1074

Journal of Food Protection, Vol. 66, No. 6, 2003, Pages 10741078

Copyright q, International Association for Food Protection

Research Note

Antimicrobial Properties of Commercial Annatto Extracts

against Selected Pathogenic, Lactic Acid, and
Spoilage Microorganisms
VERONICA GALINDO-CUSPINERA, 1 DENNIS C. WESTHOFF, 1 AND SCOTT A. RANKIN2*

1 Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742-2311; and 2 Department of Food Science,
1605 Linden Drive, University of Wisconsin, Madison, Wisconsin 53706-1565 USA

MS 02-235: Received 24 July 2002/Accepted 4 January 2003

ABSTRACT
Annatto preparations are used to impart distinctive avor and color to foods and are a primary colorant in dairy foods
such as cheese and butter. There are several reports indicating that certain fractions of the annatto plant have biological activities
against microorganisms of signi cance in food fermentation, food preservation, and human health. However, little is reported
describing the nature of the antimicrobial compound(s) or their potential presence in commercial annatto colorant preparations.
This study was conducted to determine whether commonly available annatto extracts are capable of in uencing the outgrowth
of selected lactic acid, spoilage, and pathogenic microorganisms. Disk diffusion and tube macrodilution techniques were used
to determine the MICs and MBCs of double-strength water-soluble annatto extracts. Standard antibiotic disks were used as
controls for the disk diffusion assay. The results demonstrate that annatto has an inhibitory effect on Bacillus cereus, Clos-
tridium perfringens, and Staphylococcus aureus, with MICs of 0.08, 0.31, and 0.16% (vol/vol) and diameters of inhibition of
9 to 10, 12 to 13, and 15 to 16 mm, respectively. A concentration of 0.63% (vol/vol) inhibited the growth of Streptococcus
thermophilus, Lactobacillus casei subsp. casei, Lactococcus lactis, and Paenibacillus polymyxa. The MICs for Listeria mon-
ocytogenes and Enterococcus durans were 1.25 and 2.5% (vol/vol), respectively. No activity was detected against Lactobacillus
plantarum, Bi dobacterium bi dum, yeasts, or selected gram-negative bacteria.

The term annatto refers to a series of preparations
containing the carotenoid-type pigments cis-bixin and nor-
bixin. Annatto is considered essentially nontoxic; in the
United States, annatto is classi ed as a natural colorant ex-
empt from certi cation as such. Although annatto is used
in many foods, the foremost use is as a colorant in cheese.
Added directly to cheese milk at a rate upward of 0.06%
(vol/vol) with typical usage rates of 0.01 to 0.02% (vol/
vol), annatto provides the distinctive orange hue of Ched-
dar, Colby, Cheshire, and many other colored cheeses (22).
Additionally, in some Muenster-type cheeses, annatto may
be applied directly to the nished cheese to provide a dis-
tinctive orange/red surface color.
In African and Latin American countries, annatto has
long been used for medicinal purposes. Annatto is used as
a folk remedy in the form of an oil suspension for the
treatment of diabetes in the West Indies (14). South Amer-
ican natives use annatto to promote the healing of wounds,
to treat skin infections, to heal burns, to act as an antipyretic
(23), to treat measles, (1) and to subdue diarrhea and asth-
ma symptoms (16).
Annatto plant extracts have demonstrated some speci c
antimicrobial activities. The leaves of Bixa orellana exhibit
an inhibitory effect against Neisseria gonorrhoea (4). Leaf
* Author for correspondence. Tel: 608-263-2008; Fax: 608-262-6872;
E-mail: sarankin@facstaff.wisc.edu.
extracts have been shown to exhibit signi cant antimicro-
bial activity against standard strains of gram-positive bac-
teria including Bacillus subtilis, Streptococcus faecalis, and
Staphylococcus aureus (14). A recent study of the volatile
pro le from commercial annatto extracts identi ed several
mono- and sesquiterpenes (10), some of which have been
reported as having antimicrobial activities (17, 18).
Although no speci c reports are available in the liter-
ature, it is generally considered that the addition of annatto
to cheese does nothing more than in uence the color (22).
Given the anecdotal and research-based evidence of its an-
timicrobial activity and the complex micro ora of dairy
foods, there is suf cient cause to warrant a more thorough
investigation of the potential antimicrobial properties of
commercial annatto extracts against pathogenic, lactic acid,
and spoilage microorganisms of signi cance to dairy foods.
MATERIALS AND METHODS
Microorganisms and culture media. Seven pathogenic mi-
croorganisms were tested in this study and include S. aureus
(ATCC 25923, American Type Culture Collection, Manassas, Va.)
(17), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853), Bacillus cereus (ATCC 11778), Listeria mono-
cytogenes (ATCC 19115), Clostridium perfringens (ATCC
13124), and Salmonella Typhimurium (donated by Dr. Jianghong
Meng, University of Maryland, Department of Food Science).
Lactic acid bacteria included Lactobacillus plantarum (ATCC
J. Food Prot., Vol. 66, No. 6
700210), Lactobacillus casei subsp. casei (ATCC 39539), Strep-
tococcus thermophilus (ATCC 14485), Lactococcus lactis (ATCC
11454), and the probiotic strain of Bi dobacterium bi dum
(ATCC 15696). Spoilage bacteria included Pseudomonas uore-
scens (ATCC 13525), Paenibacillus polymyxa (ATCC 842), Aci-
netobacter baumannii (ATCC 19606), and Enterococcus durans
(ATCC 11576). Spoilage yeast included Candida parapsilosis
(ATCC 22019), Debaryomyces hansenii (ATCC 10623), Kluyver-
omyces marxianus (ATCC 8554), Saccharomyces cerevisiae
(ATCC 2601), and Yarrowia lipolytica (ATCC 9773). All culture
media were purchased from Difco (Sparks, Md). Microorganisms
were grown overnight on deMan Rogosa Sharpe (MRS) broth
(lactic bacteria), reinforced clostridial broth (anaerobes), Emmons
Sabouraud agar (fungi), and Trypticase soy broth (all other mi-
croorganisms) under optimal conditions. The standard inoculum
was adjusted following the principles developed for the standard
disk diffusion technique (2). Overnight cultures were adjusted to
match a 0.5 McFarland standard (BBL; Becton Dickenson, Cock-
eysville, Md.) and further diluted 1:100 with Mueller-Hinton
broth, Yeast Nitrogen Base with glucose and asparagine (for
yeasts), or Anaerobe Broth (for C. perfringens and B. bi dum).
The dilution obtained served as the inoculum for disk diffusion
tests and broth dilution tests. Standard plate counts were per-
formed for this inoculum. See Results for the actual amount of
bacteria present in the tubes and plates tested; the original inocula
were corrected to account for the nal dilution.
Disk diffusion technique. The diffusion test was performed
following standard procedures (3, 26). The objective was to mea-
sure any halo of inhibition caused by a speci c annatto solution.
In practice, 0.10 ml of the inoculum was spread with glass sticks
onto either agar antibiotic 1 (most bacteria), MRS (lactic bacteria),
Wilken-Chalgrens (anaerobes), or Emmons Sabouraud (fungi)
plates. Sterile 6-mm paper disks were soaked with 25 ml of a 5.0%
double-strength annatto (DSA; 2.8% norbixin, DSM Food Spe-
cialties, Menomonee Falls, Wis.) solution and placed on the pre-
viously inoculated plates. Plates were maintained at optimal con-
ditions, and the halo of inhibition was measured after 18 to 24 h
of incubation. A disk containing known amounts of penicillin,
gentamicin, or chloramphenicol (BBL) was placed in one quadrant
of the plate as a comparative standard.
Antimicrobial dilution test. A stock solution of annatto was
prepared by diluting 5.2 ml of DSA (2.8% norbixin) with 99 ml
of phosphate buffer (pH 7.0; VWR, Westchester, Pa.). This solu-
tion was adjusted to pH 7.4 with 2 N HCl and ltered through a
0.2-mm pore-size membrane (ZapCap, VWR, Bridgeport, N.J.),
yielding the annatto stock solution. The stock solution was used
to prepare serial twofold dilutions to a nal concentration of
0.08% of annatto. A 2.0-ml aliquot of the microbial inoculum was
placed in each tube containing annatto. A positive blank contain-
ing 2.0 ml of the microbial inoculum and 2.0 ml of medium was
prepared for each series. At least three replicates were performed
for each microorganism. Tubes were incubated for 18 to 24 h at
optimal conditions, after which the MIC was determined. The
MIC was de ned as the lowest concentration of annatto in a tube
with no visible growth when viewed against standard uorescent
light. To corroborate the results, the microbial density was mea-
sured with a Novaspec II spectrophotometer (Pharmacia, Cam-
bridge, UK) at 540 nm (absorbance .0.05 was considered a pos-
itive).
After the MIC was determined, each tube showing no visible
evidence of microbial growth was then subcultured to determine
whether viable organisms were present. Using a calibrated loop,
0.01 ml was subcultured to a quadrant of Mueller-Hinton agar
ANTIMICROBIAL PROPERTIES OF ANNATTO 1075
plate (MRS agar for lactics, Wilken-Chalgrens agar for anaerobes,
or Emmons Sabouraud agar for yeasts) to determine the MBC.
After 18 to 24 h of incubation under optimal conditions, the plates
were examined, and the number of CFUs was determined. The
MBC was de ned as the lowest concentration that resulted in
99.9% kill (2).
Data analysis. To correct for variation among replicates and
outlier values, the median MIC and MBC were calculated by SAS
version 8 statistical software. For this analysis, the median was
chosen because it re ects the actual concentration necessary to
inhibit growth. With twofold dilutions, the concentration range
between tubes is different, and the arithmetic mean tends to in-
crease the MIC and MBC when the overall tendency is for a
smaller value.
RESULTS AND DISCUSSION
Pathogenic bacteria. According to a report released in
1990 on the microbiological safety of cheese made from
heat-treated milk (15), between 1948 and 1988, the most
frequent causative factor in U.S. and Canadian cheese-re-
lated outbreaks was postpasteurization contamination.
Three organisms, Salmonella spp., L. monocytogenes, and
enteropathogenic E. coli, were considered high-risk threats
to the cheese industry, while S. aureus was considered low
risk because growth and toxin production are readily sup-
pressed by adequate refrigeration and pH control. In a re-
cent study based on data collected from 1970 to 1997, Sal-
monella spp., S. aureus, and L. monocytogenes were the
most common organisms in cheese-related disease out-
breaks (9). The current study evaluated the activity of an-
natto against common foodborne pathogens (Table 1). An-
natto did not affect the growth of E. coli, Salmonella Ty-
phimurium, or P. aeruginosa. L. monocytogenes outgrowth
was weakly inhibited. A 1.25% solution was capable of
inhibiting the growth of L. monocytogenes; however, no
bactericidal effect was detected at the concentrations tested.
The outgrowth of S. aureus, B. cereus, and C. perfringens
was substantially inhibited in the presence of annatto ex-
tracts. The highest activity was detected against B. cereus,
where an 0.08% solution was suf cient to inhibit growth,
and a 0.16% solution exhibited a bactericidal effect. It is
important to note that in the case of B. cereus, the inoculum
used was smaller than the one used with other bacteria. S.
aureus was inhibited with a 0.16% DSA solution and
showed the largest zone of inhibition among all microor-
ganisms tested.
Cheese commonly presents anaerobic conditions and
pH levels favorable for clostridial growth, although clos-
tridia outbreaks in cheese are rare (9). In this study, C.
perfringens, another well-known food pathogen, was inhib-
ited with a 0.31% annatto solution, and a 0.55% solution
was capable of killing vegetative cells.
Lactic acid bacteria. It is widely recognized that the
avor of cheese results from the interaction of starter bac-
teria, milk and rennet enzymes, and secondary micro ora
(25). The microorganisms involved in cheese making and
ripening can be divided into starter lactic acid bacteria and
nonstarter lactic acid bacteria. L. lactis and S. thermophilus
are common starter cultures, while typical nonstarter lactic
1076 GALINDO-CUSPINERA ET AL. J. Food Prot., Vol. 66, No. 6

TABLE 1. Antimicrobial activities of double-strength water-soluble annatto

Average 5% DSA disk Antibiotic disk
inoculum MIC MBC inhibitiona inhibitionb
Microorganism (log CFU/ml) (% DSA) (% DSA) (mm) (mm)

Pathogenic bacteria
Bacillus cereus 4.40 0.08 0.16 910 P 912
Clostridium perfringens 5.23 0.31 0.55 1213 P 3032
Escherichia coli 6.98 .2.5 nb ni C 1315
Listeria monocytogenes 5.91 1.25 nb 1213 P 1821
Pseudomonas aeruginosa 6.89 .2.5 nb ni G 13
Salmonella Typhimurium 5.29 .2.5 nb ni G 89
Staphylococcus aureus 5.98 0.16 1.60 1516 P 3031
Lactic acid bacteria
Bi dobacterium bi dumc 5.91 .2.5 nb 89 P 2225
Lactobacillus casei subsp. casei 5.46 0.63 0.94 89 P 1517
Lactococcus lactis 6.80 0.63 1.04 9 P 2931
Lactobacillus plantarum 5.30 .2.5 nb 9 P 2126
Streptococcus thermophilus 5.08 0.63 2.5 910 P 1820
Yeasts
Candida parapsilosis 4.97 .2.5 nb ni N 2225
Debaryomyces hansenii 4.68 .2.5 nb ni N 1517
Kluyveromyces marxianus 4.78 .2.5 nb ni N 2931
Saccharomyces cerevisiae 4.66 .2.5 nb ni N 2126
Yarrowia lipolytica 4.67 .2.5 nb ni N 1820
Spoilage bacteria
Acinetobacter baumannii 5.62 .2.5 nb ni G 11
Enterococcus durans 5.38 2.5 nb 810 P 1719
Paenibacillus polymyxa 4.85 0.63 1.25 89 P 1618
Pseudomonas uorescens 5.72 .2.5 nb ni G 913
a DSA, double-strength annatto in water, pH 7.4, % (vol/vol); nb, nonbactericidal at 2.5% DSA; ni, no inhibition.
b P, penicillin 2 U; C-chloramphenicol 5-U disks; G, gentamicin 10 U; N, nystatin 2 U.
c Probiotic bacterium was included in this classi cation because of its association with fermented dairy foods.

acid bacteria include several lactobacilli species such as L.
casei, L. plantarum, L. paracasei, and L. curvatus (8). In
this study, four strains of lactic acid bacteria were tested.
Additionally, because fermented dairy foods are recognized
as a source of health-promoting or probiotic bacteria (21),
B. bi dum, a common probiotic strain, was also included.
Annatto exhibited limited activity against L. lactis, L.
casei subsp. casei, and S. thermophilus. A 0.63% DSA so-
lution was suf cient to inhibit the growth of these organ-
isms (Table 1). The MBC concentration had more variation
among these three organisms; S. thermophilus proved to be
the most resistant at 2.5%. No antimicrobial activity was
detected against B. bi dum or L. plantarum.
In uence on yeasts. Yeasts ful ll an important role in
the manufacture and spoilage of many dairy foods. In pas-
teurized dairy products, yeasts typically originate from
postpasteurization contamination and are common contam-
inants of cheeses (7). Among the most frequently occurring
yeasts in dairy products are D. hansenii, S. cerevisiae, K.
marxianus, C. parapsilosis, and Y. lipolytica. These ve
strains have been isolated from cheese and yogurt and oc-
casionally have been found in ice cream (7, 20). Annatto
did not inhibit the growth of any of the ve yeasts tested
at the concentrations used (Table 1). Growth was consistent
throughout the dilution series. Annatto even appeared to
promote the growth of yeasts. Some growth was detected
on the disk itself, and the tubes that contained annatto ap-
peared slightly more turbid than the positive blank.
Spoilage bacteria. Psychrotrophic bacteria affect the
quality and shelf life of numerous milk products (13, 24).
Pasteurized dairy products can be spoiled by gram-negative
bacteria resulting from postprocess contamination or by
gram-positive organisms, especially spore-forming bacilli,
that survive pasteurization. Psychrotrophs have proteolytic
and lipolytic activity and may produce malodorous metab-
olites in milk. In a study made to classify the spoilage ora
of raw and pasteurized milk, Pseudomonas spp. and Bacil-
lus spp. were the most frequently encountered spoilage bac-
teria. P. uorescens and P. fragi are commonly associated
with raw and pasteurized milk spoilage (24). Bacillus po-
lymyxa and B. cereus are described as the second most im-
portant bacteria in the spoilage of pasteurized milk. Bacilli
spores withstand pasteurization and can grow at low tem-
peratures, thus becoming a limiting factor in the shelf life
of pasteurized milk products (6). B. cereus is also a well-
known foodborne pathogen. It is capable of rapid growth
during the manufacture of Cheddar cheese between the end
of cooking and the curd-milling step and has been isolated
J. Food Prot., Vol. 66, No. 6
from cheese samples (5). Some psychrotrophic strains of B.
cereus have been shown to produce toxins and thus are of
importance from a public health standpoint (6).
Other bacteria important in dairy food spoilage are aci-
netobacter and enterococci. Acinetobacter spp. have low
spoiling potential because they lack important food spoilage
biochemical activities such as proteolysis or production of
unpleasant odors. However, they are of particular note be-
cause of their potentially high levels in raw milk and their
ability to produce a capsular polysaccharide resulting in
ropy milk and whey as well as slimy surface defects in
cheese (12). Enterococci ful ll many roles in dairy products
as starter, probiotic, and spoilage bacteria (11). Some strains
seem to play an important role in the development of cheese
avor; nevertheless, they are not considered GRAS (gen-
erally recognized as safe) organisms (11). Enterococci are
thermoresistant bacteria that withstand lactic acid and are
commonly found in levels ranging from several thousands
to tens of millions per gram in a variety of cheeses pro-
duced from raw and pasteurized milk (19).
The antimicrobial activity of annatto was tested against
four spoilage bacteria commonly found in dairy products
(Table 1). There was no inhibitory effect detected against
A. baumannii or P. uorescens, both gram-negative bacte-
ria. Conversely, annatto showed activity against gram-pos-
itive E. durans and P. polymyxa (also referred to as B.
polymyxa). A 0.63% solution of annatto was capable of
inhibiting the growth of P. polymyxa, while a 1.25% so-
lution had a bactericidal effect. In the case of E. durans, a
more concentrated solution of 2.5% annatto was necessary
to inhibit growth; no bactericidal activity was detected at
the concentrations tested. Because of the spreading char-
acteristic of bacilli, it was not possible to detect a consistent
halo of inhibition with the disk diffusion technique. Also,
it was dif cult to obtain an inoculum higher than 104 be-
cause of the turbidity caused by the capsule. However,
growth inhibition was clearly detected against both bacilli
strains in the tube macrodilution technique.
Although annatto is generally considered an inert col-
oring agent in dairy products, these results suggest that an-
natto may also have some ability to in uence micro ora of
signi cance to dairy foods, namely certain gram-positive
species. In each case, the inhibitory effects were manifested
at substantially higher concentrations than those found in
cheese manufacturing practices. However, little is known
about the properties of the antimicrobial agent(s) in annatto
or its fate during the cheese making process. It is possible
that the inhibitory in uence is ultimately increased in
cheese because of the concentrating effect manifested dur-
ing the separation of curd and whey. Conversely, the active
agent(s) may be entirely water soluble and partition with
the whey, thus further diminishing its potentially antimi-
crobial effect in the curd. When annatto is applied directly
to the outer surface of cheese, as in the case of Muenster
cheese, annatto may be present at concentrations suf cient
to serve as a surface-associated antimicrobial barrier. Fur-
ther research is necessary to identify the compound(s) re-
sponsible for this activity and to determine the effectiveness
in an actual food matrix.
ANTIMICROBIAL PROPERTIES OF ANNATTO 1077
ACKNOWLEDGMENTS
The authors thank the University of Maryland, Department of Ani-
mal and Avian Sciences, the CONACYT/Fulbright Garcia-Robles pro-
gram, the Banco de Mexico/FIDERH for nancial support, and DSM Food
Specialties USA Inc. for the contributions of annatto samples.
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