoculated into oysters showed a gradual decline in
plaque-forming units (PFU) during frozen storage at - 36C (DiGiro
lamo, Liston, and Matches 1970). About 10 percent of the original PFU
survived twelve weeks of storage.
Vibrio parahaemolyticus is a potential pathogen associated with fishery
products. It is generally stated that this organism does not survive freez-
ing and frozen storage. V. parahaemolyticus cells were inoculated into
oysters, fillets of sole, and crabmeat (Johnson and Liston 1973). Although
there was a sharp reduction in vibrios during freezing, these organisms
persisted in these seafoods stored at either -15 or - 30C, with better
survival at - 30C. They were found in oysters after 130 days, the end of
the experiment.
The microbiology of frozen egg products is similar to that of other
foods. During freezing, there may be microbial growth and, in poorly
frozen product, spoilage may occur. With proper freezing, there is usu
ally a reduction in the microbial load. During frozen storage at -15 to
-18C, the microbial load may show a slight decline. There is a 50 to 85
percent reduction in bacteria within sixty days of frozen storage.
The microbial load on raw vegetables may reach 1Q6/g. Washing and
blanching (95C for 1 to 3 min) will reduce the number of microorgan-
isms. In commercial practice, the vegetables are recontaminated by
equipment, so that the microbial load at the time of freezing may be
higher than that of the raw vegetables.
When slow cooling rates are used for freezing vegetables such as corn
or peas microbial growth may occur before the product is frozen. How-
ever, the inactivation of cells during the freezing process helps compen
sate for any increase in numbers. Overall, there may be a slight decrease
or increase in the microbial load. Fast cooling rates and freezing result
in essentially no change in the microbial count of vegetables. The 30C
aerobic plate count of frozen vegetables was reported as 45,000/g of cauli-
flower, 8,500/g of corn, and 6,800/g of peas (Barnard et al. 1982). The
highest aerobic plate count of various frozen potato products was
3,600/g (Duran et al 1982).
There is a greater reduction in the bacterial load if the product is
stored at -12C rather than at either -18 or - 23C. However, quality
may suffer at the warmer temperature. A temperature of - 4C is not
sufficiently low to inhibit microbial growth in vegetables such as peas.
Some of the dominant organisms in frozen vegetables are species of
Micrococcus, Flavobacterium, Streptococcus, Pseudomonas, Lactobacillus, and
Enterobacter.
When frozen fruits are held at -4C or higher, yeasts and molds have
a tendency to grow. Growth usually does not occur if the fruit is packed
in an airtight container. At these temperatures, the respiration of the
fruit replaces the oxygen with carbon dioxide. This, along with the low
pH, inhibits the growth of most types of microorganisms.
DRYING
Drying is thought to be the oldest method used to preserve food. Early
in time, man observed that naturally dried seeds could be stored from
one season to the next. The sun drying of meat, fish, and fruit has been
practiced for many centuries. These dried foods tended to have their
own characteristic flavor, aroma, and texture. The drying process caused
irreversible changes; for example, grapes became raisins, and the rehy-
drated food was different from its fresh counterpart.
In order to compete with fresh foods or with other preservation meth
ods, irreversible reactions of dried foods should be kept to a minimum.
Most commercially dried foods are produced with controlled conditions
of temperature, humidity, and airflow. Since removal of moisture by heat
is relatively expensive and uses much energy, systems such as screening,
pressing, centrifuging, settling, filtration, reverse osmosis, osmotic solu
tions, or freeze concentration may be used to eliminate water prior to
final drying of the food.
Reasons for Drying
The obvious reason for drying food is to prevent microbial growth.
However, there are other reasons, including (1) to preserve the product
from physical or chemical changes induced or supported by excess mois-
ture; (2) to reduce the packaging, storage, and transportation costs; (3) to
prepare a material for a process when only dry materials can be used; (4)
to remove moisture added in previous operations; (5) to bring the prod-
uct to a moisture level at which it is normally graded, bought, and sold;
(6) to recover waste products; and (7) to provide convenience and a sav-
ing of time to users.
Dried foods weigh less and have reduced bulk. A thirty dozen case of
shell eggs weighs about 26 kg, while the dried product weighs about 5.1
kg. The dried eggs occupy about 16 percent of the space of the fresh
product. Thus, costs of transportation and storage are lower.
If properly packaged, most dried foods can be stored at ambient con-
ditions, with no special freezing or refrigeration requirements. One ad
vantage is that the package of dried food can be opened and part of it
removed for use, and the remainder can be expected to have an accept
able storage life. Due to their concentrated character, dried foods can be
used as ingredients to create new types of foods.
The reduced weight and bulk, the good storage life, and the versatility
of dried food are important, especially during wartime. In the past, dried
foods were exploited during wartime and then more or less forgotten.
However, after World War II, although the amount of drying decreased,
the technology that had been developed was used to produce consumer
goods, such as instant dried milk and dried potato products. Also several
mixes, such as various dried soups and cake mixes, have been developed.
Pretreatments
Only food of good quality should be used for drying. Generally, qual
ity is not improved by drying.
The usual and necessary washing, cleaning, breaking, peeling, slicing,
and other operations are carried out prior to drying. The operations
needed depend upon the type of food to be dried.
Some foods require a heat treatment prior to drying. Quite often,
foods such as meats are cooked before dehydration. Cooking meat can
reduce the moisture content by 20 percent or more. Cooking before dry
ing adds a useful convenience factor to dried foods. Also, cooking reo
duces the number of viable microorganisms.
Potatoes are cooked prior to drying, so the dried product is ready for
use merely by adding water and mixing. Instant rice is. prepared by a
cooking process and redrying of the product.
Blanching is an important pretreatment for dehydrated vegetables.
This process inactivates enzymes, expels air from tissues, yields a more
tender product on reconstitution, minimizes loss of palatability, delays
the development of odors, reduces color loss, helps retain carotene and
ascorbic acid during dried storage, and lengthens the storage life.
The peel, as well as the waxy coating on fruits, inhibits the removal
of water from fruits. Dipping fruits and berries into a weak sodium car
bonate or lye solution produces fine cracks in the skin, removes the out
side waxy covering, and facilitates drying.
Fruits are given a sulfite treatment to prevent browning during dry
ing and storage. Antioxidants may be added to foods to help prevent
oxidative changes in the dried product.
For eggs and egg white, the reducing sugars are removed by fermenta
tion or treatment with glucose oxidase. This is to prevent the Maillard
reaction in the dried products.
Other pretreatments may consist of concentration by reverse osmosis,
ultrafiltration, or other methods. Milk may be concentrated to 20 to 40
percent solids by evaporation in vacuum pans before spray drying. The
osmotic concentration of potatoes in a 45 percent sucrose/I 5 percent salt
solution before solar drying resulted in a two- to fivefold increase in the
throughput of product (Islam and Flink 1982). This system can be used
for various foods.
Various ingredients may be blended before drying to impart desired
functional traits to the dried product.
Drying Aspects
Water is associated with foods as (1) water containing solids in solu-
tion (dissolved salts or other solutes); (2) water containing solids in sus-
pension; (3) water occupying the pores or interstices of a solid; (4) water
adhering to the surface of a solid; (5) chemically combined water (water
of hydration); and (6) hydroscopic water that is naturally absorbed by a
substance from an atmosphere containing water vapor. Generally, water
is considered to be bound or free (Chung and Chang 1982; Rizvi and
Benado 1984).
The removal of water during drying requires the addition of heat for
evaporation. The water migrates within the material to the surface. Here
it is evaporated into the atmosphere surrounding the product and then
removed from the vicinity of the food_ The rate of removal of moisture
from the surface is dependent upon the temperature, humidity, and
movement of the surrounding air. The size, shape, and other characteris-
tics of the food particle will influence moisture migration from the cen-
ter to the surface.
The usual drying curve shows a constant rate period followed by a
falling rate period (Fig. 11.2). According to Szentgyorgyi and Orvos
(1985), four periods are involved: a period of constant drying rate, a first
falling rate period, a second falling rate period, and a final period of
drying. During the constant rate period, moisture travels to the surface
of the food quite rapidly so that the food is kept moist. When the rate of
migration of water from the interior cannot maintain a sufficient flow to
the surface to sustain the initial rate of evaporation, the constant rate
period is terminated. The drying rate declines, becoming very small as
the water content of the food approaches equilibrium.
During the drying process, some portions of the food will dry more
quickly than others. The wet areas will be at lower temperatures than the
dry portion, due to evaporative cooling.
In dehydrated foods, the sugars, acids, and inorganic salts are concen-
trated, which also lowers the a
w
of the foods and increases the preserva-
tive action.
2
2.4
2.2
2.0
... 1.8
'!
'"
'" 1.6
I L4
! 1.2
i
~
1.0
CIt
0.8
s
0.6
0.4
0.2
0
200
\_---..-Slope dw/de' 2.2/402
."

~
600
(minl
""-.
800 1000 1200
Figure 11.2. Drying curve showing slope for constant rate period.
Courtesy of Charm (1978).
Chemical Stability
Dried foods may have adverse quality characteristics when they are
compared to fresh foods. The alterations in quality may be due to enzy-
matic activities, protein denaturation, oxidation of lipids, or other reac-
tions during drying, storage, or reconstitution. The three factors that gen-
erally determine the type and extent of deterioration of dried food are
residual moisture, oxygen content, and storage conditions (time and tem-
perature) (McWeeny 1980). Quite often, a number of preservation tech-
niques must be used to obtain the desired dried food. For example, a
food may be heated to inactivate enzymes, dried to a low moisture,
treated with a chemical antioxidant, and packaged in an inert gas such
as nitrogen in a sealed container.
There is an optimum moisture content for the best storage stability
of each food. Complete dehydration may result in destruction of the
food. The monomolecular layer of moisture, referred to as bound water,
may be regarded as a protective film that protects the particles of food
from attack by oxygen.
Dried foods must be packaged in moisture impermeable materials or
stored in an atmosphere with a relative humidity similar to that of the
food. In mixtures of various dehydrated foods, moisture is transferred
from items of high moisture vapor to those of lower moisture vapor until
equilibrium is attained. Quite often, dried mixes include smaller pack
ages of ingredients within the main package in order to maintain the
components at the desired moisture level (Hong, Bakshi, and Labuza
1986; Iglesias, Viollaz, and Chirife 1979).
Drying preserves food as long as the material is kept dry. Reconsti
tuted dry products should be used as soon as possible since, after recon
stitution, these foods are perishable, being subject to microbial spoilage.
Types of Drying Systems
There are several systems that can be used to remove water from
foods. Some types of drying systems are listed in Tables 11.9 and 11.10,
and several are depicted in Figures 11.3 through 11.7. The choice of the
method depends upon the qualities that must be maintained, the sensitiv
ity of the food to heat damage, the rehydration characteristics, and the
cost of the process. In some cases, only one of the systems can be used
to obtain a commercially acceptable or marketable product.
The four principal methods of drying are (1) hot air drying, for foods
such as vegetables; (2) spray drying for liquids and semiliquids; (3) vac
uum drying for juices; and (4) freeze drying. There are various types of
TABLE 11.9. SOME SYSTEMS FOR THE DRYING OF FOODS
Types of Drier
Natural (solar)
Bin
Kiln
Cabinet or compartment
Tray or pan
Tunnel
Continuous belt (atmospheric or vacuum)
Fluidized bed
Roller or drum (atmospheric or vacuum)
Spray
Foammat
Freeze vacuum
Microwaves
Usual Food Types
Grapes (raisins), meats, fish
Grain, peanuts
Apples, some vegetables
Fruits and vegetables
Egg white
Fruits and vegetables
Vegetables
Potato granules, coffee whiteners, spices
Milk, fruit and vegetable purees
Milk, eggs, pureed foods
Fruit and vegetable juices
Chicken, shrimp, meat, coffee
Pasta products, finish drying of foods
TABLE 11.10. COMPARISON OF SOME DRYING SYSTEMS
Type
Continuous belt
(conveyor
convection)
Fluidized belt
Spray
Freeze vacuum
(freeze dry)
Microwave
Advantages
Relatively low cost; high produc
tion rate; continuous process
Gentle to product; lowenergy
operation
For liquid products or high
moisture content; can be used
for heatsensitive foods, short
drying time, and retention of
flavor, color, and nutritive val-
ues
High retention of color, flavor,
and nutritive values; rapid re-
hydration of product
Efficient energy usage; very
short drying time; high pro-
duction rates; space savings
Disadvan tages
Slow drying; large space needed;
slow product rehydration
Difficult cleaning of equipment
May require product concentra-
tion prior to drying and prod-
uct must be in solution, paste,
or slurry form.
Time consuming and costly;
batch operation
Delicate products may puff; less
economical for high-moisture
(over 50 %) products; may be
difficult to work with micro-
waves
driers that use these principal methods. For some products, combina
tions of these methods are used.
A combination of explosion puffing with hot air drying increases the
efficiency of dehydration as well as rehydration of the dried product
(Sullivan and Craig 1984). Also, the addition of low-frequency sound
waves allows the use of lower temperatures during drying (Swientek
1986).
NATURAL. Natural air drying is simply the exposure of the product to
the sun and the wind. .
Fish and meats are dried by natural methods in some areas of the
world. Since natural drying of fish is a slow process, if the temperature
is not satisfactory, microbial growth can occur and cause spoilage before
the moisture content is reduced to a level that will inhibit growth. It is
difficult to preserve fatty fish by this method.
Raisins can be produced by sun drying of grapes. Besides the simple
placement of grapes in trays in the sun, solar driers have been developed
for various agricultural products so that the food is protected from rain
(Eissen, Muhlbauer, and Kutzbach 1985; Wagner, Coleman, and Berry
1984).
To prevent spoilage of coconut during sun drying, a thin coat of
acetic acid was applied to the kernel surface (Nathan et al. 1979).
CONTROLLED. Controlled air drying is used to reduce the moisture
content of various crops to a level acceptable for storage. Warm or hot
air is blown through bins of the product. High temperatures affect the
enzymes, germination, and nutritive value of grains. Barley may become
unacceptable for malting when used in beer making if it is subjected to
very high air temperatures.
Air-suspension drying is used as a finishing stage of some spray-dry-
ing operations to obtain very low moisture levels of food. This is similar
in principle to the fluidized bed dryer.
The vibrating conveyor dryer is used to redry powdered products that
are moistened in the instantizing process.
Vacuum Drying. In this process, the food is placed in a chamber that can
be evacuated. Water is evaporated at a lower temperature than at atmo-
spheric pressure. At lower temperatures, less damage to the quality of
the food occurs.
Tunnel Drying. This system uses drying tunnels through which carts or
trucks containing trays of product to be dried are moved (Fig. 11.3). Hot
air is blown parallel or countercurrent to the movement of the food_
Fluidized Bed Drying. Fluidized beds are used for freezing, blanching, trans-
porting, and other procedures, as well as for drying (Fig. 11.4).
Fluidized bed dryers hold granular material in a turbulent suspension
by aeration. Hot air supports and dries the food particles. Due to the
intimate mixing, the process has a high thermal efficiency. It is particu-
larly suited for the final drying of partially dried particles.
To overcome problems of the fluidized bed, centrifugal fluidized beds
have been developed (Hanni, Farkas, and Brown 1976). In this system,
the food particles are subjected to a centrifugal force greater than gravi-
tational force. This increases the apparent density of the particles and,
by varying the centrifugal force, achieves smooth fluidization of the food
at any air velocity. With the increased air velocity, heat transfer is im-
proved, which helps prevent scorching or surface heat damage of the
food.
Drum Drying. In this system, the liquid or liquefied food is run as a thin
layer or film onto a revolving heated drum. The moisture is removed
almost instantaneously. The dried product is scraped off in sheets or bro-
ken into flakes as the roller is turned slowly (Fig. 11.5).
The drum drier is used in the production of dried foods, such as sweet
potato flakes, tomato flakes, instantized breakfast cereals, starch, and
soup mixes (Manlan et al. 1985).
Spray Drying. In this process, a liquid is sprayed with heated air into a
drying chamber. The atomized substance gives up its water very rapidly.
in
Dry

II aut
o. Countercurrent Tunnel Dryer
-....
...0(---- - - - - -.-(---
Trucks
Exhaust-air stack'
b. Parallel Current Tunnel Dryer
8/awtlr Htloltlr Frtlsh-oir inltlts --_,a". Htlottlr Fan
pppp , IIl1liq ----=""-....,,.. ........ --.
;f
.:0.. ... ---------,...-._
" .... , t
,:
\
Wtlt
mottlrio/
Dry
+--l+---l+---H-
in
Exhaust-air stock' "-Movobltl portilion
c. Center Exhaust Tunnel Dryer
Figure 11.3. Three types of tunnel driers.
courtesy of Charm (1978).
out
The dry powder falls to the floor or is separated by a bag or cyclone
collector, and the air stream goes out of the drying chamber carrying the
water vapor (Fig. 11.6).
Since the drying occurs almost instantaneously (5 to 30 sec), spray
drying is useful for heat-sensitive as well as other liquids. The very rapid
evaporation of water from the atomized particles produces a cooling ef-
fect that helps counterbalance the heating effect of the hot air.
Spray drying can be used to dry any liquid that can be atomized. The
main foods are milk and eggs, but coffee, tea, fruit juices, cake mixes,
flavorings, garlic, extracts, whiteners, and other foods have been spray
dried.
Foam Spray Drying. This process is applicable to liquids and slurries. The
injection of an inert gas into the feed line of a conventional spray drier
produces a foam that dries rapidly.
Figure 11.4. Fluidized bed drier.
Courtesy of USDA.
MOIST AIR OUTLET
INSULATED
tiOOO
GRANULES
Foam spray drying is said to increase the efficiency and capacity of a
spray drier. The product has enhanced flowability, is bulkier, and recon-
stitutes readily, even in cold water. Milk and eggs have been dried by this
system.
Foam-Mat Drying. In this process, the liquid food is concentrated, con-
verted to a stable foam, spread in a thin layer on a supporting surface,
cratered by blowing air or an inert gas through the foam, and dried with
warm air.
The process has been used for drying a wide range of foods, including
fruit juices, egg white, whole egg, whole milk, fruit purees, potatoes, and
tomato juice. Most fruit juices should be concentrated to 40 to 45 percent
solids prior to foaming.
Freeze Drying. In this process, frozen food is dried under a high vacuum.
This is also called sublimation drying, lyophilization, or vacuum contact
plate drying (Fig. 11.7).
Sublimation is the conversion of ice to water vapor without passing
through the liquid phase (Fig. 11.8). At normal atmospheric pressure,
sublimation is a very slow process, but it can be speeded up by reducing
the pressure. As ice is sublimed, energy is used_ Therefore, it is necessary
to supply heat to the food to compensate for this latent heat of sublima-
tion. The heating must be carefully controlled so that the food remains
Figure 11.5. Vacuum roller drier.
Courtesy of Farrall (1976).
frozen. Sunderland (1982) discussed the use of microwaves as a source of
heat for sublimation of the ice. This system is reported to be more rapid
and also less expensive than conventional freeze drying.
Due to the low temperatures involved in freeze drying, the method is
especially suited to the dehydration of heat-sensitive foods. Freeze drying
occurs with a minimum of discoloration and loss of flavor and nutrients,
such as vitamins. Foods retain more of their volatile constituents when
freeze dried than when dried by other systems.
Microwave Drying. In this process, microwaves are used to heat the product
being dried. With microwave heating, the material being treated is ex-
posed to a high-frequency field. This causes polar molecules of the mate-
rial to oscillate rapidly. It is this molecular motion that produces heat
within the material rather than in the air surrounding the material. The
internal heating permits drying from the inside out and avoids extreme
surface hardness (case hardening).
Liquid water selectively absorbs the radiation. Hence, as the water is
removed from the food, there are fewer water molecules to absorb the
2b.
4.
AIR
SPRAY
I
3.
AIR
AIR
SPRAY
---,
5. PRODUCT
.][
~
PRODUCT
Figure 11.6. Types of spray driers.
Courtesy of Van Arsdel and Copley (1973).
microwave energy, and the process becomes self.regulating. In conven
tional drying with hot air, the entire food mass, even when dry, absorbs
the heat energy.
Heat loss to the surrounding air is minimal, since the heat generated
is inside the food product. Maurer, Tremblay, and Chadwick (1971) indio
cated that the cost of microwave drying was only three fourths of the cost
of conventional drying. Another study (anon. 1974) reported that
the energy used was only half of that in conventional drying. Due to the
potential for case hardening, conventional air drying of noodles, maca
roni, or other pasta products requires from 5 to 10 hr. With microwave
heating and drying, this time is reduced to between 15 and 36 min
(Maurer, Tremblay, and Chadwick 1971).
A.
B.
c.
w
a::
:::>
CJ)
CJ)
w
a::
0...
vapor condenser
SCHEME A
32
vacuum chamber with
vapor condenser and
radiant baffle
SCHEME B

TEMPERATURE (OF)
Figure 11.7. Freeze-
drying relationships:
(A) the freeze-drying
cycle; (8) process and
cycle plant flow; (C) ar-
rangement of vacuum
freeze-drying chamber
and condenser.
Courtesy of Farrall (1976).
Figure 11.8. Triple point
of water. Considerations
in freeze drying.
Courtesy of Desrosier and
Desrosier (1977).
581
There may be some adverse reactions that occur during and after the
drying of food. Thijssen (1979) considered these to be material losses,
physical and chemical changes, and microbial growth and toxin produc
tion.
Food Products
Dried milk products and potato products account for most of the
commercially dried foods produced in the United States. Milk represents
the main dehydrated food in countries in which dairies are prominent.
There are many other types of dehydrated foods, including both animal
and plant products. Condiments, such as spices, garlic, and onions, along
with modified starches, coffee, and beans are prominent dried foods.
Even when dried meat, poultry, and fish products are microbiolog
ically stable, they are subject to oxidative and browning reactions. The
removal of all fat and the packaging of the dried product in an inert gas
or vacuum helps retard oxidative rancidity. Browning can be controlled
in some dried foods by removing the reducing sugars before drying.
The browning of dried fruit may be accompanied by the impairment
of flavor, texture, and nutrient value. The rate of browning is dependent
on the moisture content, availability of oxygen, storage temperature, and
concentration of added S02. If the fruit is treated with S02, enzymatic
browning is inhibited and nonenzymatic browning is retarded. The dehy
dration and storage of food was reviewed by Cunningham (1982). The
drying and storage of dried milk (Ford, Hurrell, and Finot 1983; Hurrell,
Finot, and Ford 1983), apples (Bolin, Steele, and Davis 1985; Magee, Has
saballah, and Murphy 1983) and blueberries (Yang and Atallah 1985)
have been discussed more recently.
Microbial Aspects
In considering the microbial aspects of dried foods, we should exam
ine the growth or death of cells before drying, during drying, during
storage, during rehydration, and in the reconstituted product.
BEFORE DRYING. Foods that are to be dried should be considered
perishable foods. They should be handled so that the microbial load is
kept as low as possible before drying. The finding of salmonellae in dried
milk was related to the isolation of these organisms in the milk plant (J arl
and Arnold 1982). Obviously, the sanitary condition of the processing
environment will influence the presence not only of salmonellae, but
also of other organisms, in dried foods. The contamination of dried fruit
with mold spores is difficult to prevent because of the prevalence of
molds in air around fruitprocessing plants.
Heat treatments, such as the blanching of fruits and vegetables, the
cooking of meat, poultry, or fish, or the pasteurization of liquid eggs or
milk reduces the total microbial count. Freezing in the freezedrying sys
tem and atomization during spray drying may lower the number of viable
microorganisms in foods.
DRYING. Drying removes the moisture from a food and lowers its wa
ter activity (aw). If no death occurred during drying, there would be a
higher microbial load per gram of dried product than in the original
food, due to the loss of water. Usually, the dried product has a lower
microbial level than the original food due to the death of microorgan
isms. Of those organisms that survive drying, some have been stressed
and have sublethal lesions.
The lethal action during drying is influenced by the organism (spe
cies, strain, physiological age, state of the cells, cell concentration), the
condition of drying (freeze, spray, roller, air, moisture, temperature, time,
the rate and extent of water loss, residual water content), and the type of
food or suspending medium (protective factors, pH, inhibitors).
If drying proceeds at a slow rate and at acceptable temperatures, the
microorganisms may multiply during the process. In general, foods being
dried should be either at a high or low temperature, so that bacterial
growth cannot occur until the water content is reduced to a level that
will inhibit microbial action. This is not always possible because some
products become overheated and lose their physical qualities if temper
atures are too high.
Although the viable microbial load is decreased during drying, the
dried product is not sterile, regardless of the system used for drying. In
fact, drying is used to preserve cultures of microorganisms.
Freeze drying is the most gentle of the drying methods, with respect
to both foods and the microbial population. A high microbial count in
the frozen product will be reflected in the dried food. During freezing,
drying, storage, and rehydration, there can be a shift in the microbial
population. Gramnegative bacteria such as Pseudomonas, Escherichia, and
Vibrio do not survive freeze drying as well as do Grampositive cocci and
the sporeforming bacteria.
Freeze drying reduced the total viable count of 10
4
/g in acceptable
shrimp to 10
3
/g (Moorhouse and Salwin 1970). Shrimp of questionable
quality or spoiled shrimp with total counts of 10
6
/g had viable counts of
10
3
to 104/g after freeze drying. Although no specific microorganisms
were determined, it can be assumed that the spoiled shrimp contained
pseudomonads.
Freeze drying includes a number of stresses that may cause injury to
microorganisms. Cells of Lactobacillus acidophilus that survived freeze dry
ing revealed damage to the cell membrane and cell wall (Brennan et al.
1986). The formation of mutants by freeze drying indicates that cellular
DNA is altered or damaged. Asada, Takano, and Shibasaki (1979) sug-
gested that the removal ofunfreezable water from the cells during drying
might induce DNA strand breaks_
During spray drying, aerosols of the liquid food are dried in hot air.
Microorganisms are affected by the aerosolization, heating, and drying.
When cells are aerosolized there may be changes in the outer layer of
the cell envelope, cytoplasmic constituents may leak out, and they may
lack control of ion transport. The exact nature of the lesions resulting in
permeability changes is unclear.
The lower the temperature of spray drying liquid egg, the better the
physical qualities ofthe powder, but the greater is the survival of bacteria,
including the salmonellae. Over 99 percent of the salmonellae in liquid
egg are killed during spray drying if the inlet air temperature is 121 DC
and the outlet temperature is about 60
D
C. There is a difference in the
survival rate of various strains or serotypes of Salmonella. Rapid cooling
of the powder is essential to maintain the quality of the powder. How-
ever, quick cooling also allows greater survival of the microorganisms.
The microorganisms associated with various dehydrated foods were
determined by Powers, Ay, and EI-Bisi (1971). Of 129 samples, 93 percent
had less than 10,000/g (total aerobes), 98 percent had less than one col-
iform in 109, and 99 percent revealed no fecal coliforms. Of 104 sam-
ples, all of the 5-g subsamples were negative for coagulase positive S.
aureus, and 98 percent of the 10-g subsamples yielded no Salmonella. Only
102 samples were tested for fecal streptococci, and 93 percent of these
had less than 100/g.
Graikoski (1973) reviewed the microbiology of dried fish. The dried
product could be produced with less than 100 microorganisms per gram.
However, the analysis of commercial dried fish revealed aerobic bacteria
at 10
4
to 10
6
/g (Ito and Abu 1985), or 10
6
to 10
7
/g (Valdimarsson and Gud-
bj6rnsd6ttir 1984). The majority of surviving bacteria were Gram-positive
heat-resistant micrococci, with Gram-negative cocci comprising the re-
mainder.
The plate count of dried vegetables is usually 10
3
/g or less. The flora
varies with the vegetables. The organisms include molds, actinomyces,
micrococci, staphylococci, bacilli, enterococci, and coliforms. Clostridia
may be found in some samples_
STORAGE. The removal of moisture lowers the aw of a food. The aw is
related to the spoilage rate and type of spoilage that may occur in a food
during storage. Lowering of the aw can result in the selective growth of
microorganisms. Thus, it is common to see mold growth on dried prod
ucts stored in humid conditions.
At 0.70 aw and below, most microbial growth is inhibited. However,
some xerophilic fungi can grow at a
w
of 0.65 and osmophilic yeasts at an
aw of 0.60. To recommend safe aw limits for foods, consideration must be
given to the nature of the food, its history, processing, packaging, and
conditions of storage and handling.
Foods are usually dried below the moisture levels that will inhibit mi
crobial growth in order to retard chemical deterioration, which occurs
more readily at 0.65 to 0.70 aw than at lower aw levels.
The unequal distribution of moisture can occur in various types of
dried foods. Thus, there may be areas or pockets that contain more mois
ture than is indicated by the analysis of the entire sample. Molds, or even
bacteria, might grow in these pockets of moisture.
Factors affecting survival include the species and strain of the or
ganism, morphology (spores), age and mass of the culture, treatments
before storage, moisture content of dried material, composition of the
food or substrate, and the conditions of storage (temperature, time, and
atmosphere).
Although organisms causing spoilage tend to lose viability during
storage, components of the cells, such as enzymes, remain active and con
tribute to undesirable changes in food. Hence, keeping the initial micro
bial count low is important in the maintenance of quality during storage
of dried food.
Species of Salmonella that survive drying of eggs die during storage of
the dried product, even at low temperatures. The storage of dried albu
men at elevated temperatures (50 to 60C) will inactivate any salmonel
lae in a matter of a few days to a week. The time needed for this inactiva
tion is influenced by number of survivors, the temperature and the
moisture content, or the aw of the dried powder. Inactivation is greater
at 10 percent moisture than at 2 percent moisture.
The initial death rates of salmonellae in dried fish meal were not
influenced appreciably by aw from 0.54 to 0.71 (Doesburg, Lamprecht,
and Elliott 1970). Lowering the a
w
to 0.34 reduced the death rates, espe
cially in meal stored at 15 or 20C.
During storage of dried milk, the viable bacteria decrease in number.
Streptococcus durans dies quite rapidly in spraydried milk powder, while
S. thermophilus remains viable for longer periods. Micrococci survive bet
ter than streptococci and, as the storage time is lengthened, aerobic spore
formers (bacilli) tend to dominate the viable population. Storage of
dried milk for three months will inactivate more than 70 percent of the
bacteria.
Survival is highest for organisms stored in vacuum and lowest for
those stored in air or oxygen (Cox and Heckly 1973). Following dehydra
tion, oxygen can kill Serratia marcescens (Cox, Gagen, and Baxter 1974).
The site for the toxic action of oxygen lies in the interspace between
the cytoplasmic membrane and the cell wall. The presence of reducing
substances helps prevent the lethal action of oxygen.
Because microorganisms die during the storage of dried foods, it is
difficult to correctly assess the microbial quality of these products. This
is especially true if there is an unknown interval between drying and
examination for viable microorganisms. A total microscopic count may
be useful in determining the microbial character of dried foods.
REHYDRATION. Normally, no bacterial growth occurs in dried food.
Reconstitution or rehydration of such food results in a perishable prod
uct. This rehydrated food should be used immediately or refrigerated as
any other perishable food.
The growth of microorganisms in rehydrated food is influenced by
the initial number and types that are present, as well as by the temper
ature and time for rehydration.
The microbial population of dried animal products becomes essen
tially mesophilic types. When rehydrated and held at 4C, there is a shift
in types so the psychrotrophs dominate. If the rehydrated food is held at
20 to 30C, mesophilic types-including potential pathogens-will
grow and become prominent.
With the total viable population reduced in dried food, any surviving
potential pathogen will have less competition and fewer microbial inter
actions in the rehydrated food. If not properly handled before use, the
food may become a vehicle for foodborne illness.
Due to osmotic forces and plasmolysis, rehydration can cause the ap
parent death of some cells. Plasmolysis is considered to be the result of
the retraction of the plasma membrane away from the cell wall due to
osmotic forces.
Sublethal injury prevents some cells from growing as readily as others
in the rehydrated food. Since rehydration is necessary for microbial anal
ysis, the effect of rehydration on these injured cells or other cells is im
portant.
The microbial analysis of dried food should be interpreted with cer
tain facts in mind. If inhibitory media are used, injured cells will not
grow and be detected. Bacteria play no significant part in the microbio
logical deterioration of dried foods under normal conditions. This is be-
cause there is not sufficient water for their growth. The bacteria that
dominate the microbial flora are important only when the food is rehy-
drated and stored at temperatures acceptable for their growth.
INTERMEDIATE-MOISTURE FOODS
An intermediate-moisture (1M) food is one that is preserved by lower-
ing the water activity by drying or adding solutes, often in combination
with chemical preservatives, anaerobic packaging, or both. 1M foods have
been defined as having moisture levels ranging from 15 to 50 percent
and 0". values of 0.6 to 0.9. Regardless of the exact moisture or 0". value,
1M foods can be eaten without rehydration, but they are microbiolog-
ically stable without refrigeration.
1M foods have certain advantages:
1. They are relatively low in moisture. Hence, they can be considered
concentrated in regard to bulk, weight, and caloric content.
2. Their plastic characteristic allows them to be molded into uni-
formly shaped cohesive blocks to facilitate packaging and storage.
3. They can be consumed with no preparation by the user.
4. They are similar to normal foods in texture.
Foods with 0". 0.60 or less are essentially free from microbial growth.
If solutes are used to obtain the low 0"., the food may have unacceptable
flavors as a result of the high concentration of solutes needed.
Foods with 0". over 0.85 readily support the growth of microorgan-
isms. Between 0". 0.60 and 0.85, most of the microbial growth is due to
molds, with some yeasts growing in this range. By adding antimycotic
agents to the food, the microbial growth can be controlled. Since molds
are primarily aerobic, reducing the availability of oxygen will limit their
growth. Thus, there can be good storage stability at room temperature.
The types of 1M foods include high-moisture dried fruits (figs, dates,
raisins, prunes, apricots, apples), fig newtons, marshmallows, soft can-
dies,jams,jellies, molasses, honey, syrups, fruit cake, brownies, fruit-filled
toaster products, diet bars, pepperoni and other dry sausages, pemmican,
jerky, and the semimoist pet foods.
Most of these foods are well known. With the creation of semimoist
pet foods, there has been an effort to develop more human foods that
have 1M characteristics. The effect of blanching and sulfite treatment on
1M bananas was discussed by Levi, Ramirez-Martinez, and Padua (1980).
Substances that have been added to lower the 0". to obtain 1M foods in-
clude glycerol, propylene glycol, sodium chloride, sucrose, dextrose, corn
syrup, and sorbitol, as well as lactose, sodium lactate, lactic acid, and
other hydroxy acids. Various 1M foods were reviewed by Erickson (1982).
There are many ways in which an 1M food can be developed. So far,
systems that have been used are the following:
1. Mix ingredients so that an acceptable moisture level and aw are
obtained. Usually antimycotic agents and solutes to keep the aw
low are included in the formula.
2. Dry a food below aw 0.85 and, if needed, add antimycotic agents
for preservation.
3. Mix ingredients, such as sugar added to fruit, and then evaporate
water to a desired consistency and sugar concentration, as in the
manufacture of jam.
4. Dry a food and then rehydrate it in a solution containing solutes
and antimycotic agents so that the final food has a normal mois
ture content, but a lowered aw and preservatives.
5. Soak small pieces of food in a low aw solution of various chemicals
and antimycotic agents. Equilibration will produce a food with
lowered aw and preservatives.
6. Add waterbinding agents to maintain a higher water content but
a lower aw.
7. Use mild drying conditions to lower water content in conjunction
with the waterbinding capacity of natural plant colloids (Gee, Far-
kas, and Rahman 1977).
By incorporating an effective antimycotic agent, heating to destroy
vegetative microorganisms, and adjusting the aw to 0.85, pet foods sealed
in plastic pouches have an excellent record for stability under market
conditions.
Equilibration of foods by soaking in solutions of low aw was discussed
by Brockman (1970) for tuna, pork, beef, carrots, celery, pineapple, and
macaroni. Water activities of 0.81 to 0.86 were obtained for these foods.
The usual antimycotic agent is potassium sorbate. However, other
substances also have antimycotic activities. Propylene glycol acts as a
plasticizing humectant for texture of the food as well as contributing to
the soluble solids of the aqueous phase. It is also an antimycotic agent.
Together, potassium sorb ate and propylene glycol act as an antimycotic
system to protect 1M foods from mold and yeast deterioration. Calcium
propionate also may be used to aid in antimycotic activity. The addition
of sorbic acid to the surface with a zein coating, and lowering the surface
pH were used to control microbial activity of an 1M cheese analog (Torres
and Karel 1985).
With 1M foods, small temperature changes may cause moisture con-
densation inside the package. Microbial growth can occur in these
condensates which have high a
w
and usually sufficient nutrients.
One problem with the bacteriological picture ofIM foods is the possi-
bility of growth of S. aureus. This organism can begin to grow at a
w
0.83
to 0.84, and to produce enterotoxin at 0.86 (Tatini 1973; Troller 1973).
The growth and toxin production depend upon other growth factors be-
ing at acceptable levels_
A reduction in the bacterial load has been reported for 1M foods
stored at 38C (Brockmann 1970; Kaplow 1970)_ S_ aureus at a level of
104fg was inoculated into both chicken a la king and ham sauce at aw 0.85.
The number of S. aureus was reduced to less than 2,000fg in one month
and to about O.4fg after four months. Inoculation with E. coli, C. per-
fringens, or Salmonella gave comparable results.
Labuza, Cassil, and Sinskey (1972) challenged 1M foods with various
microorganisms. The 1M foods were prepared by direct mixing or by
freeze drying, and then absorption of moisture to the desired level. The
microorganisms showed better growth in the direct mix than in the
freeze-dried food system, even though both foods were at the same a
w
S.
aureus grew at 25C in the direct-mix food at aw 0.84. In the freeze-dried
system at 0.84, death occurred, but survivors were evident for one month.
Pseudomonads showed growth at aw 0.84 in direct-mixed food, but no
growth in food adjusted after freeze drying, even at aw 0.90. These data
indicate that microbial growth is affected by more than the aw of a food.
Presumably, the moisture content must be considered.
CHEMICALS
The chemical preservation of food has a long history. The preserva-
tive action of acids, salt, and sugar was recognized by primitive peoples.
As discussed in Chapter 4, there are chemical inhibitors naturally present
in some foods.
Public Concern
Some consumer groups have argued against the presence of chemi-
cals in foods. There will always be chemicals in foods, simply because
foods are composed of chemicals. Some chemicals naturally present in
foods are toxic to humans (Chapter 1).
Food Additives
There are many definitions for food additives. According to the U.S.
Food, Drug, and Cosmetic Act, a food additive is a substance the intended
use of which results or may be reasonably expected to result directly,
or indirectly, in its becoming a component or otherwise affecting the
characteristics of any food. In 1958, Congress amended the act to restrict
food additives to substances specifically approved after that date by the
FDA. Many substances had been used for years without FDA approval, so
Congress exempted substances that, through long use, were "generally
recognized as safe" (GRAS) by experts in the field. Since then, those
chemicals that have been used outside the United States and whose expe
rience has established safety can be included as GRAS. In 1969, President
Nixon called for an evaluation of GRAS substances, and in 1979, the
FDA began a comprehensive program to determine the safety of these
chemicals through information already published.
Food additives have been defined by the FAOIWHO as substances that
are added intentionally to food, generally in small quantities, to improve
the appearance, flavor, texture, or other storage properties. This defini
tion does not include substances that are not deliberately added but
nonetheless find their way into food.
Additives may be intentional or incidental. Intentional additives are
added on purpose to perform specific functions. Incidental additives
have no function in the finished food but become part of it through some
phase of production, processing, storage, or packaging (Davis 1979;
Karas and Hopfenberg 1979; Larsson et al. 1983; Miller 1985; Mock and
Cress 1986; Pariza 1982). Normally, these residues are present in foods
only in trace amounts.
Chemicals Added to Food
There are several major classes of chemicals that are added to foods.
These have been listed variously, but usually the function in food is can
sidered. Examples are acidulants, antioxidants, colors, enzymes, flavors
or flavor enhancers, functional protein additives, leavening agents, nutri
tive agents or supplements, preservatives, stabilizers or thickeners, sur
factants, and miscellaneous (including anticaking agents, dough condi
tioners, and sequestrants). According to an FDA press conference in
1973, the FDA uses thirtyone categories for food ingredients or addi
tives. Of the total food additives, chemical preservatives comprise only 3
percent or less on either a weight or a dollar basis.
According to Middlekauff (1974), sugar is the main substance added
to foods, being used at an annual rate of about 46 kg per person. Of the
other major substances per person, we use salt (6.8 kg per year), corn
syrup (3.8 kg per year), and dextrose (1.9 kg per year). All of the other
substances total about 4.1 kg per year for each person. Of this amount,
the next thirty two additives, including nutrient supplements (vitamins
and amino acids), account for 3.5 kg per year. Although there are thou
sands of additives, most of them account for a very small portion of the
910 kg of food that a person living in the United States consumes each
year.
A more recent estimate is that we consume an average of 681 kg of
food per year (Emerson 1981). Of this amount, food additives account
for about 500 g. According to Rehwoldt and Van der Have (1986) the
food additive used most often is salt, although its use has decreased in
recent years. The researchers did not include sugar, corn syrup, or dex
trose in their list of the ten most heavily used food additives.
Reasons for Using Additives
There are certain reasons for adding and for not adding chemicals to
foods. The reasons for adding chemicals are to
1. Maintain or improve the nutritional quality of food.
2. Reduce waste by enhancing the keeping quality.
3. Make foods more attractive to the consumer.
4. Provide essential aids in food processing.
5. Minimize public health hazards.
With our present technology of food preservation, the use of chemi-
cals is essentiaL Without food additives, the cost of food would increase.
Besides the increased cost, most foods would be less wholesome and lack
their present keeping quality.
Reasons to Not Allow Additives
Chemicals cannot be added to foods if they
1. Disguise faulty processing or handling methods_
2. Deceive the consumer.
3. Reduce the nutritive value.
4. Replace good manufacturing practices that could accomplish the
same effect.
5. Are unsafe for human consumption.
Strong flavors cannot be added to foods to disguise spoiled or par-
tially spoiled food.
Safety of Chemicals
The safety of chemicals in foods is very important, regardless of
whether the chemical is added or is naturally present in the food. The
absolute proof of safety of a chemical for human use is difficult to dem-
onstrate. It is much easier to show that a chemical may be unsafe than to
prove absolute safety. For this reason, consumer action groups have a
definite advantage over the food industry in the battle against food addi
tives.
To petition for the use of a chemical additive for foods, one must
submit certain information to the regulatory agency. This information
includes the chemical identity of the substance, the purity, a quantitative
method of assay for the amounts that would be expected in a food, its
reactivity and stability, solubility, manufacturing process, its intended
purpose, and, if it accomplishes this purpose, an environmental impact
analysis concerning its manufacture, use, and consumption, suggested
tolerances for use, toxicology, biochemistry, and allergic responses
(Clausi et al. 1982; Lechowich 1981; NAS 1980).
According to the safety decision tree developed by NAS (1980), the
first step in toxicity testing is for acute toxicity, then genetic toxicology
as well as metabolism and pharmacokinetics, followed by subchronic tox
icity and reproduction, and finally, chronic toxicity studies. Some tests,
such as the Ames mutagenicity test, may use microorganisms or cell cuI
tures, while others use one or more species of animals, such as rats, mice,
dogs, or hamsters. Even if these tests show a chemical to be positive for
toxicity, it does not necessarily mean that the chemical is toxic to humans.
The level of feeding to animals is generally considerably higher than
would be used in human food. In some cases, one test might indicate
that the chemical is carcinogenic in animals, but if the experiment were
repeated, a negative result might be obtained. Thus, the safety of a chemi-
cal is difficult to guarantee. It is obvious that some chemicals should
never be used in foods, while others may be used safely at low levels.
Almost anything at a very high level has a toxic effect. There have been
instances of people dying from kidney failure after drinking too much
water. If a chemical passes the acute and subchronic tests, the chronic
effects are determined next.
The chronic feeding tests require a minimum of two years but may
go on forever. Two species, such as the rat and dog, are used. A reproduc-
tion study through three generations may be needed_ As in the subacute
studies, complete work-up is done of biochemical and histopathological
effects. The metabolic fate of the chemical should be determined, such
as the absorption, distribution, concentration in organs or tissues, and
metabolic products that result. The mutagenic, carcinogenic, and terato-
genic properties of the chemical are determined. The possible interac-
tions with other chemicals in foods or drugs, as well as dietary restric-
tions, must be known.
It should be evident that to acquire all of this information requires
considerable time and money. Even if the tests indicate that the chemical
is safe, there is no assurance that it will receive favorable consideration.
When a chemical is deemed to be safe, a tolerance is established. The
tolerance or limitation does not exceed the smallest amount needed for
the intended purpose, even though a higher amount would be safe. After
determining the amount of the chemical that produces any undesirable
effect on the test animals, only 11l00th of that amount is normally the
maximum allowed for humans. It is likely that humans might react differ
ently than the test animals to the chemical.
Since there are certain chemicals that do have a real value when
added to food, and since humans cannot be used as test animals, it is
necessary to develop acceptable tests, extrapolate the data to human be
ings, and determine the risks as well as the benefits that could result from
the use of the chemical (Anon. 1978; Casciano 1982; Devoret 1979;
Flamm and Winbush 1984; FSC 1978; Gibbs and Kahan 1986; Hattan
and Rulis 1986; Schramm 1984; Smith 1984; Taylor 1982; Wodicka 1980).
Because regulations for food additives of various countries differ, there
is much interest in having some type of international regulations to help
world trade of food products (Feldberg 1983; Gangolli 1983; Kroes and
Feron 1984; Wonnacott and Jukes 1986).
Chemical Preservatives
A chemical preservative can be defined as a substance that is capable
of inhibiting, retarding, or arresting the decomposition of food. This def
inition includes microbial inhibitors, as well as antioxidants, acidulants,
and sequestrants. In this text we are concerned mainly with controlling
the deterioration of foods by microorganisms. Besides the requirements
described for all chemical additives, there are requirements for food pre
servatives. Chemical preservatives should
1. Provide an economical means of preservation.
2. Be used only when other preservation methods are inadequate
or not available.
3. Extend the storage life of food.
4. Not lower the quality (color, flavor, odor) of the food.
S. Be readily soluble.
6. Exhibit antimicrobial properties over the pH range of the partic
ular food.
7. Be safe at levels that are needed.
8. Be readily identified by chemical analysis.
9. Not retard the action of digestive enzymes.
10. Not decompose or react to form compounds of greater toxicity.
11. Be easily controlled and uniformly distributed in the food.
12. Have a wide antimicrobial spectrum that includes the spoilage
types of organisms associated with the food to be preserved.
Such factors as how the food is to be processed, packaged, stored, and
distributed, composition of the food, other preservation processes, the
types and number of organisms to be controlled, shelf life, and cost
should be considered when selecting the antimicrobial agent or agents
to be used.
Chemical preservatives should be used only when other methods to
control microorganisms are lacking, damaging to the product, or very
expensive. It has been suggested that, except in a limited number of
cases, the use of chemical preservatives would be unnecessary if strict
attention were paid to sanitation and hygiene during processing and ade
quate refrigeration were used during the various stages after processing.
Chemical preservatives do add a margin of safety from possible abuses
by people involved in any of the postprocessing stages. This is applicable
especially to the actions of the consumer.
In considering the possible hazards associated with the use of chemi
cal preservatives, it is important to consider the foods in which they are
used. The possible adverse effects to humans are likely to be greater if
such preservatives are used in foods such as bread, meat, milk, or fresh
fruits and vegetables, which may be consumed in substantial amounts
daily, than in foods that are eaten only occasionally in small amounts.
Ideally, the chemical will inhibit or kill the important microorganisms
and then break down to harmless, nontoxic substances. The chemical
should not decompose so fast that it is ineffective. Slow inactivation of
microorganisms may lead to unsuccessful preservation. Nonsterilizing
amounts of antimicrobial agents are selective and influence the type of
microbial flora that can survive in a food system. The degree of inhibi
tion varies with the preservative, and the amount of inhibition is deter
mined by the concentration of the chemical.
ACTIVITY OF PRESERVATIVES. The factors that affect the microbial
activity of chemical preservatives include such things as the type of chern
ical and its concentration, the type of organism and its physiological
state, the numbers of organisms, the composition of the food and its pH,
and the temperature of storage.
Bacterial spores are the most resistant type of microorganism. Fungal
spores are more resistant than vegetative cells to the action of preserva
tives. In many cases, molds are more susceptible than yeasts to inhibitors.
There is a variation in resistance to chemicals between species and be
tween strains of the same species. The higher the microbial load, the
greater the amount of chemical preservative needed to accomplish inhi
bition or death of the cells.
Actively growing microbial cultures are susceptible to killing agents.
As the culture ages and becomes inactive, the cells tend to become more
resistant to antimicrobial conditions. Sporicidal agents can be consid
ered as chemical sterilizers, since they have the potential to destroy all
microbiological forms of life. The apparent lethal or inhibitory action
on microbial spores may be due to either inhibition of germination or
of outgrowth. Only a few agents are capable of killing all types of micro
organisms, including spores.
Many preservatives have an increased activity in acid foods. Organic
material of the food may react with the preservative, rendering it less
toxic or inert to microorganisms. Liquid foods allow better contact be
tween the inhibitor and the microorganisms than do solid foods. In cases
in which the microorganisms are inside food particles, they are protected
from the effects of the chemical preservatives.
In general, increasing the temperature increases the effect of preser
vatives on microorganisms. If, however, a low temperature is increased
toward the optimum of the organism, the stimulatory effect on growth
may outweigh the increased action ofthe preservative. If the temperature
is above the optimum for growth, the increased preservative effect is
more pronounced.
Time is an important factor in the use of chemical agents. Chemicals
may react with the organisms quite rapidly, or they might require several
hours to achieve the desired action on the microorganisms. Hence, the
longer the contact time, the more effective will be the action of the chem
ical preservative.
Chemical preservatives may inhibit the growth of microorganisms
(bacteriostat, fungistat), or kill them (bactericide, fungicide, sporicide, or
virucide). Whether a chemical is bacteriostatic or bactericidal may be reI
ative. In very dilute amounts some chemicals may act as food sources for
the microorganisms. Increasing the amount may be inhibitory, and still
higher levels may kill some or all of the microbial cells. In general, the
more concentrated the chemical agent, the more effective will be its ac
tion. Usually, very high levels are not desired due to the potential adverse
effect on food quality and toxicity to humans consuming the food.
We determine the viability of a microbial cell by its ability to multiply.
The apparently lethal action on cells may be reversible, so that there is
only a bacteriostatic effect. In some cases, the number of killed cells is
balanced by the number of cells that are able to multiply, so that, al
though there is a lethal effect, the action appears to be only bacterio
static.
The bacteriostatic agents most frequently used are those that lower
the a
w
below the level needed for bacterial growth. Sugar and salt are two
of these important compounds. Besides retarding growth by affecting the
chemical or physical nature of foods, preservatives affect microorgan
isms by several mechanisms, including the denaturation of protein, in
hibition of enzymes, alteration or destruction of DNA, the cell wall, or
the cytoplasmic membrane, suppression of cell wall synthesis, or compe
tition with essential metabolites.
Interference with Genetic System. In this case, the chemical enters the cell.
Some chemicals can combine with, or attach to, the ribosome and inhibit
protein synthesis. If genes are affected by the chemical, enzyme synthesis,
which the genes control, will be inhibited.
If the logarithm of surviving bacteria is plotted against time, at least
a portion of the curve is a straight line. This indicates that the lethal
action is a result of the reaction of a single molecule of the cell. The
alteration or destruction of the DNA fits the monomolecular reaction
requirement. Hence, the effect of inhibitors on the genetic mechanism
was thought to be the primary action on the cell. The fact that the curve
is not exactly a straight line function indicates that other actions occur.
Cell Walls or Membranes. The chemical does not need to enter the cell to
inhibit growth. A reaction on the cell wall or membranes can alter the
permeability of the cell. This can impair the passage of nutrients into the
cell, as well as allow leakage of cellular constituents from the cell.
Damage to the cell wall alone does not kill the microbial cell. Due to
the increased permeability, the chemical is able to enter the cell. Once
inside, the chemical may lyse the cell membrane or coagulate the cyto
plasm of the cell. Damage to the cell membrane can occur by preserva
tives reacting with chemically active sites or by dissolving lipid constitu
ents.
Since cell walls are complex polymers, some chemicals can interfere
with cell wall production, by affecting synthesis of simple components
or by inhibiting the polymerization of the components. Or, the cell wall,
when developed, may not be able to satisfy the requirements of the cell.
Inhibition of Enzymes. Since enzymes are proteins, they can be denatured
by heavy metals, alcohols, phenols, or surface active substances. Gener
ally, these chemicals are too toxic for food preservatives.
Wetting agents alter the colloidal properties of enzymes and interfere
with enzyme action. High concentrations of salts will curtail the biologi
cal activity of enzymes. Sufficient alteration of the pH, either up or down,
will inhibit the action of enzymes and prevent the multiplication of mi
croorganisms. Oxidizing agents can inactivate certain enzyme systems
that contain active sulfhydryl (SH) groups. These SH groups, when oxi
dized, form disulfide (S-S) bonds that cause the enzymes to become inac-
tive_ Reducing agents split the S-S bonds and reproduce the -SH form_
The halogens, hydrogen peroxide, and ozone, can oxidize the -SH group_
Other mild oxidizing substances also may inhibit microbial cells in this
manner-
On the other hand, certain active systems have S-S bonds. These en-
zymes can be inactivated by reducing agents.
The hydroxyl (OH) group is essential for the activity of certain en-
zymes. This group can react with chemicals and cause inactivation of the
enzyme. Other groups on enzymes that can react with chemical inhibitors
include the amine, amide, and carboxyl groups, as well as metals in respi-
ratory enzyme systems.
Another action on enzyme systems is due to antienzymes, such as the
trypsin inhibitor of soybeans. These antienzymes are found in a number
of natural products, especially in cereals and legumes.
Some chemicals selectively inhibit certain enzyme systems. Although
both microorganisms and humans can perform a given enzymatic reac
tion, the actual enzymes involved, and even the associated coenzymes,
can be quite different in many features. Thus, it is possible to selectively
inhibit specific enzyme reactions of microorganisms without affecting
those of humans.
Binding of Essential Nutrients. Since microorganisms differ in their nutrient
requirements, the binding of essential nutrients affects different organ-
isms in different ways. If an organism has few requirements, it will be
less affected than an organism that needs many preformed nutrients.
Although hundreds of antimetabolites (analogues of vitamins, amino
acids, purines, and pyrimidines) have been synthesized, only a few have
been useful to inhibit microbial growth. The sulfonamides that prevent
the conversion of para-aminobenzoic acid to folic acid are the classic
example of these antimetabolites.
ACTION ON HUMANS. The toxicity of chemicals to humans may be
either due to the same mechanism that causes the effect on microorgan-
isms, or the person can develop a hypersensitivity to the compound.
This results in an allergic reaction. Inevitably some people will develop
a hypersensitivity to any type of substance that is introduced into the
body.
When a foreign chemical enters the body, it has three possible fates:
(1) The chemical may undergo spontaneous reactions to other products
without the intervention of enzymes. (2) The chemical may be metabo-
lized and transformed into other compounds by means of enzymes of
the body. This is the fate of most compounds. The enzymes that metabo
lize foreign chemicals are located in the liver, intestines, kidneys and
lungs. Bacteria in the gut playa role in this metabolism. (3) The inhibitor
may be excreted with no alteration. These compounds are usually highly
polar types. Many other foreign compounds are metabolized to highly
polar types, which are then eliminated from the body.
ACTIVITY OF COMBINATION TREATMENTS. In certain circum
stances, combinations of two or more chemical preservatives are more
effective than indicated by testing each one individually. For the treat
ment of diseases, combinations of antibiotics may be used, since, if there
is resistance to one antibiotic, there may not be resistance to the second
or third antibiotic.
Combinations of chemicals and other treatments may increase the
potential for control of microbial spoilage of food. Using low water activo
ity to control bacteria and antimycotic agents to control fungi is dis
cussed for 1M foods.
TYPES OF CHEMICAL PRESERVATIVES. Some chemicals that help
prevent bacterial and fungal spoilage have been used for centuries. So
dium chloride was used by our primitive forebears to prevent spoilage
of meat and fish. Sugar has been used for many years in the making of
jams and jellies.
Mold inhibitors, such as the sodium or calcium salts of propionic acid
or sodium diacetate, are helpful in controlling mold in bakery products.
Mold control in bread was a serious problem for the baker, both in the
bakery and after the bread left the bakery. Chemical agents give a margin
of safety in cases such as this.
Besides chemicals that are added directly to food, some are applied
to the wrapping material to control contamination on the surface of the
food. Antimycotic agents that have been used for food wraps include
sodium benzoate, sodium and calcium propionates, methyl and propyl
parahydroxybenzoates, sorbic acid, and diphenyl.
Some chemicals are added to water that is used to wash food. In these
cases, some chemical residual may remain on the food. Fumigants, such
as S02, used for grapes and in the drying of fruit, leave a residual mate
rial on the food.
Some chemicals that are used as preservatives, such as salt and sugar,
are not classed as preservatives from a legal viewpoint. Chemicals that
are antioxidants and mayor may not have antimicrobial properties, are
listed as chemical preservatives. There are other chemicals that are added
to foods for various reasons that have some antimicrobial properties.
ACIDS. Acids have many functions in foods. An acid condition is unfa
vorable for the growth of many microorganisms. The preservative effect
of acids may be due to the pH, the undissociated acid molecule, or the
anion. With short chain fatty acids, a decrease in pH enhances the activo
ity to a greater degree than is the case for acids with longer chains. At
low pH levels, the undissociated molecules of the weak, shortchain or
ganic acids enter the cell where they dissociate and affect the pH of the
cell. This can interfere with the mechanism that transports chemicals
across membranes. Also, these acids can interfere with intracellular en
zymes. The ionized acid does not enter the cell as well as does the undis
sociated form.
Branchedchain fatty acids are less active than the corresponding
straight chain acids. Unsaturated acids have been reported to have the
same activity or to be more active than the corresponding saturated
acids. The substitution of a hydroxyl (OH) group for a hydrogen has been
reported to both increase and decrease the activity of the fatty acids.
Researchers reported that the OH group was particularly active, but a
SH group caused loss of activity (Kabara et al. 1972). They found that
amine derivatives of fatty acids showed activity against both Gramposi
tive and Gramnegative microorganisms.
It has been suggested that short chain fatty acids can inhibit or kill
both Grampositive and Gramnegative bacteria. The long chain fatty
acids affect primarily Grampositive bacteria since they cannot penetrate
the lipopolysaccharide layer of Gramnegative bacteria.
The growth inhibition of bacteria by these lipophilic acids has been
attributed to the effect on membrane transfer systems of amino and keto
acids (Freese, Shev, and Galliers 1973). The longchain lipophilic acids
are more readily partitioned into the cell membrane. This attachment is
reversible.
It is well established that an increase in acidity or in hydrogen ion
activity inhibits the growth or even destroys certain microorganisms, reo
duces the germination and outgrowth of spores, and reduces the heat
resistance. The thermal death point of yeasts and molds is less affected
by low pH than is that of bacteria.
The pH and the type of acid are important in the inhibitory or lethal
action of these chemicals. Acetic acid has greater inhibitory and lethal
effects on the basis of pH than either hydrochloric or lactic acids.
The exact order of effectiveness depends upon various factors such
as the type of microorganism, whether inhibition or death is determined,
the pH, temperature, and other environmental conditions of the sub
strate, as well as the concentration of acid that is used. Comparisons have
been made on molarity, normality, percentage, as well as at specified pH
levels. These various systems influence the relative amounts of the acids
that are present to inhibit the microorganisms. By selecting the correct
microorganism, conditions of study, and the basis for comparison, any
acid may be or may not be as effective as any other acid as an antimicro
bial agent. Comparisons should be made on the basis of molarity of un-
dissociated acids, and not on the percentage of the acids added to the
system_
Respiratory viruses are generally acid labile. Acids may be quite im-
portant in preventing the transmission of viruses by foods. Acids with
only one carboxyl group are less active against viruses than those with
more than one carboxyl group (Poli et al. 1979).
Acetic Acid (CH3COOH). This acid is soluble in water and has a pK value
of 4.76. The reasons for using acetic acid as a food preservative are its
low cost, availability, and low toxicity. Acetic acid as well as its salts-
sodium acetate, calcium acetate, sodium diacetate, and calcium diace-
tate-are GRAS. They can be added to food in accordance with good
manufacturing practice. They are effective at pH 4.5 or less. Acetic acid
is effective against a wide spectrum of bacteria, being bactericidal to
some, such as coliforms and salmonellae. It is less effective against molds
than against yeast.
Acetic acid is the principal organic component of vinegar. Vinegar
is used in many foods, including mayonnaise, salad dressing, prepared
mustard, pickles, marinades, catsup, and pickled beets.
Although acetic acid is an effective inhibitor, it cannot be used on
some foods because of its pungent odor. Moreover, it can cause discolora-
tion and textural changes when applied to poultry or meat (Bell, Mar-
shall, and Anderson 1986). However, acetic acid is permitted as an acidi-
fier in processed meat and poultry.
The addition of 0.1 percent acetic acid to poultry scald water reduced
the DS2 values of two salmonellae and C. jejuni (Okrend, Johnston, and
Moran 1986). At the 1 percent level, acetic acid caused instantaneous
death of these organisms. At 0.1 percent acetic acid inhibits most organ-
isms causing foodborne illness, and at 0.3 percent it inhibits molds that
produce toxins.
Dehydroacetic acid inhibits bacteria, yeasts, and molds at concentra-
tions of 0.005 to 0.4 percent. It appears to be a better fungistatic agent
than a bacteriostatic agent. It can be used as a fungistatic agent in cheese
wrappers. Autoclaving of this acid causes only a slight decrease in biolog-
ical activity.
Dipping certain fruits and vegetables in a solution of 0.1 to 0.2 per-
cent dehydroacetic acid extends the storage life of the produce. At 0.1
percent it inhibits undesirable secondary fermentations in alcoholic bev-
erages, such as beer and wine_ In bread and baked pastries, a level of 0.2
percent inhibits microbial growth.
The sodium salt of dehydroacetic acid at pH 5.0 is twice as active as
sodium benzoate against Saccharomyces cerevisiae and twenty-five times
more effective against Penicillium glaucum or Aspergillus niger.
Sodium diacetate is a loose, equimolecular compound of acetic acid
and sodium acetate. It tends to enhance the antimicrobial activity of
acetic acid. The action of sodium diacetate is not as pH dependent as is
that of acetic acid. This compound is listed as GRAS and can be used at
0.4 parts per 100 parts of flour for use in bread. It is effective in the
bread making industry as an inhibitor of mold and of preventing ropi
ness of bread. This inhibitor does not interfere with the yeast fermenta
tion. It also may be applied as a dust to the outer surface of bacon or
ham or other cuts of red meat or poultry. It inhibits storage molds that
commonly occur in foods and feeds (Glabe and Maryanski 1981).
Ascorbic Acid. This is vitamin C. It is present in many foods, especially
citrus fruits, and is safe to use as a food additive.
According to one study, ascorbic acid stimulated diacetyl but not acid
production by lactic acid bacteria in milk (Richter et al. 1979). Ascorbic
acid inactivated enteroviruses (Salo and Cliver 1978), inhibited growth
and toxin production of C. botulinum type B (Notermans, Dufrenne, and
Keijbets 1985), enhanced the antibotulinal effect of nitrite in canned
cured meat (Tompkin, Christiansen, and Shaparis 1978a), and showed
some inhibitory properties toward C. jejuni (Fletcher et al. 1983). The
standard plate count of ground beef increased significantly during five
days in a display case, even when treated with 0.1 percent ascorbic acid
(Shivas et al. 1984). It is evident that ascorbic acid has some inhibitory
properties against certain types of microorganisms.
The antibacterial action may be due to breakdown products of oxi
dized ascorbic acid, or ascorbic acid may cause inhibition in a manner
similar to other organic acids.
Benzoic Acid. Besides benzoic acid, sodium benzoate, potassium benzoate,
and the parahydroxy esters of benzoic acid (parabens) are used as chemi
cal preservatives in food. Due to its better solubility, sodium benzoate is
preferred to benzoic acid, but to reduce the sodium level in foods, the
potassium salt is used.
The FAOIWHO unconditional acceptable daily intake of benzoic acid
for humans is 5 mg/kg body weight. Cats and rats are particularly suscep
tible to high levels of benzoic acid. This chemical is excreted by many
mammalian species either as hippuric acid or as benzoyl glucuronide.
Benzoic acid is naturally present in many plant products, especially cran
berries. The acid and the sodium and potassium salts are GRAS with
maximum usage in food set by the FDA at a level of 0.1 percent. Since
potassium is heavier than sodium, for the same benzoate content and
antimicrobial effect, a higher level of potassium benzoate is needed than
of sodium benzoate or benzoic acid.
The antimicrobial activity of benzoates is dependent upon the pH of
the substrate, since it is the molecular form that is effective in preserva
tion. Using the formula
(ionized)
pH = pK + log --:---:--
(molecular)
and a pK of 4.19, the pH at which various amounts of undissociated acid
are present can be calculated, and is listed in Table 1 L 1 L For benzoic
acid, the inhibition of microorganisms was due to both the dissociated
and undissociated acid (Eklund 1985). However, the optimum antimicro
bial activity is between pH 25 to 4.0. At low pH values, the dissociated
acid is present in larger amounts than at higher pH levels.
Benzoates have been used in fruit juice, jellies, jams, soft drinks, pu
rees, condiments, fruit syrups, margarine, puddings, pickles, and pre
pared sauces. Benzoic acid suppresses the formation of trimethylamine,
a chemical indicator of spoilage of fish. Besides having legal limitations,
the use of benzoates may be limited by flavor considerations.
Primarily, the benzoates are used to inhibit yeasts and molds, since
most bacteria are inhibited by the low pH levels at which these chemicals
are effective. The benzoates cannot be used to correct poor sanitation
because they are not effective in highly contaminated foods. Although
considered to be fungistatic, Romano and Suzzi (1985) reported the
death of S. cerevisiae cells at a concentration of 0.05 percent benzoic acid.
The yeast spores were not affected. The addition of sodium benzoate to
the substrate reduced the heat resistance of yeasts (Beuchat 1981).
Combinations of benzoate with other preservatives may be beneficial.
Sulfur dioxide helps prevent discoloration and flavors attributed to ben
TABLE 11.11 THE pH VALUES NEEDED FOR VARIOUS LEVELS
OF UNDISSOCIATED ORGANIC ACIDS
Undissociated Acid
(%)
99
95
90
80
70
60
50(pK)
40
30
20
10
1
0.5
Benzoic
2.19
2.91
3.24
3.59
3.82
4.01
4.19
4.37
4.56
4.79
5.14
6.19
6.49
Acids
Propionic
2.87
3.59
3.92
4.27
4.50
4.69
4.87
5.05
5.24
5.47
5.82
6.87
7.17
Sorbic
2.75
3.47
3.80
4.15
4.38
4.57
4.75
4.93
5.12
5.35
5.70
6.75
7.05
zoate. Combinations of benzoate and sorbate are believed to be more
effective than either chemical used alone.
The esters of parahydroxybenzoic acid have been suggested for pres-
ervation of foods. It is only the esters and their salts that are of value as
preservatives. The free parahydroxybenzoic acid is of little use in practi-
cal concentrations. The antimicrobial activity of the parahydroxyben-
zoates increases with the length of the alkyl chain of the alcohol moiety,
up to the amyl ester. Some mixtures of the esters show greater activity
than would be expected by their individual action. These esters are called
parabens. The methyl and propyl esters are GRAS for use up to 0.1 per-
cent and the n-heptyl ester can be used at 12 ppm in malted beverages
and at 20 ppm in noncarbonated fruit-based beverages.
The parabens have an advantage over the benzoates in that they are
more effective at higher pH levels, with an active range of pH 3 to 8. They
are extremely stable in food systems and can be autoclaved or retorted or
frozen without losing their effectiveness.
The parabens inhibit yeasts and molds but are oflimited value against
Gramnegative bacteria. The inhibition of yeast precludes their use in
yeast-leavened baked products.
At levels of 0.03 to 0.06 percent, a 3:1 ratio of methyl to propyl ester
is used in cakes, pastry, toppings, or fillings. At 0.03 to 0.05 percent, a
2:1 ratio of methyl to propyl ester is used in soft drinks, fruit extracts,
and flavorings. A 0.07 percent level of a 2:1 ratio of methyl to propyl
ester is used in synthetic jam, jelly, or preserves. Antimicrobials are not
permitted in standardized sugar-sweetened jam, jelly, or preserves.
Parker (1969) suggested the parabens inhibit germination rather than
the outgrowth of bacterial spores. The methyl and propyl parabens allow
a limited degree of germination of B. subtilis spores followed by some
outgrowth of vegetative cells.
Citric Acid. Citric acid (COOHCH
2
C(OH)(COOH)-CH
2
-COOH) is a com-
mon constituent of citrus fruits. It is an acidulant with unique flavoring
characteristics and is water soluble.
Although citric acid has been tested as an antimicrobial agent, it is
considered to be not as effective as other acids or preservatives. However,
at 0.3 percent, citric acid significantly lowered the level of inoculated
salmonellae remaining on poultry carcasses (Thomson et al. 1967). The
microbial load of fish flesh was lowered to a greater extent by citric acid
than by potassium sorbate (Debevere and Voets 1972). Citric acid (0.1 or
0.3 percent) added to fish flesh inhibits the formation of total volatile
nitrogen and trimethylamine. The storage of hard-cooked, peeled eggs
in a 0.75 percent citric acid solution (pH 5.0) reduced the microbial pop
ulation and inhibited the growth of inoculated organisms (S. typhimurium,
E. coli, Y. enterocolitica, or S. aureus) (Fisher et al. 1985).
Lactic Acid. This acid (CHg-CHOH-COOH) is a natural constituent of
some foods and is formed by lactic acid bacteria in several fermented
foods.
Depending upon the substrate and microorganisms, lactic acid has
been reported as having very good, just average, or rather poor antimi-
crobial properties.
Using lactic acid solutions as a wash for livers or as a spray for car-
casses resulted in reduced microbial load on these meats (Smulders and
Woolthuis 1985; Woolthuis and Smulders 1985; Woolthuis et al. 1984).
Gill and Newton (1982) observed that the inhibitory effect oflactic acid
on Gram-negative psychrotrophs on meat appeared to be due to the de-
crease of pH and not to the undissociated acid. According to Van Netten,
Van der Zee, and Mossel (1984), the lethality of cells due to lactic acid
has been overestimated because many cells exposed to the acid can re-
pair injury due to the acid in a rich infusion agar containing catalase.
Malic Acid. This is hydroxysuccinic acid (COOH-CHOH-CH
2
-COOH), a
natural component of apples. It is permitted by the FDA as an optional
acidifying ingredient in mayonnaise and other salad dressings. In these
products, it has a microstatic action against yeasts and certain bacteria.
Peracetic Acid. This is known also as peroxyacetic acid (CHg-COOOH) or
acetyl hydroperoxide. It is a strong oxidizing agent and will explode if
heated to 110
a
C.
It is freely soluble in water, and the aqueous solutions are quite stable.
It reacts slowly with water to form acetic acid and hydrogen peroxide but
shows greater bactericidal action than either of these. A 1 percent solu-
tion is active against vegetative bacteria, bacterial spores, fungi, and vi-
ruses.
The main problem with the use of this chemical is the pungent odor
of its vapors and the off-flavor associated with acetic acid that occurs due
to residues.
Propionic Acid. This acid (CH3"CH
2
-COOH) is soluble in water and has a
pK of 4.87. It is produced by propionibacters during the aging of Swiss
cheese. Propionic acid is a constituent of human body fluids as the free
acid or its salts. The propionates are GRAS to be used in accordance with
good manufacturing practice. In bread and rolls, propionates are limited
to 0.32 percent of the weight of the flour; in whole wheat products, the
limit is 0.38 percent; and the limit in cheese products is 0.3 percent.
The bacteriostatic and fungistatic activity is greater in acid than in
neutral or alkaline substrates, indicating the antimicrobial action is due
mainly to the undissociated acid. Eklund (1985) reported that some inhi
bition is due to the dissociated acid.
The propionates are effective against molds and have a limited anti
bacterial activity and relatively no effect on yeasts. Since propionates do
not affect yeast fermentation, but do inhibit molds as well as Bacillus spe
cies causing ropiness, they are ideal for yeastleavened bakery products.
Propionates are used to control mold in various types of cheese, malt
extracts, chocolate, fruits, vegetables, bread, cakes, pies, jellies, and pre
serves.
Sorbic Acid. This unsaturated carboxylic acid (CH3CH = CH CH = CH
COOH) is water soluble (0.25 percent at 30C). The sodium and potas
sium salts are more soluble than the acid, and the potassium salt is usu
ally selected for use. Sorbic acid is more soluble in fat or oil than in
water. The sorbates are more effective against fungi than bacteria and at
pH values of 6.5 or less. However, Eklund (1983) reported that bacterial
inhibition is due to both the undissociated and dissociated acid, and the
effect of dissociated acid becomes appreciable with increasing pH levels.
Since sorbic acid sublimes when heated, it should be added to food after
heat processing. Other precautions for its use have been listed by King
(1981).
The sorbates are listed as GRAS. These chemicals may be added to
foods in accordance with good manufacturing practice. They are used in
foods such as baked goods, toppings (fruit, icing), beverages (fruit juice,
carbonated), cheese products, dried fruit, jams, jellies, preserves, marga
rine, dry sausage, cucumbers, sauerkraut, olives, smoked and salted fish,
syrups, wine, and cake (batter). Usually, it is used at levels ranging from
0.01 to 0.1 percent, although higher levels are used in some foods, such
as cheese products and fats.
Sorbic acid may be applied to foods in a variety of ways, including
direct incorporation into the food, dipping the food in a solution or
slurry of the acid, spraying a solution onto the food, application by
means of impregnated packaging materials, or addition as a solution in
ethanol, propylene glycol, or vegetable oil.
The antimicrobial effect of sorbates and their use in foods has been
reviewed (Dziezak 1986; Liewen and Marth 1985; Sofos et al. 1986). The
effectiveness of sorbates is influenced by the type of food, pH, water ac
tivity, types and numbers of microorganisms, type of atmosphere, and
temperature.
To inhibit mold on food such as bread, a 3 percent solution of potas
sium sorbate is sprayed over the surface of the freshly baked product.
The heat from the product evaporates the water leaving the sorb ate as
an inhibitor. With propionates inside and sorbates outside, the usual
moldfree shelf life can be extended significantly. Spraying a 5 or 10 per
cent potassium sorbate solution onto cured hams protected them from
mold growth for sixty days (Baldock et al. 1979).
Yousef and Marth (1981) reported that potassium sorbate (200-300
ppm) delayed growth of A. parasiticus but did not inhibit biosynthesis of
aflatoxin. Similar results were reported for higher levels of potassium
sorb ate (0.05 to 0.15 percent: 500-1,500 ppm) on the growth of P. roque
forti and the production of patulin (Bullerman 1984).
The addition of potassium sorb ate at 500 to 1,000 ppm to the heating
menstrum significantly reduced the D value of yeasts (Beuchat 1981).
The dipping of beef steak into a 10 percent potassium sorbate solu
tion extended the shelf life for two days (Greer 1982).
The use of potassium sorb ate increased the shelf life of poultry (Cun
ningham 1981; Robach 1979), and seafoods (Bremner and Statham
1983a; Shaw, Bligh, and Woyewoda 1983). Potassium sorbate inhibited
the growth of S. aureus (Elliott, Tomlins, and Gray 1982; Pierson,' Smoot,
and Stern 1979).
In an effort to reduce the nitrite level of cured meats, various chemi
cals were tested for their ability to inhibit growth and toxin production
of C. botulinum. The sorbates were found to be an effective additive for
this purpose (see sodium nitrite).
Treatment of salad vegetables by immersion in potassium sorbate (0.2
percent) solution is not effective and, in some cases, is detrimental in
maintaining these foods during refrigerated storage (Priepke, Wei, and
Nelson 1976).
The dipping of dates in a 2 percent solution of potassium sorbate
does not prevent yeast growth during storage (Bolin et al. 1972). This is
due to the pH of dates being near 5.9, so that the sorbates are less effec
tive than in other fruits. The addition of methyl bromide in conjunction
with the sorbate dip is effective in preserving dates.
The growth of yeasts in apple juice is retarded by addition of 0.025
to 0.035 percent sorbic acid. However, this level does not control certain
species of Acetobacter. For protection of apple juice, levels of 0.05 to 0.10
percent potassium sorb ate in conjunction with refrigeration have been
recommended.
Sorbic acid was added to wine to inhibit yeasts (De Rosa et al. 1983).
However, in sparkling wine the researchers found an odor described as
pineapplecelery, due to the formation of ethyl sorbate. Hence, they did
not recommend the use of sorbates in sparkling wines.
Some microorganisms are able to grow in the presence of large
amounts of sorbic acid or its salts. Some organisms, especially molds, can
metabolize this additive. Hence, sorbates are not a substitute for good
plant sanitation.
Succinic Acid. This acid (HOOCCH
2
CH
2
COOH) was as effective as acetic
acid in its ability to inhibit microorganisms on poultry carcasses (Mount
ney and O'Malley 1965). Although treatment with a 3 or 5 percent suc
cinic acid solution at 60C effectively reduced the microbial load ofpoul
try, the appearance was impaired (Cox et al. 1974).
Succinic acid occurs naturally in foods such as asparagus, beets, broc
coli, rhubarb, sauerkraut, and cheese.
Acetaldehyde. This chemical (CH3CHO) has a boiling point of 21C and is
naturally present in some fruit tissues. The vapors are irritating to mu
cous membranes. The chemical has a general narcotic action. Large
amounts may cause respiratory paralysis and result in death.
Acetaldehyde vapor has bactericidal and fungicidal properties. It is
effective in the control of various microorganisms causing postharvest
decay of fruits and vegetables, as well as yeasts associated with spoilage
of fruit juice (BarkaiGolan and Aharoni 1976). The extent of fungicidal
properties depends upon the concentration of the vapor and the length
of exposure.
Ethyl Alcohol. This is one of the most widely used substances to control
microorganisms. It acts by denaturing protein.
Alcohol is bactericidal but not sporicidal. It has poor penetrating
power and is readily inactivated by organic matter. It is not considered
to be a food preservative, although it can increase the antimicrobial effec
tiveness of other substances such as sorbic acid in wine. Shapero, Nelson,
and Labuza (1978) found ethanol effective against S. aureus and suggested
that it might be useful in foods with lowered aw
Fungicides. Certain fungicides are used for the postharvest treatment of
fruits and vegetables to control spoilage. Also, they may be used in foods
with low a
w
to control mold growth. Fungicides were discussed by Hall
(1979). The fungicides include benzimidazole derivatives (benomyl, thia
benzadole), and polyene macrolides (nystatin, natamycin [pimaricin]).
Benomyl is the name given to methyl1(butylcarbamoyl)2benzimi
dazolecarbamate. Benomyl dips reduce the rotting of apples by Gloeospo
rium species. Applied to tomatoes as a dipping treatment, benomyl con
trolled Botrytis, Penicillium, and Cladosporium (Domenico, Rahman, and
Westcott 1972). Benomyl did not control Alternaria, which seemed to
grow better with benomyl treatment. This was believed to be due to reo
duced competition from the inhibited fungi.
Thiabenzadole has been widely adopted by apple growers to reduce
rotting by Gloeosporium species (Edney 1973).
The FDA has permitted the use of natamycin on the surface of cuts
and slices of cheese when the standards of identity allow antimycotic
agents. Application can be made by dip, spray, or incorporation into edi
ble or plastic coatings.
Antioxidants. Antioxidants such as butylated hydroxyanisole (BHA) and
butylated hydroxy toluene (BHT) also have certain antimicrobial proper
ties. Both of these chemicals are classified as GRAS at levels currently
used. In fact, there is evidence that antioxidants prevent the formation
of carcinogens in foods such as fried hamburger. They retard the devel
opment of oxidative rancidity and extend the shelf life of foods contain
ing fat. Hence, they are chemical preservatives.
One team of researchers reported that BHT is toxic to rodents at
doses of 0.25 or 0.50 percent ofthe diet (Vorhees et al. 1981). These levels
are about 100 times higher than would be used in any food.
In their reviews, Kabara (1981) and Davidson and Branden (1981) reo
ported the antimicrobial effects of BHA on bacteria (P. jluorescens, E. coli,
V. parahaemolyticus, S. typhimurium, C. perfringens, C. botulinum, S. aureus) and
molds (A.jlavus, A. parasiticus, Penicillium, Geotrichum) and BHT on bacteria
(S. senftenberg, S. typhimurium, Mycoplasma synoviae) and viruses, as well as
tertiary butylhydroquinone (TBHQ) on S. aureus. Pediococcus pentoaceus
was inhibited by TBHQ with the minimum inhibitory concentration
(MIC) at 15 ppm (Raccach and Henningsen 1981). According to Post and
Davidson (1986) two of the most resistant species to BHA were S. aureus
and P. jragi, while two of the most susceptible they tested were C. per-
jringens and P. jluorescens.
Protease secretion of Aeromonas hydrophilia was inhibited by BHA
(Venugopal, Pansare, and Lewis 1984). When S. aureus was treated with
BHA, there was a leakage of nucleotides (Degre and Sylvestre 1983) and
inhibition of respiratory activi,ty (Degre, Ishaque, and Sylvestre 1983).
Chlorine. The use of chlorine in the sanitation of processing plants and
equipment is discussed in Chapter 10.
There have been many hypotheses regarding the site of action of hy-
pochlorite on the bacterial cell. It is generally agreed that the action of
chlorine involves the penetration of the cell. There are suggestions that
once inside, chlorine reacts with cellular protoplasm, which causes inacti-
vation. There are other suggestions that the chlorine reacts with key en-
zyme systems and prevents normal respiration. These reactions may in-
volve oxidation or denaturation of the protein. The -SH groups of
essential enzymes are possible sites of action. The oxidation theory has
been questioned, since oxygen from other sources does not kill bacteria
as readily as does chlorine.
There have been suggestions that cell membranes are altered or dis-
rupted by chlorine. This allows diffusion of cell contents out of the cell
or toxic materials into the cell.
Sodium hypochlorite preferentially inhibits polymerase deficient
bacteria. Polymerase I is active in repair of damaged DNA. Rosenkranz
(1973) suggested that the hypochlorite enters the cell and reacts with cel
lular DNA. According to Roller, Olivieri, and Kawata (1980), the primary
site of action is not dehydrogenase enzymes, the proteinsynthesizing
complex, or DNA.
Using chlorine (1,000 ppm) as a dip for fresh fish extended the stor
age life of the fish (Kosak and Toledo 1981). When added to chiller water
for poultry, the microbial load was reduced in the water and on the poul
try carcasses (Lillard 1979; Thiessen, U sbome, and Orr 1984).
Diphenyl. This is also biphenyl or phenylbenzene (C12HIO). It is colorless
with a pleasant but peculiar odor and is insoluble in water.
In experimental animals, the diphenyl is excreted mainly as 4hydroxy
diphenyl. High levels cause an intoxication, with increased respiration
rate, loss of appetite and body weight, muscular weakness, paralysis, and
convulsion. Respiration difficulties can terminate in coma or death. The
LD50 for rats is 2.2 g/kg.
Diphenyl is effective against molds but is not very active against yeasts
or bacteria. It is used on the inside of shipping containers of citrus fruits,
especially oranges. The diphenyl gradually volatilizes and inhibits the
growth of molds on the fruit surface.
Although diphenyl may migrate into the peel, very little is found in
the juice (usually less than 1 J.tg/ml). The amount allowed in the whole
fruit (including the peel) is 110 J.tg/g (0.011 %).
Ethylenediaminetetraacetic Acid (EDTA). This chemical is a chelating agent.
The chemical formula is (HOOCCH
2
hNCH
2
CH
2
N(CH2COOHh. The
principal function of EDT A is to chelate trace metals that have an adverse
effect on the quality of foods. EDTA is colorless, odorless, and tasteless.
EDTA is not GRAS, but it has been approved as a food additive by the
FDA. The toxicity of EDT A was reviewed by Kabara (1981). There is only
a low order of toxicity in animals, and no effects were noted in humans.
Apparently EDTA is not carcinogenic.
The main effect of EDTA on microorganisms is as a synergist for
other antibacterial compounds. By che1ating the trace metals, EDTA de
prives the microorganisms of these essential cellular components as well
as affecting the metalcontaining enzymes.
Although EDTA had only a slight effect on the total bacterial count
of fresh fish (Baltic herring and rainbow trout), it had a favorable effect
on the keeping quality (Kuusi and Loytomaki 1972). The volatile basic
nitrogen, trimethylamine and hypoxanthine, increased at a slower rate
in EDTA-treated fish than in the untreated controls.
A combination of lysozyme and EDTA inhibited microbial growth on
shrimp (Chander and Lewis 1980) and S. typhimurium on broiler parts
(Samuelson, Rupnow, and Froning 1985). At 500 ppm EDTA, B. cereus
was inhibited, but the addition of iron, zinc, and calcium reversed the
inhibition (Bulgarelli and Shelef 1985). Houben and Krol (1985) stated
that EDTA was promising for use in pasteurized ham to control Streptococ
cus faecium spoilage.
Hydrogen Peroxide (H20 2). Hydrogen peroxide is available as stabilized solu
tions at various concentrations. Aqueous solutions of H
2
0
2
gradually de
teriorate to water and oxygen. Excess H
2
0
2
can be removed from food
by adding catalase to produce water and oxygen. The addition of H20 2
to certain foods is a simple and efficient method to inactivate certain
undesirable microorganisms.
Hydrogen peroxide has been approved by the FDA for use as an anti
microbial agent in raw milk to be used to make certain types of cheese,
and in whey processing. Also, it can be used for sterilizing food-contact
surfaces made with polycarbonate resins. The milk can be treated with
no more than 0.05 percent H
2
0
2
When added to liquid egg white, H20 2
is an aid in pasteurization to destroy salmonellae. Milk or liquid egg
white treated with H
2
0
2
can be pasteurized at a lower temperature than
that normally used for this purpose.
The effectiveness of H
2
0
2
is influenced by the concentration, the mi-
crobial population, the temperature, pH, inorganic ions, the time of con-
tact, and other treatments given the microorganisms, such as heating,
ultraviolet light, or other preservatives.
The actual effect ofH
2
0
2
on cells is not completely known, other than
its oxidative action. According to Rosenkranz (1973), H
2
0
2
is a mutagen
and an inducer of chromosomal aberrations. It is capable of reacting
with DNA and its bases. The results of Hoffmann and Meneghini (1979)
favored the hypothesis that H
2
0
2
reacts in the cell, producing a radical
that causes the formation of singlestrand breaks ofthe DNA. Reportedly
H20 2 affects certain enzyme systems, and it can affect the ultrastructure
of certain spores. According to Baldry (1983), H20 2 is more effective as
a sporicide than as a bactericide. The sporicidal effects of H
2
0
2
were
discussed by Stevenson and Shafer (1983) and Wang and Toledo (1986).
Lysozyme. This is an enzyme that is naturally present in various biological
systems. Egg white lysozyme was suggested as a preservative for sake (Ya
jima, Hidaka, and Matsuoka 1968). The minimum inhibitory concentra-
tion against lactobacilli in sake varied from 1.25 /-tg/1I.lI for L. fermentum
to 100 /-tg/ml for L. acidophilus. In sake, lysozyme was very stable. Lysozyme
has its optimal activity near pH 7.0.
Systems have been suggested that allow lysozyme to attack the cell
walls of Gram-negative bacteria. The use of EDTA in combination with
lysozyme was suggested as a means of controlling salmonellae (Anon.
1976). Miller (1969) suggested that treating Gram-negative bacteria with
a mixture of H
2
0
2
and ascorbic acid renders the organisms sensitive to
lysozyme.
The relatively high cost of lysozyme limits the use of this enzyme as
an antimicrobial agent for food preservation.
Methyl Bromide. This chemical is used for fumigation of dried fruits to
prevent insect infestation. Methyl bromide has weak bactericidal proper-
ties. It is active only on fungi and vegetative bacterial cells.
At a level at which methyl bromide protected dates against microorgan-
isms, the dates developed an off-odor (Bolin et al. 1972). A combination
of sorb ate and methyl bromide gave good protection of dates with no
detectable odor or flavor changes. Methyl bromide is allowed in dates if
the residual inorganic bromide is below 100 J-tg/g.
The fumigation of poultry feed with methyl bromide eliminated sal-
monellae from the feed. There were no adverse effects on the chicks fed
this treated feed (Tucker, Brown, and Goodship 1974). Methyl bromide
can cause mental and neurological disturbances in humans and concen-
trations above 500 mg/kg can be fatal.
Propylene Glycol. This chemical (CH3-CHOH-CH2-OH) has many functions
in the food industry. It has some antimicrobial properties and has been
used as a food preservative. The chemical is relatively nontoxic.
Propylene glycol exhibits activity toward Gram-positive and Gram-
negative bacteria. Acott, Sloan, and Labuza (1976) found that the only
approved chemical effective in controlling aspergilli and staphylococci
in an 1M food was propylene glycol. Usually a mixture of 2 percent pro-
pylene glycol and 0.3 percent potassium sorb ate is used as the antimy-
cotic system in 1M meat products.
Sodium Chloride (NaCl). Treating food with salt was one of the earliest
methods of food preservation. Salt preservation has been important in
the development of the fishing industry. In some areas of the world,
salted fish remains an important food product. Besides its preservative
function, salt is added to foods to enhance flavor, to aid in the develop-
ment of color in processed meats and sauerkraut, to aid in the texture
of processed meats, yeast-raised baked goods, and certain cheeses, to sol-
ubilize myofibrillar proteins that act as binders in cured meats, and to
help in the regulation of the rate of fermentation in pickles, sauerkraut,
sausage, cheese, and yeast-leavened dough.
In some individuals, the ingestion of sodium has been associated with
hypertension. As a result, there is an effort to reduce the consumption
of salt by various means. One possibility is to substitute KCl or MgCl
2
for
TABLE 11.12. SALT CONCENTRATION IN THE WATER PHASE
(OR BRINE) IN FOODS WITH VARIOUS WATER CONTENT
Salt Added
Brine Concentration with Water Content of Food (%)
(%) 40 50 60 70 80
2.5 5.88 4.76 4.00 3.45 3.03
3.0 6.98 5.66 4.76 4. II 3.61
3.5 8.05 6.54 5.51 4.76 4.19
4.0 9.09 7.40 6.25 5.41 4.76
5.0 ILl I 9.09 7.69 6.67 5.88
6.0 13.04 10.71 9.09 7.89 6.98
7.0 14.89 12.28 10.45 9.09 8.04
8.0 16.67 13.79 11.76 10.26 9.09
9.0 18.37 15.25 13.04 11.39 10.II
NaCI in cured meats (Barbut, Tanaka, and Maurer 1986; Keeton 1984;
Pelroy et al. 1985; Sofos 1983; Wagner and Busta 1985; Whiting, Bene-
dict, and Cangialosi 1986). In some cases, it appears that sodium can be
reduced or partially replaced with other chemicals or metallic ions, but
further research is needed before widespread changes are made in food
formulations. As discussed by Sofos (1983), there are many factors that
influence the effect of salt on microorganisms in a food. The level of salt
presently used does not completely inhibit sporeforming organisms, but
other things must be considered (refrigeration, pH, heat treatment, and
other chemicals or treatments).
Microorganisms vary in their tolerance to salt. The least tolerant are
organisms such as Spirillum, which can tolerate only about 1 percent
NaCl. Most normal organisms have a slightly greater salt tolerance. Gen-
erally these are inhibited by 3 to 10 percent salt. The mesophilic Gram-
negative rods and psychrophiles are inhibited by 4 to 10 percent salt.
The lactic-acid-producing bacteria vary in tolerance from 4 to 15 percent
salt. Sporeforming bacteria can tolerate from 5 to 16 percent salt. The
organisms in these upper ranges are called halotolerant. Although they
grow well in low levels of salt, they can tolerate these high levels of salt
and even display some growth. These organisms include the micrococci,
staphylococci, and sporeforming bacteria.
The halophilic (salt-loving) bacteria need relatively high concentra-
tions of salt for growth. Some may grow at levels approaching saturation
(26.4 percent). Most of the halophiles contain red, pink, or other pig-
ments and are classified in the genera Halobacterium or Halococcus.
Yeast species vary greatly in their tolerance to N aCl. The two most
important factors are thought to be a mechanism to prevent entrance of
NaCI to retain a low concentration of salt within the cells and the ability
of enzyme systems to react in the presence of high levels of salt. A num-
ber of yeast types are able to propagate in pickle brine with 19 to 20
percent NaCl. They are more resistant to salt at pH 3.0 to 5.0 than at
higher or lower values.
Salt has been reported to both increase lethality due to heat and to
have a protective effect for the cells. Generally, low levels (I to 2 percent)
of salt may protect, while higher levels (6 to 10 percent) may increase
lethality of heat.
Although salt at a level of 10 percent in glucoseyeastsalt medium
inhibited A. parasiticus, it did not prevent the formation of aflatoxin pro
duction over a tenday period at either 13 or 28C (EIGazzar, Rusul,
and Marth 1986).
There are three main types of microbial spoilage of salted fish. These
are sliming, pink (red), and dun. Sliming is characterized by a semigreasy,
sticky, glistening layer that is yellow gray or beige and results in a pun
gent odor. The dominant organisms involved are Gramnegative rods,
although Grampositive and Gramnegative cocci also are present. These
organisms grow optimally with 6 to 8 percent salt.
The pink or red defect is due to growth of red halophilic bacteria,
which are species of Halobacterium or Halococcus. Besides the red color, an
off.odor is evident due to the production of indole.
The defect called dun consists of peppered spots ranging in color
from chocolate to fawn. It is caused by a halophilic mold, Sporendonema
epizoum, which has optimum growth at 10 to 15 percent salt.
Sodium Phosphates. Sodium and phosphate combine to form a number of
compounds such as sodium hexametaphosphate, sodium tripolyphos
ph ate, and tetrasodium pyrophosphate. These are referred to as poly
phosphates. The polyphosphates are added to foods for various reasons.
The antimicrobial effects are mainly due to the chelation of metallic
ions.
Soaking chicken carcasses in solutions of 8 percent polyphosphates
retards the production of off odor, slime, and discoloration during refrig
erated storage. Lipolytic types of bacteria on the poultry seem to be in
hibited to a greater extent than proteolytic types.
Dipping of cherries in 10 percent polyphosphate solution inhibited
fungal growth up to thirty days at 1.1 C (Post et al. 1968). Untreated fruit
showed fungal growth at fourteen days. Sodium tetraphosphate ap
peared to be the most effective antimycotic of the polyphosphates that
they tested.
The type of phosphate and pH influenced the inhibition of S. aureus
(Jen and Shelef 1986). Inhibition was reduced or eliminated by the addi
tion of magnesium, calcium, or ferrous salts. Sodium acid pyrophosphate
improved the antimicrobial properties of ground meat with reduced salt
content (Madril and Sofos 1986). Various other antimicrobial activities
of the polyphosphates were reviewed by Sofos (1986), Tompkin (1983),
and Wagner (1986).
Sucrose. Although there are many sugars, the common name sugar denotes
sucrose. Low levels of sucrose are utilized by many microorganisms as a
source of energy, but high levels of this chemical can have inhibitory
properties. The preservative effect of sucrose is due to the development
of osmotic forces and a lowered water activity.
Sugar is used in the preservation of several foods, including sweet
ened condensed milk, jams, and jellies. Sweetened condensed milk is
made by adding up to 40 percent sucrose to fresh milk. The milk is
heated in evaporating pans to remove water and then canned as a prod-
uct with 70 to 75 percent solids. Yeasts and molds are killed by the heat
treatment during evaporation, the can keeps out further contamination,
and the high sugar content prevents the growth of surviving microorgan-
isms.
The main preservative factor in normal jams is the combination of
sucrose concentration and low pH. In an acid medium, sucrose is in-
verted to glucose and fructose. This increases the number of solute mole-
cules, which assist in lowering the aw of the product. The low aw, along
with a pH of about 3.0, prevents the growth of most microorganisms.
The control of microbial growth is helped by filling these products
while still hot, by heating the surface by steam injection, spraying the
surface with a chemical fungicide, or using a cover paper impregnated
with a fungicide.
Sulfites. The GRAS list of FDA has included sulfiting agents. The sulfites
include sulfur dioxide, sodium sulfite, and the sodium and potassium
salts of bisulfite and metabisulfite.
Besides their antimicrobial effects, the sulfites are added to foods to
control enzymatic and nonenzymatic browning, for bleaching, antioxi-
dant properties, and as dough conditioners. Hence, they have been
added to many foods, but in the United States they have not been allowed
as additives of fresh red meats or of foods known to contribute more
than 10 percent of the RDA of thiamin.
There have been reports of more than 1,000 adverse reactions, includ-
ing some deaths, presumably caused by the ingestion of sulfites. These
chemicals are not harmful to most people, but some asthmatic people,
particularly those who are on steroid drugs, may be hypersensitive to
sulfites and experience anaphylactic shock or acute asthma attacks after
ingestion of these chemicals. Most of the problems with the chemical
have been a result of the consumption of sulfites used on fruits and vege-
tables in salad bars. As a result, in 1986, the FDA issued regulations ban-
ning the use of sulfites on fresh fruits and vegetables, including those in
salad bars. Furthermore, since January 1987, the presence of sulfiting
agents in any food at levels of 10 ppm or over must be declared on the
label, whether the chemical was added directly or indirectly. Besides legal
limits of sulfites, there are technical limits, because, at high levels, they
may produce off.odors or off.flavors in certain foods.
There are other chemicals that can be used to help maintain the ap
pearance and retard browning of fruits and vegetables in salad bars.
The antimicrobial action is pH dependent. Sulfur dioxide combines
with water to form sulfurous acid (S02 + H
2
0 H
2
S0
3
). This acid has
two dissociation constants. These are near pH 2 and pH 7. At pH 7, the
HS0
3
- and S03 = are in about equal proportions. At pH 5, most of the
compound is in the form of HS0
3
- . At lower pH values, protonation of
the bisulfite ion results in molecular sulfur dioxide. The lower the pH,
the higher is the proportion of S02. The free, molecular S02 is about
sixty times more inhibitory than is the bound form. The molecular S02
form has the greatest and the S03= the least antimicrobial effect. The
undissociated H
2
S0
3
is the only effective molecular species against yeast
(King et al. 1981).
Sulfites inactivate certain enzyme systems, such as cytochrome oxi
dase. The cytoplasmic membrane of bacterial cells is attacked by S02,
which alters the permeability.
Sulfites combine with acetaldehyde, the normal hydrogen acceptor
required for glycolysis, and inhibit fermentation. There is evidence that
bisulfite reacts with pyrimidine bases, so the antimicrobial activity may
involve the RNA and DNA of microbial cells.
The sulfites show antimicrobial activity against both fungi and bacte
ria. Gramnegative bacteria are more susceptible than Grampositive
types. Some lactic acid bacteria are fairly resistant to the action of S02,
even at high concentrations. Yeasts are more resistant to S02 than either
bacteria or molds.
Antibiotics. The therapeutic value of antibiotics in medicine stimulated
research to determine their effectiveness as food preservatives. This re-
vealed that broad-spectrum antibiotics, such as chlortetracycline (CTC)
or oxytetracycline (OTC), had greater potential for use in foods than anti-
biotics such as penicillin. In November, 1955, a tolerance of 7 /-tg/g for
CTC in or on uncooked poultry was established. The following year, the
same tolerance was established for OTe. The petition requesting the es-
tablishment of the tolerance stated that the cooking of poultry destroyed
residues of CTC when used at the accepted level. In 1959, the authoriza-
tion to use CTC or OTC was extended to raw, whole, or gutted fish,
shucked scallops, and unpeeled shrimp. A maximum tolerance of 5 /-tg/g
was established for these products.
The original tolerances were established due to the antibiotics having
a useful function in the food and because their use in food was seen as
safe. However, due to the hazard of the development of resistant strains
of pathogens, the increasing resistance of these organisms, the potential
for human hypersensitivity to the antibiotics, the finding of residual anti
biotics even after cooking (although these levels were very low and prob
ably insignificant), the lack of significant benefits when used in commer
cial operations, and the costs and difficulties involved in monitoring all
of these aspects, the tolerance of CTC, OTC, or similar antibiotics on or
in food was rescinded, and they are no longer permitted in foods in the
United States. Antibiotics were never approved for use in fresh meat.
Although antibiotics are not permitted as direct additives, this does
not mean that there are no antibiotics in our food, nor does it mean that
all antibiotics will be banned in the future.
Several antifungal antibiotics have been suggested to control mold or
yeast damage on crops in the field or on fruits and vegetables immedi
ately postharvest. Since the antibiotics are on the surface, they can be
washed off quite readily, so there should be little, if any, residual in the
food.
As long as microorganisms are associated with food, there is a possi
bility that antibiotics are present, since, after all, microorganisms pro
duce antibiotics.
One antibiotic that occurs naturally in food products such as cheese,
and has attracted much attention, is nisin. This antibiotic is a polypep
tide produced by certain strains of Streptococcus lactis. It has inhibitory
properties against Grampositive microorganisms.
Some advantages concerning the use of nisin in foods are that it is
not toxic to humans, it has no use in medicine, and it does not form
crossresistant mutants. Its medical value is of no significance because it
is practically insoluble in blood at physiological pH. It is digested by in
testinal enzymes and bacteria, and the large size of the molecule pre
cludes absorption if intramuscular injection is used. It is unlikely that
nisin has any effect on the intestinal flora. Nisin has no effect on Gram
negative bacteria and does not inhibit all of the Grampositive bacteria.
Some bacteria produce the enzyme nisinase, which inactivates nisin.
The primary application of nisin is in heat processed foods. This is
due to the action of nisin on spores. Heat fixes nisin on spores and
thereby reduces their heat resistance. This makes it possible to heat steri
lize foods with reduced heat treatments.
Nisin does not influence the heat resistance of some spores, but its
continued presence in the recovery medium reduces the count of survi
vors. In sufficient concentration, nisin inhibits either the germination of
spores or the outgrowth of the cells. According to the review by Hurst
(1972), nisin prevents the outgrowth rather than the germination of
spores.
Clostridium botulinum is one ofthe more nisinresistant species. Somers
and Taylor (1981) found that nisin was less effective in preventing the
outgrowth of C. botulinum spores in cooked meat medium than in TPYG
broth. They suggested that nisin binds to meat particles. Rayman, Malik,
and Hurst (1983) reported that up to 550 ppm nisin in combination with
60 ppm nitrite failed to prevent outgrowth of C. botulinum spores when
added to pork slurries at pH 5.8. Hence, with canned foods, a sufficient
process (time and temperature) is needed to assure that C. botulinum
spores are controlled. The use of nisin would need to be evaluated for
each type of heatprocessed food.
Nisinsensitive cells of lactobacilli were killed within 1 min of contact
with the antibiotic, apparently because of the loss of ATP (Ogden and
Waites 1986). Ogden and Tubb (1985) found that most Gramnegative
bacteria and none of the brewing yeasts were resistant to nisin. They
suggested the use of nisin to prevent spoilage of worts or beers by lactic
acid bacteria.
Nisin is not allowed as a food additive in the United States; however,
some countries allow its use in one or more foods.
Since antibiotics can become associated with foods in many ways,
some countries, and the Food and Agricultural Organization of the
World Health Organization of the United Nations, have set tolerances
for some of these agents. For the FAO, these include ampicillin (0.06
/-tg/g) , penicillin (0.06 /-tg/g), oxytetracycline (0.25 /-tg/g), and neomycin
(0.50 /-tg/g).
Sodium Nitrite (NaN02 ). The history of meat curing by nitrites and nitrates
was discussed by Binkerd and Kolari (1975). Besides these chemicals, var
ious ingredients, including NaCl, are used to produce cured meats. In
recent years, many chemicals have been tested as adjuncts to or substi
tutes for nitrites in these foods.
An additive should have a useful function in the food and be safe for
the consumer. In cured meats, nitrites are involved with the development
of the typical color and flavor as well as having antimicrobial activity,
especially against C. botulinum. However, the safety of nitrites has been
questioned, mainly due to the formation of carcinogenic nitrosamines.
Nitrite is added to the curing mixture to develop the typical color of
cured meat. In acid conditions, nitrite forms nitrous acid, which is reo
duced to nitric oxide. By a series of reactions, the nitric oxide combines
with the muscle pigment myoglobin to form nitric oxide myoglobin.
Upon heating, this pigment forms the more stable red pigment nitrosyl
hemochrome, associated with cooked ham.
According to Kemp, Fox, and Moody (1974), goodquality hams can
be produced without either nitrate or nitrite. Hams cured with 182 p,g/g
nitrite had a more typical ham color than those cured with 91 p,g/g nitrite
(Brown, Hedrick, and Bailey 1974). Eakes and Blumer (1975) obtained
hams with acceptable color when 90 p,g/g nitrite was used in the cure.
Higher levels of nitrite produced higher concentrations of the cured pig
ment, but these higher values were not considered to be necessary for
color acceptability.
Hence, relatively low levels of nitrite are sufficient for color develop
ment. Nitrite levels of 10 p,g/g and 50 p,g/g of meat have been suggested
as sufficient for complete color development of cured meat. Reducing
agents such as ascorbic acid accelerate color formation and increase
color uniformity and stability.
A cured meat flavor in ham was developed by as little as 50 p,g/g so
dium nitrite (MacDonald et aL 1980). This level of nitrite was as effective
as 500 p,g/g in retarding the development of off-odors or offflavors duro
ing aerobic storage at 4C for seven days. The role of nitrite in cured
meat flavor has been reviewed in detail (Gray et aL 1981).
The antimicrobial action of nitrite is due primarily to the presence
of undissociated nitrous acid (HN0
2
). The antimicrobial activity is pH
dependent. Above pH 7.5, nitrite may aid bacterial growth. Between pH
6.0 and 7.0, only a small portion of undissociated HN0
2
is present, so
there is only a slight antibacterial effect. Between pH 4.5 and 5.5, the
presence of nitrous acid shows a definite antimicrobial effect. Below pH
4.5, the nitrous acid appears to decompose to nitric acid, nitric oxide,
and water. Neither nitric oxide nor nitric acid shows the same activity
against microorganisms as nitrous acid. In the presence of oxygen, the
nitric oxide is oxidized to nitrogen dioxide. This combines with water to
form nitric acid and nitrous acid. In canned or vacuumpackaged meat,
as well as in the interior of meat, there would not be sufficient oxygen
for these reactions to occur. Below pH 4.5, nitrite is less effective, but pH
effects help control bacterial growth.
The main benefit of nitrite is the inhibition of C. botulinum in canned
cured meat. If sufficient heat is used to destroy C. botulinum spores, the
quality of ham may be affected. The nitrite concentration in vacuum
packed hams delays the onset of toxin formation and spoilage. Despite
the fact that both aerobic and anaerobic spores can be grown from
canned cured meats, according to an FDA consumer memo, there have
been no known outbreaks of botulism caused by processed food contain
ing nitrites.
The inhibition of C. botulinum involves the interaction of the concen
tration of salt and sodium nitrite, the pH, the amount of heating, the
number of spores that are present, and the temperature of storage. Ap
parently, heat injury is not necessary for NaN0
2
to inhibit toxigenesis of
C. botulinum (Pivnick et al. 1967). Curing salts do not alter the heat resist
ance of spores, but they tend to inhibit the subsequent outgrowth of sur
viving spores.
Tompkin, Christiansen, and Shaparis (1978b) reported that iron af
fects the inhibition of C. botulinum in canned cured meat. Their hypoth
esis is that nitric oxide reacts with iron, such as in ferredoxin, in the
germinated spore and prevents outgrowth of the cells. When the avail
able iron was increased, the antibotulinal effect was reduced (Tompkin,
Christiansen, and Shaparis 1979). The addition of ferric chloride or myo
globin decreased the effect of nitrite on C. botulinum (Vahabzadeh et al.
1983). For both studies, the addition of EDTA, which chelates iron, re-
stored the antibotulinal effect of nitrite. Reddy, Lancaster, and Cornforth
(1983) also suggested that the inhibition of C. botulinum by nitrite is due
to the inactivation of iron-sulfur enzymes (especially ferredoxin) by the
binding of nitric oxide.
Another team of researchers stored cured bacon inoculated with C.
botulinum at 7 and 27C (Christiansen et al. 1974). No toxin was found
in samples stored at 7C for eighty-four days. However, samples formu-
lated with 120 p,g/g nitrite or less became toxic when held at 27C. At low
levels of inoculation (210 spores before processing and 52 spores/g after
processing), levels of 170 p,g/g nitrite or more prevented toxin formation.
At higher levels of inoculation (4,300 spores/g after processing), toxin was
detected even with 340 p,g/g nitrite added to the meat. Increased levels of
nitrite decreased the rate of toxin production. The level of nitrite needed
to prevent toxin production depended upon the number of C. botulinum
spores.
The importance of a low pH on nitrite inhibition of toxin production
has been established (Christiansen et al. 1975). At normal pH levels (pH
5.8-5.9) in a summer sausage, nitrite levels of 150 p,g/g did not prevent
toxin production. At lower pH (4.2-4.7), no toxin was evident with nitrite
at 50 p,g/g.
Sorbic acid or potassium sorbate, at 0.2 percent or higher, alone or
in combination with nitrite, has been effective in retarding spore germi-
nation, outgrowth, and toxin production (Huhtanen et al. 1983; Ivey et
al. 1978; Roberts, Gibson, and Robinson 1982; Sofos et al. 1980; Wagner
et al. 1982). With the addition of sorbate, the level of nitrite can be re-
duced and still be effective in inhibiting C. botulinum. There have been
several reviews on the effect of nitrites on C. botulinum in cured meats
(Benedict 1980; Holley 1981; Sofos, Busta, and Allen 1979).
Many organisms, such as some strains of lactobacilli, pediococci,
enterococci, and various Gram-negative species, including salmonellae,
are relatively resistant to nitrite. Under anaerobic conditions, L. plan-
tarum was sensitive to nitrite (Collins-Thompson and Thomson 1986)_ Ni-
trite at 150 ppm did not inhibit Y. enterocolitica at 5C and B. cereus or
salmonellae at 10 to 12C (Nielsen and Zeuthen 1984). Nitrite did inhibit
active transport, oxygen uptake, and oxidative phosphorylation of P. aeru-
ginosa (Rowe et al. 1979).
The concentration of nitrite decreases during storage of cured meat
and may disappear after several months. Sodium nitrite is considered to
be heat labile, and some of it is destroyed during the heat processing
used in cooked or canned cured meats. Growth of organisms in stored
cured meat may be due to a tolerance for nitrite, or it can be due to the
loss of nitrite to such a low level that there is essentially no antibacterial
activity.
The loss of nitrite is important not only because there is a reduction
in the amount of inhibitor, but also because the type of compounds that
are formed may be desirable or undesirable_ In a meat model system,
about 74 percent of the nitrogen from added nitrite was found as nitrite,
nitrate, nitrosothiol, denatured nitrosomyoglobin, and gaseous nitrogen
(Miwa et al. 1978). The other 26 percent was found in unidentified com-
pounds. Other researchers suggested that about 30 percent of the added
nitrite is bound to protein (Woolford et al. 1976). Woolford and Cassens
(1977) reported that between 10 and 15 percent of the nitrite was in the
adipose tissue of bacon.
Besides the consumption of cured meats, there are other dietary
sources of nitrate and nitrite. According to Rooma (1971), many vegeta-
bles contain higher levels of nitrate than do cured meats. High levels
of nitrate and nitrite in vegetables were reported by Eerola, Varo, and
Koivistoinen (1974), Lin and Yen (1980), and Hall, Stall, and Hicks (1978)-
Well water and rainwater runoff from fertilized cropland may contain
high levels of nitrate (Nicolson 1979; Super et al. 1981). The major source
of nitrate is vegetables, while nitrite is primarily from cured meats, ce-
reals, baked goods, milk, fruit juice, and water (McCormick 1982).
According to Emerick (1974), nitrite is ten times as toxic as nitrate.
Bartholomew and Hill (1984) stated that ingested nitrate is absorbed
from the upper digestive tract and is secreted in sweat and saliva. How-
ever, they also stated that 65 to 70 percent of nitrate is excreted in the
urine within 24 hr.
Some nitrite reacts with hemoglobin in the blood, oxidizing it to met-
hemoglobin (CDC 1975). In this form, it cannot carry oxygen to the tis-
sues. The normal reducing systems of the blood can handle some in-
crease in methemoglobin, but when these systems become overtaxed,
cyanosis occurs, followed by methemoglobinemia, which can be fatal. Nu-
merous cases of infant methemoglobinemia have been related to exces-
sive nitrite in the diet.
In recent years, the main concern about sodium nitrite has been the
reaction of nitrite with secondary and tertiary amines to form nitrosa-
mines_ Some, but not all, nitrosamines are mutagenic, teratogenic, and car-
cinogenic The mutagenic and carcinogenic aspects of nitro sa mines were
reviewed by Olajos and Coulston (1978)_ Various carcinogenic and non-
carcinogenic nitrosamines have been listed (Nagao et aL 1977)_
The widespread use of nitrites, coupled with the natural occurrence
of amines in foods, has resulted in the possibility that these carcinogens
are present in our food supply or are produced in the digestive tract-
There are many nitrosamines, but the common ones are N-nitropyr-
rolidine, N-nitrosodimethylamine, and N-nitrosopiperidine (Fig_ 11-9).
Nitrosamines have been detected in rubber baby bottle nipples and paci-
fiers (Havery and Fazio 1983; Osterdahl 1983), vegetable oils and marga-
rine (Hedler, Schurr, and Marquardt 1979; Sen and Seaman 1981 a), malt
and beer (Goff and Fine 1979; Havery, Hotchkiss, and Fazio 1981; Sen,
Tessier, and Seaman 1983), seafoods (Fong and Chan 1976; Kndlchel and
Huss 1984), dairy products (Havery, Hotchkiss, and Fazio 1982; Lakritz
and Pensabene 1984; Libbey, Scanlan, and Barbour 1980; Sen and Sea-
man 1981b; Weston 1983), various Chinese foods (Fong and Chan 1977;
Gough et aL 1977), and animal feed (Ragelis et aL 1983). Generally, the
nitrosamines are at very low levels or in trace amounts. Also, research
has shown the sources for some of these compounds so that contamina-
tion can be reduced further (Havery and Fazio 1985; McWeeny 1983; Sen
and Baddoo 1986).
When nitrosamines are found in cured meats, they are usually at lev-
els below 10 ng/g (Fiddler et aL 1975; Panalaks et aL 1974). However,
some products had levels of nitrosodimethylamine from 90 to 105 ng/g.
o
I
NO
Nitrosopiperidine
CJ
I
NO
Nitrosopyrrolidine
C H 3 ~ C H 3
NO
Dimethylnitrosamine Figure 11.9. Nitrosamines.
Due to the reduction of the amount of nitrite in cured meats, as well as
other procedures, most cured meats now have either nondetectable levels
of volatile nitrosamines or only trace amounts. However five of nine sam
pies of cured meats contained the moderately volatile nitrosothiazolidine
(Sen, Seaman, and Baddoo 1985).
The analysis of spice-cure mixtures yielded nitrosamines at levels of
2 Jlg/g (Havery et al. 1976) to 25 Jlg/g (Sen et al. 1974). Due to findings
such as these, the FDA and USDA ordered meat packers to quit mixing
sodium nitrite with spices in advance of placing the curing mixture into
meat products. Federal regulations prohibit the marketing of spice-cur-
ing mixtures unless the nitrites and spices are separated by chemical
buffer or by packaging.
Although various foods and cured meats have been found to contain
low levels of nitrosamines, bacon has been the greatest concern in this
regard due to the presence of relatively high levels of nitrosodimethyl-
amine and nitrosopyrrolidine, both of which are carcinogenic. The forma-
tion of nitrosamines in cooked bacon is influenced by preprocessing pro-
cedures, pH, salt concentration, nitrite concentration, the presence of
other additives, ratio of lean to fat, slice thickness, smoking, and method
and temperature of cooking (Bharucha, Cross, and Rubin 1979; Lee,
Gray, and Pearson 1983; Pensabene and Fidolt:r 1983, 1985; Pensabene
et al. 1980; Reddy et al. 1982; Robach et al. 1980; Sen, Seaman, and Miles
1979; Skrypec et al. 1985; Spinelli-Gugger et al. 1981; Theiler et al. 1981).
One team of researchers reported different levels of nitrosamine for-
mation in bacon cooked by various methods (Pensabene et al. 1974). Bak-
ing of bacon resulted in the detection of 12 to 35 ng/g, while microwave-
cooked bacon contained from 0 to 3 ng/g. Broiling, pan frying, and the
baconer system resulted in levels of nitrosamine between 7 and 20 ng/g.
There has been concern that the mere presence of nitrites and amines
in food can result in formation of nitrosamines by reactions in the food,
by microorganisms, or in the acidic environment of the stomach. It has
been suggested that these precursors should be at a low level in the diet.
Lijinsky (1984) found an increase in tumors in rats fed nitrosatable
amines, and he suggested reducing the intake of nitrite-containing cured
meat. However, Wagner and Tannenbaum (1985) reported that humans
on a low-nitrate diet excreted significant levels of nitrosoproline in their
urine. When they compared the endogenous production of nitrite, they
found that the amount consumed in cured meats was not significant.
Another study did not find an increase in the number of tumors in mice
fed nitrite with pyrrolidine (Pearson et al. 1982).
To reduce or eliminate nitrosamines, there has been an effort to
lower the nitrite level of cured meats without sacrificing the organoleptic
characteristics or the antibotulinal effects. Widdus and Busta (1982) de-
scribed six antibotulinal alternatives (a-tocopherol and ascorbate, sor-
bate, acidulation by lactic acid bacteria, sodium hypophosphite, fumarate
esters, and irradiation)_
Bacon with acceptable quality can be made with no added nitrite
(Tichivangana, Morrissey, and Buckley 1984; Williams and Greene 1979)_
Researchers found that sodium ascorbate, ascorbic acid, or sodium ery-
thorbate (isoascorbate) markedly inhibit nitrosamine formation in amodel
system as well as in frankfurters (Fiddler et aL 1973)_ Both ascorbic acid
(vitamin C) and alpha-tocopherol (vitamin E) act as reducing agents or
antioxidants and are effective in blocking the formation of nitrosamines.
Vitamin C is water soluble and acts in the lean tissue, while vitamin E is
fat soluble and acts in the fatty tissue of bacon. Alpha-tocopherol is an
effective inhibitor of nitrosamine formation in bacon (Bernthal et aL
1986; Gray et aL 1982). The presence of up to 1,000 ppm of tocopherol
did not retard the antibotulinal action of nitrite (Tanaka 1982). Up to a
level of 500 ppm, alpha-tocopherol has been approved for use in cured
bacon to inhibit nitrosamine formation.
Besides inhibiting nitrosamine formation, according to Raevuori
(1975), erythorbate increases the antimicrobial effect of nitrite. Tompkin,
Christiansen, and Shaparis (1978c) reported that 50 p,g/g nitrite with ery-
thorbate was as effective as 156 p,g/g of NaN0
2
alone to inhibit C. botu-
linum.
Potassium sorbate has been proposed as an adjunct with nitrite to
control the growth and toxin production of C. botulinum. Reports that
0.26 percent potassium sorbate with either 40 or 80 ppm nitrite inhibits
C. botulinum as well as does 120 ppm nitrite were reviewed by Robach
and Sofos (1982). According to this review, different types of bacon pro-
duced with any of these chemicals were equivalent in organoleptic qual-
ity. Although sorbates are listed as GRAS by the FDA, they have not been
approved as additives in bacon by the USDA.
Wierbicki and Heiligman (1973) suggested using lower levels of ni-
trite in conjunction with radiation pasteurization (radappertization). It
was determined that the radiation level would need to be less than 0.8
megarad or the treatment should be done at - 51C or less to reduce the
adverse effects of the treatment (Terrell et aL 1981, 1982). The irradiation
treatment of foods for microbial destruction has not been approved in
the United States.
Treating an inoculated meat model with 3,000 ppm of sodium hypo-
phosphite closely resembled the antibotulinal effect of 150 ppm of nitrite
(Wood et aL 1986). At 1,250 ppm, monomethyl fumarate scored better
than did other treatments tested.
Studies have reported that a system using a lactic acid producer (Pedio-
coccus acidilactici) with 40 or 80 ppm nitrite and 0.7 percent sucrose added
to bacon (Wisconsin Process) resulted in a comparable and safer product
than bacon produced with 120 ppm nitrite (Tanaka et al. 1985a, 1985b).
Lowering the aw of bacon to 0.92 or less will inhibit C. botulinum
growth and toxin production and allow lowered nitrite levels in the cur
ing mixture (Konstance and Panzer 1985).
Compounds such as sodium bisulfite, tannic acid, hydroquinone, am
monium sulfamate, methionine, cysteine, glutathione, and propyl gallate
can block the nitrosation reaction (Gray and Dugan 1975). Treatment of
bacon with 1,000 ILg/g of propyl gallate, piperazine, ascorbyl palmitate,
or sodium ascorbate just prior to frying reduces the formation of nitros
amines during heat treatment (Sen et al. 1976).
The legal limits of nitrite have been lowered in recent years. Origi
nally, the limit was 200 ILg/g residual nitrite. With this limit, higher levels
of nitrite could be added, as long as the residual was 200 ILg/g or less.
After research showed that nitrosamines were formed in cooked bacon
and may be present in other cured meats, an expert panel was estab
lished to advise the Secretary of Agriculture.
With the panel's recommendation, the USDA proposed various levels
of nitrite, depending upon the type of cure and the specific product.
Since bacon is the cured meat of most concern as a source of nitrosa
mines, the acceptable nitrite levels are most stringent for this food. In
1986, the USDA specified alternative procedures for bacon. Processors
may now use anyone of the following: (1) 120 ppm of sodium nitrite (or
148 ppm of potassium nitrite) and 550 ppm of sodium ascorbate or so
dium erythorbate (isoascorbate); (2) 100 ppm of sodium nitrite (or the
equivalent amount of potassium nitrite) and 550 ppm of sodium ascor
bate or sodium erythorbate, with an approved partial quality control pro-
gram to precisely control the level of nitrite and other factors; or (3) 40
to 80 ppm of sodium nitrite (or the equivalent amount of potassium ni-
trite) and 550 ppm of sodium ascorbate or sodium erythorbate plus
added sugar and starter cultures (lactic-acid-producing bacteria), with an
approved partial quality-control program to precisely control the level
of added nitrite, added starter cultures, and other factors.
In 1978, the USDA established a lO-ppb violative level for volatile
nitrosamines in fried, cure-pumped bacon. Hence, any process used must
result in a level of volatile nitrosamines of less than 10 ppb.
Smoking. In ancient times it was noticed that meat exposed to smoke from
a fire had improved keeping qualities. Since the beginning of refrigera-
tion, the preservation of meat by smoking has not been as important.
However, smoking is used to develop distinctive and desirable flavors
in certain foods, such as meat, fish, poultry, and cheese. The chemical
composition of wood smoke depends upon factors such as the type of
wood, the amount of air, the temperature, and the time. Hickory or other
hardwoods are preferred. Soft woods cause unpleasant flavors in the
food.
The preservative effect is a result of the combination of drying and
the deposition of the chemicals resulting from the thermal decomposi
tion of wood. The amount of smoke deposited on a food is a complex
function ofthe composition and concentration of the smoke, the temper
ature and humidity, and the nature of the food surface. To enhance the
deposition of particles of wood smoke onto the food surface, electrostatic
charges can be used.
Several hundred chemicals have been identified in the smoke
condensate. These chemicals include acids, alcohols, aldehydes, ketones,
phenols, carbonyl compounds, waxes, resins, and tars. The acids, phe
nols, and carbonyls are recognized as the components that contribute
most significantly to the odor and flavor of smoke. Volatile phenolic com
pounds in the vapor phase of the smoke are primarily responsible for
the smoke flavor.
Several of the chemicals found in smoke or on smoked foods nor
mally would not be allowed as food additives. Hence, smoking is one
means of using these chemicals in foods.
A number of investigators have detected 3,4benzopyrene and 1,2,5,6
dibenzanthracene in wood smoke. These polycyclic hydrocarbons are
carcinogenic to laboratory animals. Although it is thought that smoked
foods are unlikely to be a health hazard, according to Moodie (1970),
there is a relatively high incidence of carcinoma of the alimentary tract
in populations in which smoked foods are consumed in large quantities.
The presence of a nitrosamine, nitrothiazolidine, found in bacon is reo
lated to the smoking process. The exact mechanism for the formation of
this compound has not been determined.
In a survey, researchers detected polycyclic aromatic hydrocarbons in
many smoked foods at low levels (0.5 to 7.0 ng/g) (Malanoski et al. 1968).
Fretheim (1976) reported benzopyrene at levels of 0.15 ng/g, as well as
several other polycyclic hydrocarbons in smoked sausages. The carcino
genic polycyclic hydrocarbons are derived from the lignin portion of
wood. Their production is minimized when the temperature of combus
tion is maintained below 350C.
U sing liquid smoke is one means of developing a smoked flavor in
meat. In liquid smoke production, the smoke is fractionally distilled and
the selected fraction diluted with water. This results in a product report
edly essentially free of the water insoluble 3,4benzopyrene. However,
White, Howard, and Barnes (1971) found concentrations of benzopyrene
ranging from 25 to 3,800 ng/g in condensates that settled out of liquid
smoke flavors during storage.
Formaldehyde, acids, and phenols are antimicrobial agents. They are
regarded as being responsible for much of the antibacterial action in
smoked meats. As would be expected, bacterial spores are quite resistant
to smoking. Many vegetative bacteria are destroyed by smoking for two
or three hours, but most of the spores survive smoking for seven or more
hours. The destruction of bacteria is correlated to some extent with
smoke density, duration of smoking, temperature of the meat, fish, or
poultry, and the humidity. Spoilage of smoked meat is often due to con
tamination that occurs after processing, with subsequent exposure to
conditions that permit microbial growth.
Since smoking does not control spores, including those of C. botuli
num, the good manufacturing practices for smoked fish (GSA 1977) reo
quire heating at 82.2C for 30 min.
Liquid smoke at 0.08 percent retards but does not stop growth of
bacteria. At a level of 0.1 percent liquid smoke has a preservative effect
and gives a mild smoke flavor. Together, liquid smoke with NaCI effec
tively inhibited outgrowth and toxin production of C. botulinum in white
fish (Eklund et al. 1982).
Other Chemicals. There are many other antimicrobial agents. Some of
these may be acceptable, while others may not be acceptable as food
additives. These chemicals include calcium (Conway and Sams 1985; Mc
Guire and Kelman 1984), diacetyl (Jay, Rivers, and Boisvert 1983), di
methyl dicarbonate (Porter and Ough 1982), fumaric acid, methyl and
ethyl fumarates (Huhtanen and Guy 1984; Islam, Lirio, and Delvalle
1984), glutaraldehyde (Collins 1986; Gorman, Scott, and Russell 1980;
Gorman et al. 1983; Mast and MacNeil 1978), hydroxycinnamic acid (hy
droxycinnamates) (Baranowski and Nagel 1984), monolaurin (Beuchat
1980; Kabara 1984), poly(hexamethylenebiguanide hydrochloride) (Thom
son et al. 1981), and xylitol (Makinen and Soderling 1981). Some of these
inhibit only certain types of microorganisms. Although calcium does not
inhibit organisms, it tends to make the treated produce more firm and
less susceptible to attack by spoilage types.
Combinations. In meat curing, a combination of NaCI and NaN0
2
as well
as ascorbate or erythorbate is used. There are other cases in which a
synergistic antimicrobial effect is noted when two or more chemicals are
added together. Doty (1968) reported that a mixture of 10 percent propi
onic acid, 10 percent lactic acid, and 5 percent citric acids in 75 percent
water killed salmonellae. When this mixture was added to meat meal at
the rate of 5 percent, all salmonellae were destroyed.
Webster, Fowler, and Cooke (1985) studied a combination of sodium
citrate and sodium benzoate at various a,. and pH levels. A synergistic
effect with lactic and acetic acids was noted by Rubin (1978). Sodium salts
of these acids plus sodium propionate were synergistic in their effect on
yeast (Moon 1983). Potassium sorbate has been used in combination not
only with nitrite in meat curing, but also with antioxidants, acids, salt,
and EDTA (Davidson, Brekke, and Branen 1981; Kondaiah, Zeuthen, and
Jul 1985; Lahellec, Fung, and Cunningham 1981; LaRocco and Martin
1981; Restaino, Lenovich, and Bills 1982; Robach and Stateler 1980).
CONTROLLED ATMOSPHERES
The gaseous environment surrounding a food can influence the types
of microorganisms associated with the food. The alteration of the air may
result in a controlled atmosphere (CA.), modified atmosphere (MA), CO
2
,
enriched atmosphere, vacuum packaging (VP), or a hypobaric environ
ment.
If a chemical preservative is added to a food, its presence is listed
with the ingredients. However, since gases such as CO
2
are naturally pres
ent and leave the food when the package is opened, they do not need to
be included as an ingredient.
Fruits and vegetables respire during storage, and this respiration con
tributes to the loss of quality. As the temperature of storage is decreased
toward OC, the rate of respiration is reduced, and storage life is in
creased. During respiration, O
2
is used and CO2 is given off. The regula
tion of the concentration of O
2
and CO
2
for storage of fresh food is called
CA. storage.
Developing the desired atmosphere by natural respiration is satisfac
tory for fruits such as apples. However, this is not satisfactory for perish
able vegetables. Deterioration occurs before the optimum atmosphere is
obtained by respiration. Rather than depending upon the natural con-
version of O2 to CO2 through respiration, the gases can be altered by
adding CO2 and removing O
2
until the desired concentrations are ob-
tained. This is useful for produce as well as for nonrespiring foods such
as meat, poultry, and fish. The use of controlled or modified atmospheres
to store fresh produce was reviewed by Brecht (1980) and Kader (1986).
Different types of fruits and vegetables, and even the varieties of the
same type, have different optimum concentrations of O
2
and CO2 for
storage. When the O
2
concentration is reduced low enough to apprecia-
bly reduce mold growth, many fruits are affected adversely.
High CO
2
and low O
2
concentrations become a safety hazard for
workers. Therefore, controlled atmospheres are not practical in ware-
houses when produce is constantly being moved in and taken out.
The low O
2
level in CA affects the growth of some molds, and the CO2
level limits the growth of some microorganisms. In addition, microorgan
isms are less able to attack ripening fruit than to attack overripened
fruit. Hence, the microbial spoilage is retarded by the slowing of the
ripening process. The direct inhibition of microorganisms due to gas
concentrations seems to be secondary.
Carbon Dioxide
When the concentration of CO
2
is increased slightly above normal,
microbial growth is usually stimulated. At high concentrations, CO2 has
been recognized as a means to control the growth of microorganisms in
carbonated beverages, beef, poultry, fruits, vegetables, and shell eggs.
Edney (1973) reviewed the storage of apples in CA. Refrigeration and
CO
2
will retard the rate of apple rotting. However, if the CO
2
concentra
tion is increased beyond the optimum, increased rotting may result.
There were fewer rotten apples at 8 percent CO
2
than at either 4 or 12
percent CO
2
The lowering of the O
2
level from the 21 percent in air to
10 percent reduced the rotting of apples, and a further reduction in rots
was noted as the O
2
concentration was reduced from 10 to 3 percent.
The storage of blueberries in CO
2
enriched atmospheres (10 to 20
percent) significantly reduced decay (Ceponis and Cappellini 1985). Stor
age of grapefruit in low O2 resulted in increased alcohol in the juice
and an undesirable stale flavor (Barmore, Purvis, and Fellers 1983). An
atmosphere of 2 percent O
2
and 5 percent CO
2
retarded the decay of
immature apricots but was oflittle benefit when ripe apricots were stored
(Brecht et al. 1982). They also reported that different results were ob
tained for different varieties of peaches when stored in a 2 percent O
2
,
5 percent CO
2
environment. Hence, the benefits of CA storage depend
not only on the type of fruit or vegetable, but also on the variety. Besides
the composition of gases, the humidity, temperature, and permeability
of the package are important considerations (Geeson et al. 1985; Wilson,
Jay, and Hill 1985).
The packaging of foods subjected to mold spoilage, such as bread, in
high CO
2
or N2 atmospheres with no O
2
, may be beneficial in extending
the shelf life.
Wilson, Huang, andJay (1975) stored highmoisture corn (19.6 or 29.4
percent moisture) inoculated with Aspergillus jlavus in various mixtures of
O
2
and CO
2
They found that reduced O
2
, increased CO
2
, or both delayed
deterioration, but growth was not completely stopped for A. jlavus. In
their modified atmospheres, the level of aflatoxin in the corn was always
less than 20 J-tg/kg. They suggested that, in naturally infested corn, the
A. flavus might do better than these inoculated cultures in the modified
atmospheres.
Lillehoj, Milburn, and Ciegler (1972) studied the effect of CO2 on ger-
mination and growth of Penicillium martensii on corn. At 30
0
C, the germi-
nation fell from 36 percent in air to 2 percent in 60 percent CO2, and
no germination was found with the combination of lOoC and 60 percent
CO
2
With either 40 percent or 60 percent CO
2
, and storage at 50 or
lOoC, there was essentially no penicillic acid formed by this mold. Peni-
cillic acid is a carcinogenic mycotoxin.
The shelf life of meat, poultry, and fish products is extended by stor-
age in a CO
2
-enriched atmosphere. The greatest antimicrobial effect is
obtained at a level of 100 percent CO
2
However, this concentration af-
fects the color of red meat, but not poultry or fish. Even at 10 percent
O
2
, beefsteaks did not maintain their bright red color during storage
(Bartkowski, Dryden, and Marchello 1982). According to a review by
Finne (1982), CO
2
at 15 to 20 percent partially inhibits bacterial growth
and maintains the bright red color of fresh meat. Luiten, Marchello, and
Dryden (1982) suggested a combination of 60 percent C02/40 percent O2
to increase the shelf life and maintain the color.
Broilers stored at 2C in 65 percent CO
2
had an increased shelf life
of five days over broilers in ice, but this was only one day more than the
shelf life of broilers stored in 20 percent CO
2
(Bailey et al. 1979). How-
ever, Statham (1984) stated that microbial growth in a CO
2
-enriched envi-
ronment is inversely related to the CO
2
tension in the atmosphere.
For fishery products, rather high levels of CO
2
have been used. Sten-
strom (1985) suggested a 50 percent C02/50 percent O2 mixture for retail
packages of cod fillets. In a CA with 100 percent CO
2
, the bacterial counts
were lower, but weight losses were higher, than for spotted shrimp stored
in air (Matches and Layrisse 1985)_ Storage in CO
2
did not prevent toxin
production in fish fillets inoculated with C. botulinum (Post et al. 1985).
In some cases, toxin was present in the fillets before they were rejected
by sensory evaluation_
In muscle foods, high CO
2
atmospheres inhibit the normal spoilage
types (pseudomonas, Acinetobacter, Moraxella) but allow the growth of lactic
acid bacteria, which results in a sour and acid condition of these foods.
King and Nagel (1967) found a linear correlation between CO2 con-
centration and the amount of inhibition on the rate of growth of Pseudo-
monas aeruginosa. The inhibition by CO
2
was observed only when other
environmental factors (0
2
tension, pH, nutrients, and temperature) were
not optimum. Compared to growth in air, the relative inhibition of 100
percent CO2 for those organisms tested was greatest for B. cereus, Brocho-
thrix thermosphacta, and Aeromonas hydrophilia (Molin 1983). Further inhibi-
tion by 100 percent CO
2
was lowest for E. coli, S.faecalis, and Lactobacillus.
The effect of CO
2
on microbial growth and food quality was reviewed by
Daniels, Krishnamurthi, and Rizvi (1985).
Vacuum Packaging
This type of package is evacuated before sealing, either by inserting
a vacuum probe into the neck of the package, or by placing the package
into a chamber and evacuating it. With flexible films, this results in a
package that is drawn tightly around the contents. The development of
packaging materials impermeable to oxygen and other gases has made
vacuum packaging possible. The integrity of the package must be main
tained, or the vacuum will be lost. It should be evident that a complete
vacuum is not attained, but any residual oxygen is soon utilized by the
microorganisms on the food and replaced with CO
2

Vacuum packaging has been the method for packaging tableready
processed meats. These meats have a longer shelf life than when pack
aged with air. There is a tendency for a greenish discoloration of vacuum
packaged cured meats. The use of nitrogen packaging retarded the
greening of meat loaves and frankfurters (Lee et al. 1984b; Simard et al.
1983a). Vacuum packaged fresh beef tends to become purplered rather
than bright red. According to Lynch, Kastner, and Kropf (1986), con
sumers will accept the purplered color of vacuum packaged meat.
As with packaging in CO
2
atmospheres, the microbial flora of meat
is altered by vacuum packaging. At the beginning, pseudomonads and
Brochothrix are dominant, but during refrigerated storage, lactobacilli be-
come dominant (Lee et al. 1984a; Nielsen 1983; Simard et al. 1983b).
Molds do not grow on vacuum-packaged meat (Carr and Marchello 1986).
Vacuum packaging extends the refrigerated storage life of fresh fish
(Clingman and Hooper 1986; Scott, Fletcher, and Summers 1984), as well
as poultry (Anderson et al. 1985; Jones et al. 1982; Patterson, Gillespie,
and Hough 1984). Notermans, Dufrenne, and Keijbets (1981) reported
that potatoes inoculated with C. botulinum spores, then cooked and vac-
uum packed, became toxic unless stored at temperatures below 4C.
Hypobaric
Hypobaric storage refers to a system with a controlled environment
involving low pressure, low temperature, high humidity, and ventilation.
As compared to conventional coolers, this system can extend the shelf
life of beef and lamb carcasses, ham, pork loins, and broilers (Mer
melstein 1979, Restaino and Hill 1981).
Ozone
Ozone is formed by electric discharge, such as an electric arc through
air or by UV radiation at wavelengths of 200 to 210 nm. This production
occurs especially at higher altitudes.
Ozone contains three atoms of oxygen. The third atom is loosely
bound to the other two. This third atom unites easily with cellular mate
rial, resulting in inhibition or death.
Since ozone is a powerful oxidizing agent, it has been postulated that
part of the cellular damage is due to the oxidation of SH bonds. It is
likely that the ozone reacts with the double bonds of cell wall lipids, since
ozone causes leakage of cellular components and lysis of some microbial
cells. The leakage or lysis of the cells depends on the extent of the reac
tion with cell wall lipids.
Ozone inactivates viruses by breaking the protein capsid into sub
units (Kim, Gentile, and Sproul 1980) or by damaging the viral RNA (Roy
et al. 1981).
The lethal threshold concentration of ozone for washed cells of Bacil
Ius cereus was 0.12 mg per liter and for E. coli and B. megaterium, it was
0.19 mg per liter. For spores of the bacilli, the lethal threshold concentra
tion was 2.29 mg per liter (Broadwater, Hoehn, and King 1973). Un
washed cells were not killed during five minutes exposure up to 0.71 mg
per liter of ozone. Apparently, organic material associated with un
washed cells affected the activity of the ozone.
A part of the microbial effect of UV lamps in meat storage rooms is
due to the production of ozone and the resultant inhibition or death of
bacteria. An ozone level of 0.1 mg per liter has little effect on microorgan
isms in air, but on moist meat surfaces, it exerts a bacteriostatic effect.
The continual exposure of the organism to sublethal doses of ozone may
eventually cause the death of the cell, particularly at low temperatures.
Ozone was effective in destroying microorganisms in water used for chill
ing broiler carcasses and those on the carcasses as well (Sheldon and
Brown 1986). Generally, ozone is not recommended for use on fresh pro
duce.
The ozone level that is toxic to small animals is about 3 to 12 mg per
liter. At 0.02 to 0.04 mg per liter, most people can detect the odor of
ozone, and at 0.1 mg per liter, it may cause headache, dryness, and irrita
tion of the throat, respiratory passages, and eyes. Ozone is a powerful
mutagenic and oxidizing agent.
MICROBIAL INTERACTIONS
The use of microbial fermentations as a means of preservation is well
known and is discussed in Chapter 9. Besides the production of acids
or alcohols, some microorganisms form bacteriocins and antibiotics that
inhibit other bacteria (Chapter 4).
The inoculation of foods with certain microorganisms to control
other microorganisms might be unique, although this has been used in
fermentation such as in dairy products. The addition of a lactic culture to
ground meat significantly retarded the growth of Gramnegative spoilage
bacteria (Reddy, Chen, and Patel 1975). There was a less dramatic effect
when the lactic culture was used on beefsteaks. Besides a lower count of
spoilage bacteria, the pH, volatile nitrogen, and free fatty acids were
lower in inoculated than in control samples of meat. The inoculation of
lactic acid bacteria onto scallops did not retard spoilage (Bremner and
Statham 1983b).
This type of inhibition is limited to certain types of foods. In most
instances, it would probably be more beneficial to add the chemicals di
rectly than to depend on inoculated microorganisms to produce them in
the food.
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by Destruction
12
The death of some microbial cells occurs during refrigeration, freezing,
drying, or chemical treatment. However, these systems are not expected
to produce a sterile food. Sterilization is considered to be a process by
which all forms of life are destroyed. When subjected to a lethal process,
a bacterial culture is reduced at a rate that is approximately logarithmic.
This indicates a first order reaction. The inactivation or death of cells
can be determined by the formula
K = log a log b
--where K is the death rate, a is the initial number of organisms, and b is
the number of cells remaining at time t.
Considering the microorganisms of public health significance, C. bot-
ulinum is the most difficult to destroy. The main objective is to eliminate
this organism from processed and packaged foods.
The inactivation of microorganisms has been attributed to gases,
thermal processing, ionizing radiation, and light waves.
GAS TREATMENTS
Gases can be used to sterilize materials that cannot withstand the high
temperatures of heat sterilization. Many organic compounds begin to de-
grade at 71 DC and this degradation accelerates as the temperature is in
creased to 121 DC. Volatile food flavors can be altered by heat. Some plas
tic materials are softened by heat.
Gaseous sterilization offers a means for packaging heatsensitive
products in an essentially microbe-free condition. A gas can not only
affect airborne and exposed surface bacteria, but it can also penetrate
porous materials to attack the microbial cells. Each gaseous substance
has certain advantages and disadvantages.
It is necessary to evaluate the physiological effectiveness and the toxic
651
hazards, as well as the retained residuals of any particular gas. Important
aspects in the use of gas sterilization are comparative efficiency of differ
ent methods of application, effect of humidity and atmospheric gases,
resistance of microorganisms, synergism or potentiation of gas mixtures,
the mode of action, reaction kinetics, physical and chemical sorption,
effects on food quality, photodecomposition, air pollution, worker safety,
permeability of materials such as packages, and the nature and physio
chemical properties of the material being treated (Berck 1965).
Besides the gases used to inactivate microorganisms, fumigants such
as phosphine, ethylene dibromide, and methyl bromide are employed to
destroy insects in stored grain.
Ethylene Oxide
Ethylene oxide is a cyclic ether with the structural formula
Ethylene oxide is a gas at ambient temperature and pressure. It boils
at 1O.7C and freezes at -111C. It dissolves in and reacts with water to
form ethylene glycol. Mixtures of 3 to 100 percent ethylene oxide in air
are highly flammable and explosive. To eliminate this hazard, ethylene
oxide is mixed with dichlorodifluoromethane, carbon dioxide, or formic
acid methyl ester.
For an effective sterilization process, the gas concentration, time, tem
perature, and humidity are parameters that must be considered and con
trolled. Factors such as stratification effects of various gases, diffusion
barriers, moisture reducing effects, temperature gradients, and ancillary
gaseous reactions can enhance or limit sterilization. The sterilizing ac
tion of ethylene oxide requires three hours or more but is effective
against spores as well as vegetative bacteria, yeasts, molds, and viruses.
Some advantages and disadvantages of ethylene oxide treatment are
listed in Table 12.l.
The ability of ethylene oxide to penetrate certain packaging materials
(paper and some flexible plastics) allows foods to be sterilized in sealed
containers. The packaging material must be gas permeable, but imperme
able to bacteria.
The mechanism by which ethylene oxide inactivates microorganisms
is not completely known. A commonly accepted hypothesis is that the
microbicidal action is directly related to alkylating activity. The enzymes
and other proteins essential for cellular metabolism contain carboxyl,
amino, sulfhydryl, hydroxyl, and phenolic groups. Ethylene oxide reacts
with these groups by replacing an available hydrogen atom with the alkyl
CONTROL OF MICROORGANISMS BY DESTRUCTION 653
TABLE 12.1. ETHYLENE OXIDE STERILIZATION
Advantages
Good penetration of materials
Attacks and kills all microorganisms
Little, if any, damage to materials (com
pared to heat)
Effective at room temperature
Effective with dry products
Sterilizes packaged items
Disadvantages
Flammable
Process is slow
Residues (analysis may be needed)
Expensive
Each process must be closely monitored
Toxic
May alter nutrients and other quality fac
tors of foods
hydroxyethyl group. The bacterial cell cannot utilize the new chemical
and therefore it dies. According to Ernst (1974), the biologically critical
reaction occurs with RNA and DNA within the cells. Winarno and
Stumbo (1971) observed an impairment of RNA and protein synthesis.
They considered this to be an indirect effect resulting from alkylation of
DNA components. This alkylation reaction is thought to be important in
the inactivation of spores. Spores are usually several times more resistant
than vegetative cells to chemical agents. With ethylene oxide, the resist
ance of some spores is comparable to that of some vegetative cells, while
the more resistant spores are only about five times as difficult to inacti
vate as the less resistant vegetative cells.
Bacillus subtilis var niger spores are used to determine the effectiveness
of ethylene oxide sterilization. However, these are not the most resistant
spores (Dadd and Daley 1980). The apparent resistance of bacterial
spores is influenced markedly by environmental conditions that exist
during spore production and their preparation and evaluation (Dadd,
McCormick, and Daley 1983). Rupture of the spore coat did not sensitize
the spores to ethylene oxide (Dadd and Daley 1982).
Ethylene oxide and propylene oxide have been used to sterilize
spices, cereals, colloid gums, fruits, dried yeasts, and other items. For the
sterilization of paprika, gamma irradiation was more effective than was
treatment with ethylene oxide (Franco et al. 1986).
The use of ethylene oxide to sterilize food has been questioned. Ethyl-
ene oxide is chronically toxic at levels not detected by smell. The toxicity
of ethylene oxide was reviewed by Glaser (1979). Human exposure to
ethylene oxide can cause irritation of the skin, throat, and eyes as well
as respiratory problems, headaches, lachrymation, nasal discharge, saliva-
tion, and labored breathing. Further effects may include nausea, vomit-
ing, diarrhea, lung edema, paralysis, convulsions, and, according to
White (1977), death. Rats exposed to ethylene oxide developed various
neoplasms (Lynch et al. 1984). A mutagenic effect on human fibroblasts
was noted (Star 1980). Embree, Lyon, and Hine (1977) reported muta-
genic effects on various cells, including mammalian cells.
Besides formation of the toxic ethylene glycol when hydrated, Wesley,
Rourke, and Darbishire (1965) reported that ethylene oxide reacts with
naturally occurring chlorides in food to form chlorohydrins. These are
toxic and nonvolatile and persist in the food during processing. The reo
searchers reported ethylene chlorohydrin at levels of over 1 mg/g in
spices sterilized with ethylene oxide.
Ethylene oxide sterilization should be used only when other steriliza-
tion techniques are not practical.
Propylene Oxide
Propylene oxide has the structural formula
It is a colorless liquid with a boiling point of 35C. It is used like
ethylene oxide since it must be diluted with inert gases for safety. It has
weak penetrating ability, but it is microbicidal. It requires a higher tem-
perature or longer treatment time to accomplish the same microbial kill
as ethylene oxide.
Propylene oxide has been used in various foods to kill 90 percent
or more of the microorganisms. Propylene oxide treatments of pecans
reduced the surface flora by 80 to 92 percent and the internal microorgan-
isms by 64 percent (Blanchard and Hanlin 1973). Even with high con-
centrations of propylene oxide, neither bacteria nor fungi could be elimi-
nated completely.
The hydration of propylene oxide yields propylene glycol. This
should be acceptable since propylene glycol is used to lower the aw in 1M
foods.
However, as with ethylene oxide, propylene oxide can react with chlo-
rides to form chlorohydrins in foods (Wesley, Rourke, and Darbishire
1965).
Propylene oxide can be used as a package fumigant on or in dried
prunes or glace fruit. It can be used on bulk quantities of cocoa, gums,
processed spices, starch, and processed nutmeats (except peanuts) when
these foods are to be further processed. There are limitations on temper-
ature, time, and residues (FDA 1977).
Formaldehyde
Formaldehyde (HCHO) gas is present in wood smoke, especially in
that used for smoking ham or fish. It is flammable in air at concentra-
tions of 7 to 73 percent by volume. Like ethylene oxide, it is an alkylating
agent. As such, bacterial spores are only slightly more (2 to 15 times)
resistant than are vegetative cells. Optimum activity occurs at about 80
to 90 percent relative humidity (RH).
Formaldehyde vapor is not very penetrating when used under the
normal conditions of fumigation. Its action is confined primarily to air
borne bacteria and those on exposed surfaces. Even a thin film of organic
matter will protect microorganisms from formaldehyde.
HEAT
The most common method of killing microorganisms is to subject
them to a heat treatment. When properly done, this is an effective means
of improving the microbiological quality of foods. Sometimes, relatively
mild heat treatments are used in conjunction with other processes, such
as refrigeration, freezing, drying, or acidification. Since the destruction
of microorganisms is incomplete, poor sanitation and mishandling of
these mildly heat treated products may result in a higher total load of
microorganisms than was present before the heat treatment.
The destruction of spoilage microorganisms increases the storage life
of the product. However, if this food is contaminated with pathogenic
types of microorganisms, the natural competing flora is not present, and
the food may become a health hazard.
Compared to other means of sterilization, heat may be considered
more efficient and convenient. Besides killing microorganisms, heating
will inactivate enzymes (tissue or microbial) that can cause deterioration
of food during storage. The one condition needed in using heat is that
the material being sterilized must be resistant to heat damage.
Food microbiologists must be concerned not only with the preserva
tion of foods, but also with the sterilization of laboratory materials. The
heat treatment of instruments used for sampling and analysis is discussed
in Chapter 2. Moist heat (steam or water) is much more effective in de
stroying microorganisms than is dry heat. The effect of heat on microor
ganisms is generally believed to be due to enzyme inactivation, protein
denaturation, or both.
There are many processes in which foods are subjected to heat. Each
of these systems may influence the microbial population. The extent of
this influence depends on the type of heat treatment (heat source, tem
perature, time), the type of food, and the types of microorganisms that
are present.
Heat may be transferred to and through foods by conduction, convec
tion, or radiation. The type of heat transfer depends upon the source of
heat and the type of food being heated.
Heating Systems
The processes in which heat is applied to foods include cooking,
scalding, pasteurizing, blanching, and canning, as well as drying, distilla-
tion, evaporation, and concentration_
In most of the processes employing heat, complete destruction of all
microbial forms is neither attempted nor attained. Only in thermal pro-
cessing of foods with steam under pressure, such as in canning, is an at-
tempt made to approach a sterilized product. When sufficient heat is
applied to assure a sterile food, the organoleptic and nutritional proper-
ties of the food suffer. In many cases, the food would no longer be accept-
able to the consumer.
COOKING. Although some fresh fruits and vegetables are eaten raw,
most of our foods are given a heat treatment prior to consumption.
Cooking is an example of temporary preservation.
Normal cooking may destroy most of the vegetative bacteria, except
for thermophiles. Most of the spores will survive normal cooking proce-
dures. The guidelines established by the USDA for cooking roast beef
suggest a minimum end-point temperature of 63C or an alternate proc-
essing procedure as listed in Table 12.2. These processes will destroy
some pathogenic organisms but will not affect bacterial spores, the enter-
otoxin of S. aureus, or the neurotoxin of C. botulinum. The data of Blan-
kenship (1978) revealed that some salmonellae may survive in beef roasts
TABLE 12_2_ ALTERNATIVE PROCESSING PROCE-
DURES FOR COOKED BEEF AND ROAST BEEF
Minimum Internal Temperature
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
54.4
55.0
55.6
56.1
56.7
57.2
57.8
58.4
58.9
59.5
60.0
60.6
61.1
61.7
62.2
Minutes"
121
97
77
62
47
37
32
24
19
15
12
10
8
6
5
"Minimum processing time after minimum temperature
is reached.
heated to a temperature of 64C. Heating beef in a water bath to an
internal temperature of 60C and holding for 12 min decreased the level
of C. perfringens by about three log cycles (Smith, Evans, and Buck 1981).
The cooking of various foods lowers or eliminates pathogenic cells but
not spores (Bryan et al. 1982; Cornforth et al. 1982; Parry and Gilbert
1980; Ritter, O'Leary, and Langlois 1979).
The D values for S. typhimurium in ground beef were reported as 61
to 62, 3.8 to 4.2, and 0.6 to 0.7 min at 51.6,57.2, and 62.7C, respec
tively (Goodfellow and Brown 1978), or 7.1, 5.1, 1.2, 0.9, and 0.6 min
at 50, 55, 60, 65, and 70C, respectively (Tamminga, Beumer, and
Kampelmacher 1982).
The effect of cooking oysters in destroying inoculated polioviruses
was reported by DiGirolamo, Liston, and Matches (1970). Stewing (plac
ing oysters in boiling milk and stewing for 8 min), frying (placing oysters
in oil at 177C and frying for 10 min), baking (20 min in an oven at
121.5C), and steaming (held in flowing steam for 30 min) did not elimi
nate the inoculated viruses. From 7 to 10 percent of the viruses survived
these treatments. Coxsackievirus B2 and poliovirus 1 were inactivated by
broiling hamburger patties to 60C and holding at room temperature
for 3 min (Sullivan et al. 1975). At high inoculation levels (10
7
pfu/g),
Sabin type 1 poliovirus survived in ground meat held at 80C for 5 min
(Filppi and Banwart 1974).
It is important that foods are cooked sufficiently to destroy certain
pathogenic organisms, but they should not be heated to the extent that
the nutritional value is reduced significantly. Also, the longer certain
foods are heated at an excessively high temperature, the more likely is
the formation of mutagens (Bjeldanes et al. 1982; Knize et al. 1985; Lin,
Lee, and Huang 1982).
Since cooking does not eliminate all microorganisms, it is essential
that the cooked food be held above 50C or promptly cooled below 4C
to prevent the growth of potential pathogens.
MICROWAVE HEATING. Microwave ovens are prominent in home
kitchens throughout the United States. Also, there are many industrial
applications for microwaves. Microwave ovens usually operate at either
915 or 2,450 megacycles. At 915 megacycles, the electric current is reo
versed 915 million times each second.
Polar molecules, such as water, have positive and negative charges
concentrated at opposite ends of the molecule. As the microwaves pass
through food, these molecules attempt to become aligned with the alter
nating positive and negative field of the microwaves. This rapid move-
ment back and forth creates molecular friction, which appears as heat.
Microwave heating differs from the more conventional methods of
heating. Heating occurs internally rather than from the outside. This
type of heating system is extremely rapid.
Other heating sources must be at a higher temperature than the food
so that the heat will move from the source to the food. Microwaves, per
se, are not hot, so by using suggested cooking procedures, there is less
chance for microwaves to burn the food surface. Only the polar mole
cules become hot directly due to the microwaves. The nonpolar mole
cules are heated indirectly by conduction or convection from the heated
polar molecules.
There have been several reports implying that microwave heating is
more bactericidal than conventional cooking methods. Conversely, some
reports infer that microwave cooking could result in a health hazard due
to survival of microorganisms that might cause illness. An explanation
of these diverse results is that there is a lack of uniform distribution of
microwaves, which results in hot or cool spots. These areas cause more
or less destruction of microorganisms than would be expected by deter
mining the average temperature of the food.
Rosen (1972) very convincingly reported that the killing of microorgan-
isms is due only to thermal effects. Janky and Oblinger (1976) found no
apparent difference in the microbial population of turkey rolls heated
in either hot water or in a microwave oven. Fruin and Guthertz (1982)
found no significant difference in the distribution of bacteria by micro
wave, conventional oven, or slow cooker heating of meatloaf.
A major concern is the survival of Trichinella spiralis larvae in pork
heated in a microwave oven (Kotula et al. 1983; Zimmermann 1983).
However, there is no problem if microwaves are used to reheat cooked
pork products.
PASTEURIZATION. The heat treatment of foods below temperatures
needed for sterilization may be referred to as pasteurization. Quite often,
a temperature below 100C is called pasteurization, while above 100C,
the process is sterilization. In most of the pasteurization processes, the
food is heated to between 60 and 85C for a few seconds up to an hour.
Generally, the heat treatment given to a food is designed to inactivate
specific types or groups of microorganisms. With pasteurization, some
organisms are killed and some are attenuated (sublethal injury), while
the spores may be stimulated to germinate. The lethal effect depends
upon the heat resistance of the organisms. Pasteurization can be used
for foods in which quality is affected adversely by a more severe heat
treatment.
The pasteurization process is named for Louis Pasteur, the French
chemist who found that heating wine to 50 or 60 C for a short time
inactivated spoilage microorganisms without seriously affecting the qual-
ity of the wine. Since his work, the main objective of pasteurization has
evolved to destroy certain pathogenic microorganisms in specific foods.
When spoilage organisms are not very heat resistant, pasteurization can
extend the storage life of products, especially if the treated food is refrig
erated, frozen, or otherwise treated to control surviving microorganisms.
Pasteurization is used to control microorganisms in foods such as liq
uid egg products, dairy products, alcoholic beverages (beer, wine), crab,
smoked fish, and certain highacid products (fruit juice, pickles, sauer
kraut, vinegar).
Egg Products. The USDA regulations stipulate that all liquid, frozen, and
dried whole egg, yolk, and white be pasteurized or otherwise treated to
destroy all viable salmonellae. Depending on the nature of the liquid egg
product, various times and temperatures are used, as listed in Table 12.3.
Salmonellae are less heat resistant in egg white than in whole egg or egg
yolk. Higher temperatures are needed to pasteurize egg yolk products
than liquid whole egg. A variety of egg products are produced, and they
have different degrees of heat sensitivity. Salted eggs used in salad dress
ings do not have to be pasteurized. They must be properly labeled, and
the salad dressing must contain not less than 1.4 percent acetic acid and
have a pH of 4.1 or lower. The final product must be held for 72 hr.
Salmonellae will die at room temperature under these conditions.
These pasteurization conditions ordinarily will reduce the standard
plate count by about 99.9 percent and the number of salmonellae essen
tially to zero. The heat sensitivity of salmonellae is affected by the pH.
When 60C is used, the D value at pH 9.0 is about 0.1 min and at pH 5.5,
it is about 1.0 min. Hence, pasteurization of egg white at pH 9 requires
less heat treatment than at lower pH levels. The addition of 10 percent
salt or sugar to egg yolk increases the heat stability of salmonellae by five
to ten times. Fortunately, the stability of the egg proteins to heat also is
increased by sugar or salt.
TABLE 12.3. PASTEURIZING CONDITIONS FOR EGG PRODUCTS
Temperature Average Holding
Product
(0C)
Time (min)
Whole egg, plain 60 3.5
Yolk
Plain
60 7.0
or 62.2 3.5
Sugared or salted 62.2 7.0
or 64.4 3.5
Egg white
Plain, pH 9.0 56.7 3.5
Plain, pH 9.0, treated with H
z
0
2 51.7 3.5
Stabilized with Alz(S04)" at pH 7.0 50 3.5
Successful pasteurization is based on a critical time-temperature rela-
tionship_ If the temperature is lowered by 1 C, the efficiency of pasteuri-
zation is decreased_ If the temperature is allowed to increase, there is
danger of coagulating the egg, forming a film on the heat-exchanger sur-
faces (Fig_ 12_1), or damaging the functional properties of the treated
egg_
Figure 12_1. Plate heat exchanger opened to show arrangement of plates and
gaskets.
Courtesy of CREPACO, Inc.
As liquids flow through the holding tubes, the flow may be laminar,
turbulent or transitional. The latter has some characteristics of both lami-
nar and turbulent flow_ In any flow, the material in the center of the tube
is flowing at a faster rate than that near the side of the tube_ Hence,
not all of the liquid egg passes through the pasteurizer at the same rate_
Researchers suggested that the holding tube requirements should be
based on the fastest, rather than the average, particle to traverse the tube
(Scalzo et al. 1969)_ The pasteurization specifications (USDA 1969) re-
quire that every particle be held for at least a specified time and temper-
ature. The normal control of plant operations uses the average holding
times. These times are considered to be twice that of the fastest particle
time. Hence, the minimum time for whole egg at 60C would be 1.75
min, and the average time 3.5 min.
Liquid egg white is more heat sensitive than whole egg or yolk. If egg
white is heated above 56 to 57C, there are problems of coagulation.
The addition of hydrogen peroxide to egg white reduces the heat resis-
tance of the salmonellae so that lower pasteurization temperatures can be
used.
Except for conalbumin, the proteins in egg white are sufficiently heat
stable at pH 7.0. Adding an aluminum salt such as aluminum sodium
sulfate stabilizes the conalbumin so that it can be heated to 60C without
coagulation (Cunningham and Lineweaver 1965)_
Shafi, Cotterill, and Nichols (1970) found Micrococcus, Alcaligenes,
Pseudomonas, Bacillus, and unidentified bacteria in pasteurized egg white.
These genera, as well as species of Staphylococcus, Streptococcus, Arizona (Sal-
monella) and unidentified yeasts and molds, were reported in pasteurized
whole egg (Shafi, Cotterill, and Nichols 1970). The researchers reported
similar types in pasteurized egg yolk. Payne, Gooch, and Barnes (1979)
found Microbacterium, various cocci and coccobacilli, and some Bacillus in
pasteurized (65C for 3 min) whole egg. The presence of egg yolk pro-
tected S. aureus from the effects of heat (Verrips and van Rhee 1983).
Milk. Before milk pasteurization was a requirement, diseases due to
drinking contaminated milk were a common occurrence. There were epi-
demics of typhoid fever, scarlet fever, diphtheria, and infant diarrhea, as
well as outbreaks of tuberculosis, brucellosis, and other illnesses. Today,
pasteurized milk is one of our safest foods. Although the elimination
of diseased animals and better sanitation and refrigeration have helped
improve our milk supply, much of the credit should go to pasteurization.
Originally, the pasteurization of milk was designed to destroy Mycobac-
terium tuberculosis. Since the beginning, the process has also been used to
eliminate Coxiella burnetii, the causative agent of Q fever. Pasteurization
destroys pathogenic organisms, reduces total microbial numbers, inacti-
vates certain enzymes, and extends the storage life of milk. Since milk
proteins are much more heat stable than egg proteins, a higher temper-
ature can be used for pasteurization_
In the vat process, the temperature is increased to 62_7C and main-
tained for 30 min_ In the high temperature short time (HTST) process,
the milk is heated rapidly to 7L7C and held for 15 sec. Equivalent tem-
peratures and times for HTST approved by the FDA (1977) are (1) 89C
for 1 sec; (2) 90C for 0.5 sec; (3) 94C for 0.1 sec; (4) 96C for 0.05 sec;
or (5) 100C for 0.01 sec.
Ultrapasteurized (UHT) dairy products are heated at or above 138C
for at least 2 sec (FDA 1977). These products have an extended refriger-
ated shelf life. With proper heating for sterilization and aseptic packag-
ing, this milk may be held at ambient temperature (Hansen, Swartzel,
and Giesbrecht 1980;] elen 1982; Mehta 1980; Perkin 1985; Zall, Chen,
and Fields 1986)_ However, storage time is never indefinite, since there
might not be complete sterility or destruction of enzymes, which can re-
sult in biochemical and physical changes (Burton 1984; Keogh and Pettin-
gill 1984; Mitchell and Ewings 1985). The time-temperature relationships
of various pasteurization systems are shown in Figure 12.2.
Some particularly heat-resistant micrococci and microbacteria as well
as spores of Bacillus and Clostridium may survive UHT. Heating milk to
149C is expected to kill even some of the most heat-resistant spores_
Pasteurization of milk destroys several enzyme systems. Milk is ana-
lyzed for residual alkaline phosphatase to determine the adequacy of
heat treatment. One problem with using this enzyme assay is the reactiva-
tion of the enzyme following heating by the HTST system. The reactiva-
tion is small compared to the activity in raw milk.
The contamination of milk after pasteurization can affect the shelf
life and even the safety of this food (Griffiths and Phillips 1984; Schroder
1984).
Animal Tissue. Crabmeat may be given heat treatments somewhat below
those used for cooking in order to eliminate a sizable proportion of the
microbial population_
Cockey and Tatro (1974) found that pasteurization at 85C for 1 min
was sufficient to reduce the level of inoculated C. botulinum type E spores
from 10
8
1100 g to six or fewer per 100 g. The pasteurized meat was not
toxic after storage for six months at 4.4C. The D
s2
.
2
o
c
varied from 0.49
to 0.74 min for four strains of C. botulinum type E spores (Lynt et al. 1977).
For a 12D treatment, this means the crabmeat would need to be heated
to 82.2C for 8.9 min.
The average plate count of pasteurized crabmeat is reportedly 3,000
bacteria per gram. In retail samples, Lee and Pfeifer (1975) found crab-
meat to range from 2.1 X 10
4
to 8.2 X 10
7
bacteria per gram. The anaer-
obes plus facultative anaerobes were reported at about lOs/g for pasteur-
CONTROL OF MICROORGANISMS BY DESTRUCTION
663
U. H. T.
320
I
,
Steam inject ian uperisation
., :
.
300
Small diameter tube (Mallorizer)
,.
280
Modified tubular (Roswell) .. :
~

:1

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240

II
Vacuum (Vacreatar)
I

:1

220
IL
Quick time
1
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0
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!I
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11 I,
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100 I
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80 /
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II
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I
I
40
30 15 2 1'1/2-4 3-4 <I
Min. Sec. Sec. Sec. Sec. Sec.
HOLDING TIME AT MINIMUM TEMP.
Figure 12.2. Pasteurization time and temperature.
courtesy of FarraJ/ (1976).
ized crabmeat (Ward, Pierson, and Van Tassell 1977). The prominent
genera in canned crabmeat samples obtained from retail stores were Mor-
axella, Pseudomonas, Acinetobacter, Arthrobacter, and Micrococcus (Lee and
Pfeifer 1975). Equivalent processes for crabmeat in cans and pouches
were reported by Ward, Pierson, and Minnick (1984).
The problems associated with pasteurized fishery products became
very apparent in 1963 with seventeen cases of botulism and five deaths
due to consumption of smoked fish that had been given a pasteurization
treatment. C. botulinum spores were not killed and, due to mishandling
and holding at warm temperatures, the spores germinated, multiplied,
and produced the lethal toxin.
The pasteurization process for ham causes the destruction of most
vegetative cells, except for heatresistant strains of S. faecalis, S. faecium, L.
viridescens, and micrococci. Ham appears to act as a heat protective agent
for L. viridescens (Milbourne 1983). The surviving microflora of pasteur
ized chubpacked luncheon meat was dominated by species of Lacto
bacillus, Brochothrix, and Micrococcus (Bell 1983).
Alcoholic Beverages. Pasteurization is used to destroy spoilage organisms in
beer and wine. Heating of beer in the final container is effective, but it
is also costly and inefficient. Overpasteurization can adversely affect the
flavor of beer (Fricker 1984).
Pasteurization just before bottling controls the growth of microorgan
isms in wine. The heat treatment needed often has an adverse effect on
highquality wines. As with any heat treated product, there is no residual
effect as there is with chemical additives.
Acid Foods. Microorganisms are less heat stable as the acidity of foods is
increased. Hence, pasteurization is an effective means of destroying the
microorganisms in acid foods.
For fruit juices, the heat treatment selected for pasteurization often
is based on inactivation of enzymes rather than destruction of microorgan-
isms. The microbial population usually consists of yeasts, molds, lactic
acid, and acetic acid bacteria that are generally heat sensitive. Other organ
isms usually are inhibited by the acid in fruit juices. Even intermediate
acidities are suitable for the growth of only a few sporeformers, such as
B. thermoacidurans (B. coagulans) or C. pasteurianum.
In grape juice, yeasts are destroyed in a few minutes at 55 to 65C.
Mold spores are more resistant, with some withstanding temperatures of
74 to 80C for up to 15 min. Commercially, grape juice is flash heated
to 75 to 85C.
Fresh pickles packed in solutions of acetic or lactic acids are called
fresh-pack pickles. These pickles are given a heat treatment to aid in pres-
ervation. Underpasteurization results in spoilage, while overpasteuriza-
tion can cause a softening of the pickles. Heating to a temperature of
74C for 15 min was recommended for pasteurization of acidified fresh
pack pickles. Pickles covered with acetic or lactic acids at pH 3.7 re-
mained firm when heated to 74C for 30 min (Bell, Turney, and Etchells
1972).
DRY HEAT. Moist heat is more effective than dry heat for destroying
microorganisms. Dry heat is believed to cause death of the cells by the
destructive oxidation of cell components.
There are significant reductions in the total count, and salmonellae
can be eliminated when dried egg white is stored at temperatures of 50
to 70C. Commercially, the dried albumen is stored at 51.6 to 54.4C
for five to seven days, which not only eliminates salmonellae, but also
improves the functional performance of the product.
BLANCHING. This is a heat treatment given to many foods prior to
drying, freezing, or canning. Blanching inactivates enzymes, aids in
cleaning, expels internal gases, removes mucilagelike substances that
contribute to off.flavors, helps preserve the organoleptic quality during
storage, aids in filling of containers by wilting, shrinking, or softening
the food, helps assure adequate can vacuum, is essential when the spe
cific gravity of certain products is used in grading, and destroys many
vegetative microbial cells.
There are disadvantages, including the loss of nutrients, especially
vitamin C, and the need for water and energy during blanching of fruits
and vegetables (Poulsen 1986; Williams et al. 1986).
Blanching can reduce the total microbial load of raw vegatables by
99.9 percent. The product generally has a lower microbial count when
steam blanched than when hot water blanched. A steam blancher is
shown in Figure 12.3.
CANNING. This is the main system used for the long term preservation
of food. The food is packaged in hermetically sealed containers of metal,
glass, thermostable plastic, or a multilayered flexible pouch (Adams, Pe
terson, and Otwell 1983; Berry and Kohnhorst 1983; Beverley, Strasser,
and Wright 1980; Chia, Baker, and Hotchkiss 1983; Tandon and Bhow
mik 1986, either before or after heat treatment. Aseptic packaging is used
for those products packaged after heating (Carlson 1984; Ito and Steven
son 1984; Teixeira and Manson 1983; USDA 1984; von Bockelmann and
von Bockelmann 1986).
Flowing steam is at a temperature of 100C or less. The temperature
of the steam increases as the pressure increases. The usual temperature
for sterilizing media in a bacteriology laboratory is 121.1 C. This corre
sponds to a gauge pressure of 103.5 kPa or absolute pressure of 207 kPa.
At this temperature (121.1C), steam will kill bacterial spores in fifteen
min. Saturated steam is more penetrating than hot air. Because of its
dryness, superheated steam is about as effective as hot air for sterilizing.
It is important to remove all of the air from the sterilizing chamber.
If 50 percent of the air remains, a pressure of 207 kPa will be only 112C
instead of 121C.
The temperatures used for heat processing or canning vary from
100C for highacid foods to 121C for lowacid foods. For HTST pro
cesses, temperatures in the range of 120 to 150C or higher may be used.

i I
I I
: : Oplional
I I
I
__ ...J
Damper
Cover pi t ched to drain condensat e to side gullers
Figure 12.3. Controlled-temperature steam blancher.
Courtesy of Tressler. Van Arsdale. and Copley (1968).
A short time at a high temperature generally results in a product of better
quality than equivalent processes at lower temperatures.
The use of heat for food preservation and the history of canning was
reviewed by Goldblith (1971, 1972).
Fully sterilized canned products generally are less palatable and less
nutritious than less severely heat treated products. Complete sterilization
of food may not be attained, and generally it is not needed. The heat-
processed products are considered to be commercially sterile. This
means that all organisms that might be a health hazard (c. botulinum), as
well as spores that might cause spoilage under normal nonrefrigerated
storage conditions, are destroyed. Some extremely heatresistant thermo
philic spores might remain in the product. With normal storage temper
atures, these will not germinate and grow. However, there may be prob
lems in tropical areas, in vending machines that dispense hot foods, or
when heatprocessed food is not cooled properly before warehousing.
The time necessary to obtain commercial sterility at a specified tern
perature depends upon the number and type of sporeforming organisms
that are present. By practicing good sanitation and keeping the heat
resistant spores at a low level, less time is needed to obtain a commer
cially sterile product.
When the food is processed in a jar or a can, a good vacuum inside
the container is desired. A vacuum reduces the strain on the container
during heat processing, holds the ends in a collapsed concave position
during subsequent storage, and reduces the amount of headspace oxy-
gen. The vacuum is obtained by exhausting with steam or by vacuum
sealing. The loss of vacuum or springer formation is one of the principal
types of pack failure. The evolution of gas within the container causes
the can ends to become distorted beyond the normal concave position.
The formation of gas may be either from hydrogen produced by corro-
sion on the inside of the can by the food product or by microbial action
on the food product. Microbial degradation of the food is usually recog-
nized by the odor or appearance of the product when the container is
opened. If gas formation due to microorganisms occurs in canned prod-
ucts stored below 38C, the food is not commercially sterile, since the
spores that germinate and grow at these temperatures should have been
destroyed.
It is not the intent of this text to suggest times and temperatures for
processing foods. Such things as the type of retort or process used for
heating, the size of the container, the composition of the product,
thermal conductivity of the food (rate of heat penetration), the number
and heat resistance of bacterial spores, the initial food temperature, and
the temperature of the retort must be considered when developing a
thermal process to obtain a commercially sterile product.
There are various types of retorts for heat processing canned foods.
These include still retorts (vertical or horizontal) (Fig. 12.4), agitating re-
torts, hydrostatic retorts (Fig. 12.5), rotary cookers, helical pump can ster-
ilizers, as well as aseptic canning systems and flame sterilizers.
The food may be heated directly by steam either before filling into
the container or by injection of steam through the open top of the con-
tainer prior to closing and sealing. When heated prior to filling, the food
product is flash sterilized by direct steam injection or in a high-pressure
continuous heating system at 120 to 150C or higher for brief periods.
This process is particularly valuable for food to be packed in large cans
which, with still retorting, require a long process time. Heat sterilization
before filling is limited to foods that can be pumped. The heated foods
are usually cooled slightly and filled into sterile containers in an aseptic
manner.
Certain canned foods can be heated directly with a flame. In this pro-
cess, the cans are rotated rapidly to prevent burning of the food and to
induce convection heating. The direct flame process reportedly yields
processed products with improved quality as compared to those pro-
cessed conventionally (Leonard et al. 1983; Seet et al. 1983).
Mathematical evaluations can be used to determine either the steriliz-

REliEF VALVE
r BLEEDER
;J
VALVE
COVER CLAMP
f\{ONTROLLER
OVERFLOW
LEG
'\ RELIEF
BASKET
SUPPORTS
WATER
IN
DRAIN
STEAM
SPARGER
,
, L'::::=:::-':: ::it .. .::-:.:,-;:: :.::.::.] ,
I'
I:
Figure 12.4. Conventional retort.
Courtesy of Nickerson and Ronsivalli (1979).
O
VALVE
t
AIR
IN
o 0 S
Ar
FIL'tEgrJ

VALVE
AUTOMATIC
VALVE
tl STEAM
IN
ing effect of a heat process or the necessary heat process that will pro ..
duce a specific sterilizing effect. Many suggestions have been presented
for estimating either of these two considerations, as well as heating and
other aspects of thermal processing of foods (Dail 1985; Hayakawa 1978;
Merson, Singh, and Carroad 1978; Pflug and Christensen 1980; Smith
and Tung 1982; Spinak and Wiley 1982; Young and Steffe 1985).
After processing, the cans are cooled in water to a temperature of 36
to 42C. If cans are cooled much below 36C, they may not dry thor-
oughly and rusting will result. If the cans are cased at temperatures much
over 42C, thermophilic spoilage may occur.
(CANS
CANS
IN
OUT
\
,
COOL
w
STEAM
WATER
HOT
WATER fiN
IN
OUT
,,-,
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Figure 12.5. Hydrostatic retort.
Courtesy of Nickerson and Ronsivalli (1979).
Heat Penetration
Foods do not become hot or cold instantly. Therefore, a process call
ing for a retort temperature of 121C for 15 min means that the product
is heating during part of the process and cooling after the process. The
temperature in part of the food may never reach 121C, but it ap
proaches this temperature during the 15 min process. The temperature
attained depends upon the rate of heat penetration and other factors.
The transfer of heat into foods depends upon the thermal properties of
the food, the geometry of the container of the food, and the thermal
processing conditions (Fig. 12.6). Liquid foods or particulate matter in
HEATING FOOD IN A CAN
CONVECTION HEATING
(LIQUID IN CAN)
STEAM
OR
HEATING
MEDIUM
\

I
"'"
cold
,.-
---
X
--
"
point
"'-
I
t
,
CONDUCTION HEATING
(SOLID FOOD IN CAN)
Figure 12.6. Cold point of heating for convection and conduction
products.
Courtesy of Desrosier and Desrosier (1977).
fluids is heated in a much shorter time by convection than solid or semi
solid products that are heated by conduction.
Heat Resistance
The heat resistance of microorganisms in a food is a major consider
ation in establishing processing temperatures and times.
Several methods have been described and used to determine the heat
resistance of microorganisms. These include the thermal death time tube
or can methods, the rate of destruction method, capillary tube, special
heat exchangers, and the miniature retort system. These various methods
attempt to minimize the comeup time and cooling effects on destruction
of the microorganisms.
Vegetative cells of bacteria, molds, and yeasts are destroyed by tem-
peratures 10 to 15C above the optimum temperatures for growth. For
appropriate times at temperatures of 60 to BOC, most vegetative cells,
as well as viruses, are destroyed. Somewhat higher temperatures may be
needed for thermophilic or thermoduric microorganisms.
All vegetative cells are killed in 10 min at 100C, and many spores are
destroyed in 30 min at 100C. However, some spores will resist heating at
100C for several hours.
Spores are as much as 100,000fold more heat resistant than the corre
sponding vegetative cells (Gould and Dring 1975a). Some mold spores are
only slightly more resistant, while others are much more resistant than
the mycelium cells. Reportedly, spores of Byssochlamys julva can survive 5
hr at 88C. Bacterial spores are the most resistant entities. Some of these
spores, such as those of Bacillus stearothermophilus, may survive at 100C
for up to 20 hr with an initial population of 10
5
to 10
6
spores/g. The
spores of B. stearothermophilus are of particular interest to the canning
industry because of their high heat resistance and the flat sour spoilage
of low-acid canned foods caused by this organism.
It has been suggested that the heat resistance of bacterial spores may
result from dehydration of the central protoplast (Algie 1980; Gould and
Dring 1975a, 1975b). This dehydration is brought about and maintained
by osmotic activity, and the cortex may act as an osmoregulatory organ-
elle. Mineralization increased the heat resistance of bacterial spores (Bea-
man and Gerhardt 1986; Marquis and Bender 1985), but dehydration was
the dominant factor. Acids may interfere with osmoregulation and ren-
der the spores heat sensitive (Gould 1977).
Different microorganisms differ in their heat resistance, and differ-
ent strains of the same species also show differences. The conditions un-
der which the organisms are grown, the nature of the product in which
the microorganisms are suspended during the test, and the methods used
to determine the survivors can influence the apparent heat resistance of
a microbial culture.
Effect of Heat on Microbial Cells
Although moist heat has been used for many years as a method of
sterilization, the primary lethal event in thermal inactivation of microor-
ganisms has not been fully determined. The denaturation or coagulation
of proteins, breaks in deoxyribonucleic acid (DNA), lesions in ribo-
nucleic acid (RNA), and damage to the cytoplasmic membrane have been
suggested as possibilities.
The denaturation or coagulation of proteins involved in cell respira-
tion or cell multiplication usually is suggested as a cause of death.
In the range of 50 to 60C, the leakage of cellular components into
the suspending medium indicates that there is damage to the permeabil-
ity barrier of the cell. However, at higher temperatures, death precedes
leakage. Scheie and Ehrenspeck (1973) suggested that heat caused denat-
uration of protein(s) in the cell envelope of E. coli. This resulted in a
weakened peptidoglycan layer, which would be sufficient to prevent mul
tiplication with the conditions they provided. Further injury results from
internal osmotic pressure rupturing the cell membrane at the weakened
areas. As long as heat is applied, the rupture allows cellular material to
escape. Hence, it might be assumed that destruction of cells at temper
atures that cause sublethal injury may involve the cell membrane. If
death precedes leakage at higher temperatures, apparently other mecha
nisms are involved.
Russell and Harries (1968) reported that for nonsporeforming cells
such as E. coli, RNA degradation is closely related to heatinduced death.
The heating of E. coli caused the release of part of the outer membrane,
resulting in a partial disruption of the permeability barrier function of
the membrane (Tsuchido et aL 1985). Both RNA and membrane lesions
were reported in Pseudomonas jluorescens (Gray, Witter, and Ordal 1973).
For commencement of growth, repair of both the cell membrane and
RNA damage was required.
The heating of spores at 70 to 100C results in a loss of dipicolinic
acid (DPA), proteins, and other cellular constituents. This indicates dam
age to the spore coat. However, reportedly, the death of spores proceeds
faster than the release of DPA.
The apparent death of spores may be due to the inability of the spore
to germinate or, after germination, the inability for outgrowth to occur
with the production of more cells. Hashimoto, Frieben, and Conti (1972)
suggested that the primary cause of thermal inactivation of spores is due
to physical and chemical alterations that interfere with the imbibition of
water into the core during germination. The data of Flowers and Adams
(1976) suggested that a spore component destined to become cell memo
brane or wall was the site of injury.
Other researchers concluded that thermal inactivation of phage or
viral infectivity was due to protein coagulation (DiGioia et aL 1970).
Protein coagulation, if not the primary lethal event, is important in
the thermal destruction of microorganisms. The denaturation of protein
can occur in several areas of the cell or spore.
Factors Affecting Heat Resistance
The heat resistance of microorganisms is influenced by the type of
heat treatment, the type of cell, as well as the conditions of growth and
age, the suspending medium, and the method for enumerating the sur
VIvors.
TYPE OF CELL. Spores are more heat resistant than vegetative cells,
usually by a factor of 10
4
to 10
5
Although there are many theories for
the high resistance of spores, the exact mechanism is still conjectured.
Even with spores, the method of production can affect the apparent heat
resistance. Yeast ascospores were 30 to 350fold or 100fold more heat
resistant than the corresponding vegetative cells (Put and De] ong 1982;
Splittstoesser, Leasor, and Swanson 1986).
GROWTH OR SPORULATION MEDIUM. According to a review by
Gould and Dring (1974), calcium and manganese ions in sporulating me
dia increase the heat resistance of the spores. They reported no direct
evidence implicating monovalent ions in increasing the thermal proper
ties of intact spores. No increase in heat resistance was observed with
manganese ions in the sporulation medium of Bacillus stearothermophilus
(Cook and Gilbert 1968).
The pH of the sporulation medium affects the heat resistance of reo
sultant spores (Pang, Carroad, and Wilson 1983).
The types of peptone, carbon source, and fatty acids in the medium
influence the apparent heat resistance of microorganisms. Spores are
more resistant when produced in cooked meat medium than in raw meat.
Doyle and Marth (1975) found that Aspergillus conidia produced on me
dia low in protein and high in glucose were more heat resistant than
those produced in highprotein-Iowglucose media.
According to Roberts (1984), cadmium in the growth medium in
duced heat resistance in E. coli. The preincubation of arizonae at 35C
in a medium containing NaC1 resulted in an increased heat resistance
(Ng 1982).
TEMPERATURE OF GROWTH. Spores produced at high temper
atures are generally more heat resistant than those produced at low tern
peratures. Hence, spores produced by thermophiles tend to be more heat
resistant than spores produced by either mesophiles or psychrophiles.
Although heat resistance is directly related to the growth and sporulation
temperature, there are some limitations and exceptions. Phages react
similarly to spores. A higher propagation temperature enhances the ther
mostability of viruses.
Increasing the incubation temperature increased the heat resistance
of vegetative cells of bacteria and yeasts (EIBanna and Hurst 1983; Katsui
et al. 1982; McAlister and Finkelstein 1980; Roy, Busta, and Thompson
1981).
AGE OF CELLS. Young, actively growing cells are more senSItIVe to
almost any stress than are older, more mature cells. Young spores tend
to be less heat resistant than older spores.
SUSPENDING MEDIUM. The chemical and physical characteristics of
the suspending medium in which microorganisms are heated influence
the heat resistance. It is well established that spores are significantly
more resistant when heated in distilled water than when heated in var
ious buffers.
Perhaps the most important aspect that influences heat resistance is
the pH of the suspension. Besides this, the aw, the presence of various
solutes, fatty materials, and proteins affect the heat resistance of microor
ganisms.
Since foods vary in their chemical and physical characteristics, the
heat resistance of a microorganism would be expected to vary when sus
pended in different foods. At 70C, the D value for Bacillus licheniformis
spores was almost doubled when 4 percent salt was added to the heating
medium (Bell and DeLacy 1984). Compared to the addition of NaC1,
some solutes increased heat resistance, while others decreased heat resis
tance (Smith, Benedict, and Palumbo 1982). juven, BenShalom, and
Weisslowicz (1983) suggested that pectin in tomato juice protected Lacto
bacillus fermentum from the effects of heating.
Effects of pH. Generally, microorganisms have a greater heat resistance
near pH 7.0 than above pH 8.0 or below pH 6.0 (Fig. 12.7).
To take advantage of the lower heat resistance at low pH values, acids
are added to certain food products prior to processing. Due to the vari
able pH of foods such as tomatoes, acids are added to adjust the pH to
acceptable levels. At pH 3.9, tomatoes can be processed at 100C for 34
min to kill a normal spore load, but at pH 4.8, a process of 110 min at
100C is required.
Part of the preservative effect of low pH may be due to damage to
the cells or spores with sublethal treatments, and part may be due to the
inability for growth of these damaged cells at low pH values.
Spores derived from Bacillus that grew at pH 10 but not at pH 7 had
an optimum pH for heat resistance at pH 8 to 10 (Kudo and Horikoshi
1983). Increasing the pH of chicken scald water to 9.0 from 7.0 lowered
the D value (52C) of S. typhimurium from 34.5 min to 1.25 min (Hum
phrey, Lanning, and Beresford 1981).
Water Activity. The water activity of the suspending medium in which the
organisms are heated influences the heat resistance. In general, the mi
croorganisms are more susceptible to heat in foods with high aw. In foods
with very low a
w
, the heat effect is similar to that of dry heat.
One of the effects of heat on microbial cells is the coagulation of
protein. Coagulation occurs more readily at high aw and is reduced as
the aw is lowered.
With moist conditions, spores of B. stearothermophilus are about 50,000
100
pH 5 to pH 7
10
KILLING
TIME
(min.)
1.0
pH 4.5
pH 3.5
0.1
210 230 250

TEMPERATURE
(OF)
Figure 12.7. Influence of pH of heating medium on heat resistance of
spores. The more acidic the substrate, the less heat-resistant the spore
suspensions.
courtesy of Desrosier and Desrosier (1977).
times more heat resistant than spores of C. botulinum type E. However, at
a
w
values less than 0.5, the ratio falls to about ten times (Murrell and Scott
1966). The heat resistance of spores of B. stearothermophilus in egg powder,
fish protein concentrate, and wheat flour was greater at aw 0.33 than at
0,0.68, or 0.99 (Harnulv,johansson, and Snygg 1977). In an egg macaroni
product, Hsieh, Acott, and Labuza (1976) found that both Salmonella ana-
tum and Staphylococcus aureus had a maximum heat resistance in the aw
range of 0.75 to 0.80. As the aw is reduced from 0.4 to 0, the heat resis
tance of spores decreased (Alderton, Chen, and Ito 1980).
The water activity of the suspending medium is often adjusted by the
addition of salt, carbohydrates, or other solutes. These substances can
influence the heat resistance of microorganisms. Hence, the effect of
these adjusted suspensions on heat resistance may be due to the solutes,
to lowered aw, or to both. When salmonellae were heated in solutions of
sugars and polyols, there was no direct relationship between heat resis
tance and a
w
(Corry 1974).
Carbohydrates. These substances can influence the heat resistance of mi
croorganisms (Fig. 12.8). The thermostability of C. botulinum spores is di
10,. sucrOIt added
KILLING
TIME
na
(min.)
TEMPERATURE (oF)
Figure 12.8. Influence of sugar on the heat resistance of bacterial
spores. High concentrations of sugar protect spores.
recdy related to the concentration of sucrose in the suspending medium.
As the concentration of sucrose increases, the heat resistance increases.
The protection afforded the cells by sucrose may be due to the reduc-
tion of the Ow, the medium surrounding the cells, as well as the extraction
of water from the cell. Corry (1976b) found that the degree of protection
of the solutes was correlated with plasmolysis or cell shrinkage.
Corry (1976a) determined the heat resistance of Saccharomyces rouxii
and Schizosaccharomyces pombe in solutions of sugars and polyols. The re-
sistance was maximum with sucrose, less in sorbitol, and least in solutions
of glucose, fructose, or glycerol. The higher heat resistance of S. cerevisiae
in sucrose than in either fructose or glucose was reported by Torreggiam
and Toledo (1986). Sucrose increased the heat resistance of S. cerevisiae
spores, but alcohol decreased it (Splittstoesser, Leasor, and Swanson
1986). Sucrose protects various vegetative bacteria from heat (Kwast and
Verrips 1982; Smith et al. 1983).
The presence of higher carbohydrates, such as starch and pectin, reo
portedly gives some protection to spores and cells and increases the heat
resistance.
Proteins. There is a tendency for proteins to protect microorganisms from
the lethal effect of heat. Pep tones, yeast extract, and albumin can provide
protection for certain organisms. When ground beef was supplemented
with 30 percent textured soy protein, the heat resistance of salmonellae
was increased (Craven and Blankenship 1983).
Antimicrobial Substances. Various antibacterial substances tend to increase
the destruction of microorganisms by heat. An example is hydrogen per
oxide used in conjunction with heat pasteurization of liquid egg white.
The addition of sulfur dioxide to fruit and fruit products tends to
lower the heat resistance of the microorganisms. The effect is less appar
ent with yeast than with bacteria. Yeasts are relatively tolerant of sulfur
dioxide. Potassium sorbate and sodium benzoate acted synergistically
with heat to inactivate various molds (Beuchat 1981).
Lipids. Microorganisms suspended in fat or oil are more difficult to de
stroy than when they are in an aqueous medium (Fig. 12.9). Because of
the poor heat conductivity of oil and the reduction in moisture, microor
ganisms in a fatty medium have a much greater resistance to heat than
those heated in an aqueous medium without fat. The heat destruction in
fats resembles dry heat sterilization.
Increasing the fat content of hamburger increased the D value of
KILLING
TIME
(min.)
TEMPERATURE ("F)
Figure 12.9.. Influence of oil on heat resistance of yeasts. (Organ
isms trapped in oil phase are killed by dry heat and have much heat
resistance in comparison with organisms in water phase.
poliovirus (Filppi 1973). This protection of the virus was more evident
at 80C than at SOC.
ENUMERATION. When spores or vegetative cells receive a sublethal
heat treatment, they may be injured in some manner. The spores may
remain dormant for extended periods before germination. Dormancy
can be reduced or eliminated by using a suitable recovery medium. The
addition of starch is thought to absorb fatty acids that inhibit outgrowth
of germinated spores. Vegetative cells that are sublethally damaged reo
quire special environmental conditions for repair, growth, and multipli
cation.
Since the recovery medium can affect the number of survivors of a
heat treatment, the apparent heat resistance of an organism may vary to
a significant extent.
Spores that survive sublethal heat treatments generally are recovered
better at a lower temperature than the optimum for unheated spores.
The addition of lysozyme to recovery media aids the germination and
colony formation of spores surviving extended heat treatments. The ex-
posure of heated spores to EDTA sensitized the spores to lysozyme and
increased the number of recovered survivors (Adams 1974). Calcium di-
picolinate also can initiate germination of heat-treated spores.
Survival Curve
Since microorganisms differ in their resistance to heat and this resis-
tance can vary according to age of cells, growth medium, and suspending
medium, we need a system to mathematically express survival. Such a
system makes it possible to compare the heat resistance of different spe-
cies at the same temperature, or the heat resistance of one species at
different temperatures, and in various suspending media. It also enables
the establishment and evaluation of thermal processes for foods in rela-
tion to the types of organisms that must be controlled.
By plotting the logarithm of the number of survivors at each time
of sampling, a survival curve is obtained. Generally, bacteria exhibit a
logarithmic survival curve when exposed to heat or other destructive
agents. If a microorganism is either alive or dead, then death is a first-
order reaction. However, various shapes of survival curves have been re-
ported. Various explanations have been given for nonlogarithmic death
(Han, Zhang, and Krochta 1976; Cerf 1977).
Another aspect is the tendency for the survival curves to show a
tailing effect (Fig. 12.10). As survivors approach a low number, they ap-
pear to be very heat resistant. This can result in microorganisms surviv-
7
en
5
E
.!!
c
as
Logarithmic death
g)
4
..
0
-
0
..
CD
.Q
3
E

c
g>
...I
2
1
2 4 6 8 10
Time in minutes
Figure 12.10. Survival curve showing a shoulder and tailing.
ing a heat process that is in excess of that calculated to destroy the entire
microbial population. The exact reason for the tailing effect is not
known.
Although deviations from the logarithmic order of death do occur,
for the purposes of further discussion, we will assume that a straight line
for the survival curve is obtained (Fig. 12.11).
The number of survivors is directly proportional to the initial num-
ber present for any given treatment. The higher the initial number of
cells, the longer the time necessary to cause their complete destruction.
Since the logarithm of survivors never reaches zero, theoretically com
plete destruction of a microbial suspension can never be achieved.
4
3
~ 2
>
~
~
(/)
'0
E
..c:
;t:
;
OJ
.3
o
-1
.----2
\
\
-. ..
i\
~
.
0
0
>
M
"
U)
~
u
M
~
.1
~
.<:
M
.
~
-2
-3
o
-
P. A. 3679 in Strained Peas
Temperature = 240F
1\
'\
1\
'\
\
\
!\
~
11\
1'\
-I
i 1\
1
\
1
1 1
1
11\
1 1
\
10240 =4.9
1\
\
5
10 15 20 25
Time in Minutes
30
Figure 12.11. Death rate curve showing the determination of D.
Courtesy of National Canners Association Research Laboratories (1968).
Decimal Reduction Time (D)
The decimal reduction time is the time required to reduce the micro
bial population by 90 percent at a specified temperature. The decimal
reduction time is designated by D. This is also the time required for the
survival or death rate curve to traverse one logarithmic cycle (Fig. 12.11).
In this case, D is 9 min. D can be determined mathematically by the for-
mula
D=----
loga-log b
where a is the initial number of cells and b is the number remaining after
time t. When the difference between a and b is one log cycle, then (log a
- log b) is equal to 1, and D = t.
The D value is not constant, but varies with species and strains of
microorganisms, temperatures, and other factors that affect the heat reo
sistance. The temperature at which the D is determined may be desig
nated by a subscript. For example, D
12JOC
= 1.0 min means that 90 per
cent of the spores are destroyed in 1.0 min at 121C, and 99 percent are
destroyed in 2.0 min. The D values for several microorganisms deter
mined in various suspending media are listed in Table 12.4. As can be
seen in this table, the D values vary due to type of cell, temperature,
and suspending medium. The growth conditions, recovery medium, and
testing different strains of the same organism can result in different D
values (Doores and Westhoff 1981; Feeherry, Munsey, and Rowley 1987;
Feig and Stersky 1981; Grieme and Barbano 1983; Hyun et al. 1983;John
son, Nelson, and Busta 1982; Lovett, Bradshaw, and Peeler 1982; Lynt,
Solomon, and Kautter 1984; Scott and Bernard 1982, 1985).
The destruction of S. aureus is accomplished more readily in whole
milk than in skim milk. In skim milk at 60C, the D value for S. aureus
strain 161C was 1.30 min (Walker and Harmon 1966). In whole milk,
the D value for strain 161C was 0.75 min. FirstenbergEden, Rosen, and
Mannheim (1977) reported a D60 (D value at 60C) in whole milk of 0.87
min for S. aureus when salt-free media were used to detect heated cells.
When salt was present in the recovery medium, the apparent D60 was 0.62
min. Sublethal heat treatments cause S. aureus to lose salt tolerance.
These latter workers reported a z value of 9.46C so that if D60 is 0.87
min at 50.54C, the D is 8.7, and at 69.46C, it is only 0.087 min.
According to Bean and Roberts (1975), levels of 8 percent salt (w/v)
in the heating medium protect S. aureus from heat. They reported the D60
for a meat emulsion at pH 6.5 was 4.61 to 9.62 min. When 8 to 8.5 percent
salt was added to the meat emulsion, the D60 increased to 18.62 to 26.38
min. The addition of sodium nitrite had little effect on the heat resist
ance of this organism.
In thermally processed canned foods, the destruction of spores of C.
botulinum is a major concern. To have reasonable assurance that the
spores of C. botulinum are destroyed, a 12 D concept was established. This
means that the number of spores will be reduced by 12 log cycles. Hence,
with an original level of 10
6
C. botulinum spores, the 12 D thermal process
would reduce this to 10
5
spores. It is evident that the lower the original
contamination, the lower the number of spores after the process and the
TABLE 12.4. DECIMAL REDUCTION TIMES (D) FOR HEATING VARIOUS
MICROORGANISMS
D Value
Organisms
Vegetative Cells
Salmonella senftenberg
775W
Salmonella manhattan
Staphylococcus aureus
Escherichia coli
Spores
Aspergillus flavus
(conidia)
Type A
Type B
Type E
Bacillus stearothermophilus
Suspending Medium
Custard
Chicken it la king
Custard
Chicken it la king
Custard
Chicken it la king
Raw milk
Ice cream mix
Phosphate buffer
pH 7.0
Phosphate buffer
pH 7.0
Tomato juice
pH 4.2
Phosphate buffer
pH 7.0
Water
4% NaCI
Buffer-pH 7.0
Buffer-pH 5.0
Temp.
(0C) (min.)
60 11.3
60 9.6
60 2.4
60 0.40
60 7.7-7.8
60 5.2-5.4
57.3 1.3
57.3 5.1
50 16.2
55 3.1
104.4 17.6
110.0 4.4
115.6 1.3
121 0.2-0.4
104.4 6.0
110.0 1.6
115.6 0.4
110.0 0.7
70.0 29.3-37.5
80.0 0.4-3.3
115.0 17.5-18.3
115.0 11.3-12.7
115.0 9.2-11.3
115.0 4.2
SOURCES: Cook and Gilbert (1968,1969); Doyle and Marth (1975); Odlaug and Pflug (1977);
Read, Schwartz, and Litsky (1961).
less the chance that a can of food will contain a potentially viable spore.
Even if one spore does survive the process, there is a chance that it is
injured or damaged and is not able to germinate, outgrow, and produce
the neurotoxin in the environment of the canned food. The spores of C.
sporogenes and B. stearothermophilus are more heat resistant than those of
C. botulinum.
The thermal inactivation of viruses has been difficult to characterize.
It has been suggested that, like bacteria, viruses are inactivated by denatu-
ration of protein. If first-order kinetics prevail, the survival curve should
be a straight line. The tailing effect, or decrease in inactivation rate as
the time is extended, is a common occurrence in viral survival curves.
The formation of aggregates or clumps of viruses is a prominent sugges-
tion for the occurrence. Clumping tends to protect the viruses located in
the middle of the clump from the heating effect. During analysis, the
clumps are disintegrated to release the surviving viruses (Cliver 1971;
Sullivan et al. 1971b). It is not likely that viruses can withstand the heat
treatment given to thermally processed canned foods.
Thermal Death Time
The thermal death time (TDT) is the time required to achieve sterility
of a suspension containing a known number of cells or spores at any
predetermined temperature. At any particular temperature, the TDT de
pends upon the resistance of the cells, as well as the number of cells in
the suspension.
Thermal death times are determined for suspensions of a particular
organism at several temperatures. When the times necessary for killing
are plotted on a logarithmic scale and the corresponding temperatures
plotted on a linear scale, a straight line is obtained. This is the TDT curve
for that particular organism.
When the D values are plotted on a logarithmic scale, with the corre
sponding temperatures on a linear scale, a phantom TDT curve is ob
tained. This should be a straight line (Fig. 12.12). These lines can be de
scribed by a D or TDT at some reference temperature (usually 60 or
121C) and the slope (z). The symbol z is defined as the temperature
necessary to bring about a tenfold change in the TDT or D value. The
value of z corresponds to the number of degrees of temperature passed
over by the curve in traversing one logarithmic cycle. In Figure 12.12, the
z is listed for each curve. The symbol F is used to designate the time
necessary to destroy a given number of microorganisms at a reference
temperature, usually 121C for spores, or 60C for vegetative cells. To
avoid confusion, the temperature can be added as a subscript to F. Hence,
F60 is the TDT at 60C and F121 is the TDT at 121C. In Figure 12.12, OF
are used, and the F250 for curve Al is 5.8 min and for curve A2 is 7.4 min.
For spores of C. botulinum suspended in phosphate buffer, the value
of z is about loC. Researchers reported that z values ranged from 5.7
to 7.6C for five strains of C. botulinum type E in fish paste (Crisley et al.
1968).
For the microorganisms of most importance in canned foods, the z
values are usually between 6 and 14C. The z value varies for different
strains and types of microorganisms, as well as the chemical and physical
characteristics of the suspending medium. A strain of Clostridium thermo
saccharolyticum had a z of 6.94C in distilled water and loC in molasses
200
0 Survival Time
100
Destruction Time
80 z = 15.8 F. oo = 5.8
z = 18.8 F,oo = 7.4
60
z = 17.3 F. oo = 6.6
~ Q )
Q ) ~
~ ::l
40
80
1-1-
30
0 0
1-1-
.... e
00
20
u,_
-c
(1)1
cu.t::.
;;0..
c ....
-0
: : : E ~
10
CQ)
8
-::l
<D-
e"'
->
6
1- 0
4
3
2
1
245
Temperature in Degrees F
Figure 12.12. Thermal death time curve.
courtesy of National Canners Association Research Laboratories (1968).
(Xezones et al. 1965). The z values for three strains of Byssochlamys varied
from 3.9 to 7.8C (Hatcher et al. 1979).
The work by Esty and Meyer in 1922 showed that the F was 2.78 min
for spores of C. botulinum. Due to this, the generally accepted minimum
heat treatment for a 12 D destruction of spores of C. botulinum in low
acid foods is considered to be 3 min at 121C, or its equivalent. This
is termed the minimum safe process or botulinum cook. Commercially
canned cured meat products are given thermal processes well below this
treatment. In these products, the heat treatment is supplemented by the
effects of curing salts on the outgrowth of the spores, so that the treated
products are not likely to be a health hazard. Low acid noncured meats
are subjected to a treatment of Fl2l = 6 min.
Thermal Processes
With D, F, and z values, the rate of heat penetration, the pH of the
food, the retort temperature, and other information, a thermal process
can be calculated. Experimental inoculated packs are processed to deter
mine the validity of the calculations. It is evident that the determination
of acceptable, safe processing schedules for foods can become quite com
plex. Thermal processing schedules can be obtained from the National
Canners Association, Washington, D.C. The Food and Drug Administra
tion also has guidelines for acceptable processes. Due to the complex
interactions of factors concerning the determinations of processing
schedules, computers have become an important tool for the calculation
of needed processes.
Contaminants and Spoilage
Heatprocessed canned foods are subject to various types of spoilage.
The appearance of the container will indicate probable spoilage. The
internal formation of gas will cause conditions of the can termed flipper,
springer, soft swell, or hard swell.
For a flipper, only one end of the container is slightly bulged, or the
end of the can bulges when the container is struck on a hard surface. In
either case, by applying finger pressure, the end may be pressed back to
remain flat.
For cans that are swelled, both ends bulge and cannot be pressed flat.
Bulged ends of soft swells yield slightly to finger pressure, but those of
hard swells do not.
Even if there is no bulging of the can, the contents may be spoiled
due to flat sour, sulfide, or mold spoilage.
Spoilage may occur prior to processing or it may be due to inade-
quate heat treatment, improper holding temperature, or leakage through
defective closures, other can defects, or rough handling of the cans.
The organisms causing spoilage of under processed products are usu-
ally heat-resistant spores of bacteria that survive the heating process, ger-
minate, and grow during storage of the canned food. Inadequate cooling
or high storage temperatures can result in spoilage by the growth of ther-
mophilic bacteria. The normal commercial heat treatment is not de-
signed to destroy the spores of all obligate thermophilic organisms.
Spoilage due to leakage involves nonsporeforming bacteria, yeasts,
and molds that could not have survived the heat process. They enter the
can after processing. Also, organisms involved in foodborne illness may
enter cans after processing. Leakage into the container occurs when the
hot cans are cooled in contaminated water. When hot, the sealant for the
lids and seams is soft and not set, so organisms can enter. This entrance
of organisms is aided by the vacuum created in the can during cooling.
Pflug, Davidson, and Holcomb (1981) examined 1,104 swollen cans of
food obtained from supermarkets. They found that 314 ofthese cans had
major container defects. Of the remaining 790 cans, 86 percent had typi
cal leaker spoilage. The other defects were due to underprocessing (7
percent), nonmicrobial spoilage (6 percent), and thermophiles (l per
cent). From their data, it is obvious that postprocess leakage of organisms
into the cans is very important. Put, Witvoet, and Warner (1980) listed
the three main factors causing spoilage from postprocess operations as
the condition of the double seams, contamination of cooling waters, and
can abuse during handling. The effectiveness of the addition of germi
cidal agents to the cooling water was reported by Ito and Seeger (1986).
Their use depends upon the operation of the processing plant.
The two organisms that can cause flat sour spoilage of canned foods
are Bacillus stearothermophilus and Bacillus coagulans (Ito 1981; Thompson
1981).
To prevent the postprocessing contamination of retort pouches, it is
important that they be dried immediately after processing, be stored dry,
and be handled carefully (Michels and Schram 1979).
Sterility Testing
Canned foods should be commercially sterile. That is, there should
be no microorganisms present that have public health significance or
that are capable of reproducing in the food under normal nonrefriger
ated conditions of storage and distribution.
Contamination of the canned product during sampling and analysis
has caused many erroneous results. One of the main sources of contami
nation is the investigator (Denny 1972; Evancho, Ashton, and Briskey
1973). These workers discussed the necessary precautions that must be
taken for sterility testing of canned foods, as well as the media, equip
ment, and procedures for this test.
IONIZATION RADIATION
Shortly after Roentgen discovered Xrays in 1895, it became known
that ionizing radiations kill microorganisms. However, it was not until
the early 1940s that research employed these radiations as a means of
food preservation.
Ionizing radiation consists of corpuscular and electromagnetic radia
tions (Fig. 12.13) with sufficient energy to strip or cause ejection of elec
trons from atoms or molecules. Corpuscular radiation is due to sub-
atomic particles of various types which can transfer their kinetic energy
to anything they strike. The energy of these subatomic particles is based
upon their velocity. Beams of electrons that have been accelerated to
speeds approcahing that of light are called cathode rays. These are re-
lated to the beta particles emitted from atomic nuclei. The other sub-
atomic particles are not usable in food preservation. Alpha particles do
not have sufficient penetrating ability (Fig. 12.14). Neutrons cannot be
used because they can induce radioactivity into the food.
Electromagnetic waves produced by high-voltage equipment are
called X-rays. Gamma radiation is an electromagnetic wave from atomic
nuclei and is essentially the same as X-rays. The electromagnetic waves
have an energy that is inversely related to the wavelength of the radia-
tion. Thus, the shorter the wavelength, the higher the energy content.
The longer wavelength electromagnetic radiations (infrared, microwave,
radio) are nonionizing.
The process of food irradiation uses gamma rays from radioactive iso-
topes, such as cobalt-60 or cesium-137, or electrons from linear accelera-
tors. The radionuclides emit both gamma and beta rays. However, the
energy level of the beta rays is relatively low for food sterilization, so
gamma rays are usually the only ones considered. The electrons are lim-
ited in their ability to penetrate food materials. The depth they penetrate
depends upon their energy level. Electron beams over 10 million elec-
tron volts may induce radioactivity into some of the food constituents or
the container, so they are not considered to be practical for food preser-
vation. Cobalt-60 has been the most acceptable radiation source because
of its availability, price, and properties.
The energy of radiation is expressed in either rads or grays. A dose
of 1 rad is obtained when 0.01 joule u> of energy is absorbed per kilogram
of material, or one rad is equal to 100 ergs of energy absorbed per gram
of material. When 1.0 joule is absorbed per kilogram of material, this is
1 gray (Gy)_ Hence, 1 Gy is equal to 100 rads. For convenience, the kilorad
(Krad) for 1,000 rads and megarad (Mrad) for 1,000,000 rads, are used. A
typical sterilizing dose is 4.5 Mrad or 45 kGy, which are equal to 45 kj/kg.
For any method of food preservation to be successful, it must be
proved to be technically advantageous and economically competitive (the
benefits are greater than the costs), and the treated food must be whole-
some and safe for human consumption. For this purpose, the biological
688
VISIBLE
SPECTRUM
RADIO
WAVELENGTH
inCM
FREQUENCY
CYCLES/SEC
10-
12
COSMIC RAyS
10
22
GAMMA RAyS
10-10
10
20
MEDIUM X-RAYS
10-'
101
SOFT X-RAYS
10-6
ULTRA- VIOLET
10
16
{
10-4
10
14
INFRA-RED
10-2
10
12
MICRO
WAVES
10
0
SHORT
10
1
0
WAVES
10
2
BROADCASTING
WAVES
10'
10
4
LONG
WAVES
10
6
10
6
10
4
10'
DOMESTIC 10
2
ALTERNATING
CURRENT
10
10
10
0
Figure 12.13. The electromagnetic spectrum.
Courtesy of Robert Taft Sanitary Engineering Center.
alpha He"
beta e
gamma photon
Sheet of
paper
Sheet of
aluminum
Block of
lead
Figure 12.14. Relative penetrating power of alpha, beta, and gamma radiations. Use-
fulness of various radiations is dependent upon requirements. Where depth of pene-
tration is a factor, gamma radiations and high-energy electrons must be used.
courtesy of Desrosier and Desrosier (1977).
effects, the stability of the treated foods in storage, the organoleptic and
nutrient effects, the safety of the treated food, and the relative costs of
the process are discussed.
Biological Effects
The lethal action of ionizing radiations on living cells is due to either
a direct action on genetic material or to an indirect effect. With indirect
action, there is an initial change in the suspending medium or some non
genetic molecule.
Ionizing radiation causes breaks in the DNA. Restitution can occur in
most of the single-strand breaks, as long as the repair process is operative
(Hittelman and Pollard 1982; I wan ami and Oda 1985). Within the first
hour after irradiation, about 90 percent of the DNA-breaks are rejoined
(Graubmann and Dikomey 1983). According to Bresler, Noskin, and Sus-
lov (1984), most double-strand breaks occur due to enzymatic incision of
primary damages.
Generally, the more complex the organism, the more sensitive it is to
ionizing radiations (Table 12.5). Living cells have always been subjected
to natural radiation from cosmic and terrestrial sources. The extent of
this radiation depends upon the geographic location (altitude and distri
bution ofradionuclides). When shielded from natural ionizing radiation,
the cell growth rate of blue-green algae was reduced (Conter, DuPouy,
and Planel 1983).
Uses in Foods
Ionizing radiation can inhibit sprouting of vegetables (potatoes, on-
ions); delay ripening of fruits and vegetables; destroy insects, their larvae,
TABLE 12.5. APPROXIMATE LETHAL
DOSES OF IONIZING RADIATION
Organism
Human being
Insects
Bacteria
Fungi
Bacterial spores
Viruses
Dose (Gy)
6-10
250-1,000
1,000-10,000
1,000-10,000
10,000-50,000
30,000-50,000
and eggs in grains, spices, fruits, and vegetables; destroy parasites in
pork, beef, or fish; eliminate pathogens in meats and poultry; reduce the
number of viable microorganisms in dried products such as spices and
herbs; destroy organisms to extend the shelflife of various foods; or com
mercially sterilize various foods. Besides these processes, other actions of
gamma radiation include the reduction of insecticides (Cin and Kroger
1982), the reduction of polychlorinated biphenyls (Cichy, Zabik, and
Weaver 1979), the reduction or elimination of flatulence factors in green
gram (Rao and Vakil 1983), the alteration of seeds resulting in plants
resistant to diseases (Utkhede and Rahe 1982), and the reduction of seed
germination (Wang et al. 1983). Ionizing radiation could be used to steri
lize packaging materials used in aseptic processes.
The potential usage of ionizing radiation on foods gained momen
tum in 1980 when the Joint Expert Committee of the Food and Agricul
tural Organization, the International Atomic Energy Agency, and the
World Health Organization concluded that the irradiation of any food
up to an overall average dose of 10 kGy presents no toxicological hazard.
Further, this level of treatment causes no special nutritional or microbial
problems. Even then, relatively few nations have approved the use of
ionizing radiation on foods.
The United States FDA approved the radiation of grain for insect
control in 1963 and of potatoes for sprout inhibition in 1964. However,
there have been no commercial applications of these approved uses in
the United States.
In 1981, the FDA published an advance notice of proposed proce
dures for the regulation of irradiated foods for human consumption.
Since then, the organization has approved ionizing radiation for various
food uses. These include the reduction of the microbial level and control
of insect infestation of dried herbs, spices, and vegetable seasonings as
well as blends of these; the control of microorganisms in enzymes used
in food processing; the killing of the parasite Trichinella spimlis in fresh
pork; and the control of insects on and the delay of ripening of fresh
fruits and vegetables to extend shelf life.
It is expected that there will soon be further approvals, probably for
the elimination of pathogens on poultry and meat and the elimination
of parasites in meat and fish.
For most approvals, there is a limit of 1 kGy, but for dried spices, 30
kGy can be used. Since the safe level for international trade has been
approved at 10 kGy, the level approved by the FDA may be increased.
Mangoes were the first irradiated fruit sold in the United States (Sep'
tember 1986). Only about 1 percent of the spices sold are being irradi
ated, and even less pork is being treated.
The irradiation of food does not prevent enzymatic degradation, oxi
dative reactions, or recontamination by organisms. However, after a heat
treatment such as blanching, to inactivate enzymes, the food can be irra
diated after sealing in an oxygen-impermeable package so that recontam-
ination does not occur. It is expected that radiation will not supplant
other methods offood preservation, but rather, it will be used in conjunc-
tion with these systems.
INHIBITION OF SPROUTING. One of the major problems of storage
of foods such as tubers or bulbs is sprouting.
The irradiation level needed to inhibit sprouting ranges from 50 to
150 Gy, depending on the cultivar. Limited sprouting has been observed
on some types of potatoes receiving 5 to 10 Krad of irradiation.
High-level irradiation tends to increase the tendency of potatoes to
rot. The rotting progressively increases as the irradiation dose increases.
There is a tendency for gamma-irradiated potatoes to darken after
cooking_
. When performed within one or two weeks after harvest, the irradia-
tion of onions with as little as 20 Gy inhibits sprouting. However, if stored
for several weeks before treatment, irradiation even at higher levels will
not prevent sprouting, but will soften the onions. The usual gamma ray
treatment is with doses of 70 to 150 Gy. In one study, a level of 30 Gy
prevented sprouting of garlic bulbs for 300 days (Croci and Curzio 1983),
although Kwon, Byun, and Cho (1985) reported that 75 Gy is the minimal
optimum dose.
Although irradiation was approved in 1964 to prevent sprouting of
potatoes, the process has not been used commercially in the United
States.
DELAY RIPENING. Irradiation can be used to extend the storage life
of many fresh fruits by controlling the rate of maturation. To transport
fruit to the market, it is necessary to pick it while it is in the green stage
and sell it before it becomes overripe. Often, the ripening of the fruit is
so rapid that the area of marketing is limited.
Irradiation with 250 to 500 Gy will delay ripening and extend the
market life of most tropical and subtropical fruits. Temperate fruits reo
quire higher doses (1,000 Gy) to delay ripening, but this treatment may
cause uneven ripening and excessive softening.
DESTRUCTION OF INSECTS. Insects can consume or ruin 25 per
cent or more of the grain stored in some countries. Chemicals can be
used to destroy the insects, but there has been public concern that resi
dues of fumigants and insecticides may be a health hazard. Irradiation
not to exceed 1 kGy has been approved by the FDA for insect disinfesta
tion of food. However, for herbs and spices, up to 30 kGy can be used.
The adult state of insects is the most resistant and the egg phase the least
resistant to radiation.
A dose of 250 Gy has been suggested as a quarantine treatment for
fresh fruits and vegetables. According to Sommer and Mitchell (1986),
enormous logistic problems, irradiation induced injury, and adverse
changes in quality will limit the number of foods that can be treated.
DESTRUCTION OF PARASITES. The most common parasitic dis
eases transferable from meat to human beings are cysticercosis of cattle
and trichinosis of pigs. In 1985, the FDA approved gamma radiation at
0.3 to 1.0 kGy, to control trichinae in pork. This action, also approved by
the USDA, allows the irradiation of fresh pork carcasses and cuts such as
pork roasts and pork chops but does not allow the treatment of pro
cessed products. At the present time, radiation cannot be used as an alter
native to curing, canning, or freezing of fresh pork for destruction of
trichinae. Since Trichinella spiralis is killed by 0.3 kGy (Brake et al. 1985),
this is the lower limit. A higher level gives a margin of safety.
Cysticerci of Taenia solium and Taenia saginata require higher doses of
radiation for destruction. They were still infective after treatment with
doses of 0.2 to 1.2 kGy. Various nematode larvae found in fish can infect
the intestinal tract of humans. In fish with 6 percent salt, irradiation with
only 10 Gy can result in a 100 percent kill of these larvae.
DESTRUCTION OF MICROORGANISMS. Ionizing radiations, espe
cially from linear accelerators, are not necessarily uniform. Therefore,
all treated microbial cells might not be exposed to equal levels of these
random radiations. The death or injury of microorganisms is due to
either direct or indirect effects. The direct effect is due to a direct hit
with the radiation causing ionization within the microorganism while the
indirect effect results from ionization and diffusion of free radicals and
peroxides produced in the vicinity of the organism.
The ionizing radiation doses for destruction of microorganisms vary
with the type and concentration of cells, the type of substrate or suspend-
ing medium, oxygen tension, temperature, or the presence of protective
compounds_ Certain pre- and postirradiation treatments can influence
the inactivation of microorganisms_
Cellular death from irradiation is related directly to lesions in the
nuclear DNA. The damage caused to DNA is in the form of single- or
double-strand breaks. The resistance and survival of microorganisms de-
pend upon the capacity to repair any damage to the genetic material or
other parts of the cell.
Death due to radiation is considered to be a first-order reaction, so
that if the logarithm of survivors is plotted with the corresponding radia-
tion dose on a linear scale, a straight line should be obtained. As with
destruction by heat, various types of survivor curves are obtained, includ-
ing shoulders at the beginning of irradiation and a tailing effect.
The D values should be relatively constant, regardless of dose. How-
ever, researchers found considerable variation with different levels of ir-
radiation (Anellis et al. 1969). The D values can be used to reveal the
relative resistances of microorganisms. Some of the reported D values
RELATIVE RESISTANCES. The radiation resistance of strains and
species of microorganisms can vary widely. Bacterial spores and viruses
are among the more resistant of the microbial contaminants of foods.
Generally, Gram-positive bacteria are more resistant than are Gram-nega-
tive types. Certain streptococci, deinococci, and Acinetobacter-Moraxella
have very resistant vegetative cells. The D values for yeasts and molds are
similar to those of most bacterial cells. Young cells are more sensitive to
ionizing radiation than are old cells, with certain exceptions (Hastings,
Holzapfel, and Niemand 1986; Keller and Maxcy 1984). The presence of
oxygen during irradiation enhances cellular damage, apparently due to
a reduction of a repair mechanism (Vinicombe, Moss, and Davies 1978).
Chemicals such as cysteamine, sodium formate, glycerol, and thiourea
can protect cells from ionizing radiation (Antoku 1983; Billen 1983). Ex-
cept for cured meats, higher doses of radiation are needed to destroy
microorganisms in foods than in buffer solutions.
The removal of water generally increases the resistance of microorgan-
isms to irradiation. This is thought to be due to reduced formation of
H* and *OH radicals, the reduction in the mobility of free radicals that
are formed, and to the protection given the cells by the dried substances.
Some of the least resistant bacteria are species of Pseudomonas, Proteus,
Escherichia, and Vibrio. Ito and lizuka (1971) isolated from rice a pseudo-
monad which they named Pseudomonas radiora because of its unusual re-
sistance to radiation.
TABLE 12.6. REPORTED D VALUES FOR IRRADIATION OF SOME
MICROORGANISMS
Microorganism
Spores
Type A
Type B
Type E
Type F
C. sporogenes
C. perfringens
Bacillus cereus
B. stearothermophilus
Vegetative Cells
Escherichia coli
Salmonella typhimurium
Salmonella oranienburg
Staphylococcus aureus
Enterobacter cloacae
Pseudomonas sp.
Aeromonas hydrophilia
Yersinia enterocolitica
Campylobacter jejuni
Deinococcus radiodurans
Vibrio parahaemolyticus
Molds
A.flavus (conidia)
Penicillium citrinum
(conidia)
(mycelium)
Yeasts
Saccharomyces cerevisiae
Candida sp.
694
Suspending Medium
Cured ham
Bacon
Cured ham
Pork loin
Chicken
Water
Water
Water
Water
Broth
Buffer
Buffer (anoxic)
Buffer
Buffer (air)
Buffer (anoxic)
Dried bone meal
Dried fish meal
Broth
Dry
Buffer
Crab meat
Broth
Broth
Dry
Dry
Fish (22C)
Fish (-15C)
Beef (2C)
Broth
Beef (25C)
Beef (- 30C)
Beef ( - 30C)
Turkey (- 30C)
Turkey (30C)
Broth (30C)
Buffer
Raw beef
Buffer, pH 7.0
Water
Dry
Cacao beans
Wet
Dry
Cacao beans
Buffer
Buffer
D Value (kGy)
2.18-2.35
2.14
1.64-1.75
2.86-3.36
3.70
0.80-1.60
2.50
1.60-2.20
1.20-3.40
2.50
2.52
4.25
0.09
0.20
0.60
0.91-1.00
1.70
0.28-0.56
0.37
0.20-0.25
0.40
0.22-0.29
0.47-0.69
0.18
0.38
0.11-0.15
0.22-0.34
0.14-0.19
0.097-0.118
0.195
0.388
0.315
0.293
0.162
0.174
1.94-3.08
3.5-6.0
0.05-0.14
0.38
0.50-0.55
0.66
0.18
0.55
0.88
0.36
0.32
TABLE 12.6 (Continued)
Viruses
Coxsackievirus B2 Eagles medium 4.40-6.90
Water (-90C) 5.30
Cooked beef (16C) 7.00
Cooked beef (- 90C) 8.10
Adenovirus 2 Eagles medium 4.10
Adenovirus 3 Eagles medium 4.90
Echovirus 18 Eagles medium 4.40
Toxins
Botulinum
Type E toxin
washed cells Buffer 17-21
cell extract Buffer 2.10
purified Buffer 0.40
Type A toxin
purified Buffer 0.50-2.00
purified Broth 40
purified Cheese 60
Staphylococcal
Enterotoxin B purified Buffer 27
Milk 97
SOURCES: Anellis et al. (1965,1967,1969); Ito, Iizuki, and Sato (1974); Kopelman, Markakis,
and Schweigert (1968); Miura et al. (1970); Read and Bradshaw (1967); Roberts (1968); Sui
livan et al. (1971a, 1973); Hastings, Holzapfel, and Niemand (1986); Tsuji (1983); Restaino
et al. (1984); ElZawahry and Rowley (1979); Lambert and Maxcy (1984); Thomas, Ouwer
kerk, and McKercher (1982); Palumbo et al. (1986).
Escherichia coli is quite sensitive to ionizing radiation, although there
are resistant strains. Due to the rapid demise of E. coli, this organism is
a poor indicator of sanitation for irradiated food. Nearly all of the organ-
isms involved in foodborne illness have a higher resistance to irradia-
tion.
Serotypes of Salmonella vary in their resistance to irradiation. The rel-
ative resistance of serotypes shows that the mechanisms are different for
the destructive action by heat and radiation. S. senftenberg 775W is the
most heat-resistant strain, while the radiation resistance of this organism
is more similar to other strains and serotypes of salmonellae.
A radiation-resistant coccus was isolated from irradiated meat (An-
derson et al. 1956). Although originally named Micrococcus radiodurans,
this organism, like some other radiation-resistant cocci, is distinct from
other micrococci. Therefore, the name Deinococcus radiodurans was sug-
gested by Brooks and Murray (1981). Reportedly, the high radiation re-
sistance of these organisms is due to a very efficient system to repair
DNA lesions.
Moraxella and Acinetobacter are more resistant than pseudomonads to
radiation. Ito, Sato, and Iizuka (1976) reported the D values for Moraxella
species to vary from 44 to 54 Krad and Acinetobacter to be 12 Krad. These
values are much lower than the 477 to 1,000 Krad for Moraxella and 405
to 814 Krad for Acinetobacter reported by Welch and Maxcy (1975).
C. botulinum spores are generally more resistant than other clostridial
spores. This is very important if ionizing radiations are used for steriliza
tion of food. Spoilage types of clostridia could be eliminated from the
food, while C. botulinum spores could remain viable and produce toxins.
Hence, the consumer might not be forewarned of possible danger by gas
formation or putrid odors normally developed in contaminated food by
spoilage types.
The required minimum radiation dose to achieve commercial sterility
of food is based on the resistance of C. botulinum spores. In general, type
A spores are more resistant than type B spores. Type A and B spores are
more resistant to radiation than are types C, D, E, or F spores.
The temperature at which irradiation occurs influences the resistance
of C. botulinum spores. From 65C down to -196C, the spore resistance
increases with decreasing temperature (Grecz et al. 1971).
The presence of curing salts (sodium nitrite, sodium chloride, and
sodium nitrate) reduces the irradiation dose needed to inactivate spores
of C. botulinum. This is evident by the lower D values for C. botulinum in
ham or bacon than in pork loin or chicken.
Viruses tend to be more resistant than other organisms to gamma
irradiation. Viral inactivation demonstrates a one-hit or first-order reac-
tion curve, when the suspending medium contains free radical scaven-
gers or is in a frozen state. Freezing increased the resistance in water and
cooked beef, but this increase was not observed in raw beef (Sullivan et
al. 1973). Fortunately, viruses are not found in most foods. If present,
they tend to be at low levels.
One team of researchers suggested that the irradiation inactivation
of Newcastle disease virus at low temperature (2.2C) was due to nucleic
acid degradation, while at higher temperatures (60C), it was due to pro-
tein denaturation (DiGioia et al. 1970). Survival is enhanced in the pres-
ence of impurities. These impurities apparently bind free radicals and
peroxides giving protection to the viruses.
Radappertization. Radiation sterilization or radappertization is the de-
struction of all or practically all of the organisms. To accomplish com-
mercial sterility, relatively high levels of irradiation are required. Of all
the microorganisms of concern that are associated with food, the spores
of C. botulinum are the most resistant to irradiation. The spores of C. botu-
linum are more resistant to ionizing radiation than spores of organisms
that have a high heat resistance and cause spoilage.
For ionizing radiation as with heat treatment, a 12D concept is used
for producing commercially sterile foods that are reasonably safe from
C. botulinum spores or toxin. If the D used is 3.75 kGy, for commercial
sterility, a 12D treatment is 45 kGy. According to Josephson (1969), the
minimum radiation dose (MRD) needed for commercial sterilization var-
ies from 23 kGy for bacon irradiated at ambient temperature to 57 kGy
for beef irradiated at - 80C. The 12D irradiation doses at - 30C listed
by Rowley and Brynjolfsson (1980) varied from 25.5 kGy for pork sausage
to 43.7 kGy for pork, with beef at 41.2 kGy.
Five spices with bacterial levels of 10
2
to 10
5
/g were sterilized with 7.5
to 10 kGy (Sharma et al. 1984). However, for commercial sterility of sev-
eral spices, the treatment varied from 6 kGy for poultry seasoning to 13
kGy for black pepper and thyme (Grecz, AI-Harithy, and Jaw 1986).
Radiation doses for radappertization can cause losses in the organo-
leptic quality of food. The higher the dose, the greater is the potential for
development of undesirable flavors, odors, and colors, as well as losses of
nutrients.
Of the common meats, beef is the most sensitive and pork the least
sensitive to the production of off-flavor. Although these changes in foods
due to irradiation have been of concern, often they are less than the
alterations due to heat processing.
To minimize alterations of foods due to irradiation, various combina-
tion processes have been suggested. These processes include addition of
chemicals that are free radical acceptors, use of anaerobic conditions,
elimination of moisture, or irradiation at freezing temperatures. Each of
these systems reduces the objectionable activity of the free radicals.
The amount of nitrite needed in cured ham can be reduced when
radappertization is used to control C. botulinum (Wierbicki and Heilig-
man 1973). For this treatment, the ham is frozen and treated at - 30C
with 3_7 Mrad. The nitrite was reduced to 25 p,g/g, but to prevent fading
of the cured color, 100 p,g/g of nitrate was added.
Even though nearly all of the bacteria are killed by radappertization,
enzymes are not destroyed as readily. Hence, enzyme activity continues,
especially when the food is held at ambient temperature.
Radappertization of meat involves a preirradiation treatment with
heat (65 to 80C) to inactivate proteolytic enzymes, sealing in a package
under a partial vacuum, freezing to - 30C or lower, and exposing the
packaged food to ionizing radiation until the desired dose is absorbed.
Freezing prior to irradiation reduces the rate at which free radicals
diffuse through the tissue. This minimizes the side reactions of irradia-
tion and reduces the alteration of odor and flavor. Generally, the lower
the temperature of irradiation, the more the quality factors are pro-
tected. However, as the temperature is lowered, the microorganisms, in-
cluding C. botulinum spores, have increased resistance to irradiation, so
that higher doses are needed to sterilize frozen meats than meats at ambi-
ent temperatures.
Radappertization appears to be beneficial for sterilizing and storage
of foods when refrigeration and frozen storage are not available. There
are disadvantages and problems associated with this treatment. These
include the need for heating and freezing during the process, the alter-
ations in organoleptic and nutrient quality, and the cost of the operation.
Thus, it will be difficult for radappertization to compete with the present
preservation procedures used in the United States. However, radiation-
sterilized foods may be produced for hospital patients with suppressed
immune systems. Also, radiation-sterilized foods have been used to feed
astronauts during space flights.
Radurization. The use of low doses of radiation to destroy a sufficient
number of microorganisms and enhance the storage life of foods is called
radiation pasteurization or radurization.
The doses of radiation used for radurization (1 to 10 kGy) will destroy
from 90 to 99 percent of the microorganisms. Some resistant vegetative
cells and spores will survive. Hence, refrigeration or some other combi
nation treatment is needed to retard growth and possible toxin produc
tion of the survivors.
The dose used for radurization is a compromise. High doses prolong
storage life, but deleterious effects and costs increase as the dose in-
creases. Quite often, the dose used is the maximum amount of irradia-
tion that produces no detectable change in the product. The other possi-
bility is to determine the storage life needed to market a product and fit
the dose of irradiation to this requirement.
The psychrophilic pseudomonads are the main spoilage organisms of
protein foods (meat, poultry, fish). These bacteria are very sensitive to
irradiation, being essentially eliminated by doses used for radurization.
Enterobacteriaceae, pseudomonads, and enterococci are eliminated
from vacuum-packed beef when irradiated with 2 kGy (Niemand, van der
Linde, and Holzapfel 1981). During storage of this beef at 4C, radiation-
resistant lactobacilli grew and became dominant. The majority of strains
isolated from minced beef radurized with 5 kGy were Lactobacillus sake
(Hastings and Holzapfel 1987). After storage for 15 days at 3C, spoilage
odors were detected in ground beef treated with 1.03 kGy but not in that
irradiated with 1.54 kGy (Dempster et al. 1985).
The treatment of fishery products with 1 to 6 kGy can increase the
refrigerated storage life (Angel et al. 1986; Licciardello et al. 1984; Venu-
gopal et al. 1982). The effectiveness of radurization varies with the type
and initial quality of the product and the associated microbial flora. One
problem is the presence of C. botulinum type E and their potential radia-
tion resistance in fishery products. However, according to Rowley,
and Shattuck (1983), any type E spores surviving 1 to
4 kGy are injured and do not produce toxin at lOoe or less.
The foods that rank highest in merit for radurization are shrimp, fin
fish (haddock, flounder, cod), blue crab, strawberries, and mushrooms.
These foods are perishable and have high spoilage rates during market
ing. They are of relatively high value and, at some marketing stage, they
have large volume concentrations.
Radicidation. This is a lowlevel irradiation treatment to destroy viable
nonsporeforming pathogenic organisms to reduce or eliminate the prob
lem of foodborne illness. Spores of C. botulinum or C. perfringens are not
destroyed at levels normally used for radicidation. This term was sug
gested to describe the ionizing radiation treatment needed for elimina-
tion of salmonellae from food and feed.
The D values in ground beef ranged from 0.55 to 0.78 kGy for four
serotypes of salmonellae, from 0.10 to 0.21 kGy for three strains of Y.
enterocolitica, and from 0.14 to 0_16 kGy for three strains of C.jejuni (Tar-
kowski et al. 1984). In frog legs, the D values for three serotypes of salmo-
nellae ranged from 0.18 to 0.30 kGy (Nerkar and Lewis 1982), which is
considerably lower than the D value reported for salmonellae in ground
meat.
Tarkowski, Beumer, and Kampelmacher (1984) stated that a dose of
1 kGy eliminates or reduces some pathogens in raw ground meat to ac-
ceptable levels. Mulder (1984) suggested that to decrease potentially
pathogenic organisms and to increase shelf life of poultry, doses ranging
from 2 to 9 kGy are needed, but doses over 5 kGy may cause undesirable
side effects in this food. In a review by Dempster (1985), a level of 7 kGy
is recommended to eliminate salmonellae.
Besides eliminating pathogens, this low-dose irradiation will destroy
parasites such as tapeworms and trichinae.
Heat treatment can be used to eliminate salmonellae from dried feed
such as meat, bone, fish, and blood meal, but there is a potential for
recontamination prior to packaging, and heating decreases the available
protein. A dose of 8 to 10 kGy can reduce salmonellae in packaged dry
feeds to below detectable levels. Irradiation of animal feeds to eliminate
salmonellae would be an important step in breaking the salmonellae
cycle and might reduce the involvement of animal foods in human salmo-
nellosis.
Dose levels of 2 kGy are sufficient to destroy all expected levels of
Staphylococcus aureus in most food products. The problem with S. aureus is
the recontamination by people handling the food during preparation for
serving. Radicidation cannot prevent this from occurring.
Paster, Barkai-Golan, and Padova (1985) found enhanced production
of ochratoxin by Aspergillus ochraceus after irradiation with 0.5 to 2.0 kGy.
No growth occurred when a dose of 3 kGy was used. These researchers
suggested that care should be taken when agricultural products on which
mycotoxigenic fungi can grow are irradiated with low doses.
These low levels are not expected to control sporeforming bacteria.
Hence refrigeration of foods so treated is needed to prevent germina-
tion, outgrowth, and toxin production by organisms such as C. botulinum.
Since both radurization and radicidation utilize essentially the same
intermediate levels of radiation, some authors prefer to use the term radi-
ation pasteurization for both processes. Although this level of irradiation
is included in the international level of usage of 10 kGy, the present FDA
level of 1 kGy is not sufficient to allow the treatment of foods to eliminate
salmonellae and many other pathogens.
Thermoradiation. This is a combination treatment of heat and radiation.
Enzymes and spores of C. botulinum are relatively resistant to radiation
but are more sensitive to heat. Conversely, thermophilic spores are very
heat resistant, while relatively more sensitive to radiation. A combination
of heat and radiation, either in sequence or simultaneously, may be of
value in food preservation.
Preheating to sublethal temperatures can sensitize vegetative cells but
not spores to irradiation. Preirradiation sensitizes spores to subsequent
heat treatments. In some cases, simultaneous heating and irradiation are
more effective than applying the treatments in sequence. Heat and irra-
diation are synergistic when used together for killing spores of anaer-
obes. Thus, it is possible that this synergism can be used to sterilize food
so that the quality of the food is less affected than with the level needed
for either treatment applied alone. The synergistic effect is evident only
in certain temperature and radiation dose ranges, and these depend
upon the organism being treated.
Shrimps subjected to heat at 121C for 8 min and irradiated with l.0
kGy can be stored up to two months at ambient temperatures (Savagaon
et al. 1972). Canned products with excellent quality can be obtained by
thermoradiation.
Synergistic effects of temperature and radiation have been deter-
mined for several organisms, including B. stearothermophilus. For this or-
ganism, the D of 2.10 kGy at 27C was reduced to 1.57 kGy at 60C (Tulis,
Fogarty, and Sliger 1973).
INACTIVATION OF TOXINS. Microbial toxins reportedly are inacti
vated at radiation levels comparable to bacterial spores. However, the
amount of radiation needed to inactivate botulinum toxin is greater than
that needed to destroy the organisms (see Table 12.6).
The purer the toxin, the more sensitive it is to irradiation (Miura et
al. 1970). This indicates that impurities protect the toxin in much the
same way that cells are protected. Miura and researchers (1970) found
that serum albumin, casein, DNA, and RNA protected purified and actio
vated toxin against radiation inactivation. Sugars or ascorbic acid showed
little or no protection.
Staphylococcal enterotoxin is more heat stable than botulinum toxin.
This higher stability tends to be true for inactivation by ionizing radia
tion. With the D values for inactivation of enterotoxin determined by
Read and Bradshaw (1967), it would be impossible to rid a food of signifi
cant amounts of this toxin without destroying the quality of the food.
Low doses of irradiation would have little or no effect on either botuli
num or staphylococcal toxins. A dose exceeding 10 kGy was needed for
total destruction of aflatoxin BJ (Van Dyck et al. 1982).
INACTIVATION OF ENZYMES. The complete, or at least partial, in
activation of enzymes has been considered essential for preservation of
foods. The inactivation of enzymes requires higher doses of radiation
than that for microbial destruction.
To determine if milk has been heat pasteurized, researchers test for
the presence of phosphatase enzyme. During irradiation of milk, phos
phatase is not affected.
Heat treatment of meats to inactivate the enzymes prior to irradiation
improves the storage stability of these products. This preheating treat
ment applies not only to red meats, but also to poultry and seafoods.
Even 45 to 52 kGy of radiation will inactivate only about 75 percent of
the proteolytic activity of beef (Losty, Roth, and Shults 1973).
The heat treatment needed to inactivate enzymes in fruits is sufficient
for preservation, so the use of ionizing radiation is unnecessary. There
is little advantage in using radiation for the preservation of vegetables.
Since enzymes are more resistant than microorganisms to irradiation,
it is possible to use this process to sterilize or at least significantly reduce
the microbial level in commercial enzyme preparations.
Quality of Irradiated Foods
At some level of ionizing radiation, microorganisms, toxins, and even
some enzymes can be destroyed or inactivated. However, at the doses
necessary to destroy viruses, spores, or even some vegetative cells, the
quality of the food may suffer.
The effect of ionizing radiation varies with the type of food. Some
foods, such as milk, are very susceptible to even low doses of radiation.
Other foods, such as cured meats, can be treated with relatively high
doses of radiation without the quality being seriously affected. According
to a review by Reineccius (1979), odor changes in meats result from the
formation of carbonyl compounds due to reactions of components of
fats and from side chains of proteins, as well as from carbohydrates.
Ionizing radiation can cause undesirable effects in food; however,
other systems of preservation, especially heating, can change the organo
leptic and nutrient factors in food. In many cases, the effect of radiation
is less drastic than is that of heating.
Undesirable effects resulting from radiation are generally due to free
radicals produced when ionizing radiations react with water in the food.
Thus, the effects on quality can be reduced by irradiation in the frozen
or the dried state, by the use of free radical acceptors, or by irradiation
in inert atmosphere or in vacuum. Although these procedures will help
reduce, they do not necessarily prevent changes in food.
The irradiation of wheat with low levels may increase the resultant
bread volume and overall quality (ParedesLopez and Covarrubias
Alvarez 1984). However, they found that the overall bread quality was
reduced at medium doses of radiation. MacArthur and D'Appolonia
(1983) found a reduced quality of bread at 1.0 kGy treatment of wheat.
Wholesomeness
In the United States, as well as many other countries, statutes require
that proof of safety for consumption (wholesomeness) must be adequate
to convince the appropriate healthregulating officials before irradiation
of foods will be approved. The factors considered are nutritional ade
quacy, microbiological safety, absence of induced radioactivity, and abo
sence of carcinogenic or other toxic substances that may be formed by
exposure of the food to ionizing radiations.
Under provisions of the Food, Drug and Cosmetic Act, the FDA must
approve the use of ionizing radiation for each food given this treatment.
The USDA has legal responsibility for meat and poultry and their prod
ucts.
The wholesomeness of irradiated food is determined by laboratory
analysis of constituents in the food, as well as by using animal feeding
studies. The many wholesomeness studies have revealed that irradiated
foods are as wholesome and nutritious as thermally processed foods
(Brynjolfsson 1985). Hospital patients taking immunosuppressant drugs,
people with immune system disorders, and astronauts have consumed
irradiated foods with no apparent bad effects.
NUTRITIONAL ADEQUACY. There is concern about the effect of ra
diation on the proteins, energy sources, and vitamins. In general, the loss
of any nutrient is dependent on the dose of radiation, as well as the
environment during irradiation. The low doses of radiation used for
sprout and insect control would be expected to cause insignificant nutri
tional damage. At high doses (45-55 kGy) needed for sterilization, the
nutritional adequacy of irradiated foods is comparable to conventional
heatprocessed foods.
In some cases there is an apparent increase in the nutritional value
of food due to irradiation Gami, Pubols, and McGinnis 1980; Reddy, Pu
boIs, and McGinnis 1979). One concern is the loss of thiamin due to irra
diation (Skala, McGown, and Waring 1987).
MICROBIOLOGICAL SAFETY. With radappertized foods that are
shelf stable at ambient temperatures, it is essential that all cells and
spores of C. botulinum be destroyed. This is also true of thermally proc
essed canned foods. In either case, residual, viable spores can result in a
toxic food.
There is a possibility that food sterilized by radiation may contain
or develop toxin after processing. The amount of radiation required to
inactivate the toxin is greater than that needed to destroy the organisms.
Inasmuch as radiation sterilization does not have the many years of
widespread distribution and consumer use as does thermally processed
food, it is difficult to compare the potential hazards of the two treat-
ments. In general, it is agreed that with proper controls throughout pro
cessing and distribution, irradiated foods will present no microbiological
hazards to the consumer.
By virtue of eliminating organisms such as salmonellae, S. aureus, Vi-
brio parahaemolyticus, and shigellae, irradiation can be used to reduce the
number of foodborne illnesses.
INDUCED RADIOACTIVITY. No radioactivity is induced when suit-
able radiation sources are used. Electron accelerators with energy levels
above 10 million electron volts (Mev) may induce small, but significant,
amounts of radioactivity. Therefore, this treatment is considered unsuit-
able for irradiation of food. Below 10 Mev no induced radioactivity can
be detected. Great Britain limits the accelerator level to 5 Mev.
Irradiation of food with gamma rays from cobalt
60
, even at high dose
rates, induces less radioactivity than can be detected with the most sensi-
tive instruments available.
CARCINOGENS AND OTHER TOXIC FACTORS. The effect of ioniz-
ing radiation on the chemical components in food and an estimation of
the concentration of radio lytic products has been reported (Diehl and
Scherz 1975; Merritt 1980; N awar 1978). Numerous animal feeding stud-
ies have revealed no toxic effects of foods when irradiated at doses below
68 kGy (Renner et al. 1982; Thayer et al 1987).
Most of the radio lytic products that have been identified also occur
naturally in a wide variety of foods. The concentration of radiolytic prod-
ucts is such that they are generally not considered to be of toxicological
significance.
Animals fed irradiated foods have developed tumors. However, the
incidence of tumors has been no higher with a diet of irradiated than
unirradiated foods. Proof of the absence of carcinogens or other toxic
substances in food due to irradiation is extremely difficult. No demon-
strable carcinogenicity has been shown in the extensive tests that have
been done.
Economic Aspects
The irradition cost per unit depends to a large extent upon the an-
nual volume of an irradiation plant, the type of irradiation source (in-
cluding its efficiency and price), the capital investment, and the proxim-
ity of the processing plant to the food source.
The centralization of a food industry is necessary before irradiation
can be accomplished at a reasonable cost per unit. On the other hand,
the equipment needed for thermal processing or freezing is available for
various sized operations. Only certain foods are accumulated in large
volume in one location.
According to Swientek (1985), cost estimates have ranged from less
than 2 cents per kilogram to more than 20 cents per kilogram of food.
Beattie and Wiblin (1984) reported that, although the cost of fruit and
vegetable treatment could be quite low, there is a lack of demand for the
process, and an irradiation facility could not be operated economically
in Australia.
Less energy is needed for irradiation of foods than for heat treatment,
refrigeration or freezing (Diehl 1983). However, radiation-pasteurized
foods must be refrigerated, and radappertized foods must be heated to
inactivate enzymes and then frozen to prevent undesirable reactions dur-
ing irradiation, which must be considered part of the overall energy reo
quirements for this treatment.
There are some benefits of radiation treatments. No other processing
system can eliminate salmonellae from frozen meat or poultry products.
No one system will eliminate salmonellosis completely, but if 50 percent
of the cases of foodborne illness could be eliminated by irradiation of
certain foods, a considerable savings could be envisioned.
Some benefits that have been suggested for irradiation of food in-
clude the money saved by reducing food spoilage, the extension of stor
age life, a saving in transportation and storage costs, and a reduction in
the number of cases of foodborne illness. It has been suggested that, with
irradiation, there would be a larger variety of foods essentially free of
pathogens and parasites. The losses due to insects and sprouting would
be reduced. There would be an increased availability of foods to under
nourished peoples, expanded export potential, and market stabilization.
These advantages would be for both the processor and the consumer.
The energy requirements for radappertized and radurized foods
should be compared with thermal processing, freezing, drying, or other
preservation methods. If radiation treatments save energy, they may be
necessary in the future if we hope to maintain some semblance of our
present standard of living. When all of the benefits are considered, for
some foods they outweigh radiation costs by a wide margin. However,
for other foods, irradiation is not practical due to quality alterations or
the cost involved.
Summary
Preservation methods, including irradiation, will not improve the
quality of a food, but they will tend to retain the attributes of highquality
food. Irradiation has the potential to reduce the enormous loss of food
so that more food will be available to the ever-increasing population.
Radiation is not expected to have any substantial effect on the current
systems of processing and distribution. However, radiation has the poten-
tial for reducing food loss due to sprouting, insects, and microorganisms,
of decreasing hazards, of increasing food quality and convenience, and
of decreasing costs of storage, distribution, and marketing.
Radiation will be of considerable importance in eliminating salmo-
nellae from meats and poultry products, as well as from bulk animal
feeds. No other process we now have can accomplish this task. In 1976,
the World Health Organization approved the radiation of poultry with
doses up to 700 Krad. In Canada, levels up to 750 Krad are approved for
controlling salmonellae on frozen poultry. The Netherlands has ap-
proved irradiated poultry with doses up to 300 Krad. It is expected that
the FDA and the USDA will approve the use of irradiation to eliminate
salmonellae from poultry and red meats. Presently these foods are con-
taminating kitchens throughout the United States with salmonellae.
In the United States, we have highly acceptable frozen, dried, and
thermally processed foods. However, in less developed areas in which
these or other systems are not as adequate, radappertized foods have
a greater importance. With the continued problem of energy sources,
irradiation of food might become an important preservation method in
the future of the human race, regardless of the country.
ULTRAVIOLET RADIATION
The antimicrobial effect of ultraviolet (UV) light has been known for
about 100 years. The lethal action ofUV light varies with the intensity of
the light and the time of exposure. The temperature, pH, relative humid
ity, and amount of microbial contamination influence the effectiveness
of UV light. According to Severin, Suidan, and Engelbrecht (1983), UV
disinfection is relatively insensitive to temperature changes.
Useful Aspects
The energy of UV light is much lower than that of ionizing radiations.
This means that UV light has low penetrating power. Ultraviolet light is
effective against microorganisms in air, liquids that are clear or in thin
films, and on surfaces that do not produce shadows. Microorganisms are
protected by dust in the air, as well as by dust on UV light bulbs. Food
particles or other substances that UV cannot penetrate protect the micro
organisms. Clumping of microbial cells allows those in the central part
of the clump to escape the biocidal effect of UV light. Over 50 percent
of the radiation energy is lost at a depth of 5 cm in clear water. Penetra
tion into milk is only about 1 or 2 millimeters.
The suggested or actual uses of UV light include the control of micro
bial growth in bulkstored maple sap, tenderizing, aging, and retailing of
meat, the removal of ethylene from banana storage areas, sterilization of
packaging materials used in aseptic packaging, control of surface growth
on bakery products, treatment of water and wastewater, destruction of
thermophiles in refined sugars intended for canned foods, sanitation of
equipment, and air purification (Brown and Russo 1979; Gilpin et al.
1985; Huang and Toledo 1982; Mans 1987; Qualls and Johnson 1983;
Robinson and Adams 1978; Severin 1980; Stannard, Abbiss, and Wood
1985; Stermer, LasaterSmith, and Brasington 1987). The production of
ozone by certain wavelengths of UV light gives an added germicidal ef.
fect.
Relative Microbial Resistance
With some exceptions, UV irradiation is about equally effective
against yeasts and either Grampositive or Gramnegative bacteria. Cer
tain species of Deinococcus, such as D. radiodurans, have exceptionally high
resistance to UV as well as ionizing radiations, apparently due to a very
efficient repair system. E. coli is more resistant to UV than either Y. enter
ocolitica or C. jejuni (Butler, Lund, and Carlson 1987). Of the pseudomo
nads tested, P aeruginosa was the most sensitive to UV radiation (Abshire
and Dunton 1981). They also reported increasing resistance from S.
aureus through E. coli, S. Jaecalis, Candida albicans, to D. radiodurans. How
ever, C. albicans was only about 25 percent as resistant as D. radiodurans.
Researchers found that UV inactivation of E. coli was comparable to that
for S. typhi, Shigella sonnei, and S. aureus (Chang et al. 1985). Poliovirus and
rotavirus were three to four times as resistant, and spores of B. subtilis
about nine times as resistant as E. coli.
Light rays can cause chemical changes and biological damage only if
they are absorbed. Light that passes through a cell has no effect. Due to
the high absorption of light with wavelengths between 210 and 300 nm,
there is a strong biocidal effect. Several "most effective" wavelengths
have been listed in the literature. The wavelength most often suggested
is 253.7 nm; however, the wavelength with optimum biocidal effect varies
with different microorganisms.
Ultraviolet radiations between 210 and 300 nm are absorbed by pro
teins and nucleic acids. As a result of chemical reactions, there is chromo
some breakage, genetic mutation, and enzyme inactivation which can reo
sult in cellular death. There is a variety of genetic alterations including
base-pair substitution and frameshift mutations, deletions, mitotic cross-
ing over, and mitotic gene conversion induced by UV light (Hoffman and
Morgan 1976).
Ultraviolet light produces potentially lethal photoproducts in the cel-
lular DNA. A large portion of the bactericidal activity of UV results from
nucleotide dimer formation. These dimers are the most abundant and
stable of the photoproducts. They inhibit DNA synthesis and, to a lesser
extent, RNA and protein synthesis.
Repair of Lesions
The irradiation of bacteria with ultraviolet light produces a variety
of pyrimidine dimers in the DNA. The repair of these lesions can be
accomplished by photoreactivation, excision (dark) repair, or postrepli-
cation repair. Both photoreactivation and dark repair mechanisms have
been described in a variety of microorganisms.
Photoreactivation is the reversal of short-wavelength UV damage by
postirradiation exposure to longwavelength light. Photoreactivation is
enzymatic and functions by splitting the dimers in situ. The photoreacti-
vation of damage caused by UV light clearly establishes a major role of
pyrimidine dimers in the inactivation of various cells, especially
radiation-sensitized mutants. The enzymes involved in this repair are
called the photoreactivating enzymes.
Excision repair of pyrimidine dimers is considered to be a sequence
of enzymatic steps. This involves incision breaks, removal of the dimers,
resynthesis of the excised regions, and bonding between the newly syn-
thesized and preexisting DNA to restore the continuity of the repaired
DNA strand_ Strains deficient in excision repair are more sensitive to UV
radiation than are repair-proficient strains_
At high fluences, lethality may be enhanced by damage to the excision
and recombination repair systems (Webb and Brown 1976)_ Certain en-
zymes can be inactivated by light. The inactivation of photoreactivating
enzymes by UV light has biological significance_ One of the causes of
death of cells is the disappearance of the enzyme responsible for the final
step in the repair of DNA_
The rate of inhibition and death depends, at least partially, on the
rate ofrepair. With large doses ofradiant energy, the repair cannot main-
tain a balance and photoinhibition occurs_ The occurrence of sigmoidal
inactivation curves could be related to such effects_
The repair of DNA damaged by UV light has been discussed in several
reports (Adkins and Allen 1982; Friedberg 1975; Pollard and Fugate
1978; Prakash 1976; Sutherland 1977)_
Protective Agents
Plasmids may confer partial protection against the bactericidal effects
of UV irradiation_ Siccardi (1969) reported that of thirty-one plasmids,
fifteen protected, eleven had little or no effect, and five increased the
susceptibility of E. coli K-12 to UV irradiation_ How these plasm ids afford
protection is not known_ However, the authors offered suggestions as to
the possible mechanisms for this occurrence_
E. coli was protected from mutagenesis and inactivation by broad
spectrum near UV light when catalase was incorporated into the plating
medium (Sammartano and Tuveson 1984)_ Glycerol protects bacterial
cells against near UV (365 nm) but not against 254 nm UV light (Peak
and Peak 1980; Peak, Peak, and Foote 1982)_
Interactions
The lethal interaction of UV radiation and mild heat on E. coli was
studied by Tyrrell (1976)_ Irradiation with most wavelengths sensitized
the cells to temperatures of 45 or 48C. He proposed that, in addition
to DNA damage, both mild heating and near UV treatment interfere with
DNA repair mechanisms_ Hence, the combination of the two treatments
leads to a strong positive interaction_
The simultaneous use of UV and ozone produced synergistic effects
on molds but not on bacteria (Kaess and Weidemann 1973)_
The interaction of UV light and ionizing radiation was studied by
Schneider and Kiefer (1976). Submitting Saccharomyces cerevisiae to UV
light followed by Xrays, and also in the reverse order, revealed differing
reactions. With UV, then Xray treatment, synergism was observed with
all strains tested. With the reverse treatment, differences in synergism
were obtained with different strains.
The simultaneous treatment of hydrogen peroxide and UV light has
a synergistic killing effect on both sporeforming and nonsporeforming
bacteria (Bayliss and Waites 1980, 1982).
Other studies have revealed an Xrayultraviolet synergism for diploid
yeast, certain strains of E. coli, Aspergillus nidulans, Clostridum botulinum,
and Serratia marcescens.
OTHER SYSTEMS
There are other systems that can inactivate microorganisms, but they
have not been developed for commercial usage. This is probably because,
at present, there are few, if any, practical applications for these processes.
Visible Light
The effects of visible light on microorganisms were reviewed by Har
rison (1967). He listed the types of damage induced by light as loss of
colonyforming ability, photolysis, lengthening of lag period, loss of fer
mentative ability, and reduction of ability to support phage growth. The
presence of carotenoid pigments in the bacteria sometimes enhances the
survival of bacteria when exposed to light.
Strains of E. coli inactivated by near UV light also are sensitive to inac
tivation by visible light (Tuveson and March 1980). Visible light kills Bacil
lus spores exponentially (Abad-Lozano and RodriguezValera 1984) as
well as prevents or retards the outgrowth of surviving spores (Quesnel
and Spencer 1985).
The photodynamic effect on bacteria can be enhanced by simultane
ously exposing the organism to light, a photosensitizing compound (usu
ally a dye) and to oxygen (Banks, Board, and Paton 1985).
Laser Energy
Pulsed laser energy effectively inhibited the growth of E. coli (Taka
hashi et al. 1975). Other research has indicated the requirement of a
photosensitizing dye for inhibition of microorganisms to occur. In the
pulsed laser energy study, however, researchers found no difference in
inhibition with or without added toluidine blue, and less inhibition when
acridine orange was present (Takahashi et al. 1975). They stated that tech
nology was just beginning to produce tunable laser irradiation powerful
enough for microbiological research.
Ultrasonic Energy
Ultrasonics is the transmission of vibrational energy above the level
of audible sound. When alternating current is applied to a crystal, the
shape of the crystal changes with the electrical field. These continuous
changes in shape cause pulsations or waves which travel through a liquid.
These waves have alternate compressions and rarefactions. If the ampli
tude is high enough, a phenomenon known as cavitation is produced.
This is the making and breaking of microscopic bubbles. These bubbles
tend to become larger and when they grow to what is known as resonant
size, they collapse instantly and violently, producing high local pressures.
This pressure is the force that alters the microbial cells.
When microorganisms are subjected to ultrasonics, the overall result
may be molecular bond breakage, physical damage by shock waves, de
naturation, reactions with free radicals, or a combination of these effects.
Microbial cells vary in their ease of ultrasonic disruption. Cells that
are relatively easy to disrupt include Pseudomonas, E. coli, salmonellae, and
vegetative cells of B. cereus. Cells that are considered difficult to disrupt
include Staphylococcus aureus, streptococci, lactobacilli, Serratia marcescens,
B. cereus spores. Candida albicans spores, and poliovirus. Generally, the
smaller and rounder the cell, the more difficult it is to disrupt. Spores
are not greatly affected by ultrasonic treatment.
In a laboratory, suspensions of cells can be inactivated after long ex
posure to ultrasonics. However, commercially, this process seldom pro
vides a 100 percent kill of microorganisms. Reportedly, another potential
use of ultrasonics is the inactivation of toxins.
The combination of ultrasonics and UV radiation acts synergistically
to destroy microbial cells. Synergism has been noted with chemicals, such
as chlorine or hydrogen peroxide and ultrasonic treatments (Ahmed and
Russell 1975). The time required for the fumigant gases ethylene oxide
or propylene oxide to kill microorganisms is reduced by simultaneous
treatment with ultrasonic waves.
Ultrasonic treatment lowers the resistance of D. radiodurans and S.fae
calis to ionizing radiation. According to Burgos, Ordonez, and Sala
(1972), the heat resistance of B. cereus and B. licheniformis spores decreased
markedly after ultrasound treatment. Ordonez and Burgos (1976) reo
ported that ultrasonic waves do not affect the heat resistance of some
strains of Bacillus species.
Electricity
Stersky, Heldman, and Hedrick (1970) used various voltages to inacti
vate microorganisms aerosolized into a chamber. As the voltage in
creased from 6,000 volts to 20,000 volts, the reduction of the bacterial
population increased. The highest mean reduction they reported was
59.2 percent.
Edebo (1969) found the microbicidal effect of transient electric arcs
on E. coli in tap water was similar to that of UV radiation, both biologi
cally and chemically.
Grampositive bacteria and yeasts were less sensitive than were Gram
negative bacteria (Hiilsheger, Potel, and Niemann 1983). Cells in the loga
rithmic phase of growth were more sensitive than were those in the sta
tionary phase. They found there was selective damage to the inner cell
membranes.
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13
Regulations and Standards
NEED FOR REGULATIONS
When the United States began to change from a rural society to an
urban one, food science was nonexistent, microbiology was in its infancy,
and little was known about the chemistry of foods. Workers in the food
business were trained in the need for sanitation. The adulteration of
food was common.
As people became aware of the problems in their food supply, they
demanded some action. In 1906, Congress passed the Pure Food and
Drugs Act, as well as the Meat Inspection Act.
Some regulations are needed to eliminate unfair competition. For ex
ample, a process such as pasteurization will help control a health hazard,
but increases the cost of production. If every processor is not required
to pasteurize a particular product, it is difficult for those willing to do it
to compete. The inspection of meat and poultry, with elimination of dis
eased animals, is a cost that an unscrupulous person would not be willing
to pay without government regulations.
PURPOSE OF REGULATIONS
The basic purpose of food laws and food regulatory agencies is to
ensure that all foods reaching the consumer are safe, wholesome, and
truthfully labeled. The production and marketing of wholesome foods
should be important to everyone affiliated with the food industry.
There is general agreement that we, as consumers, want clean, whole
some food that is produced and processed in a sound, sanitary manner
and is free of microbial pathogens, toxins, or harmful chemicals. Some
consumer advocates demand 100 percent assurance of safety of all con
sumer goods. However, there is no way that absolute safety of food or
any other commodity can be proven. The safety of food involves the eval
uation of a variety of risks. Everything we do in life involves some risk,
including eating food.
Since we have an abundant supply of food in the United States, the
present demands for food safety overshadow the hunger in many other
725
countries. A hungry person is more interested in satisfying an appetite
than in the esthetic aspects of the food that is offered.
FOOD LAWS
The enforcement of the Pure Food and Drugs Act and Meat Inspec
tion Act of 1906 was delegated to the Secretary of Agriculture in the
United States Department of Agriculture.
The Pure Food and Drugs Act made illegal the adulteration of food
entering into interstate commerce. The individual states maintained the
sole authority to regulate intrastate production and marketing of food.
The enforcement of the law improved the quality of the food supply.
However, as advances were made in technology, it became apparent that
a major revision was needed. In 1933, a bill was introduced in Congress
and, after many revisions, was passed in 1938. It became the Food, Drug,
and Cosmetic Act. This act is still the basic legislation, although it has
been amended several times. The law applies to exports, imports, and
commerce between states, in the District of Columbia, and in the territor
ies. The act prohibits adulteration of food and requires certain informa
tion on the food label. It authorizes definitions or standards for food.
The specific standards are the standards of identity, standards of quality,
and standards of fill of container.
Among the amendments to the act, the Miller Pesticide Amendment
of 1954 allows the establishment of tolerances of residues of pesticides
on raw agricultural commodities shipped in interstate commerce.
The Hale Amendment of 1954 simplifies procedures for establishing
food standards. The FactoryInspection Amendment of 1954 provides
that inspections of processing plants and other establishments are autho
rized upon presentation of credentials and a written notice to the owner,
operator, or agent in charge.
The Food Additives Amendment of 1958 requires proof of safety be
fore a chemical additive can be used in food. It allows the use of sub-
stances that are safe at the levels of intended use. This amendment put
the burden of proof of the safety of additives on the chemical and food
industries rather than on the regulatory agencies. The Delaney Clause in
this amendment forbids the use of any substance in any amount if it is
found to induce cancer in humans or animals_ This clause has resulted
in much controversy.
The Fair Packaging and Labeling Act of 1966 requires the use of retail
packages that are not deceptive. The label must contain certain meaning-
REGULATIONS AND STANDARDS 727
ful information about the value of the food and the ingredients. The
name and address of the manufacturer or distributor are required.
The Meat Inspection Act of 1906 requires the inspection of meat
processing plants that ship products in interstate commerce. The Whole
some Meat Act of 1967 requires that state inspection services for intra
state meat processors have regulations and requirements comparable to
those of the federal regulatory agency.
Prior to 1959, poultry was inspected on a voluntary basis, with the
poultry industry paying for the service. The Poultry Products Inspection
Act of 1959 made poultry inspection mandatory for any processor that
ships products in interstate commerce. The Wholesome Poultry Act of
1968 requires comparable regulations for intrastate processors.
In the past, foods not in interstate commerce were not subject to fed
eral laws and regulations. However, this was changed with the Whole
some Meat Act and the Wholesome Poultry Act. The food services on
ships, trains, airplanes, and buses, and on interstate highways are subject
to federal inspection. Many foodservice facilities are national chains,
some using foods prepared in central kitchens and shipped across state
lines.
Besides the laws directly aimed at the food industry, there are many
other federal laws and agencies that regulate and affect our food supply.
There is no limit to the number of laws and agencies that can be created
to regulate every segment of our life, including foods.
Besides all of the laws and regulations imposed at the national level,
the food processor must comply with city, county, and state laws and reg-
ulations. For those engaged in exporting and importing, there are inter-
national considerations. Through organizations such as the Codex
Alimentarius Commission and the International Commission of Micro-
biological Specifications for Foods, foods are being standardized at the
international level.
The purpose of the Codex Alimentarius Commission is to protect the
health of consumers and ensure fair practices. The primary means of
accomplishing this is through establishment of food standards and codes
of practice that we hope will be adopted by cooperating countries.
THE ENFORCERS
A number of government agencies are involved in the enforcement
of laws and regulations that affect the food industry either directly or
indirectly. According to Sloan (1976), there were fifty regulatory agencies
in the federal government, many of which exert influence upon or con
trol the food industry.
Food and Drug Administration (FDA)
This is the agency generally recognized as the main enforcer of the
regulations concerning food. The FDA is part of the Public Health Ser
vice located in the Department of Health and Human Services.
The FDA enforces the Food, Drug, and Cosmetic (FD&C) Act, as
amended. It is responsible for regulating the production, storage, and
labeling of all foods, except red meats and poultry. These foods are regu
lated by the USDA. The FDA has the responsibility to enforce tolerances
for pesticide residues.
The FDA also administers the Fair Packaging and Labeling Act as reo
lated to foods and household products.
The FDA devotes part of its effort to the sanitation of milk processing,
shellfish, restaurants, and interstate travel facilities included in the Pub
lic Health Service Act.
FDA ACTIVITIES. The food processor has the prime responsibility for
the safety, wholesomeness, and nutritional quality of his food. The role
of the FDA is to see that industry is meeting its responsibility.
About 40 percent of the FDA budget is allocated to food programs.
In these food activities, most of the effort is devoted to food safety and
the remainder to economic aspects. The food safety programs include
food sanitation control, chemical contaminants, mycotoxins, food and
color additives, and nutrition. The economic aspects include food stan
dards and controlling unfair competition due to economic cheating.
In food sanitation control, there are efforts to reduce and control the
incidence of foodborne illness due to salmonellae, the toxins of S. aureus
and C. botulinum, as well as other pathogenic and toxic agents. The food
sanitation and control program is responsible for the development and
enforcement of the good manufacturing practice regulations.
The FDA periodically inspects processing and storage plants to deter
mine if they are sanitary. The inspectors check the wholesomeness of
ingredients and finished food products and the legality of packages and
labels.
It is impossible for the FDA to regulate the entire food industry as
well as imported foods. Hence, the FDA depends upon the integrity of
responsible people in the food industry as well as upon qualityassurance
programs as described by Majorack (1982).
Another program of the FDA is to maintain surveillance on commod
ities that have demonstrated a potential for mycotoxin contamination.
Products that are contaminated above acceptable levels are removed
from the market.
The FDA enforces safe levels of use for chemical additives. Standards
are established for classes of foods, and all such products must then meet
the minimum requirements.
PRODUCTS NOT IN COMPLIANCE. When a product that is not in
compliance with the various laws and regulations is discovered, the FDA
has certain courses of action. Among the possible actions it might take
are recall, seizure, injunction, and prosecution.
Recall. Some recalls begin when the firm finds a problem. Others are
conducted at the FDA's request. Recalls may involve the physical removal
of products from the market or correction of the problem where the
product is located.
Recalling a product from the market has become the most effective
method of removing all units found to be adulterated, misbranded, dan
gerous to health, grossly fraudulent or deceptive, or materially mislead
ing to the detriment of consumer health and welfare. The food industry
can recall a product more efficiently and effectively than the FDA. If the
firm refuses to recall the product, or the recall is not effective, seizure
actions and FDA publicity are alternative considerations.
There are three classes of recalls. For Class I recalls, there is a reason
able probability that the use of the product will cause a serious adverse
health consequence. The Class II recall involves products which, when
used, will cause a temporary or medically reversible adverse health conse
quence or present only a remote possibility that there will be serious
adverse health effects. Class III recalls are those in which the food is not
likely to cause an adverse health effect.
Seizure. This is an action taken to remove a product from commerce be
cause it is in violation of the law. The FDA initiates a seizure by filing a
complaint with the U.S. District Court where the goods are located. A
U.S. marshal is then directed by the court to take possession of the goods
until the matter is resolved.
Injunction. This is a civil action filed by the FDA against an individual or
company seeking, in most cases, to stop a company from continuing to
manufacture or distribute products that are in violation of the law.
Prosecution. This is a criminal action filed by the FDA against a company
or individual charging violation of the law.
NATURAL OR UNAVOIDABLE DEFECTS. Growing crops in an open
field can result in natural or unavoidable defects. It has never been possi
ble, nor is it now possible, to prevent certain levels of contamination.
With this realization, the FDA has developed food defect action levels.
The alternative to allowing certain defects would be to use increased lev
els of pesticides. This could result in higher residues of pesticides in our
foods. Growing one's own food organically does not ensure the absence
of insect or other contamination.
United States Department of Agriculture (USDA)
The USDA has many functions in food production and processing.
The agency most directly associated with the food industry is Food Safety
and Inspection Service (FSIS). The FSIS is charged with the inspection
of domestic and imported meat, poultry, and their products, as well as
establishing ingredient standards and the approval of recipes and labels
for meat and poultry products.
Other USDA agencies involved in food include the Packers and Stock-
yards Administration, Animal and Plant Health Inspection Service, Agri-
cultural Research Service, Economic Research Service, and the Agricul-
tural Marketing Service.
National Marine Fisheries Service (NMFS)
This organization is under the National Oceanic and Atmospheric
Administration (NOAA) in the Department of Commerce. Through its
Fishery Product Inspection and Safety Program (FPISP), it offers a volun-
tary fee for service inspection to processors of fishery products. This ser-
vice includes inspection for plant sanitation, inspection of the product
for safety and wholesomeness, laboratory analyses, product grading, lot
inspections, and consulting services. The NMFS has developed memos
of understanding with the FDA and the USDA regarding the areas of
jurisdiction which each organization has with fishery products.
Occupational Safety
and Health Administration (OSHA)
This organization was created in the Department of Labor to enforce
the Occupational Safety and Health Act. OSHA develops and promul-
gates safety and health standards and regulations, investigates to deter-
mine compliance with these standards and regulations, and issues cita-
tions with proposed penalties for noncompliance. Every employer who
is engaged in interstate commerce is subject to the act and to the regula-
tions of OSHA. The law requires detailed reports from the employers
concerning every category of safety, noise, and other working conditions.
Environmental Protection Agency (EPA)
This agency was established in 1970 to coordinate programs of the
federal government concerning the environment. This includes air, wa
ter, solid wastes, pesticides, radiation, and noise. As a regulatory agency,
it establishes and enforces environmental standards. Two of the acts it
enforces are the Insecticide, Pesticide, and Rodenticide Act and the Water
Pollution Control Act. These are important to food processors.
Federal Trade Commission (FTC)
The FTC is an independent agency that enforces various laws. It in
cludes the Bureau of Competition, Bureau of Consumer Protection, and
the Bureau of Economics. In the consumer protection area, the food in
dustry is affected by rules concerning marketing practices and national
advertising.
FOOD STANDARDS
Section 401 ofthe FD&C Act provides that whenever, in the judgment
()f the Secretary, such action will promote honesty and fair dealing in
the interest of consumers, he shall promulgate regulations fixing and
establishing for any food, under its common or usual name, a reasonable
definition and standard of identity, standard of quality and/or standard
fill of container. Fresh and dried fruits and vegetables are exempt, except
for avocados, cantaloupes, melons, and citrus fruits. Also, butter is ex
empt since it was defined by an Act of Congress in 1923. Meat and poul
try products are in the domain of the USDA.
The Hale Amendment of 1954 simplified the procedures for estab
lishing food standards. A standard constitutes the official specification
for that food. Supposedly, these standards are important to the con
sumers and food manufacturers, as well as the enforcers. Standards pre
vent certain types of debasement and promote honesty and fairness to
the consumer, and serve to prevent unfair competition.
The Codex Alimentarius Commission is concerned with establishing
food standards on a worldwide basis rather than having many regional
standards. Establishing international standards for foods is complicated.
Over the years many countries have developed their own standards and
codes of practice. The differences in these standards must be reconciled
by participating countries with those being developed by the Commis
sion.
The FDA has stated that it will be its policy to accept the standards
recommended by the Codex Alimentarius Commission insofar as the re-
quirements of those standards can be shown to be reasonable and calcu-
lated to promote honesty and fair dealing in the interest of the American
consumers_
Although these standards should promote better international trade,
they, like any other standards, must be somewhat flexible and subject to
change when new technology requires a change_
The regulatory agencies attempt to improve sanitary standards
through the development of ordinances, codes, manuals, and guides to
safe practices. This is done at the local, state, and federal level. Although
the FD&C Act does not specifically mention sanitary standards, these are
written on the premise of increasing food safety. Reportedly, the sanitary
standards are used to prevent foodborne illness in the United States.
The GMP's of the FDA and the Regulations of the USDA for operating
meat, poultry, and egg products plants can be considered sanitary stan-
dards for food processors. Some food processors have in-house standards
that exceed the requirements of the federal agencies.
The sanitary standards usually include observation of the overall
plant and facilities, raw material, plant operations, clean-up operations,
and finished products. Many also include the handling and storage of
the finished products, especially if the product is perishable.
MICROBIOLOGICAL CRITERIA
A criterion is a standard that can be used to evaluate the correctness
of a judgment. When the microbial load of a food is determined, we then
need to make a judgment as to whether the food is satisfactory or not.
Hence, microbial criteria are established to help make a valid judgment
concerning the safety and keeping quality of a food.
Another reason for adoption of microbial standards for foods is to
compel people in the food industry to adopt quality-control procedures
to protect the microbial quality of food from producer to consumer.
Most microbiological criteria are developed to protect the safety of
the food supply and reduce the risk to the consumer. Since the FD&C
Act states that harmful or deleterious substances are prohibited in food,
the position of the FDA is that standards are needed to evaluate quality.
In previous chapters, several potential pathogens and toxins were dis-
cussed. Of all of these, only salmonellae are determined in foods on a
regular basis.
It is well known that salmonellae are present on much of our raw red
meat and poultry. Although these products are not under the jurisdiction
of the FDA, they are an example of foods that are known to contain
pathogens and, at present, they are tolerated.
Since the local health authorities and the FDA cannot adequately in
spect all of the food processing establishments in the United States, the
establishment of microbiological criteria for the finished product is the
only method to estimate the microbial quality of food.
The functions of microbiological criteria should be to
1. Control the risk to the consumer from pathogenic organisms or
their toxins.
2. Reveal if gross contamination has occurred.
3. Assure that a reasonable shelf life of the food is attainable.
There are various criteria that can be established for foods. A micro
biological criterion of a food should include the microorganisms and/or
their toxins of concern, the methods for analyzing the food for detection
and quantification, a sampling plan that includes the number of samples
to be obtained and the size of the sample unit, the limits for the microor
ganisms and/or toxins that are appropriate for the specific food, and the
proportion of sample units that should conform to these limits.
The microbiological load of any food is influenced by various produc
tion and processing procedures. These variations should be considered
in establishing microbial limits. The limits should be attainable by using
good manufacturing practices. There should be tolerances due to the
inherent errors in sampling and microbial analyses.
Types of Criteria
To evaluate the microbiological condition of foods, the criteria that
have been used are guidelines, recommended limits, specifications, and
standards.
GUIDELINES. Microbiological guidelines have been developed for
many foods. A guideline is used when no official standard exists for a
particular food. Guidelines might be considered as administrative stan
dards.
A guideline usually is an estimate of the microbial load that can be
attained readily in a satisfactory plant using satisfactory methods of pro
cessing and materials.
Although not an official standard, a guideline can be useful for quality
control personnel to make judgments about the acceptability of raw rna
terials, finished products, or operations during processing.
Both processors and regulators can establish microbiological guide-
lines. The FDA has used guidelines in conjunction with processing-plant
inspections. When the microbial load exceeds the guidelines, it supports
the observations of poor sanitation and failure to follow good manufac-
turing practices (Read and Baer 1974). These guidelines have been useful
at the processing level but less useful at the retail level. The analysis of
food at the retail level has merit if the consumer is considered. Abuses
that might occur between processing and retailing could influence the
consumer risk.
The defect action levels for molds or mold fragments established by
the FDA for certain fruits and various other foods can be considered
microbiological guidelines. Some defect action levels for microorganisms
TABLE 13.1. MICROBIAL DEFECT ACTION LEVELS IN SELECTED HUMAN FOODS
Product
Potato chips
Dried whole eggs
Frozen eggs
Citrus fruit juices (canned)
Pineapple (canned, crushed)
Plums (canned)
Prunes (dried and dehydrated low mois
ture)
Raisins
Strawberries (frozen) (whole or sliced)
Apple butter
Cherry jam
Black currant jam
Allspice, bay leaves, capsicum, cassia, or
cinnamon
Beets (canned)
Greens (canned)
Spinach
SOURCE: FDA (1973).
Defect Action Level
6% of the chips by weight contain rot.
Decomposed as determined by direct mi
croscopic count of 100,000,000 bacteria
per gram.
Two cans contain decomposed eggs; and
subsamples examined from cans classed
as decomposed have counts of 5,000,000
bacteria per gram.
Microscopic mold count average of 10%.
Average of 5% by count of plums with rot
spots larger than the area of a circle 12
mm in diameter.
Average of 5% by count insect infested
and/or showing mold and/or dirty fruits
or pieces of fruit.
Average of 5% by count of natural raisins
showing mold.
Microscopic mold count average of 45%
and mold count of 55% in onehalf of
the subsamples.
Average of 5% moldy berries by weight.
Average of 5% by weight of pieces with
dry rot.
Average of 10% of leaves by count or
weight showing mildew \12" in diameter.
Average of 10% leaves by count or weight
show mildew or other type of decompo
sition \12" in diameter.
RECOMMENDED LIMITS. These are the suggested maximum accept-
able number of microorganisms or of specific types of microorganisms,
as determined by prescribed methods in a food (NAS 1964). They are
similar to guidelines.
There are many recommended microbiological limits. Most of these
recommendations are based on too few samples or other considerations
to be of much value. The ranges of recommended limits for various foods
as found in the literature are listed in Table 13.2. Due to the wide vari-
ation, it is evident that most of these are not valid.
SPECIFICATIONS. A specification is a document that provides a de-
tailed description of a material. Included in a specification of a food may
be acceptable microbiological quality. A microbiological specification
has been defined as the maximum acceptable number of microorganisms
or of specific types of microorganisms, as determined by prescribed
methods, in a food being purchased by a firm or agency for its own use
(NAS 1964).
The acceptable microbiological limit that is written into a specifica-
tion is not always easy to derive. A microbiological specification should
consider both the buyer and the capabilities of the supplier. If the specifi-
cation is too strict, there may not be enough acceptable material to meet
the need of the buyer. If too lenient, the buyer may obtain rather poor
quality material. By analyzing many samples, or using information ob-
tained through guidelines, a compromise may be made between the mi-
crobial quality available and what is considered to be acceptable. To ob-
tain a high-quality product, the purchaser may expect to pay a premium
price for the material.
A practical specification should have tolerance limits set wide enough
to allow permissible deviations. However, the limits should be narrow
enough to ensure that the material will be acceptable consistently_
TABLE 13.2. RANGES OF SUGGESTED LIMITS FOR TOTAL
VIABLE AEROBES IN VARIOUS FOODS
Food Product
Frozen precooked foods
Precooked meats
Raw ground meat
Frozen whole eggs
Fish
Shellfish
Vegetables
Frozen desserts
SOURCE: NAS (1964).
Microbial Range (per g)
2,000 to 100,000
10 to 10,000
100,000 to 50,000,000
200,000 to 10,000,000
100,000 to 10,000,000
50,000 to 1,000,000
50,000 to 500,000
5,000 to 1,000,000
The specification should include sampling and analytical procedures
so that there is no question of the system that is to be used.
The microbiological limits that appear in various military and federal
specifications for food products are listed in Tables 13.3 through 13.5.
STANDARDS. Of these criteria, only microbiological standards have a
regulatory function. The National Academy of Sciences (NAS 1964) de
fined a microbiological standard as that part of a law or administrative
regulation designating the maximum acceptable number of microorgan
isms or of specific types of microorganisms, as determined by pre
scribed methods, in a food produced, packed, stored, or imported into
the area of jurisdiction of an enforcement agency.
A microbiological standard should not be established haphazardly. A
standard must be meaningful and attainable by what is considered to be
good manufacturing practice. It should be subject to reevaluation as new
technologies are developed. Above all, to be of value, a microbiological
standard must be enforceable. When enforced, a standard should reduce
the public health hazard of a food.
When the need for microbial standards for food is discussed, an ex
ample often used is how the establishment of standards eliminated or
reduced the involvement of milk in foodborne diseases. Microbiological
standards, per se, do nothing to increase the safety or quality of the food.
It is the acceptance of the standards by all segments of industry and the
enforcement by the regulators that is necessary before a standard has
value.
TABLE 13.3. MICROBIOLOGICAL SPECIFICATIONS FOR MILITARY AND FEDERAL
PURCHASES-BEEF AND PORK PRODUCTS
Aerobic Plate Salmonellae
Product Count Coliform E. coli (25g sample)
Beef, cooked, dried 150,000 60
Beef stew, cooked, dried 75,000 neg.
Beef with rice, cooked, dried 75,000 neg.
Swiss steak w/gravy, frozen 100,000 100 neg. neg.
Pork slices, cooked, dried 110,000 20
Pork sausage, cooked, dried 200,000 40
Pork loin, sliced, w/gravy, fro
zen 100,000 100 neg. neg.
Pork and beef chop suey, fro-
zen 100,000 100 neg. neg.
Meat balls and meat ball
products, cooked, dried 150,000 40
Spaghetti w/meat sauce, dried 75,000 neg.
Escalloped potatoes w/pork,
cooked, dried 75,000 neg.
Beef hash, cooked, dried 75,000 neg.
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Microbial Groups and Species
Microbiological criteria could include the determination of many
groups or species of microorganisms. Keeping the number of required
tests to a minimum has advantages. There is less expense and time in
volved in the analysis, and the interpretation of the results is less com
plex when fewer tests are needed.
The microbial limits might include the aerobic plate count for meso
philes, as well as coliforms, fecal coliforms, E. coli, salmonellae, Staphylo
coccus aureus, Clostridium botulinum, C. perfringens, group D streptococci,
Enterobacteriaceae, yeasts, and molds.
The standard aerobic plate count of mesophilic bacteria is a rough
measure of the bacterial content and the sanitary conditions during pro
cessing or temperature abuses of a food. The aerobic plate count cannot
be related unequivocally either to a health hazard or to spoilage.
Some foods normally have high microbial loads. Depending on the
raw material and the processing conditions, a particular number of organ
isms may represent a good or bad product. Precooked or other heat
treated foods would be expected to have lower counts than raw foods.
High microbial numbers in a heated food may be due to poor processing
control; contamination after heating; poor holding temperature, allow
ing multiplication; or a combination ofthese factors. The viable microor
ganisms tend to decrease in frozen or dried foods and to increase in
refrigerated foods. Hence, the microbial number that is obtained will
depend on the time of analysis after processing.
The coliform group is determined in food mainly because this group
is important in water microbiology, and researchers once believed that
this importance carried over to food products. The presence of coliforms
cannot be related unequivocally to fecal contamination. In a pasteurized
or precooked food, the presence of coliforms indicates poor processing
controls, postprocess contamination, or both.
The presence of E. coli reportedly indicates either direct or indirect
fecal contamination. However, this organism is part of the normal flora
of many raw animal products, even when prepared with acceptable pro
cessing techniques, as well as plant products that contact the soil. E. coli is
heat sensitive. When found in heat processed food, it indicates contami
nation after heating by fecal material that also could conceivably include
enteric pathogens. Certain strains of E. coli are enteropathogenic or
enterotoxigenic. Distinguishing these types from any other E. coli would
be too time consuming and costly for practical purposes regarding mi
crobial standards.
The determination of E. coli reportedly can be used to estimate the
plant sanitation, postheating contamination, poor handling practices,
crosscontamination from raw products, or a combination of these fac
tors. Being pathogenic, salmonellae should not be associated with food.
Staphylococcus aureus has been included in some microbiological crite
ria. In certain situations, S. aureus may be a pathogen, a source of enter
otoxin, an indicator of unsanitary practice, or be of little significance
and concern (Bartram 1967). Foods that are handled by people are likely
to contain coagulase positive staphylococci. Standards for S. aureus at the
processing level would have little value in preventing foodborne illness.
Clostridial spores are prevalent in the environment, especially in soil
and dust. Hence, organisms, such as C. botulinum and C. perfringens may
be present in foods. Unless the food is an acceptable environment and
is allowed to remain at temperatures that allow growth, a few clostridia
or S. aureus are not considered to be a health hazard. However, processing
and handling should be designed to minimize contamination and pre
vent the growth of these organisms.
Organisms in the group D streptococci have been considered indica
tors related to human contamination of processed meats. They have been
proposed as indicators of fecal pollution of various processed foods due
to their ability to withstand certain processing and environmental condi
tions.
The presence of yeasts and molds is used in the microbiological crite
ria for some foods. A high count (above 50/g) in cocoa powder indicates
poor sanitary conditions after roasting. The guidelines for product de
fects (Table 13.1) consider certain levels of molds unsatisfactory in foods.
Tomato products contain high levels of mold due to moldy tomatoes not
being properly sorted or trimmed or to contaminated processing equip
ment. In either case, a high level of mold indicates poor processing tech
nique. In some products, the presence of molds may indicate the possible
presence of mycotoxins that mayor may not be a health hazard.
The tests that are most useful or used in microbiological criteria are
the standard plate count of aerobic mesophiles, coliforms, E. coli, and
salmonellae.
Types of Food
There is more justification for microbiological standards for some
foods than for other foods. When there is a risk or hazard, a standard
for a food might be needed. There are some foods for which there is no
substantive need for microbiological standards.
Those foods that have demonstrated a potential health hazard should
be the first ones considered for establishment of any microbial standards.
Foods that contain animal products are the primary vehicles for organisms
that cause foodborne illness.
Frozen and chilled precooked foods that support the multiplication
of pathogenic bacteria when held at growth temperatures are considered
to be the main foods that need microbiological standards. However, even
though there is a potential hazard, these foods have been involved in
relatively few known outbreaks of foodborne illness.
Foods under consideration for international standards by the Codex
Commission include egg products, dried milk, molluscan shellfish, fro
zen frog legs, precooked frozen shrimps and prawns, food for infants
and children, and frozen desserts. Those considered for prompt atten
tion include chilled frozen poultry and meat, precooked frozen crab
meat, and lobster, cheese, and coconut.
Microbiological standards have been applied to milk for a number of
years. Standards for bottled drinking water and local standards for a few
foods have been established. Milk and water were involved in many dis
ease outbreaks before microbial standards were established. These liq
uids are homogeneous and can be subjected readily to heating, filtration,
or chemicals to control the microbial load. Hence, a microbial limit can
be attained relatively easily and the standard can be enforced fairly eas
ily. These problems are increased with heterogeneous solid foods.
In 1973, the FDA proposed microbial standards for foodgrade gelatin
and frozen readytoeat banana, coconut, chocolate, or lemon creamtype
pies. These standards were rescinded in 1978. In 1977, the FDA planned
microbiological standards for fish cakes, fish sticks, and crab cakes.
Information regarding the microbial content of various foods has been
reported by researchers of the FDA, apparently with the intent of
establishing microbial criteria. These foods include dry milk mixes and
milksubstitute infant formulas (Schwab et al. 1982), pasta products
(Swartzentruber et al. 1982), cocoa powder, dry instant chocolate milk,
nondairy creamer and frozen nondairy topping (Payne et al. 1983), crab
meat, clams, and oysters (Wentz et al. 1983), biscuit dough, snack cakes,
and soy protein meat extender (Swartzentruber et al. 1984).
Sampling Plan
Microbiological criteria should include a sampling plan, as well as
methods for analysis. Since the microbial contamination in most foods
is heterogeneous, the testing of as many samples as possible is necessary
to satisfy statistical requirements. Statistical sampling plans can ensure
that the tests that are done are sufficient to give a good assessment of the mi
crobial condition of the food. It is important to minimize the chances ofre-
jecting an acceptable lot of food (producer risk) or accepting a substandard
lot (consumer risk).
Sampling pl::ms for food have been amply described by Thatcher and
Clark (1968) and ICMSF (1974). Sampling offood is discussed briefly in
Chapter 2. The sample units should be representative of the lot and be
obtained in an unbiased manner.
The number and size of samples to be analyzed should be included
in the microbiological standard or other criterion.
Methods for Analysis
Standard methods for analysis must be selected to determine if foods
meet the requirements of microbiological standards. Methods published
by AOAC (1985), APHA (1984), the FDA (1978), or Thatcher and Clark
(1968) could be acceptable. The AOAC methods are standardized and are
accepted in courts of law in the United States.
On an international basis, the standardization of methods is more
difficult. There are different media manufacturers in different countries.
The organisms that are prevalent vary from continent to continent. Food
from a tropical country would undoubtedly contain more thermophiles
than would the food from colder climates. However, standardized meth
ods would help facilitate international trade in foods.
Whatever methods are designated, they should not require the use of
unusual and expensive equipment not found in most laboratories. The
procedure should be easy to follow by a technician and not be so com
plex that only a research scientist can master the techniques. However,
the method must give reproducible results so that the findings from dif-
ferent laboratories will agree within reasonable limits.
Methods that are applicable to many commodities or groups of com
modities are preferred to methods that apply to only one or a few foods.
Microbial Limits and Variables
There are various ways in which the limits of microbiological criteria
can be developed. One method is to make an extensive survey of the
microbiological condition of the finished product. The samples should
be obtained from several processors that operate with what are consid
ered acceptable sanitation controls. With these data, the expected level
of microorganisms in a particular product can be estimated.
A system can be used in which an inspection team visits the process
ing plant and evaluates the apparent sanitary conditions on various days.
Samples of the finished product are obtained during these visits and ana
lyzed for the microbial condition. In this way, it may be determined if
there is a correlation between sanitation and microbial condition.
The microbial limits must be achievable when good manufacturing
practices are used in processing the food. In establishing limits, there
should be a consideration of the risk associated with the presence of the
organism(s) and the number of organisms that would affect the accept-
ability of the food_
Any microbiological limit that is established by a regulatory agency
may be challenged in court. It may be difficult to prove that a certain
number of bacteria above a certain limit is a health hazard or an indica-
tion of filth and decomposition_
It is well recognized that there are variations in the microbial content
between sample units of food_ These variations must be considered when
microbial limits are applied to a food product.
The good manufacturing practices that are applied today might be
considered poor or unacceptable in a few years_ Also, the factors that are
considered as health hazards are continually changing_ To be effective,
microbiological criteria must be reevaluated and altered as experience is
gained and as technology is changed_
DECISION CRITERIA. These are part of the overall sampling plan_
However, they are used in the application of the data obtained from ana-
lyzing sample units to the microbial limits that have been established_
We can assume that the limit for the aerobic plate count (APC) is
100,000/g_ This limit is designated as m_ In sampling by variables plans,
the lot is accepted if the mean or average of the APC of the sample units
is m or less_ If the mean is greater than m, the lot is substandard. Variable
sampling requires some knowledge of the distribution of microorganisms
within the product (Thatcher and Clark 1968).
The attribute sampling plan can be used with any food that is tested.
With this system, either the APC of a sample unit exceeds m or is equal
to or less than m, the microbial limit. If ten sample units are analyzed
and the APC of eight are equal to or less than m, and two are greater
than m, what decision can be made regarding the acceptability of the lot?
The Two-Class Plan. In a two-class plan, several sample units are analyzed.
The number of sample units that must be examined from a lot of food
to satisfy the requirements of a particular sampling plan is designated as
n. If ten units are needed, then n = 10. With a two-class sample plan, a
certain number of nonacceptable or defective sample units are allowed.
The maximum allowable number of defective sample units is designated
as c. If two defective sample units are allowed of the ten that are exam-
ined, then the entire lot of a food product that has eight acceptable and
two defective units would be considered as acceptable. If there are three
defective units, the lot would be rejected.
The Three-Class Plan. In the two-class system, the sample unit is either ac-
ceptable or nonacceptable. With a three-class system, a marginal area is
included. If we keep m as 100,000, then any sample units of m or less are
still considered as acceptable. An upper limit is established which we can
assume to be 5,000,000, which is designated as M. In this example, sample
units with APC above m (100,000) and equal to or less than M (5,000,000)
are considered marginal. A product with any sample unit with an APC
over M is unacceptable. With a threeclass system, of ten sample units,
two could be between m and M, with no sample units greater than M.
Our plan can be formulated as: n = 10, c = 2, m = 10
5
, M = 5 X 10
6

For salmonellae, which are not tolerated in food, the plan would be n =
10, c = 0, m = 0. There would be other values for m, M, or c if E. coli,
coliforms, or other types of microorganisms were in the standard.
Different foods might have different types of organisms listed in the
standards, as well as different values for n, c, m, and M; these values de
pend on the type of food and the microorganisms or grou p of microorgan-
isms. The value of M should be so high that it constitutes a definite
hazard or evidence of overt spoilage. Hence, no food could be tolerated
with sample units above this level. With high potential health hazards,
the value of n would be high and c would be low. For cases in which there
is essentially no hazard and leniency is designated, n may be low and c
relatively high, or n might be 5 with a c of 3. In other words, three of five
samples could exceed the limit m without rejection of the lot.
With any sampling plan, there is a probability that acceptable prod-
ucts will be rejected and products not in compliance will be accepted.
The probability of these occurrences with various sampling plans is dis-
cussed by ICMSF (1974), Read and Baer (1974), Thatcher and Clark
(1968), Clark (1982), andJarvis and Malcolm (1986). The three-class plan
has been proposed for various foods such as chocolate (Collins-
Thompson et al. 1981), infant cereal and formula (Collins-Thompson et
al. 1980), pasta products (Rayman et al. 1981), and frozen cream-type pies
(Todd et al. 1983).
Disposition of Substandard Food
Foods that exceed the acceptable microbiological limits in a standard
are not necessarily wasted. The standards as proposed by the FDA were
for quality determination. Foods that did not qualify for the standards
were to be labeled "Below Standard in Quality-Contains Excessive Bac-
teria." It is evident that this statement on a food will decrease sales. If
there are excessive bacteria in a food, it may be subject to seizure on the
basis that it is adulterated by having been "prepared, packed, or held
under unsanitary conditions."
According to Elliot (1970), when poultry and red meat products ex-
ceed the microbiological criteria, production of further products is
halted to determine the cause of the contamination. The disposition
of the product depends upon the extent of contamination, the existence
of a hazard, presence of decomposition or adulteration, and the nature
of the microorganisms. After considering these factors, the product may
be released, sorted, reprocessed, or destroyed. If the food is not accept
able for human consumption, depending upon the contamination, it
might be fed to animals. Food should be not destroyed unnecessarily.
However, if the hazard is such that it cannot be remedied, destruction
may be the only solution.
Miscellaneous Standards
Although specifications or guidelines have been established for most
foods and food ingredients, there are very few official standards. Many
of the socalled standards in the literature are specifications, guidelines,
or suggested standards.
WATER. In the United States, the Environmental Protection Agency
develops regulations for drinking water according to the Safe Drinking
Water Act. The FDA has proposed microbiological standards for bottled
water used for drinking.
Tap Water. Standards have been established for the standard plate count
(SPC) and coliforms. The SPC limit is 500 organisms per milliliter, based
on the arithmetic average of samples collected in a calendar month.
Either the membrane filter method or the fermentation tube method
can be used to determine coliforms. For the membrane filter method, a
100ml sample is used. The arithmetic mean coliform density of all sam
pies examined per month shall not exceed 11100 m!. Per sample, the col
iform colonies shall not exceed 4/ml in more than one sample if fewer
than twenty samples are examined per month, or more than 5 percent
of the samples if more than twenty samples are examined.
For the fermentation tube method, five portions of either 10 ml or
100 ml are tested. When 10ml portions are tested, not more than ten
percent in anyone month shall show the presence of the coliform group.
The presence of coliforms in three or more 10ml portions of anyone
sample are not allowed if this occurs in either more than one sample per
month if fewer than twenty samples are examined, or more than 5 per
cent of the samples if over twenty samples are examined during the
month.
When 100ml portions are tested by the fermentation tube method,
not more than 60 percent in anyone month shall show the presence of
coliforms. The presence of coliforms in all five of the 100ml portions in
a sample shall not be allowed if this occurs in either more than one sam-
ple per month when fewer than five samples are examined per month,
or more than twenty percent of the samples when five or more are exam-
ined per month.
There are provisions for analyzing more samples if the level of organ-
isms is above the standards. Besides microorganisms, limits are set for
various chemicals. These standards are used as guidelines by many states
and cities in setting up limits for potable water.
The World Health Organization standard for drinking water includes
coliform limits. In 90 percent of the samples, there should be fewer than
one countll 00 ml, and for all samples, there should be fewer than ten
countsllOO m!.
Bottled Water. The standard for bottled water includes the coliform index.
The sample used is ten containers (sample units) of water selected from
the lot.
The coliforms can be determined by either the membrane or fermen-
tation method. For the membrane filter system, not more than one of the
sample units shall have more than four or more coliformsllOO ml, and
the arithmetic mean of the coliform density shall not exceed one col-
iformll 00 m!.
For the fermentation tube method, not more than one of the sample
units shall have an MPN of 2.2 or more coliformsllOO ml, and no analyti
cal unit shall have an MPN of 9.2 coliformsllOO m!.
MILK. Due to its widespread consumption by infants and children, and
its potential for containing health hazards, milk was the first food for
which microbiological standards were established.
The microbiological standards for milk and other dairy products are
listed in Table 13.6. The SPC of pasteurized grade A milk must not ex-
TABLE 13.6. MICROBIOLOGICAL STANDARDS FOR SOME GRADE A
DAIRY PRODUCTS
Product
Raw milk, at pick-up
Raw milk, commingled
Pasteurized milk
Pasteurized, condensed milk
Pasteurized, condensed whey
Nonfat dry milk
Dry whey
Maximum Numbr per Gram
Standard Plate
Count
100,000
300,000
20,000
30,000
30,000
30,000
30,000
Coliform Count
10
10
10
10
10
ceed 20,000 organisms per milliliter and more than ten coliforms per
milliliter. Restrictions imposed by individual dairy plants are frequently
much more rigorous than those imposed by public health requirements.
No limit is established for the SPC of fermented products, due to the
addition of a starter culture. However, there is a limit established for
coliforms.
For milk used in manufacturing, such as in making cheese, the USDA
recommended standards for a standard plate count are as follows:
Grade
1
2
3
Organisms/ml
not over 500,000
not over 3,000,000
over 3,000,000
The bacterial level in manufacturing milk is much higher than al
lowed in grade A milk.
Other countries have other standards. Quite often, a reductase test
using resazurin or methylene blue is used in place of the SPC to estimate
the bacterial quality of the milk or milk product (Cox 1970).
OYSTERS. The National Shellfish Sanitation Program established a
microbiological criterion at the wholesale level. A satisfactory sample
should not have a fecal coliform density of more than 78/100 g, with
occasional values to 230/100 g (MPN) and a standard plate count of no
more than 100,000/g, with occasional samples at 500,000/g.
Ayres (1975) stated that, for bivalve shellfish, there is no justification
for a standard based on total plate counts that often exceed 1,000,000/g.
He suggested that the relation of nonspecific bacteria growing at 20
and 37C might be useful in assessing the potential health risk of raw
shellfish.
SUGAR. This ingredient may contain spores of thermophilic bacteria
that are important in the spoilage oflowacid canned foods. In 1931, the
National Canners Association (NCA) formulated and published stan
dards for sugar for use in canning of lowacid canned foods. These stan
dards have been applied by industry, as well as federal and state labora
tories. By definition, these microbiological criteria for sugar are
specifications, since there are no official standards for sugar. However,
due to the importance of these limits, they are included in this section.
Limits have been established for total thermophilic spores, flat sour
spores, thermophilic anaerobic spores, and sulfide spoilage spores. The
sample from a lot or shipment consists of five sample units of above 225g
each taken from each of five bags or containers.
For the five sample units, there shall be a maximum of 150 total ther-
mophilic spores and an average of not more than 125 sporesllO g of
sugar. There shall be a maximum of seventy-five flat sour spores and an
average of not more than fifty sporesll 0 g of sugar. Thermophilic anaero-
bic spores shall be present in not more than three of the five sample
units and in anyone sample unit to the extent of not more than four of
six inoculated tubes. For this test, an amount of solution equivalent to
4g of sugar is distributed equally among six tubes containing liver infu-
sion broth.
The sulfide spoilage spores shall be present in not more than two of
the five sample units and not more than five spores in 10 g of any one-
sample unit.
STARCH. This ingredient can cause spoilage when added to low-acid
canned foods due to the presence of thermophilic spores. The microbial
limits for starch are the sam'e as for sugar.
PEANUTS. There are no standards for the number of microorganisms
that can be present in peanuts or peanut products. However, there is a
tolerance of aflatoxin of less than 20 p,g/g of peanuts.
REFERENCES
AOAC. 1985. Offzcial Methods of Analysis of the Association of Offzcial Analytical Chemists. 14th
ed. W. Horwitz, ed. Washington, D.C.: Association of Official Analytical Chemists.
APHA. 1984. Compendium of Methods for the Microbiological Examination of Foods. 2d ed.
M. L. Speck, ed. Washington, D.C.: The American Public Health Association.
Ayres, P. A. 1975. The quantitative bacteriology of some commercial bivalve shellfish
entering British markets.]. Hyg. Camb. 74: 431-440.
Bartram, M. T. 1967. International microbiological standards for foods.]. Milk Food Tech-
nolo 30: 349-351.
Clark, D. S. 1982. International perspectives for microbiological sampling and testing of
foods.J. Food Prot. 45: 667-671.
Collins-Thompson, D. L.; Weiss, K. F.; Riedel, G. W.; and Charbonneau, S. 1980. Micro
biological guidelines and sampling plans for dried infant cereals and powdered infant
formula from a Canadian national microbiological survey.]. Food Prot. 43: 613-616.
Collins-Thompson, D. L.; Weiss, K. F.; Riedel, G. W.; and Cushing, C. B. 1981. Survey of
and microbiological guidelines for chocolate and chocolate products in Canada. Can.
Inst. Food Sci. Technol.J. 14: 203-208.
Cox, W. A. 1970. Microbiological standards for dairy products. Chem. Ind. 1970: 223-229.
Elliot, R. P. 1970. Microbiological criteria in USDA regulatory programs for meat and
poultry.]. Milk Food Technol. 33: 173-177.
FDA. 1973. Current Levels for Natural or Unavoidable Defects in Foods for Human Use
That Present No Health Hazard. Rockville, Md.: Food and Drug Administration. De-
partment of Health, Education and Welfare.
1978. Bacteriological Analytical Manual for Foods. 5th ed. Washington, D.C.: Food and
ICMSF. 1974. Microorganisms in Foods. 2. Sampling for Microbiological Analysis: Principles and
Specific Applications. International Commission on Microbiological Specifications for
Foods. Toronto: University of Toronto Press.
Jarvis, G. A., and Malcolm, S. A. 1986. Comparison of three-class attributes sampling
plans and variables sampling plans for lot acceptance sampling in food microbiology.
I Food Prot. 49: 724-728.
Majorack, F. C. 1982. FDA's industry quality assurance assistance program. Food Technol.
36(6): 87-88, 95.
NAS. 1964. An Evaluation of Public Health Hazards from Microbiological Contamina-
tion of Foods. National Research Council Publication 1195. Washington, D.C.: Na
tional Academy of Sciences.
Payne, W. L.; Duran, A. P.; Lanier, J. M.; Schwab, A. H.; Read, R. B., Jr.; Wentz, B. A.; and
Banard, R.J. 1983. Microbiological quality of cocoa powder, dry instant chocolate
drink mix, dry nondairy coffee creamer and frozen nondairy topping obtained at
retail markets. I Food Prot. 46: 733-736.
Rayman, M. K.; Weiss, K. F.; Riedel, G. W.; Charbonneau, S.; and Jarvis, G. A. 1981.
Microbiological quality of pasta products sold in Canada. I Food Prot. 44: 746-749.
Read, R. B., Jr., and Baer, E. F. 1974. Role of the regulatory in setting microbiological
quality standards. Food Technol. 28(10): 42,44,46.
Schwab, A. H.; Swartzentruber, A.; Wentz, B. A.; and Read, R. B.,Jr. 1982. Microbiological
quality of drymilk mixes and milk substitute infant formulas. Appl. Environ. Microbiol.
43: 389-391.
Sloan, A. E. 1976. Educators, regulators, industry discuss product development-food
safety problems. Food Prod. Develop. 10(6): 66, 68, 72.
Swartzentruber, A.; Payne, W. L.; Wentz, B. A.; Barnard, R. J.; and Read, R. B., Jr. 1982.
Microbiological quality of macaroni and noodle products obtained at retail markets.
Swartzentruber, A.; Schwab, A. H.; Wentz, B. A.; Duran, A. P.; and Read, R. B., Jr. 1984.
Microbiological quality of biscuit dough, snack cakes, and soy protein meat extender.
I Food Prot. 47: 467-470.
Thatcher, F. S., and Clark, D. S. 1968. Microorganisms in Foods: Their Significance and Methods
of Enumeration. Toronto: University of Toronto Press.
Todd, E. C. D.;Jarvis, G. A.; Weiss, K. F.; Riedel, G. W.; and Charbonneau, S. 1983. Micro
biological quality of frozen cream-type pies sold in Canada. I Food Prot. 46: 34-40.
Wentz, B. A.; Duran, A. P.; Swartzentruber, A.; Schwab, A. H.; and Read, R. B., Jr.
1983. Microbiological quality of fresh blue crabmeat, clams and oysters. I Food Prot.
46: 978-981.
Index
Absidia, 401
Acetaldehyde, 444, 447-449, 462, 494,
615
preservative, 607
use in food, 607
in yogurt, 449
Acetic acid, 128,437-438,482
metabolic product, 398, 400, 408,
448, 462
microbial inhibitor, 145, 440, 537,
575, 599-601, 604, 607, 627
vinegar, 433, 468-469
Acetobacter, 55, 115, 138,464,482,606
spoilage, 55, 407-409, 461
vinegar, 434, 468-469
Acetoin, 61, 398, 400, 532
Acetylcholine, 224
Acetylmethylcarbinol (AMC). See
Acetoin
Acinetobacter, 53, 55-56, 107,401
A. calcoaceticus, 489
in food products, 51, 413-415, 418-
419, 422-426, 551, 663
radiation resistance, 693, 695-696
sources, 166, 168
Actinomucor
A. elegans, 434
Actinomyces, 584
Adenosine triphosphate (ATP), 146,
617
microbial analysis, 20, 38-39
Adenovirus, 90, 695
Adenyl cyclase, 253, 279, 288
Adulteration, 725-726
Aerobic plate count (APC), 11, 19-20,
23-28, 186, 569
criterion, 729-730
index of sanitation, 532
indicator, 372
precision, 41
in specifications, 736-738
in standards, 739, 743
Aerococcus, 51, 60, 62
A. viridans, 62
Aeromonas, 57, 60, 107, 138,482
A. hydrophilia, 608, 629, 694
A. liquifaciens, 407
enteric illness, 298
in foods, 51-52, 167-168,413-414,
418,420,422-423,551
lipolytic, 401
sources, 60
Aerosolization, 92, 149, 171-172, 584
Aflatoxins, 77, 186,300-306,312-313,
628
allowable limits, 748
biological effects, 301-304,310-311
control, 131, 311, 313, 316-320,
606,613,701
determination, 313-316
in foods, 304-306
formulae, 302
microbial inhibitor, 145
Agaricus
A. bisporus, 145
Aging, 452, 464
Alcaligenes, 51-53, 56, 107,489
in food spoilage, 401, 413-415,
418-421,423-426,452
lipolytic, 401
sources, 51-53, 56, 166, 168, 178,
551,661
Alcohol, 308, 607, 659, 676
metabolic product, 49, 61, 393, 400
precursor of acetic acid, 467-468
production, 306, 458-466, 538
751
752 INDEX
Ale, 481
Algae, 4, 70, 170, 230, 395, 484, 486,
490-491
Alimentary toxic aleukia (ATA), 300,
309
Allicin, 131
Alpha particles, 687
Alpha tocopherol, 623
Alternaria, 75-76, 401, 607
A. alternata, 76, 301
A. tenuissima, 301,407
food spoilage, 401, 406, 410, 415,
421
growth conditions, 84
source, 52, 72, 172
Alteromonas, 53, 56
A. putrefaciens, 412, 427
food spoilage, 412, 414-415, 418,
422-423,426
Amelioration, 465
Amylase, 103, 396, 459, 466, 475-477,
479
Anthocyanin pigment, 129-130
Anthrax, 65
Antibiotic(s), 3, 51, 71-72, 81, 84, 91,
126,129,144-146,148,150,
179,212,253,270,276,289,
323,444,469,481,493,578,
615-617,632
tolerance (WHO), 617
Antibodies, 127-128, 213, 216, 268
monoclonal, 50, 216, 235, 284
Antigens, 7, 50, 241, 264
flagellar (H), 50, 264-265, 292
of salmonellae, 264-265
somatic (0), 264-265, 292
virulence (Vi), 265
Antimycotic agents, 587-588, 611
Antioxidants, 481, 560, 590, 593, 608,
623, 627
Arizona, 57, 661, 673
Artemia saline, 316
Arthrobacter, 68-69,107,476,478
A. globiformis, 479
in food, 69, 166, 174,415, 425, 663
Ascaris, 8
Ascomycetes, 74-75, 83, 85-87
Ascorbic acid, 601
antibacterial action, 601, 611
in cured meats, 618, 623
Asepsis, 6, 505
Aseptic packaging, 513, 662, 665, 690,
706
Ashbya gossypii, 483
Aspergillus, 76-78, 107-108, 138,611,
673
A. candidus, 313
A. chevaliere, 313
A. clavatus, 301
aflatoxin, 77, 131, 145, 186, 300-
306,310-320,606,613,628,
701, 748
A.flavus, 77,107,145,153,186,301,
304,306,311,313,316-317,
434, 608, 628-629, 682, 694
A. fumigatus, 77, 138
A. glaucus, 107
A. halophilicus, 107
A. japonicus, 471, 480
A. nidulans, 301, 476, 709
A. niger, 77, 107, 115, 313, 319, 407,
409, 472, 475, 477-478, 480,
482-483,600
A. ochraceus, 301, 308, 311, 469-470,
480, 700
A. oryzae, 76,115,434,469-470,475-478,480
A. parasiticus, 77, 131, 153, 301, 304,
311, 313, 316-317, 606, 608,
613
A. saitoi, 480
A. soyae, 470, 480
A. tenuis, 301
A. versicolor, 301, 309
A. wentii, 480
food spoilage, 52, 401, 409-411,
415,419,427
production of citric acid, 482
production of soy sauce, 76, 470
source of enzymes, 476, 478-480
sources of, 72, 76-77, 172
Assay, 62, 68, 85, 313, 315-316, 478,
492-494
amino acids, 492-493
vitamins, 492-493
Aureobasidium, 52
A. pullulans, 415
Autolyzed yeast, 87
Available chlorine, 237
Avidin, 124-125
Azobacter chroococcum, 567
Bacillus, 51-52, 62, 65, 107, 131, 140,
513, 532, 584, 605,661-662,
709-710
B. acidocaldarius, 65
B. alcalophilus, 65
B. anthracis, 65
B. caldolyticus, 136
B. cereus, 64-65, 107, 125, 131, 138,
196, 198, 285-287, 400, 522,
610,620,629,631,694,710
B. coagulans, 65, 115,409-410,418,
428, 664, 686
B. licheniformis, 143,407,417,480,
674,710
B. megaterium, 125,405,631
B. stearothermophilus, 64-65, 409,
418,428,476,671,673-675,
682-683,686,694,700
B. subtilis, 63, 65,115,125,144-145,
149, 153, 405, 409, 411, 429,
434,475-476,480,522,603,
653, 707
B. thermoacidurans, 664
chlorine resistance, 521-522
foodborne illness, 285-287
microbial interactions, 144-145
source of amino acids, 482
source of enzymes, 475-476, 480
source of vitamins, 483
sources, 65,166-168,171,178,180
spoilage of food, 65, 140, 401, 409,
414,416,418,423-424,427-
428
Bacillus cereus foodborne illness, 285-
287
Bacon, 12, 209,415,443,601, 619-
620, 622-625, 694, 697
Bactericidal action, 127, 144,375,545,
INDEX 753
595,600,604,607,611,658,
707-708
chlorine, 519-523
iodophors, 519, 523-524
quaternary ammonium com
pounds, 519,524-525
Bacteriocin, 50, 61, 145-146,246, 632
Bacteriophage, 49, 89, 91-92, 146,
213-214,230,266,436,444,
452, 519-520, 523-526
Bacteriostatic, 127, 144, 519, 545, 595,
600, 604, 631
Bacteroides, 60, 121, 176, 180
B. fragilis, 121
Baked goods/products, 458, 466-467,
476, 565,706
Bankia setacea, 316
Bdellovibrio, 146
Beans, 12, 85, 117, 167,278,305,308-
309,381,434,582
Bee 8, 12, 14, 28, 34, 55-56, 74, 88,
112-118,123,141,175-176,188,
1 9 ~ 199, 2 ~ 2 2 ~ 241, 251-
252, 260, 296, 381, 394, 537,
547-550, 588, 606, 628, 630-
631, 656-657, 690, 696-699,
701, 736
Beer, 3, 55,62,86, 305,412,434,458-
462,477,481,494,539,576,
600,617,621,659
ale, 459, 461, 481
chill proofing, 461, 476, 480
lager, 459, 461
pasteurization of, 664
Benomyl, 607
Benzoic acid, 129-130,601-603
Benzopyrene, 625
Beta particles, 687
Bifidobacterium, 180
Biological value (BV), 487-489, 491
Biotin, 125, 493
Blanching, 147, 185,442,547,560,
565, 569,571,576,583,587,656, 665-66!
Bone darkening, 563
Botrytis, 52, 77
B. allii, 409
754 INDEX
Botrytis (continued)
B. cinerea, 77, 107, 115, 138, 406-
407,409
Botulinum toxin. See Toxins, botul
inurn
Botulism, 196-197,202,219-237,239,
250,618,663
infant, 219, 223
wound, 219, 222-223
Bound water, 105
Bread, 65, 74-75, 81,111,117,305,
404,459,466-467,475,547,
594,600-601,604-605,628,
702
Brettanomyces, 87, 411-412
Brevibacterium, 51, 68, 138,482
B. linens, 69, 145,434,456
Brine shrimp, aflatoxin assay, 316
Brochothrix, 66, 68, 153,415, 630, 664
B. thermosphacta, 68, 549, 629
Brucella, 53, 56
B. abortus, 56
B. melitensis, 56
B. suis, 56
Brucellosis, 56, 296, 507, 661
Budding of yeasts, 73, 83-88, 487
Bud fission, 99
Buffer, 116, 120, 620,674,693-695
Burri slant method, 20, 29
Burst size, phage, 92
Butter, 12, 61, 88-89, 127, 394-395,
401,426-428,449,479
Butylated hydroxyanisole (BHA), 218,
608
Butylated hydroxy toluene (BHT), 218,
608
Butyric acid, 128, 401, 455
Byssochlamys, 74-75, 684
B.fulva, 74, 123,407-409,429,671
B. nivea, 74, 301, 407-408
Cabbage, 49, 117, 129, 135, 167, 174,
278,303,393,408,434,436-
437,553,555
Cadaverine, 404
Campylobacter, 51-52,292-296
C. jejuni, 52, 196, 198, 292-296, 600-
601,694,699,706
Canduw, 87-89, 476, 482-483, 487
C. albicans, 129, 131, 707, 710
C. lipolytica, 138, 479
C. mycoderma, 88
C. utilis, 88, 476, 479, 490, 567
C. vini, 88, 407
in food, 88
growth conditions, 138
lipase of, 401, 428
sources, 87-88, 174
spoilage of food, 88, 407-409, 411-
412, 426
Canned food, 64-65,186,200,237,
512,526,536,557,617,671,
683
spoilage, 66, 166, 428-429
Canning, 6, 656-686, 692
Capsid, 89, 167, 631
Carbohydrases,472
Carbohydrates, 2, 37-38, 52, 104-105,
114,180,206,212,233,243,
293, 312, 374-375,
394-400,404-405,436,440,469,471,
485,490,675-676,702
Carbon dioxide (C0
2
), 454, 458, 464-
465,546,627-630
Carcinogenic/carcinogens, 7, 302-
303, 307-310, 318, 462, 485,
523,528,592,608-609,621-
622,625,629,702-704
Case hardening, 113, 579-580
Casein, 260, 273, 449, 481
Catalase, 54, 57-58, 60, 66, 103, 145,
148,151,286,293,295,384,
476-478,610,708
Cathode rays, 687
Cellulase, 82, 396, 476-477
Cellulomonas, 489
Cellulose, 395-396, 459, 476-477, 485,
489
Centers for Disease Control, 8, 198,
200-201,250,274,292
Centrifugation, 6, 38, 91, 323, 513,
537-538, 570
Cephalosporium, 309
Chaetomium, 52
C. olivaceum, 145
C. thermophile, 477
Champagne, 465, 539
Cheese, 3, 16,49,61,65,69,72,75,77,
84-85,89,111,117,123,127,
145, 207, 213, 215, 218, 260,
278, 280, 282, 287, 296, 298,
305-306, 380-381, 405, 426-
427,433-434,455-456,476,
479,481,483,548,551-552,
604-605,607,610-611,616,
624, 741, 747
blue, 308-309, 457
brick,306
Camembert, 306, 405, 434, 458
cheddar, 122,426,452-453
cottage, 88, 306, 426, 449, 451-452,
551
Gorgonzola, 81, 308
limburger, 69, 306, 434, 456, 458
Roquefort, 81, 308, 405, 434, 457
Swiss, 69, 122, 426, 454-456, 604
Chelating agents, 517-518, 609
Chemical preservatives, 6, 545, 589-
627
Chick embryo bioassay, 316
Chinese hamster, ovary cells of, 254,
275, 283-284, 294
Chiorella, 491
Chlorination, 269, 272, 517, 519-523,
530
Chlorine, 61, 66, 151, 219, 237, 272,
385, 440, 514, 553, 608-609,
710
breakpoint, 520-521
dioxide, 318
Chlorohydrins, 654
Cholera, 198, 254, 294
toxin, 254, 279, 284, 288, 294
Chromatography, 66, 314-316, 378
Chromobacterium, 167,401,418,426
Cider, 55, 85
Ciguatoxin, 196, 200
Citeromyces, 85
Citric acid, 61, 77, 603-604
Citrinin, 300-301, 306-307, 315
INDEX 755
Citrobacter, 57-58, 107, 167, 175, 178,
265,280,298,378,418-421
C. freundii, 372
Cladosporium, 52, 72, 77,138,172,401,
414,419,428,607
C. carpophilum, 77
C. cladosporioides, 415
C. herbarum, 415
Claviceps, 75, 482
C. purpurea, 75, 299, 301
Cleaning, 271, 507, 511, 515-516, 535,
571
agents, 150, 516-517
Clean-in-Place (CIP), 515
Clostridium, 62, 65-66, 138, 140, 149,
166,180,200,219-237,267,
269,407,411,414,428,521-
522,532,584,662
C. botulinum, 64, 66, 107, 115, 121,
123, 131, 138, 143-145, 153,
166,196,198,202,219-238,
386,509,601,608,617-619,
623-624,626,629-630,651,
656, 664, 666, 675, 681-684,
694, 696-700, 703, 709, 728,
739-740
C. butyricum, 121, 409
C. difficile, 298-299
C. pasteurianum, 409, 664
C. histolyticum, 121
C. perfringens, 51, 107, 115, 121, 138,
144,167,196,198,200,238-
250, 287, 386, 510,589, 608,
657,694,699,739-740
C. putrefaciens, 138
C. sordellii, 299
C. sporogenes, 63, 115, 121, 149,409,
414,426,683
C. thermosaccharolyticum, 138, 409,
428, 683-684
C. tyrobutyricum, 426-427
enterotoxin, 241-243, 285,298-299
Coagulase, 61, 206, 208, 211-213
Coccobacilli, 661
Codex Alimentarium Commission,
727, 731-732
Coenzymes, 103-104, 597
Cold, effects of, 149
756 INDEX
Cold shock, 149, 546-547
Cold storage, 6, 295, 311, 317
Colicin, 146, 278
Coliform(s), 19, 51, 57-58,174,176-
177,180,281,371-379,382-383,
411,426,461-462,472,509-
510, 525, 532, 551, 584, 600,
736-740,745-747
detection, 373-375
fecal, 168, 510, 584, 739, 747
IMVIC tests, 395, 398
significance, 375-376
Colletotrichum, 77, 407, 409
C. circinans, 77,
C. musae, 143
C. phomoides, 77
Colony forming units (CFU), 24-26,
282
Commensalism, 142, 449
Competitive exclusion, 270, 296
Conalbumin, 124-127,661
Controlled atmospheres, 553, 627-
631
Control of microorganisms, 247-250,
316-320, 505-540, 545-632,
651-711
Convection of heat, 558, 655, 658, 667,
670
Cooking, 6, 147,205,219,238,247,
319,323-324,376,378,451,
533,536,615,622,656-657,
662
Corynebacterium, 51-52, 68, 107, 166,
168,177-178,401,409,414-
415,422-423,482
C. rubrum, 319
Cottage cheese, 88, 306, 426, 449, 451-
452, 551
Coumarin, 302
Counterimmunoelectrophoresis, 246,
324
Coxiella, 70
C. burnetii, 70, 296, 661
Coxsackievirus, 90, 322-323, 657, 695
Crabmeat, 662-663
Critical control points (CCP), 512
Cross contamination, 533, 536, 740
Cryogenic freezing, 552
Cryoinjury of cells, 565
Cryoprotective agents, 567-568
Cryptococcus, 88
C. neoformans, 131
Cucumber, 129, 174-175,434,438,
440-441,443,537
Culture, 218, 444-445, 448, 452, 747
dried, 443, 446
frozen, 443, 446
Cultured buttermilk, 445-449
Cured meat, 51, 59, Ill, 187
Curing, 244, 454-455, 546, 692
Cyclic adenosine monophosphate
(CAMP), 254, 279, 288
Cyclic guanosine monophosphate,
280
Cytophaga, 168,418-419,423,425
Dairy products, 56, 60-61, 69, 77, 86-
87,92,126-127,169,208,227-
228,251,282,296,315,324,
436,444-458,476,482,621,
632, 659
spoilage, 56, 76, 79, 82, 88
Daphnia, 316
Dark cutting beef, 118
Dark repair, UV-treated cells, 151
Debaryomyces, 85, 107, 385, 402-404,
408-409,414,426,487
D_ hansenii, 85
Decimal reduction time (D), 219, 236,
248,273,292,296,317,600,
606,657,659,662,674,677,
680-685, 693-695, 697, 699-
701
Dehydroacetic acid, 600
Deinococcus, 693,706
D_ radiodurans, 694-695, 706-707,
710
Dekkera, 85
Delay ripening, of fruit, 691-692
Deoxycorticosterone, 129
Deoxynivalenol, 309-310, 315, 320
Deoxyribonucleic acid (DNA), 50, 64,
84-85, 89-90, 127, 146, 149,
INDEX 757
151,209,228,277-278,285,293, 394,412,478,508,512,538,
307,309-310,412,521-522, 548,551,564,570,574,577-
546,565-566,584,596,609- 578,583,585,628,659-661,
610,615,653,671,689,693, 732,734-735,738,741
701,707-708 pasteurization, 252, 677
hybridization, 50, 268 spoilage, 56, 126, 134,420-421
probe, 268 thermostabilization, 551
Desulfotomaculum, 62, 66, 428,532 white(s), 116-117, 124-126, 128,
66,409,428-429 471-472,479,659,661,665
Deuteromycetes (fungi imperfecti), yolk, 117, 135, 149,212,245,479,
74-83, 85, 87-89 659
Dextran, 404-405, 408 Eh, 120-123,208
Diacetyl, 37, 61,400, 444-446, Electricity, antimicrobial effects, 711
448, 461, 532, 601, 626 Electronic counts, 20-21, 23
Diarrhea, 181,199,201-202,204,239-240, Electrophoresis, 66, 323
242,250,275,277-279,281-
282, 285, 287-290, 295, 299-
300,303,308-309,325-327,
376,396,653
Diastase, 466
Diethylstilbestrol, 129
Differential media, 13, 30, 50, 263
Dimethylnitrosamine, 621
Diphenyl, 609
Dipicolinic acid, 64, 522, 672
DiploiOO, 407
Direct microscopic count (DMC), 20-
23, 25, 734, 737
Dried food(s), 11-12, 111, 113, 149-
150, 311, 705, 739
Drying of foods, 6, 54, 177, 179, 260,
375,539,570-589,625,651,
655-656,665
drum, 149, 574, 576
freeze, 574, 578-579, 581
hot air, 574-582
spray, 147, 149,260, 574, 577, 580
vacuum, 574, 576
Duodenum, 179-180, 243
Echovirus, 90, 322, 695
Edwardsiella, 57, 59,280
E. tarOO, 59
Egg(s), 21, 23-24, 38, 51, 82, Ill, 132-
135, 139, 177, 180, 251, 260-
261,290,306,310,320-321,
Electrostatic precipitation, 513
Elenolic acid, 131
Endomyces, 401
Endomycetales, 83
Endomycopsis, 85
Endothia, 476
E. parasitica, 481
Endotoxins, 208
Enrichment, 50, 261-264, 278, 292,
295,377,467
Enterobacter, 51, 57-58,107,144,175,
178,180,265,280,298,376,378,401,
407,413-415,418-419,482,
549,570
E. aero genes, 144,372
E. agglomerans, 383
E. cloacae, 372, 383, 694
Enterobacteriaceae, 57-60, l46, 168,
296,372,382-383,425,551,
698, 739
Enterococci, 61-62, 176, 180, 372,
383-386,532,584,619,698
E. jaecium, 429
Enteropathogenic E. coli (EEC), 57,
278,281-282,739
Enterotoxigenic E. coli (ETEC), 57,
199,258,279,281-282,739
Enterotoxins, 267-268, 275, 280-281,
283-284,286,288,290,298
cholera, 279
C. perfringens, 238-239, 241-243,
285
758 INDEX
Enterotoxins (continued)
staphylococcus, 61, 108, 153, 198,204-
205,207-217,220,237,246,
266-267, 285, 386, 588, 656,
695, 734, 740
Enteroviruses, 90-91, 166, 168, 320,
322, 387, 601
Entrapment, enzymes, 473-474
Enumeration, 71-72, 92,101,139-
140, 147-148, 150-152,211,
233, 244-246, 261-269, 282-
286, 292, 372-375, 383
Environmental Protection Agency
(EPA), 731, 745
Enzyme immunoassay, 267-268, 323
Enzyme-linked immunosorbent assay
(ELISA), 50, 91, 215-216, 235,
241, 246, 268, 284, 315, 324,
494
Enzyme(s) systems, 7, 36, 49-51, 54-
55, 64-66, 70, 72-77, 81, 87-
88,101-104,114,117,120,
124-125,127,129,131,140,
144, 148, 151, 167, 179, 211,
213, 215, 237, 321, 393-397,
401-402,405,408,433,435,
440,451-453,459,461,466,
472-481, 487-488, 493-494,
505,521,527,539,557,564,571,
573, 576, 585, 590, 596-597,
599, 608-610, 615-616, 652,
655, 661-662, 664-665, 690-
691, 697, 700-701, 703, 707-
708
glucose oxidase, 81
invertase, 87-88
pectic, 395-397, 408
pectinolytic, 54, 74, 77
proteases, 70, 73
proteolytic, 65, 77, 452, 461, 517,
564,697
rennet, 75, 77, 451, 453
Epicoccum, 52
Epidemiology, 92, 196, 198-199,202,
213-214, 225, 244-245, 265,
268, 278
Equilibrium relative humidity (ERH),
106, 110, 112
Equipment, 6, 13, 58, 88, 165,169,173,
181-187,200,210,217,257-
258,271,376,384,422,425,
507,511-512,514-525,527,
530, 532-535, 706
Eremothecium ashby ii, 483
Erwinia, 51, 57-58, 131,407,482
E carotovora, 58, 115,409-410
E herbicola, 383
E rhapontici, 411
Escherichia, 57-58, 167, 175, 178,294,
407,413,419,462,482-483,583,
693
E coli, 57, 103, 107, 115, 138, 144,
146,148-152,168,178,199,
254,258, 265-266, 270,265, 278-285,
294,297,371-372,374,376-383,
385-387, 400, 405, 427, 472,
489, 493, 510, 532, 566, 589,
604,608,630-631,667,672-
673,682,694-695,706-711,
736, 738-740
E coli enteritis, 278-285
Ethyl alcohoL See alcohol
Ethylenediaminetetraacetic acid
(EDTA), 609-610, 619, 627
Ethylene oxide, 539, 652, 654-655,
710
Eugenol, 132, 312
Euglena gracilis, 493
Excision repair, 151,707
Extract release volume (ERV), 38
Fat(s), 2, 4, 37, 103-105, 166, 180, 394-
395,398-402, 404, 413, 467,
516-517,605,608,622,702
rancidity, 514
Fatty acids, 126-127, 145,259,398,
402,428,452,457,476,479,
599, 632
Fecal coliforms, 376-379, 382
Fecal streptococci, 51,174,383-386,
584
Fermentation, 3, 6, 8, 34, 49, 52, 57,
62,69,73,84-85,87,101,114,
116, 118, 141,233,243,256,
277,308,385,398-399,404,
433,454,480,485,615,632
Fermented foods, 13, 62, 374, 435,
437, 445, 467, 604. See also Spe
cific foods
Fertilizer, microbial source, 165, 173-
174,252,278
Filtration, 6, 20, 28, 38, 91, 323, 466,
489,514,537-540,570,741
Fish, 4-5, 12,40,60, 128, 167-168,
175-176,185,199,206,227-
228, 230-232, 235-236, 258,
282,289-290,305,380,394,
402,404,412,421-425,434,
469,479,494,548,552,563-
564,575,609,615,624,626,
629-630, 654, 659, 663, 690,
692,694,698-699,735,741
microbial source, 57, 69
pH, 117, 119
salted,605
spoilage, 56,60,613
water activity, III
Fishery products, 51, 60, 168, 185,
187,200,228,241
Flat sour spoilage, 428
Flavobacterium, 51-53, 56-57, 107, 143,
166,168,171,401,413-414,
418-419,423,425-427,482,
570
F. breve, 143
Flipper, spoilage of canned food, 685
Flour, 65, 72, 111, 186,286,305,467,
526,604,675
Fluidized bed, 547,559,574-576,578
Fluming, 169, 186
Fluorescent antibody (FA), 267, 378
Food additives, 589-590
Food and Drug Administration
(FDA), 4, 16, 212, 258, 268,
310,314,316,319-320,372,
425,509-512,535,590,601,
604, 607, 609-610, 614,618,
INDEX 759
622-623,685,690-692,702,
728-734,741,744
activities, 728-729
Foodborne illness, 8, 54, 57-59, 66,
147, 165, 170, 174-175, 180,
183,195-327,371,385,505,
509-510, 527, 529, 533-535,
546, 586, 600, 686, 703, 705,
728,736,740-741
Food, Drug, and Cosmetic Act (1968),
4,269,589,726,728,731-732
Formaldehyde, 626, 654-655
Freezantspray, 559
Freeze drying, 92, 149-150,435, 574-
575, 578-579, 581
Freezer burn, 563
Freezing, 1, 6, 17, 62-63, 92, 110, 136,
149,236,261,375,383,386,
422,435,510,531,536-537,
547,557-571,576,583,651,
655,659,665,692,696-698,
704
Frozen foods, 11-12, 54,58,62,381,
512,739,741
Fruit, 52, 55, 72-73, 77, 84, 89, 116,
123, 131, 135, 167-168, 181-
182,185-186,199,206,227-
228,258,260,308,377,393-
394,406-408,420,434,436,
468,508-509,520,526-527,
534,538,548,553-554,564-
565, 571, 574, 583, 594, 600,
606-607,614-616,627-628,
653-654,656,665,677,689-
692,701,704,731,734
dried, III
juice, 39-40, 88-89, lll, 131,476,
480,539,578,602,659
pasteurization, 664
spoilage, 52, 55, 74
pH of, 117, 119
spoilage, 55,74, 78,81,85,87-88
water activity of, III
Fungal illnesses, 299-320
Fungicidal, 607
Fungicides, 595, 607, 614
Fungistatic, 595, 602, 604
760 INDEX
Fusariotoxin T-2, 145,301,309
Fusarium, 52,77-78, 107, 145, 172,
309-310,401,407,409,411
F. culmorum, 301
F. graminearum, 301, 490
F. moniloforme, 78
F. nivale, 301
F. roseum, 301
F. solani, 78, 301
F. tricinctum, 301, 311
F value, 683-685
Gamma ray, irradiation, 54, 151, 248,
274,296,317,539,653,687-
691, 696
Gaping of frozen fish, 563
Garlic, 12, 131, 149, 577, 582, 691
Gaseous atmosphere, 141-142
Gas treatments, 651-655
ethylene oxide, 652-654
propylene oxide, 653-654
Gastroenteritis, 52, 58-60, 91-92, 168,
179,242,248-249,251-252,
259, 279, 282, 293, 297-298,
320
Gastrointestinal tract, 196, 223, 283,
489,491-492
Gelatinase, 263
Gene probe, 283-285, 288
Generally recognized as safe (GRAS),
590,601,603-605,608,614,
623
Generation time, 101-102, 238
Genetic engineering, 435, 463
Geotrichum, 78-80, 84, 401, 427-428,
608
G. candidum, 79, 387, 407, 456
Germicide, 513, 519, 523-524
Giardia, 196, 199
Gibberella zeae, 301
Gloeosporium, 407, 607
Gluconobacter, 53, 55, 407-408, 411,
434, 461
G. oxydans, 55, 115
Glucose, isomerase, 70, 476, 478
oxidase, 472, 476-479, 571
Glutaraldehyde, 626
Glycogen, 38, 117
Glycollytic enzymes, 482
Good manufacturing practices
(GMPS), 529, 728, 734, 736,
742-743
Grain, 72-73, 135, 299-300, 305-306,
308, 310, 314, 317, 411, 459,
576,690,692
microorganisms in, 52, 58, 77, 80-
81
moldy, 171
rice, 78, 89, 111, 174, 286
Grape(s), 77, 85, 117, 174, 462-463,
570, 574-575, 598
juice, 393, 465
Guanylate cyclase, 280, 297
Guidelines, 319, 733-734, 740, 745-
746
Hafnia, 412, 414-415
Halobacteriaceae, 53, 55
Halobacterium, 53, 55, 422, 612-613
Halococcus, 53, 55, 422, 612-613
Halophiles/halophilic, 55, 88, 106-
111, 612-
613
Halotolerant, 106-107, 111,612
Ham, 12, 82, 84, 199, 206, 260, 415-
416, 601, 606, 610, 618, 654,
664, 694
Hanseniaspora, 85, 407-409
Hansenula, 85-86, 115, 138, 174, 407-
409,443,471,482,487
Hazard analysis (HA), 512
Hazard analysis-critical control points
(HACCP), 512, 534
Heat, 63, 147-149, 237, 261, 272, 655-
686, 698, 741
exchanger, plate, 660
resistance, 54, 64, 108, 238, 428,
658, 670-678
sterilization, 651
systems, 656-669
treatments, 205, 235
Helminthosporium, 172
Hemagglutination inhibition, 215,
324
Hemispora stellata, 422
Hemolysins, 241
Hepatic dysfunction, 341
Hepatitis, 196, 303
infectious (A), 90, 320, 324-325
serum (B), 320, 324
Heterofermentative, 67, 436
High temperature short time (HTST),
pasteurization of milk, 662,
665-666
Homofermentative, 67, 436
Honey, 84, 87,111,231,394,411,476
Hops, 459-460, 462
Human, 214, 217, 222, 243-244, 248-
249,254,257-259,277-278,
282,375,378,380,594
microbial source, 56, 58, 60-61, 88,
6 ~ 171, 176-181, 8 ~ 533
Humicola, 477
Hydrocooling, 169, 547, 553
Hydrogenomonas, 489
Hydrogen peroxide, 103, 127, 144-
145,148,151,318,476-478,
597, 604, 610, 659, 661, 677,
710
Hygiene, 181, 197,200-201,217,219,
243, 271, 274, 276, 278, 285,
326, 529, 531-532, 535, 594
Hyphomicrobium, 489
Ice, 169,412, 422,425, 509, 527, 553,
557, 567, 579, 629
Idli,457
Ileum, 179, 205, 243, 290-291, 294
Immunoassay, 50,266-268, 315
Immunofluorescent tests, 215
Immunoperoxidase, 324
Impedance, 20, 39-40, 375, 378
Imvic tests for coliforms, 378, 380
Incidental additives, 594
Index case, of shigellosis, 275
Indicator organisms, 371-387, 510
Infectious hepatitis, 90, 320
Influenza viruses, 354
INDEX 761
Ingredients, 11,65,169-170,182,201,
218,261,277,286,326,507,
514, 526, 532, 571-572, 728,
745
microbial sources, 165, 186-187
Insects, 135, 207, 257, 393, 505, 508,
511,526,689-690,692,703,
705
microbial sources, 88
Interaction of growth factors, 152
Intermediate moisture (1M) foods,
113, 587, 611, 654
International Commission of Micro-
biological Specifications for
Foods (ICMSF), 727
Intestinal tract, 90, 230, 235, 247, 259,
282, 284, 290, 294, 373, 379,
381, 385, 387, 508-509, 616
microbial source, 56-60, 69, 88,
175-176,179-180,257
Intestinal viruses, 90-91
Invertase, 103,476,479
lodophors, 61, 519, 523-524
Ionizing radiation, 64, 623, 651, 686-
706
Isohumulone, 461-462
Jejunum, 179, 205, 243, 294
Kanagawa phenomenon, V. parahaemo-
lyticus, 290-291
Keratoconjunctivitis test, 278-279,
283
Klebsiella, 51, 57-58, 107, 168, 171,
178,280,298,373,376,378,
407,426,462,472,482
K. aerogenes, 566
K. pneumoniae, 372, 383
Kloeckera, 85, 87,174,407-409,414,
487
Kluyvera, 298
Kluyveromyces, 83, 86, 115,476,487
K. lac tis, 86, 457
K. marxianus, 476, 478
Koji,470
762 INDEX
Kojic acid, 314
Kurthia, 66,68
Lactase, 477
Lactenin, 126
Lactic acid bacteria, 461-462, 466,
549,615,632,664
Lactobacillus, 51-52, 66-68, 107, 114-
115,131,138,143-144,176,
180, 401, 406-409, 411-415,
417,422-423,427,434,436,
443,445,454,462,465,471,
532,570,610,617,619,630,
664,698
L acidophilus, 115, 145, 180,444-
445,584,610
L brevis, 67, 407, 437, 439-440
L bulgaricus, 67,427,444-445,449,
454,457
L casei, 445, 493
L. delbruckii, 482
Lfermentum, 610, 674
L jructivorans, 428
L helveticus, 427, 445, 493
L lactis, 445
L leichmannii, 493
L plantarum, 68, 115, 131-132,437,
439-441,493,619-620,694
L sake, 698
L sanfrancisco, 457
L viridescens, 414, 417, 493, 664
Lactoferrin, inhibitor in milk, 126-
127
Lactoperoxidase, 126-127
Laminaria, 491
Laser energy, 709-710
Latent-zone chilling, 547
LD5o, 301, 609
Leavening, 457, 466
agents, 594
Lecithinase, 233
Legionella pneumophila, 143
Lesions of cells, 147, 316, 672
Leuconostoc, 51, 60, 62, 107, 131, 138,
143,406-408,411,414-415,
417,426,434,436,444-445,
465, 532
L citrovorum, 145
L cremoris, 62, 115,400,444,447,
452
L dextranium, 62, 405, 444
L mesenteroides, 62, 400, 405, 408,
411,437,439-440,444,493
L oenos, 115, 407-408, 465
Ligated intestinal loop, 243, 246, 283
Lignin, 166,483,625
Limonoate dehydrogenase, 479
Limulus amebocyte lysate, 20, 40
Lipase, 103, 233-234, 242, 400-401,
405,427,476,479
Lipids, 241, 398-402, 458, 573, 677-
678
Liquid immersion freezing, 559-560
Listeria, 66, 297-298
L monocytogenes, 198, 297-298
Listeriosis, 297-298
Little plate method, 20, 29-30
Liver cancer, 304, 309
Luciferase, 38-39
Luciferin, 38-39
Luteskyrin, 300-301, 307, 315
Lyophilization, 149
Lysergic acid, 75
Lysozyme, 124-126, 128-129, 212,
493, 609-611
Macrocystis, 491
Macrophoma, 406
Malo-lactic fermentation, 465
Malt, 129,459-460,475
Maltase, 103,459,476
Meat, 4, 11-12, 17,40,51,56-57,59-
60,68-69,74,77,81,105,123,
128, 139-140, 153, 178, 180-
181,183,187-188,199,208,
227, 232-234, 241, 251, 258-
259,261,270,272,282,295,
305-306, 320-321, 324, 380,
402,404,412-418,434,443,
469,476,494,512,547-551,
562-563,571,574-575,594,
600-601,604,613,616,629-
632, 657, 690-691, 697-699,
701-702,705-706, 731-732,
735, 740, 744
cured, 206, 235, 413, 562, 601, 606,
612, 617-624,630, 684, 693
frozen, 273, 704, 741
inspection, 725-727
microorganisms in, 84-85
pH, 117-118
smoked, 562
spoilage of, 82
vacuum packaging, 187, 630
water activity, III
Membrane filter (MF), 20, 30-31, 372,
374, 377, 381, 539, 745-746
hydrophobic grid, 20, 30-31, 266,
292, 374, 378
Mesophiles, 24, 37, 65, 136-141, 256,
412,422,546,612,673,739
Methyl bromide, 611
Methylococcus, 489
Methyl parahydroxybenzoate, 598,
601-603
Metmyoglobin, 122, 405
Microbacterium, 51,413-414,423,478,
482, 661-662
M. thermosphacta, 68
Microbial interaction, 142-147, 545,
632
Microbial methods, 11-41, 532-533,
742
adenosine triphosphate (ATP), 20,
38-39
aerobic plate count (APC), 11, 19-
20, 23-28, 41, 186, 327, 532,
569, 729-730, 736-739, 743
Burri slant, 29
C. botulinum, 233-235
coliforms, 373-375
direct microscopic count (DMC),
20-23, 25, 734, 737
E. coli, 380-381
electrical impedance, 20, 39-40
electronic particle count, 23
fluorescent antibody (FA), 267, 378
gas chromatography, 40
INDEX 763
limulus amebocyte, 40
little plate, 29
measurement of gas, 39
membrane filter (MF), 20, 30-31,
266,292,372,374,377-378,
381, 539, 745-746
most probable numbers (MPN), 20,
31-34, 245, 373-375, 377, 380,
509-510, 746
mycotoxin, 313-316
reductase tests, 20, 34-36
roll tube, 28-29
salmonellae, 271-272
samples, 14-20, 532, 534, 741-748
s. aureus, 210
shigellae, 278
spiral plate count, 26-27
sterility testing, 686
tube dilution, 31
viruses, 323-324
Microbial protein (MP), 4, 77, 81, 483-
492, 538
Microbial sources, 165-189
air, 170-173
animals, 173-177
clothing, 179
equipment, 182-186
humans, 177-181
insects, 175
package, 188-189
sewage, 181-182
soil, 165-167
water, 167-170
Microbiological criteria, 732-748
Micrococcus, 51, 60,107,138,145,166,
1 6 ~ 171, 1 7 7 1 7 ~ 401, 413-
419, 423-424, 426, 428, 443,
476, 482, 570, 584-585, 612,
661-664
M. luteus, 411
M. lysodeikticus, 477
M. radiodurans, 695
M. roseus, 107
Microencapsulation, of enzymes, 473-
474
Microenvironment, 101, 182
Microslide, 215
764 INDEX
Microwave, 622, 687
drying, 579-580
heating, 657-658
oven, 273
Milk, 4,12,14,17,19,22-24,28,40,
49, 51, 56, 59-61, 64, 68-69,
73,77,86,88,117,123-124,126-
127, 139-140, 4 ~ 6 ~ 177,
180, 184-185, 199-200, 206-
208, 230, 233, 251-252, 260,
263,266,294,296-297,305-
306,310,315,320-321,323,
373,376,394-395,404,425-
426,433-434,445,449,451-
452, 456-457, 467, 476-478,
489, 507, 515, 538, 548, 551-
552, 577-578, 582-583, 594,
601, 610, 614, 681, 695, 701,
706, 736-737
clotting, 476, 481
cultured, 61
dried, 214, 571, 574, 585, 741
pasteurization, 64, 69-70, 126, 185,
298, 375, 661-663, 746-747
Molds, 3, 52, 70-82, 106-107, Ill,
113-115, 123-124, 129, 131-
132, 138, 144, 148, 166, 174,
180, 186, 299-320, 387, 396,
404-407,414,418,426,429,
440, 452, 456, 458, 466, 469,
471,484,486,489-490,513,
519,522,532,536, 570,584,
587-588, 600-603, 605, 607,
609,614-616,652,661,664,
670, 677, 685, 693, 708, 734,
737-740
Monilia, 407, 409
Monilinia jructicola, 407
Monomolecular layer, 112, 574, 596
Moraxella, 138, 168,413-414,418-419,
422,424-425,629,663,693,
695-696
Moromi,471
Most probable numbers (MPN), 20,
31-34, 245, 373-375, 377, 380,
509-510, 746
Mouse, lethal dose (LD), 224
Mucor, 52, 73, 107, 115,401,409,415,
419,426-427,434,476,479
M. miehei, 481
M. mucedo, 406-407
M. pusillus, 73, 481
M. racemosus, 415
M. rouxii, 434
Mucorales, 73
Must, 462-465
Mutagenic, 302, 308-309, 463, 592,
621, 653, 708
Mycobacterium, 69, 176
M. tuberculosis, 69, 661
Mycoderma vini, 88
Mycotoxins, 7-8, 72, 74, 78, 81, 108,
299-320,411,427,552,728,
740
aflatoxin, 77, 145, 186, 300-306,
310-313,316-320, 606, 613,
628,701
citrinin, 300-301, 306-307, 315
cyclopiazonic acid, 301
ergotism, 299-300
fusarenon X, 301
fusarotoxin T-2, 145,301,309
luteskyrin, 300-301, 307, 315
neosolaniol, 301, 309
nivalenol, 301, 309
ochratoxin, 301, 308, 311, 315, 318,
320, 700
patulin, 300-301, 307-308,310,
312, 315, 318, 606
penicillic acid, 300-301, 307, 309,
311, 629
rubratoxin, 301, 310
rugulosin, 300
sterigmatocystin, 300-301, 307, 309,
315
trichothecenes, 301, 309-310, 315-
316
zearaleone, 307, 310, 315
Myoglobin, 122, 405, 617, 619
Myrothecium, 309
M. verrucaria, 477
National Marine Fisheries Service
(NMFS),730
Neisseriaceae, 53
Neosolaniol, 301, 309
Neurospora, 75, 401, 411
N. crassa, 75, 493
N. sitophila, 75, 434, 493
Neurotoxin(s), 205, 221, 228, 233, 656
Nisin, 126, 144, 616-617
Nisinase, 616
Nitric oxide myoglobin, 617
Nitrosamine(s), 443, 462, 617, 620-
622, 624
Nivalenol, 301, 309
Nocardia, 313, 483
Norwalk agent, 320
Nuclease, 206, 211-213
Nucleic acids, 488-490, 492, 707
Nut(s), 12, 72,111,135, 187, 548, 554
Oakley double gel diffusion test, 215
Ochramonas malhamensis, 493
Ochratoxin, 301, 308, 311, 315, 318,
320, 700
Ogi,457
Oidium lactis, 456
Olives, 67, 131,434, 442-443, 605
oleuropein in, 131,442
Onions, 77, 131, 145, 149, 174, 187,
582
sprout inhibition, 691
Ontjom,75
Oospora, 456
Organoleptic, 247, 393
Osmophilic yeasts, 84, 87-88, 107,
111,187
Osmotic pressure, 105, 261, 568
Ouchterlongy double diffusion test,
215
Oudin single gel diffusion test, 215
Ovomucoid, 124-125
Oxidase, 52, 54, 56-58, 60, 293, 295
Oxidation of fat, 505
Oxidation reduction (OR) potential,
120-123, 153, 232, 244, 545
Oxymyoglobin, 122, 405, 414
Ozone, 631-632, 706, 708
INDEX 765
Packazing, 13, 123, 179, 181-183,451,
460-461, 527-529, 532, 534,
536, 539, 547, 558, 570, 573,
585,590,605,628,652
microbial aspects, 165, 188-189
vacuum, 123, 232, 415, 423, 546,
549,552,564,627,630,697-
698, 702
Parabens, 601-603
Paralytic shellfish poisoning, 200
Parasites, 146-147, 195, 199-200,259,
293,690,692,699,705
Paratyphoid fever, 248
Parvoviruses, 199
Pasteurization, 6, 57, 64, 141, 147,
181-182, 200, 205, 219, 237,
323, 376, 378, 425, 435, 445,
450-451,461,464,466,471,
583, 610, 656, 658-664, 725,
739
cold (filtration), 539
dairy products, 322, 649, 652-654
egg products, 252, 564, 659-661
milk, 272, 296-297, 551, 661-663
Patulin, 300-301, 307-308, 310, 312,
315, 318, 606
Peanuts, 129, 304-305, 308, 312, 314,
317-319
aflatoxin tolerance, 748
mold in, 52, 77, 82
Pectic enzymes, 54, 395, 440, 442, 476,
480
Pectin, 65, 130, 395-397, 674, 676
Pediococcus, 51, 60, 62,131,143,411,
434,436,443,462,465,471,619
P. acidilactici, 493, 623
P. cerevisiae, 115, 132,437,439-441
P. homari, 62
P. pentoaceus, 608
P. sayae, 470
PenicilIic acid, 300-301, 307, 309, 311,
629
Penicillium, 52,72,80-82, 107-108,
115, 145, 172, 300, 306, 309,
401,406-407,409,411,414,
419,421,426-428,476,482,607
P. camemberti, 306, 381, 434, 458
766 INDEX
Penicillium (continued)
P. caseicolum, 81
P. chrysogenum, 476
P. citreoviride, 81, 300
P. citrinum, 81,300,306,694
P. crustosum, 81
P. cyclopium, 80, 301, 311
P. digitatum, 406-407
P. expansum, 301,406-407,480
P.gwucum, 478, 480,600
P. hirsutum, 415
P. iswndicum, 81,300,307
P. italicum, 406-407
P. martensii, 81, 301, 311,411,629
P. notatum, 478
P. palitans, 301
P. patulum, 301, 308
P. puberulum, 301
P. purpurogenum, 478
P. roqueforti, 3, 81, 123, 301, 308-
309,434,457,479,606
P. rubrum, 138, 301
P. urticae, 312
P. verrucosum, 301
P. viridicatum, 80-81,301,311
Peptococcus magnus, 121
Peptostreptococcus, 176
Personnel, 511, 529-534
plI, 19-20,38,64-65,71-72,74,84,
87,90,102,105,113-120,122-
127, 143, 145, 148, 153-154,
170, 178-179, 205, 208-209,
237-239, 241, 244, 253, 255,
260-262, 272, 288, 291, 293,
296,298,317, 321-322, 385,
395,406,408,413,415-417,
423,439,443-445,449-453,
459,461-462,465,467,471,
475,488-489,517,519-520,
522-523, 525-526, 539, 562,
566,570,583,588,593-594,
596,599-606,610,612-619,
622,626, 632, 659, 661, 664,
673-674,685,706
effect of microorganisms, 114-116
of food, 116-119
Phage, 146,230,266,387,445,672-
673, 709
typing, 50, 213-214,261,265
Phanerochaete chrysosporium, 490
Phoma, 52
Phomopsis, 407
Photobacterium phosphoreum, 316
Photoreactivation of DNA, 151,707
Pichia, 86, 115, 407-409, 411-412, 487
Pickle(s), 62, 67, 116,434,438-440,
602,611,659
pasteurization, 664
Picornavirus, 324
Pigment(s), 50, 54-55, 70, 136, 612
Piperazine, inhibition of nitrosamine
formation, 624
pK, 600, 602, 604
Plant(s), 257, 259, 373, 377, 730, 739
microorganisms associated with, 57,
68,77,88,165,174-175
Plaque forming units (PFU), 91, 321
Plasm ids, 50-51, 58, 61, 143, 209, 265,
280,297,436
Plate freezer, 559, 708
Poliovirus, 90, 145, 166-167,320,
322-323,522,569,657,678,
707,710
Polygalacturonase, 395, 397
Pork, 8, 12,69, 117-118,206,228,241,
251,255,416,658,690-692,
697, 736
Porphyra,491
Positive air pressure, 173, 513
Postreplication repair, 707
Potable water, 514
Potassium sorbate, 218, 260, 272, 312,
588,603,605-606,619,623,
627, 677
Potato, 117, 208, 578
dried, 571, 574, 582
rots, 78
sprout inhibition, 691
Poultry, 4, 12, 28, 40, 51, 55, 69, 111,
128,140,172,181,183-184,
187-188, 197, 199-200,206,
219,227-228,238,241,245,
251-252,258,261,270,282,
294,296,394-395,402,404,
412,418-419,424,443-444,
469, 512, 527, 548, 550-551,
563, 600-601, 606-607, 609,
613,615,624,626,628-631,
690-691, 698-699, 701, 731-
732, 738, 741, 744
cooking, 615
frozen, 260, 704-705,741
inspection, 725, 727
microbial flora, 84
microbial source, 57, 59
pH,117-119
Precision, 28, 32,40-41
Product-to-product source of contam-
ination, 165, 187-188
Propionibacterium, 68-69,115,138,178,
400,427,434,444,454-455,
482-483,604
P. acidi-propionici, 454
P_ freudenreichii
subsp_freudenreichii, 454
subsp_ shermani, 69, 454
P_ globosum, 454
P_ jensenii, 454
Propionic acid, 69, 128,454,482, 592,
602,604-605,626
Propylene oxide, 272, 653-654, 710
Proteases, 103, 402, 425, 472, 476
Protein, 2-3, 37-38, 55-56, 81-82, 89,
1 0 3 1 0 ~ 1 1 ~ 1 1 ~ 127, 1 4 ~
150, 180,208,223, 239, 241,
263-264, 312, 375, 394-395,
402-404, 433,451, 458, 461,
471-472,480-481,485-486,
516, 538, 573, 596, 607-608,
620, 653,655,659, 661-662,
672, 682, 702, 707, 741
coagulation, 671-672, 674
effects on heat resistance, 676-677
Protein efficiency ratio (PER), 487,
491
Proteus, 57, 59,138,143-144,175,178,
280,413-414,418-423,425-
426,482,693
P_ inconstans, 414
P_ vulgaris, 115, 143,421-423
INDEX 767
Protozoa, 170, 372, 486
Pseudomonas, 51-54,68, 114-115, 118,
131, 138-139, 144, 166-169,
174,178,180,372,401,405,
412-415,418-420,422-426,
428, 452, 479, 482-483, 489,
513,532,551,570,583,589,
629,661,663,693-694,706,
710
P_ aeruginosa, 54, 107, 115, 135, 138-
139,143,174,387,409,566-
567,620,629,706
P_ cepacia, 54, 409
P.jluorescens, 107, 138-139,409,418,
420, 608, 672
P_fragi, 427, 608
P_ putida, 422
P_ putreJaciens, 412
P_ radiora, 693
Psychrophile, 532, 546, 612, 673
Psychrophilic, 88, 136-141,425,568,
698
Psychrotrophs, 24, 37, 54, 65, 137-
141,169,174,177,185-186,
256,375,412,418,422,425-
426, 525, 546, 586, 604
Putrefaction, 37, 402, 404, 424
Q fever, 70, 296, 661
Quaternary ammonium compounds,
54, 519, 524-525
Racking, of wine, 464
Radappertization, 623, 696-698, 703,
705
Radiation, 6, 61, 63-64, 151, 237, 273,
558,655,731
resistance, 51, 63, 693
Radicidation, 699-700
Radioimmunoassay, 50, 215, 246, 267,
315, 324
Radurization, 698-700
Rancidity, 37, 401, 424-426, 428, 560
oxidative, 136, 401, 423, 582,608
Recall,729
768 INDEX
Recommended microbial limits, 733,
735
Redox (OR) potential, 34, 180, 457,
461
Reductase tests, 20, 34-36, 120, 747
Refrigeration, 6, 17, 54, 208, 218, 236,
247, 272, 290, 297-298, 383,
533, 536, 546-557, 571, 594,
606, 612, 651, 655, 659, 661,
698, 700
Regulation(s), 195, 319-320, 507,528,
531,533,593,614,622,725-748
Relative humidity (RH), 113, 172, 260,
313,316,321,454-455,553,
655, 706
Rennet/rennin, 481
Reoviruses, 90, 199, 320
Resistotyping, 265
Reversed passive hemagglutination,
215
Reverse osmosis, 4, 452, 514, 539,
570-571
Rhizobium, 483
Rhizoctonia carotae, 409
Rhizopus, 52, 73-74, 401, 407-410,
415,419,421,471,476,479,
482
R. arrhizus, 407
R. delemar, 477
R. nigricans, 115, 409, 411
R. niveus, 477
R. oligosporus, 434,471
R.oryzae, 313,475, 477, 480
R. stolonifer, 74, 138,407,409
Rhodopseudomonas, gelatinosa, 489
Rhodotorula, 88, 107, 174, 407, 409,
422,424,443,482-483,487
R. gracilis, 448
Ribonucleic acid (RNA), 50, 54, 64,
89-90,92,114,127,146,148,
150, 167, 522, 566, 615, 631,
653, 671-672, 701, 707
Rickettsiae, 70
Roller drying, 149
Roll tube, 20
Roquefortine, 301, 309
Rotavirus, 199, 320, 326
Rubratoxin, 301, 310
Rugulosin, 300
Saccharomyces, 86-87, 138, 174,412,
434,443,459,471
S. aceti, 87
S. bailii, 428
var. bailii, 87, 412
var. osmophilus, 87
S. bayanas, 463
S. bisporus, 87, 412
S. carlbergensis, 145
S. cerevisiae, 83, 86-87, 107, 115,
143-145, 148, 400, 407-409,
461,463,466, 469, 476,479,
482,487,490,493,538,600,
602, 676, 694, 709
var. ellipsoides, 463
S. chevalieri, 463
S. ellipsoides, 463
S. exiguus, 457
S. inusitatus, 457
S. italicus, 463
S. mellis, 408
S. oviformis, 407
S. rouxii, 84, 107, 115,408,412,470,
479, 676
S. uvarum, 86, 461, 463, 476, 493
S. vini, 463
Saccharomycetaceae, 83
Salads, 199,206,251,261,277,282, 287
Salmonella, salmonellae, 14,51,57-58,
107, 115, 131, 138, 140, 146,
153,168,173,175-178,182-
183,187-188,196,198-200,
202, 248-274,
296,375-376, 379,382-383,
420,472,508,512,532-533,
564,568,582,584-585,589,
600,603,610-611,619-620,
626, 656, 659, 661, 665, 675,
677,695, 699, 703-705, 710,
728,732,736-740
S. agona, 255, 257
S. anatum, 255, 259-260, 675
S. arizonae, 57, 257
s. bareilly, 150.
S. choleraesuis, 115, 155
S. derby, 255
S. dublin, 259-260.
S. eastbourne, 252, 260.
S. enteritidis, 255
S. gallinarum, 145, 257, 265
S. habana, 255
S. heidelberg, 255
S. infantis, 255
S. javiana, 255
S. london, 255
S. manhattan, 255, 682
S. meleagridis, 255
S. montevideo, 255
S. napoli, 252
S. newington, 255
S. newport, 255
S. oranienburg, 152, 255, 259, 694
S. panama, 255
S. pullorum, 257, 265
S. saintpaul, 255
S. sandie go, 258
S. senftenberg, 258, 273, 60.8, 682, 695
S. tennessee, 255
S. typhi, 115,255,266,269,272, 371,
70.7
S. typhimurium, 135, 148, 155, 253-
255, 259-260., 30.7, 380.-381,
566, 60.4, 60.8, tnQ, 657, 674,
694
S. typhosa, 266
S. weltvreden, 155
S. worthington, 255
Salmonellosis, 195, 248-274, 50.5
Salt (sodium chloride [NaCl]), 66,
210.-211, 232, 254, 256, 293,
298,385,415-416,437,440.-
441, 443, 452, 467, 533, 546,
587,589,591,598,611-613,
617,622,626,673,675,681,
692,696
Samples for analysis, l4-2Q, 25, 261-
262, 271-272, 313-314, 323,
655, 686, 733, 735, 741-742
Sanitation, 13, 195, 199,20.1,235,
270.-271, 289, 371, 386, 451-
INDEX 769
452,50.5-50.8,511,514-517,
525, 531-533, 535, 594, 60.2,
60.6,60.8,661,666,725,732,
740., 742
Sanitizers, 150.,210,217,50.9,530.
Sauerkraut, 49, 61-62, 67, 116,393,
434, 436-437, 439, 60.5, 60.7,
611,659
Sausage, 56, 85, 117-118, 199,434,
60.5
fermentation, 65, 67, 218
Scenedesmus, 491
Schizosaccharomyces, 87
S. malidevorans, 87
S. pombe, 87, 465, 676
Sclerotinia, 40.7
S. sclerotiorum, 40.8-40.9
Scombrotoxin, 196, 20.0.
Scopulariopsis, 82, 426
S. brevicaulis, 82, 313
Scytalidium acidophilum, 490.
Seafoods, 199,270.,290.-292, 394,
50.9-510., 621
Seizure, of food, 729
Selective media, 13, 30., 50., 151,261,
263, 266
Sequestrants, 590.
Serratia, 52, 57, 59, 280., 428, 482
S. liquefaciens, 383
S. marcescens, 59, 115, 144, 383, 411,
418,421-422,424,586,70.9-710.
Sewage, 58-59, 62, 69, 88, 165-168,
171, 181-182, 20.7, 257-258,
270., 285, 288, 321-322, 324-
325, 371, 379
Shelf life, 11, 13,24-25,118, 127,165,
187-188,417,425,449,452,
478- 479, 50.6, 511, 524, 535,
547,549, 552, 60.6, 60.8, 629-
631, 662, 690., 733
Shellfish, 57, 59,91,117,167-168,
7 ~ 181, 199, 2 Q ~ 2 5 ~ 270.,
289, 324-325, 327, 373, 376,
379,424-425,50.9-510.,548,
552, 563-564, 735, 741, 747
Shigella, 57,140.,146,175,181,196,
198-199, 258, 265, 278-281,
293, 382
770 INDEX
Shigella (continued)
S. boydii, 275, 277
S. dysenteriae, 275, 277
S. jlexneri, 275, 277
S. sonnei, 275, 277, 707
Shigellosis, 57, 274-278, 297
Shrimp, 12, 24, 35, 89, 181, 289, 292,
422,424-425, 610, 615, 629,
700, 741
pH,117-119
spoilage, 583
Single cell protein (SCP), 4, 49, 52, 65,
70,84,87-88,101,483-492,
538, 566
Smoking of foods, 6, 233, 236, 416,
443, 546, 605, 622, 624-626,
654
Sodium benzoate, 598, 601-603, 626,
677
Sodium chloride. See Salt
Sodium hypochlorite (NaOCl), 318,
519-520, 522, 524, 537, 608-
609
Sodium nitrate, 66, 443
Sodium nitrite, 415-416, 443, 617-
624, 681, 696-697
Sodium propionate, 627
Soil, 56-60,62, 65, 68-69, 7 3 7 ~ 77,
82, 85, 87, 140, 165-167, 171,
174-176, 182-183, 222, 235,
243,257,259,282,286,297,
304, 373, 375-377, 384, 408,
462, 509, 739
Sorbic acid, 598, 602, 605-606, 611,
619,623
Sources of contamination. See Micro-
bial sources
Sour dough, 88, 434, 457
Soy sauce, 76, 208, 305,434, 469-471
Specifications, 11, 186, 525, 733, 735-
738, 745, 747
Spices, 12, 65, 132, 186, 286, 305, 416,
438,443,526,582,622,653-
654,690-691,697
Spiral plate method, 26-28, 71
Spirillum, 612
Spirulinia maxima, 491
Spoilage, 5, 8, 11, 13, 24, 37-38, 49,
51-52, 54-55, 57-60, 62-63,
65-68,70, 72-75, 85, 88-89,
92,105,107, Ill, 116, 118-
119,123,132,140-141,143,
147, 169-170, 175, 181, 185-
187,232,282,402,412-429,
430,438,445,452,466,505-
506,508,511-512,514,526-
527, 529, 532, 536, 545-547,
551, 561, 568-569, 574-575,
584-585, 594, 598, 602, 607,
618, 626, 628-629, 632, 655,
658-659, 664, 666, 668, 685-
686,698,704,739,744
animal products, 412-427
canned food, 428-429, 747
dairy products, 425-427
eggs, 419-421
meat, 412-418
poultry, 418-419
seafood, 421-425
Spore(s), 53, 62-65, 70, 73-75, 79, 82-
85,87,108,143,149,153,166-
167,170-172,174,186-187,
233,235,237,241,247-248,
386,428,509,519-521,538,
583,585,594,599,610,616-
617,619,652-653,655,665-
667, 670-674,676, 681-682,
684-685, 693, 698, 701, 740,
747-748
Sporendonema epizoum, 422, 613
Sporicidal, 130, 595, 607, 610
Sporolactobacillus, 62
Sporotrichum, 82, 414, 477
S. carnis, 82, 415
S. pulverulentum, 82
S. thermophile, 82
Spray drying, 381, 385, 572, 574-577,
580, 584
Spread plates, 20, 286
Springer, 685
Stachybotrys atra, 309, 477
Standard plate count (SPC), 23-28,
140, 601, 740, 745-747
Standards, 11, 14-15,40, 507, 509,
607, 725-748
Staphylococcal intoxication, 203-219,
235
Staphylococcus, 51, 60-61, 131, 138, 140,
153,177-178,180,196,200,
202-219,238-239, 246, 249,
267,269,372,382,413,424,
426, 428, 443, 532-533, 568,
611-612, 661
S. albus, 107
S. aureus, 61, 107-109, 115, 129, 138,
143-144,148,178-179,198,
236,386,584,588-589,604,
606-608,613,656,661,675,
681-682, 694, 699, 703, 707,
710, 728, 739
S. epidermidis, 61
Starch, 34, 65, 186, 286, 305-306, 385,
396,459,466,469,476,478,
576, 582, 654, 676, 748
Sterigmatocystin, 300-301, 307, 309,
315
Streptococcus, 51, 60-62, 107, 114-115,
143,145,168,171,177,196,
287,384,408,413-414,416,
434,436,444-445,472,570,
661,693,710,739-740
S. avium, 383
S. bovis, 383, 386, 405
S. cremoris, 138, 148, 207, 426, 444-
445,451-452
S. durans, 585
S. equinus, 383, 386
S. faecalis, 138, 383-385, 407, 493,
566-567, 630, 664, 707,710
subsp. liquefaciens, 213, 383
subsp. zymogenes, 383
S. faecium, 383-385, 417, 610, 664
S. lactis, 115, 126, 138, 144,411,425-
426,444-445,451-452,457,616
subsp. diacetylactis, 444-445, 447,
451,.454
var. maltigenes, 445
S. mitis, 145, 383
S. pyogenes, 287
S. salivarius, 383, 405
INDEX 771
S. suis, 383
S. thermophilus, 138, 182, 444, 449,
454, 585
Streptomyces, 70, 166,476-477, 482-
483
S. griseus, 480
Sublethal injury, 25, 71,102,147-152,
208, 212, 261-262, 265, 374,
377, 583, 586, 658, 672, 674
Sucrose (sugar), 65,111,186,416,443,
538, 589-591, 596, 598, 614,
659, 676, 747-748
Sulfites, 614-615
Sulfmyoglobin, 415
Sulfur dioxide (S02), 463, 465-466,
582,598,602,614-615,677
Surfactants, 517, 590
Taenia saginata, 8, 692
T. solium, 8, 692
Tailing of survival curve, 693
Talaromyces, 477
Tempeh, 74, 434, 471
Temperature, 5, 19,24-25, 102, 105,
112,136-141,145,153,166,
168,172,179, 201,207-208,
218, 247, 256, 259-260, 272,
293,317,402,413,444,451,
459,462,508,517,519,536,

622, 625, 628, 652, 654-655,
657,662,664,667,672-673,
681, 683, 685, 691, 706, 739,
741
Teratogenic, 308, 592, 621
Thamnidium, 73-74, 414
T. chaetocladiodes, 415
T. elegans, 74, 415
Thermal death time, 236, 670-671,
683-685
Thermal processing, 205, 651-686,
704-705
Thermoactinomyces, 489
Thermoascus, 477
Thermodurics, 185, 385, 425, 670
Thermomonospora, 489
Thermophiles, 24, 37, 65, 137-141,
772 INDEX
Thermophiles (continued)
144, 166, 170, 177, 186,387,
656,670,673,685-686,706,
742
Torulopsis, 88,138,174,401,407-408,
411,426-427,470-471,482,
487
T. ernobi, 88, 479
T. glabrata, 144
T. halonitratophila, 88
T. magrwliae, 88
T. utilis, 88
Toxin(s), 7, 13,25, 50-51, 54, 63, 75,
90, 104, 108, 136, 167-168,
178, 180, 195, 198,200,202,
208-210, 220-239, 242, 246,
252-253, 294, 312, 546, 582,
600-601, 664, 695, 699, 701,
710,725
Trichinella spiralis, 8, 196, 658, 690, 692
Trichinosis, 197,692
Trichoderma, 82, 309, 476, 482
T. harzianum, 407
T. reesii, 477
T. viride, 82, 319, 477, 490
Trichosporon, 84, 89, 456
Trichothecenes, 301, 309-310, 315-
316
Trichothecium, 309
Trimethylamine, 404, 423, 602, 609
Typhoid fever, 248, 269, 298, 509, 661
Ultrafiltration, 4, 451-452, 539, 572
Ultra high temperature (UHT), 662
Ultrasonic energy, 710
Ultraviolet (UV) light, 54, 64, 91, 146,
151,172,301,314-315,317,
375,381,440,513,549,610,
631, 706-710
United States Department of Agricul
ture (USDA), 4, 420, 549, 622-
624,656,659,702,726-732,
747
United States Food, Drug, and Cos
metic Act (1968), 510-511
United States Public Health Service
(USPHS), 509
Ustilago, 167
Vacuum packaging, 415, 423, 546,
549,552,564,627,630,697-
698, 702
Vegetables, 40, 51-52, 54-59, 62, 67,
69,72-73,77-79,84,88, 116-
~ 135, 167-169, 7 ~ 180-
182, 185-188, 199, 206, 227-
228, 231, 258-260, 278, 282,
290, 373, 377, 380, 394-395,
408-412, 420, 436, 454, 469,
476,482,485,508-509,526-
527, 534, 548, 553, 555, 564-
565, 571, 574, 583-584, 594,
600,605-607,614-616,627-
628, 656, 665, 689-690, 692,
701, 704, 731, 734-735
Velasco flu oro toxin meter, aflatoxin
detection, 314
Verticillium, 407
Vibrio, 51, 57, 59-60, 115, 138, 167-
168,175,200,414-415,422-
423,583,693
V. cholerae, 59-60, 115, 196, 287-
289, 292, 379, 510
V. costicola, 59, 107
V. fluvialis, 287
V. mimicus, 287
V. parahaemolyticus, 59-60, 107, 115,
138, 144, 196, 198,287,289-
292,569,608,694,703
Vibrionaceae, 57, 59-60
Vibrio parahaemolyticus gastroenteritis,
289-292
Vinegar, 55, 67, 87,393, 433,467-469,
539, 600, 659
Vintage year, 462
Viral health hazards, 320-327
Virus, 49, 89-92, 103, 126, 128, 143,
4 ~ 6 ~ 168, 170-171, 176-
177,179-182,195,199,320-
327, 372, 379, 510, 513, 539,
600, 604, 608, 631, 652, 657,
670, 678, 682-683, 690, 693,
696, 701
Vitamins, 2-3, 7, 62, 68-69, 72,86,
88, 102-105, 143, 152, 180,
254,318,433,467,481,487,
489,492-493,579,590,597,
601,623,665,702
Washing, 178, 201, 235, 260, 278, 442,
449, 512, 526-527, 537-538,
571
Water, 3, 6, 40,102-113,288,508,
514-516,535,572,731
activity, 105-113, 141, 152-153,
208-209, 218, 236, 244, 254,
259,291,311-312,411-415,
545, 557-558, 572, 583, 587-
589,596,598,605,607,614,
626, 654, 674-676
potable, 169, 539
source of microorganisms, 56-58,
60, 62, 65, 68, 88, 140, 165,
167-171,174-175,177,187,
207,235,243,257-258,261,
278,282,289,291,297,321,
324,375,377,379,382,384,
739
standards, 741, 745-746
Wine, 3, 55, 62, 85, 87-88, 393, 408,
434, 458-459, 462-466, 476,
494, 539, 600, 606-607, 658-
659, 664
Wort, in beer, 459-461
INDEX 773
Xanthomonas, 52-55, 131, 138, 409, 443
X. campestris, 55, 483
Xeromyces bisporus, 107,407,411
XerophiIic fungi, 411,585
X-rays, 91, 686, 688
Yeasts, 3, 51-52, 55,74,82-89, 106-
107, 111,114-115,123,126,
131- 132, 138, 143-144, 148,
6 ~ 170-172, 174, 180, 186,
188, 233,299,407-408,414-
418, 426-429,434,437, 439-
441, 443, 452, 456-469, 472,
484-489,513,519, 523, 5 2 ~
532, 539, 566-567, 570, 585-
588, 594, 600-609, 612-616,
652- 653, 661, 664, 670, 673,
677, 685, 693, 706, 709, 711,
737-740
osmophilic, 407-408,411-412, 585
Yersinia, 57, 59
Y. enterocolitica, 59, 138, 196, 198,
296-297, 566, 604, 620, 694,
699, 706
Yogurt, 67, 180,251, 260, 306, 426,
449-450,488,551
Z value, 218-219, 681, 683-685
Zygomycetes, 73
Zygosaccharomyces, 428
Zymomonas anaerobia, 462

George J Banwart Basic Food Microbiology 1979

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George J Banwart Basic Food Microbiology 1979

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BASIC FOOD MICROBIOLOGY

Micrograph of bacteria growing on alfalfa sprouts.

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