CHAPTER 1

INTRODUCTION
1.1Review of the Literature
1.1.1 Wheat
Wheat is one of the world's most important food crops. Historic documents confirm
that wheat is the earliest field crop used for human food processing. It also became the
leading grain used for human consumption due to its nutritive profile and relatively
easy harvesting, storing, transportation, and processing, as compared to other grains.
The earliest varieties, grown 12,000-17,000 years ago in the Near East, were Triticum
monococcum (einkorn) and Triticum dicoccum (emmer). Continued breeding resulted
in the development of new varieties around the world that often became adapted to
areas previously unsuited for the cultivation of wheat.
The main wheat varieties grown today are Triticumaestivum, subspecies vulgare,
which is a hexaploid and this species includes hard red winter, hard red spring, soft
red winter, and white wheat. Another wheat durum (Triticum durum) is a tetraploid
wheat specices
Wheat (Triticum) categorizes under the grass family, Poaceae, which include the
cereal grains such as rice, corn. All of these grains can be milled into flour, but only
wheat flour has the ability to be transformed into glutinous dough, together with water
and other ingredients (Hoseney, 1998; Matz, 1989; Cauvain, 2001).

1.1.2 Types of Wheat

Hard wheat Strong whets of Canada (Manitoba) and the similar hard red spring
(HRS) whets of the US. They yield excellent bread-making flour because of their high
quantity of protein ranges 1215 % mainly in the form of Wet gluten (29-39%).
Medium type used for general purposes like bun, rolls and Donuts having protein
9-12 % with wet gluten 27-30 %.
Soft wheat less attractive bread than that achieved from strong wheat. The loaves
are generally smaller, and the crumb has a less pleasing structure. Soft wheat,
however, possess excellent characteristics for the production of flour used in cake &
biscuit manufacture
Durum Very hard, translucent, light-colored grain used to make semolina flour for
pasta
1.1.3 Wheat Flour
Wheat flour is the resultant product of wheat grains after milling. Wheat grains or
kernels are dry, one-seeded fruits, which is member of the grass family Gramineae
(Pomeranz and Shellenberg, 1971; Hoseney, 1998). The kernel is surrounded by the
pericarp within which the germ (or embryo) and the endosperm are enclosed. About
5% of the kernel consists of the pericarp, which is mainly made up of cellulose. The
germ comprises approximately 3% of the kernel and is rich in protein, B vitamins and
enzymes. Flour is mainly made up from the remainder of the kernel, which is the
starchy endosperm Starch, at about 65%, makes up the biggest portion of the flour. It
consists of linear structures namely amylose and branched structures called
amylopectin. Amylose is linked by a-1,4 glycosidic bonds, while amylopectin is
linked by both a-1,4 and a-1,6 glycosidic bonds. (Stauffer, 1998).
Protein is the second most abundant nutrient in the endosperm (Pomeranz and
Shellenberg, 1971; Kent and Evers, 1994; Hoseney, 1998).
Approximately 80 -90% of the total wheat proteins are storage proteins and they play
a major role in bread production because of its essential function in bread structure.
The gluten network forms when flour is combined with water and with some energy

input (Kent and Evers, 1994; Cauvain, 1998a; Hoseney, 1998; Cauvain and Young,
2000). It is crucial for the retention of air and carbon dioxide during bread making and
thus gives bread its structure.
The two major components of the gluten storage proteins are glutenins and gliadins.
Glutenin is known to give elasticity and gliadin viscosity to the gluten network
(Hoseney, 1998, Stauffer, 1998). About 2-3% of the total flour proteins comprise of
the water-soluble proteins. These include albumins, globulins and the water- soluble
pentosans. Other minor constituents of wheat flour are the lipids, the inorganic
compounds i.e. ash and the non-starch polysaccharides i.e. pentosans (Stauffer, 1998).
Pentosans is important because of their high water absorption capacity (Michniewicz,
et aI, 1991; Michniewicz, et aI, 1992). They occur in wheat flour at about 2.5% on a
dry flour basis (Rouau, et aI, 1994). Other beneficial effects of pentosans include
increased loaf volumes and a better crumb structure (Delcour, et al, 1991; Rouau, et
al, 1994; Qi Si, 1997) Lipids. another minor constituent of wheat flour, occur at levels
around 2% (Stauffer, 1998). Although they only account for a fraction of the flour,
they are essential in bread making. They contribute to the final texture of the baked
product and influence the flavour and the mouth feel of a product (Charley,1982).

Figure 1.1; Wheat kernel

Country

Annual Wheat Consumption (tons)

*103

China

104500

India

69000

Russia

35500

U.S.A

33203

Pakistan

18750

Srilanka

860

Table 1.1: Annual wheat consumption patterns

1.1.3.1 Types of Wheat Flour
1. All-purpose flour is white flour milled from hard wheat or a blend of hard and soft
wheat. It gives the best results for many kinds of products, including some yeast
breads, quick breads, cakes, cookies, pastries and noodles. All-purpose flour is usually
enriched and may be bleached or unbleached. Bleaching will affect nutrient value.
Different brands will vary in performance. Protein varies from 8 to 11 percent.
2. Bread flour is white flour that is a blend of hard, high-protein wheat and has greater
gluten strength and protein content than all and in some cases conditioned with
ascorbic acid, bread flour is milled primarily for commercial bakers, but is available
at most grocery stores. Protein varies from 12 to 14 percent.
3. Cake flour is fine soft wheat with low protein content. It is used to make cakes,
cookies, crackers, quick breads and some types of pastry. Cake flour has a greater
percentage of starch and less protein, which keeps cakes and pastries tender and
delicate. Protein varies from 7 to 9 percent.
4. Pastry flour has properties intermediate between those of all- purpose and cake
flours. It is usually milled from soft wheat for pastry-making, but can be used for
cookies, cakes, crackers and similar products. It differs from hard wheat flour in that
it has a finer texture and lighter consistency. Protein varies from 8 to 9 percent.
5. Semolina is the coarsely ground endosperm of durum, a hard spring wheat with a
high-gluten content and golden color. It is hard, granular and resembles sugar.
Semolina is usually enriched and is used to make couscous and pasta products such as
spaghetti, vermicelli, macaroni and lasagna noodles. Except for some specialty
products, breads are seldom made with semolina.

6. Durum flour is finely ground semolina. It is usually enriched and used to make
noodles.
7. Whole wheat, stone-ground and graham flour can be used interchangeably; nutrient
values differ minimally. Either grinding the whole-wheat kernel or recombining the
white flour, germ and bran that have been separated during milling produces them.
Their only differences may be in coarseness and protein content. Insoluble fiber
content is higher than in white flours.
1.1.4 Bread
Bread is one of the most popular yeast leavened product all over the world as well as
in Sri Lanka which made by wheat flour. The uniqueness of wheat flour to be
transformed into cohesive, viscoelastic mass is due to the hydration of gluten proteins
in the wheat together with the application of mixing energy. After the dough is
formed, the gluten structure entraps the gasses produced during the fermentation
stage. This allows the dough mass to expand and to be baked into a soft, light and
palatable product, which is known as bread (Hoseney, 1998; Matz, 1989; Cauvain,
2001).
Bread has been around for thousands of years, although its exact time of discovery
cannot be pinpointed (Pomeranz and Shellenberger, 1971; Cauvain, 1998). According
to Pomeranz and Shellenberger (1971), the history of bread is almost as old as the
history of mankind, since it has long been used as a sacred symbol in religious
ceremonies. In ancient times, the Egyptians used it as both a sacrifice and a tribute to
their gods. Today Christians portray the body of Christ at Communion with bread.
History also implies that bread has been used in a political context causing the fall of
Rome and the French Revolution to name a few. Bread has many variations
depending on the shape, size, texture, colour and taste (Cauvain, 1998a; Hoseney,
1998). The origin of the variations can be traced to the various parts of the world, for
example, baguettes from France and flat breads from the Middle East.
According to Cauvain (1998a), bread making most likely began in the Middle East,
because that is where cereal farming originated.
All over the world, bread has been regarded as a staple food (Pomeranz and
Shellenberger, 1971; Cauvain, 1998a) for the past millennia. As a single food source,
bread supplies the most nutrients (Pomeranz and Shellenberger, 1971) when
5

compared to food from other cereal grains. For sliced white bread, typical values are
7.6% protein; 1.3% fat and 46.8% carbohydrates. This is in comparison with the
typical values for cooked brown rice with a protein content of 2.6% and 1.5%; a fat
content of 1.1% and 1.1% and a carbohydrate content of 32.1% and 9.0%,
respectively (Kent and Evers, 1994).
In this twenty-first century with our busy lifestyles, people often opt for convenient
alternatives with ingredients of a more natural origin, of which bread is a good
example.
1.1.5 Additives for wheat Flour
Food additives can be defined as substances added in small amounts to something
else to improve, strengthen, or otherwise alter it. Additives are used for Wheat flour
because of they offer many advantages such as improved volume, water absorption,
texture, crumb, shelf life & slicing characteristics, aroma, shape size of the bread, the
baker could adjust the timing of fermentation and mixing, as well as proofing and
baking conditions, to adjust for variations in flour properties, yeast activity. These
adjustments are necessary to obtain the desired quality of finished baked products.
Other advantage of additives is greater when either the desired quality flour is not
available or bakery products are made from composite. Mostly weak wheat flour is
less suitable for bread making it is there for desirable to use some additives to
improve the quality of bakery product.
In the last years, diverse treatments have been applied for improving the quality of
bakery products. (Rosell, Wang, Aja, Bean & Lookhart, 2003; Aja, Wang & Rosell,
2003). Mainly use additives for improve those characteristics mentioned above.
Recently, consumers do not want artificial or synthetic additives to be used for bread
making. Therefore, the use of enzymes is very important as improvers for breamaking
(Harada, Lysenko, & Preston, 2000; Morita, Arishima,Tanaka, & Shiotsubo, 1997)

1.1.5.1 Enzymes as an additive

Enzymes are biological compounds, usually proteins, which expedite the conversion
of one substance into another. Their presence accelerates the rate of a chemical
reaction and they are often specific and act upon only one substrate, or catalyze only
one kind of reaction, in different, but related, substrates. Basically enzymes can
hydrolyse a polymeric substrate in two ways. Exo-enzymes remove a single polymer
unit from the end of the polymer chain, whereas endo-enzymes can rupture the
internal bonds in a random manner at any point along the chain. The activity of
enzymes is dependent upon temperature. Enzymes used in baking are usually stable at
room temperature and the rate of enzyme activity doubles with each 10C increase up
to the temperature of denaturation, at which the enzyme is inactivated. Most enzymes
are inactivated above 60C.
For decades, enzymes such as malt and fungal alpha-amylases have been used in
bread-making. Rapid advances in biotechnology have made a number of exciting new
enzymes available for the baking industry. The importance of enzymes is likely to
increase as consumers demand more natural products free of chemical additives. For
example, enzymes can be used to replace potassium bromate, a chemical additive that
has been banned in a Sri Lanka.
The dough for bread products consists of flour, water, yeast, salt and possibly other
ingredients such as sugar and fat. Flour consists of gluten, starch, non-starch
polysaccharides, lipids and trace amounts of minerals. As soon as the dough is made,
the yeast starts to work on the fermentable sugars, transforming them into alcohol and
carbon dioxide, which makes the dough rise. The main component of wheat flour is
starch. Gluten is a combination of proteins that forms a large network during dough
formation. This network holds the gas in during dough proofing and baking. The
strength of this gluten network is therefore extremely important for the quality of all
bread raised using yeast. Enzymes such as amylase, xylanases, lipases and oxidases
can directly or indirectly improve the strength of the gluten network and so improve
the quality of the finished bread the strength of the gluten network and so improve the
quality of the finished bread

1.1.6 Major Enzymes used in bakery industry and their functionality

1.1.6.1 Amylases
Amylase is added to dough for two purposes: (1) to aid in formation of maltose and
glucose as substrates for yeast fermentation, and (2) to interfere with starch
retrogradation and the thus enhance the shelf life of the baked product. These two
aims are accomplished at different stages of production, the first during fermentation
and the second in the oven. The amylopectin component of starch is depicted, and the
sites at which the different kinds of amylase hydrolyze the glucosidic links.
The yeast substrate produced by amylase action is maltose. Yeast uses glucose and
fructose more readily than maltose, because it must synthesize the enzyme maltose
(which hydrolyzes maltose to glucose) before it can metabolize maltose (sanderson et
al. 1983).In addition amylase requires time to hydrolyze starch, to taking these two
facts together readily shows that the use of amylase to provide yeast nutrition is
effective only in dough which receive a relatively long fermentation time and which
contain little or no added sugar. About the only bakery product today that meets these
criteria is saltine cracker dough, and the addition of diastatic malt syrup (a
concentrated water extract of malted barley and/or wheat that contains -amylase and
-amylase activity) is common in the industry. In most other bakery dough, sugar is
routinely included both for yeast fermentation and for flavor in the finished product.
The use of -amylase has been suggested to increase loaf volume (Beck, Johnson, and
Miller 1959; Rubenthaler, Finney,and pomeranz 1965;Berger and Grandvoinnet
1974).In the absence of added sugar, this increase does occur, but the effect appears to
be directly related to the stimulation of yeast fermentation by the sugars released from
starch (Berger and Grandvoinnet 1974).amylases improve the grain of the finished
bread crumb, probably because they decrease dough consistency and improve
machinability (Bernett 1975). The other reason for adding _amylase to dough is to
retard

staling

in

the

baked

product

(Conn,Johnson,and

Miller

1950;Miller,Johnson,and Palmer 1953;Zobel and Senti 1959;Waldt 1968;Dragsdorf

and Varriano-Marston 1980)

1.1.6.2 Xylanase
Wheat flour contains 2-3% pentosans. which are polysaccharides that are not
starch.about half of these are water soluble & half are insoluble. about 3/4are of these
pentosans are xylans. the significant fact about flour xylans are extremely hydrophilic,
accounting for almost one fourth of the water absorption requirement of a wheatflour
dough & loaf volume (kulp 1968;Shelton and Apolina 1985).It means that xylans
contribute to the consistency (viscosity) of dough. Xylans have an important role in
bread quality due to their water absorption capability and interaction with gluten. The
hydrolysis of pentosans using some enzymes like hemicellulase or pentosanase at the
optimal level improves the dough properties, leading to a greater uniformity in quality
characteristics. The application of xylanolytic enzymes has increased for the last few
decades owing to their potential effectiveness in breadmaking. Starch and non-starch
carbohydrate- hydrolyzing enzymes are commonly used in the breadmaking industry
as bread improvers. Enzymatic hydrolysis of non-starch polysaccharides leads to the
improvement of rheological properties of dough, bread specific volume, and crumb
firmness. Xylanase transforms water-insoluble hemicellulose into soluble form, which
binds water in the dough, therefore decreasing dough firmness, increasing volume and
creating finer and more uniform crumbs. It significantly improves manufacturing
conditions: dough is made more 'machine-friendly' as it does not stick to the
machinery parts .During gluten-starch separation process, gluten is formed first as a
result of breakdown of the gliadin-glutelin structures during mixing, followed by their
re-agglomeration. To study their effect, pentosans, enzymes,etc. can be added after
the mixing step by simple modification of Glutomatic System. It has been observed
that re-aggregation of gluten protein starts immediately after the first mixing step
during the dough dilution phase. Addition of pentosans or xylanase during this phase
can strongly affect gluten formation. The addition of xylanase prior to dough mixing
can lead to overdose effects. This is not observed when xylanase is added later during
the agglomeration phase. Pentosans and xylanase act mainly during the reagglomeration of gluten, following the breakdown of gluten structures during mixing,
which ultimately affects both gluten yield and gluten rheological properties. Effects of
pentosans and xylanase on gluten are paralleled by effects on dough, especially on
dough extensibility

Enzymes are proteins and that they are substrate specific. This means that a given
enzyme only will work on a certain substrate and only do a very particular action.
Although they take part in a chemical (enzymatic) reaction, they do not change during
that reaction. That accelerates or facilitates chemical reactions. Because they are
proteins, they are heat sensitive and all enzymes have an optimum temperature and
pH for activity. Within that range, activity increases with temperature until the
denaturation point is reached. At that point the enzyme will lose its functionality.
Apart from temperature and pH, enzymes are also dependent upon the availability of
water, amount of enzyme used, the availability of the substrate and the time allowed
for the reaction
1.1.6.3 Glucose Oxidase
Protein crosslinking or the formation of covalent bonds between polypeptide chains is
a way of modifying the protein functionality and simultaneously increasing its
applications in different processes. Oxidation induces the formation of disulfide
bonds by coupling of two cysteine residues that are adjacent within a food protein
matrix, and dityrosine crosslinks (Tilley,Benjamin, Bagorogoza, Okot-Kotber,
Prakash & Kwen, 2001; Rasiah, Sutton, Low, Lin & Gerrard, 2005), it results the
covalent crosslinking of proteins. This reaction on bread dough induces the formation
of a protein network with improved viscoelastic and structural properties, and
therefore, betters performance for breadmaking (Wikstrm & Eliasson, 1998;
Fayle,Gerrard, Simmons, Meade, Reid & Johnston, 2000). Glucose Oxidase catalysis
the oxidation of -D- glucose to gluconic acid and hydrogen peroxide.(Rosell et al.,
2003; Aja et al., 2003; Hoseney & Faubion, 1981; Haarasilta, Pullinen & Vaisanen,
1991; Nakai, Takami Yanaka & Takasaki, 1995; Primo- Martin, Valera & MartinezAnaya, 2003; Gujral & Rosell, 2004) indicate that hydrogen peroxide produced
during GO reaction causes the oxidation of the free sulfhydryl units from gluten
protein giving disulfide linkages and the gelation of water soluble pentosans,
changing rheological properties of wheat flour dough. This hypothesis was confirmed
by Velmulapalli and Hoseney (1998a), who found that free thiol groups of the water
soluble proteins of flour or dough decreased in presence of GO. The addition of GO
leads to an increase in the elastic of wheat flour dough (Gujal et al., 2004;
Vemulapalli et al., 1998b; Dunnewind, Van Vliet & Orsel, 2002) and also gives less
stiff dough than control and its addition have a strengthening effect (Martinez-Anaya
10

& Jimenez, 1997). Primo-Martn et al (2003) concluded that pentosanase/GO

combination resulted in dough with improved extensibility yielding better gluten
quality. An improvement in the wheat bread loaf volume and crumb grain has been
obtained by adding GO (Vemulapalli et al., 1998b; Xia, Jin & Liang, 1999), Van Oort
(1996) found this improved bread volume. The functional properties of bread dough
mainly depend on the proteins forming the gluten network.
1.1.6.4 Lipase
Lipases are used for their dough conditioning properties (Qi Si, 1997; Poldermans and
Schoppink, 1999) where they significantly retard bread staling. Lipase-treated flour
has an increase in gluten strength, which results in good dough conditioning ability.
Polderrnans and Schoppink (1999) reported that lipases hydrolysed triglycerides into
mono- and diglycendes. The monoglycendes interact with starch and thus reduce its
ability to retrograde, which leads to a softer crumb. Lipase also has a positive effect
on crumb colour and structure (Poldernians and Schoppink, 1999), even without
added fat. Qi Si (1997) reported a 20-30% increase in the loaf volume with the
addition of 500-2000 lipase units per kilogram (LU/kg) of flour. Too high values lead
to a negative effect on loaf volume as well as a too dry and stiff dough. A study done
by Si and Hansen (1994) in Glinas (1998 also reported lipase to be involved in
dough bleaching, though the exact mechanism is not yet fully understood. However, it
is postulated that after lipase has hydrolysed the triglycerides, the free fatty acids
generated are then oxidized by lipoxygenase according to Castello et aI (1998) in
Goesaert et al(2005).
1.1.7 The synergetic effects of enzymes
Each of the enzymes mentioned above have its own specific substrate in wheat flour
dough. For example, lipases work on the lipids, xylanase works on the pentosans, and
amylases work on the starch. Because the interaction of these substrates in dough and
bread is rather complex, the use of enzyme combinations can have synergistic effects
that are not seen if only one enzyme is used, not even at high dosages. Quite often an
overdosage of enzymes will have a detrimental effect on either the dough or the
bread. For instance, an overdose of fungal alpha-amylase or hemicellulase / xylanase
may result in dough that is too sticky to be handled by the baker or baking equipment.
It is therefore beneficial for some types of bread formulation to use a combination of
11

lower dosages of alpha-amylase and xylanase with low dosages of lipase or glucose
oxidase to achieve optimum dough consistency stability and bread quality. Another
example is to use maltogenic alpha-amylase in combination with fungal alphaamylases and xylanase or lipase to secure optimum crumb softness as well as
optimum bread quality in terms of crumb structure, bread volume, Color
1.1.8 Other ingredients in bread making
1.1.8.1 Yeast
Saccharomyces cerevisiae is the scientific name for Bakers yeast, which is generally
used in the baking industry (Jay, 1992). It is a living organism that multiplies by
multilateral budding to produce spherical spores. It comes in a number of physical
forms, namely compressed, liquid and dried yeast (Brown, 1993;Kent and Evers,
1994; Gould, 1998). Hoseney, 1998, gives the following simplified chemical reaction
for the action of yeast: This process is anaerobic and the gas (CO2) produced leavens
the bread (Kent and Evers, 1994; Williams and Pullen, 1998). Besides gas production
and leavening, yeast in baking also affects dough rheology and flavour of the bread
(Kent and Evers, 1994). The way in which dough rheology is affected can be
explained by means of the spread test. Bread dough has both viscous-flow properties
and elastic properties. Dough with higher viscous-flow properties has a larger spread
ratio and one with higher elastic properties has a smaller spread ratio. When yeast is
added to a flourwater dough with a large spread ratio, the rheology changes in that the
spread ratio becomes smaller. This means that the dough has higher elastic properties
(Hoseney, 1998). Yeast fermentation produces reducing sugars, which interact with
the dough proteins on the surface, under the influence of heat. This process is known
as the Millard reaction, which causes browning of the bread crust and contributes
greatly to bread flavour (Brown, 1993; Kent and Evers, 1994).

12

1.1.8.2 Fat / Shortening

Initially fat was only incorporated into bread formulas subject to rapid processing
(Kent and Evers, 1994; Williams and Pullen, 1998). However, even in long
fermentation bread formulas, fat has a considerable improving effect on the quality of
the bread (Hoseney, 1998; William s and Pullen, 1998). Bread with fat stays softer for
longer periods because of the antistaling properties of fat (Hoseney, 1998). Lipase that
occurs naturally in bread flour hydrolyses fats into monoglycerides and diglycerides
(Qi Si, 1997; Poldermans and Schoppink, 1999). Monoglycerides form an insoluble
complex with the amylose portion of starch, resulting in the reduction of
retrogradation (Fig. 1) and hence a softer crumb.

Figure 1.2: The effect of shortening and mono-glycerides on the firming rate of Bread
(Hoseney, 1998).
1.1.8.3 Water
After flour, water is the second most abundant ingredient in breadmaking, but Its
importance is often overlooked (Gould, 1998; Cauvain and Young, 2000). Water is of
great significance for both quality and economic concerns. From a quality
perspective, water plays a two-fold role. Firstly, water acts as a solvent during the
formation of bread dough. When all the ingredients are mixed together for dough
formation, water hydrates the flour proteins and forms the water phase, in which the
soluble solids are dissolved and the yeast is dispersed (Brown, 1993; Cauvain and
Young, 2000,). Secondly, water acts as a plasticiser during mixing and after baking
(Hoseney, 1998; Matz, 1989;Cauvain and Young, 2000). With the aid of energy

13

during mixing, the gluten proteins are transformed into the gluten network. Postbaking it continues its role as a plasticiser when it plays an important part in texture
determination of the final product (Cauvain and Young, 2000). Consumers determine
the freshness of baked bread by means of the squeeze test, therefore the higher the
amount of water remaining in the bread, the softer and the more acceptable the
bread (Gould, 1998; Cauvain and Young, 2000). UK bakers have gained a reputation
for adding excess water (Gould, 1998), which happens to be solely for economic
reasons. Increasing the amount of added water will lead to an increase in yield and
hence a cost saving for the manufacturer (Cauvain and Young, 2000). Apart from that,
increased water absorption can also provide a softer, fresher product which has an
increased shelf life.
1.1.8.4 Salt
Sodium chloride (salt) is included at levels of about 2% in breadmaking (Kent and
Evers, 1994; Cauvain, 1998b; Hoseney, 1998). The main reasons for adding salt are
to: Develop flavour without salt bread is tasteless. Salt also intensifies the bread
flavour developed by other ingredients (Kent and Evers, 1994;Cauvain, 1998b).
Retard fermentation-salt is used to control fermentation (Kent, 1994, Williams and
Pullen, 1998). Bread without salt has excessive proof and on the brink of collapse
with an uneven surface. Strengthen the gluten by suppressing the repulsion charges
and increasing the molecular interaction between protein chains (Kent and Evers,
1994; Hoseney, 1998;Stauffer, 1998). Affect the crust colourin controlling yeast
activity, salt ensure that sufficient sugar is available for the browning reactions to take
place (SA Chamber of Baking, 1995).
1.1.8.5 Ascorbic Acid
Ascorbic acid acts as an oxidising agent in the dough at levels of <200ppm of flour
weight (Williams and Pullen, 1998). It is chemically classed as a reducing agent
(Williams and Pullen, 1998; Cauvain, 2001), but in the presence of atmospheric
oxygen oxidises to dehydroascorbic acid (Kent and Evers, 1994; Williams and Pullen,
1998). The beneficial consequence of ascorbic acid comes into play during mixing of
the dough. Here the gluten network experiences disulphide-sulphydryl interchange i.e.
weakening of the gluten network due to breaking of disulphide bonds (Fig. 3).
Williams and Pullen (1998) suggested that ascorbic acid works by either oxidising S
14

 H groups to SOH making it impossible for 3 disulphide-sulphydryl interchange

disulphide-sulphydryl interchange to occur or by forming new crosslinks between the
protein chains. Secondary benefits as a result of the strengthened gluten include better
gas retention leading to improved loaf volume (Kent and Evers, 1994).

Figure1.3: Representation of disulphide-sulphydryl interchange (William and

pullen,(1998)

15

1.1.9 Water Absorption

Water absorption in baked products can be defined as the amount of water addition
required to produce a dough optimum for processing. Increasing the amount of added
water will lead to an increase in yield and consequently a cost reduction. (Cauvain
and Young (2000),)
Water is a major constituent in most food products and accounts on average for
approximately 70% of the weight of a food product (Potter, 1995). In bread
formulations water accounts for about 60%, depending on the type of processing
method used (Cauvain, 1998a). Apart from processing parameters, intrinsic
parameters (Kent and Evers, 1994) as well as additional ingredients (Cauvain and
Young, 2000) can influence the amount water addition. These intrinsic parameters
have an important role toplay in dough rheology and the characteristics of the baked
bread (Cauvain and Young, 2000)

1.1.10 Summary
The use of enzymes instead of chemical is a very interesting option to improve bread
making performance of dough and bread, because they are perceived as natural and
non-toxic food components.
Amylase; Maximizes the fermentation process to obtain an even crumb structure and
a high loaf volume Improves shelf-life of bread and cakes
Glucose oxidase; Oxidative reaction with gluten to make weak doughs stronger, drier
and more elastic
Lipase; Modifies the natural lipids in flour to strengthen the dough
Xylanase; Dough conditioning. Easier dough handling and improved crumb structure

16

1.2 Objectives
1.2.1 Main objective

The major objective was this study determined the Optimum levels of
Enzymes for chosen proportion of hard and medium wheat flour and
determined the relationship between the effect of concentrations of Enzymes
on the bread quality and dough rheology.

1.2 2 Specific objectives

Replace artificial or synthetic additives which are using for improve bread
Flour

Increase the soft wheat proportion in current bakers flour which is produce by
the company.

17

CHAPTER 2
MATERIALS AND METHADALOGY
2.1 Flour
The Flour was got from serandib flour Mills. Two types of milled wheat Flour were
used for this study. Which was containing Chosen proportion of Hard & medium
Wheat flour and current bakers flour produce by the company. It was properly packed
into bags Stored at normal room temperature.
2.2 Enzymes
Commercial source of Amylase, Glucose oxidase, Xylanase, Lipase in ppm levels
were used for this study

2.3 Ingredients
Commercial sugar, salt, yeast, improvers use as other ingredients

2.4 Analysis of flour samples

Current bakers flour (Reference), new blend of flour which containing hard and soft
wheat and enzyme supplemented flour were analyzed as below procedures

18

2.4.1 Moisture (AACC-44.19)

Amount of free water in sample was Determined using this method.
Moisture content of the flour was determined by using Rapid Moisture method this
was done in a moisture analyzer at a temperature of 135C.for 5g of flour.
2.4.2 Protein (AAcc-46.12)
Amount of protein of the flour was determined using this method.
1.00 g of sample was transferred on kjeldhal digestion tube. Catalyst powder and 25
ml of conc. H2SO4 were added to the tube. Then the tube was connected to fume trap
and attached to the pump. Sample was allowed to digest for two hours. Then the
sample was cooled. Sample was transferred to kjeldhal distillation apparatus which
has been previously conditioned by passing steam for several minutes. Then
distillation was done. Obtained ammonia trapped solution in to titration flask and it
was titrated with standard HCl solution. The endpoint is pink colour. Same procedure
was applied to a blank sample as well.
Protein % was calculated using following Formula
Protein % = (Sample titre Blank titre) x Molarity of HCl x 5.8 x100
Weight of sample taken

2.4.3 Wet gluten (ICC.13.7; AACC-38.12.A)

Wet gluten is the plastic-elastic substance. The gluten content of the flour was
determined by using glutomatic system
10g of flour +0.01g was weighted and put into the washing chambers then added
4.8ml of 2% NaCl solution after that chambers fixed in to the machine This was then
washed to isolate the wet gluten, which was weighed after the residual adherent water
was removed by centrifugation.

19

2.4.5 Ash (AACC-8.12)

Overall mineral content of the wheat flour was determined using this method 2.00g
Flour samples was weighed in to crucible and heated in muffle furnace at 600 C for 2
hours the residue was cooled to room temperature and then weighed.
Ash content was calculated by below formula
Ash%=

100 (final weight-initial wt.) *100

Sample Wt. (100-Moisture content)

2.4.6 Falling number

The time allowed for a viscometer stirrer to fall through an aqueous flour gel, was
measured in seconds. The falling number is an indication of the amylase activity
7.00g Flour samples was weighed in to viscometer tubes for 14% moisture basis.
Then 25ml of distilled water was added in to the each tube. Then dry stoppers were
fixed and shake vigorously 25 times for homogenization. Replaced the stirs in to tubes
and kept in the machine

2.4.7 Farinograph (AACC-54.21)

The Brabender Farinograph was used as a tool to measure shear (fluid) and viscosity
of a mixture of flour and water. Using this method the resistance of dough during
mixing (consistency) was measured.
Consistency in terms of the Farinograph refers to the resistance of the dough,
measured in torque and expressed in Farinograph Units (FU).
Water absorption, Stability, Development time, Breakdown time. Degrees of
softening was analyzed using this method

20

2.4.8 Extensograph (ICC Standard No. 114/1).

The extensograph determines the resistance and extensibility of dough by measuring
the force required to stretch the dough with a hook until it breaks
Dough was formed from flour, water and salt and mixed under standard condition.
From this dough, a test piece 150g was balled and then put through a molder. After a
fixed period of time (45, 90 and 135min) the dough was stretched and the force was
recorded.

21

2.6 Methodology
Each two flour samples (current bakers flour reference and new blend of flour which
containing hard and soft wheat were analyzed for moisture content, ash, protein,
farinograph, extensograph, wet and dry gluten ,gluten index, falling number
In order to have general over view of enzyme effect on flour,
Enzymes were chosen in different concentration levels (low, medium, high) and
supplemented with flour. As Table 02

Enzyme

Concentration (ppm)

Amylase

10

15

G:oxidase

10

20

30

Lipase

10

20

30

Xylanase

10

30

60

Table 2.1: Enzymes concentration levels supplemented with flour

The effect of different enzymes in different levels on, gluten quality, gluten amount,
gluten index, dough rheology, water absorption, falling number and final quality of
bread was investigated.
The effect of enzymes on gluten quality was evaluated concerning amount of wet and
dry gluten.
Water absorption, stability, development time, was analyzed using farinograph test.
Dough rheology (extensibility, resistance) was analyzed using extensograph

22

In order to find optimum levels of enzymes combination for chosen proportion of

hard and medium wheat flour blend, flour was treated with enzymes in recommended
dosage levels.
Four Enzymes combinations were prepared according to recommended range and
appropriate dosage levels.
Each enzyme was weighed out in ppm levels for 1 kg of flour basis.
Four Enzymes premixes were prepared, enzyme premixes were supplemented with
1kg of flour (four samples), and then those were sent to the mixer in order to be
properly dispersed.
Enzyme combinations activity was evaluated by the measurement of loaf volume and
in addition to that sensory analysis was done for evaluate bread quality with reference.

Enzyme

Reference

Treatment1

Treatment

Treatment3

Treatment4

239

230

233

235

237

M1

M2

M3

M4

Amylase

G-oxidase

lipase

Xylanase

Table 2.2: Enzyme combination for Bread preparation

23

2.6.1 Bread making procedure

Fermented dough procedure was done for this study
Ingredients
Flour
Sugar
Salt
Fat
Yeast
Ascorbic
Improvers
Water

Quantity
1000 g
20 g
18 g
20g
3g
acid+ 3g
610g

Ratio %
100
2.0
1.8
2.0
0.3
0.3
61.0

Table 2.3: Bread formulation

Flour (supplemented with enzymes), water, salt, yeast, fat, improvers were mixed in
above ratios and kneaded for 7minutes: Development of the gluten structure.
Then dough was kept for 90min: Incorporation of air bubbles within the dough and
Development of flavor compounds of the dough.
After that dough was divided into 450g mass pieces and kept for 10min. Then those
pieces were moulded and put into trays.
Moulde dough were kept in proofer for 2.5 hours for Fermentation and expansion of
the shaped dough pieces
Finally those were kept in oven for 25min in 185 centigrade: Further expansion of the
dough pieces and fixation of the final bread structure

24

2.6.2 Loaf volume

Loaf volume was measured by using laboratory scale
2.7 Sensory Attributes
Sample preparation for sensory evaluation
The bread/sliced bread samples were labeled with a randomly assigned 3 digit code.
Sensory parameters were assessed on reference and enzyme combination treated
bread samples.
2.7.1 Sensory Evaluation of bread
Trained Panel
10 Trained Panelists who were staff members in the Serandib flour mill used for
sensory. (Male and female). Panelists were given bread samples and asked to overall
appearance, crust colour. Symmetry of form, texture, grains, mouth feel and overall
acceptability. The panelists were asked to evaluate above characteristic using a proper
questionnaire with 5 point hedonic scale where 5 represented the highest order of
preference and 1 the least order of preference.

25

Chapter 3
RESULTS AND DISCUSSION
Table 3.1 below shows the difference in each attribute of current bakers flour which is
producing by the company and chosen proportion of hard and medium wheat flour
blend (without additives)

Parameter

Reference
(without additives)

New Bakers Flour

(without additives)

Moisture %

13.8

13.5

Ash %(as is)

0.47

0.51

Protein %(as is)

12.5

11.8

Wet Gluten %

32.5

31.7

Gluten Index %

80

80

Falling Number

530

630

Water absorption

65

64

Table 3.1: Test results of current bakers flour and chosen proportion of hard
and medium wheat flour blend

Table 3.1 was explained the differences between current bakers flour and chosen
proportion of hard and medium wheat flour blend (without additives)
It was observed in new bakers flour ash and falling number higher than reference and
lower wet gluten, water absorption values.

26

Enzyme

Level

water absorption (FU)

Control

Amylase

Glucose oxidase

Lipase

Xylanase

64
5ppm
10ppm
15ppm
10ppm
20ppm
30ppm
10ppm
20ppm
30ppm
10ppm
30ppm
60ppm

63.2
62.4
61.8
65.5
65.8
66.5
65.4
65.8
66.0
65.2
65.5
66.3

Table 3.2: water absorption of flour for each enzyme in different level

66
65
64
63
62
61
60
contol

Amylase 5ppm

Amylase 10ppm

Amylase 15ppm

Graph 3.1: water absorption of flour with amylase enzyme in different levels
According to graph water absorption of flour was decreased with amylase
concentration

27

67
66.5
66
65.5
65
64.5
64
contol

Glucose oxidase
10ppm

Glucose oxidase
20ppm

Glucose oxidase
30ppm

Graph 3.2: water absorption of flour with G:O enzyme in different levels
According to graph when increasing concentration of glucose oxidase enzyme water
absorption of flour was increased

66.2
66
65.8
65.6
65.4
65.2
65
64.8
64.6
64.4
contol

Lipase 10ppm

Lipase 20ppm

Lipase 30ppm

Graph 3.3: water absorption of flour with lipase enzyme in different levels
According to graph water absorption of flour was increased with adding lipase
enzyme

28

66.5
66
65.5
65
64.5
64
contol

Xylanase 10ppm

Xylanase 30ppm

Xylanase 60ppm

Graph 3.4: water absorption of flour with xylanase enzyme in different levels
According to graph when increasing concentration of glucose oxidase enzyme water
absorption of flour was increased

Development
Enzyme

Level
Control

Amylase

Glucose oxidase

Lipase

Xylanase

Stability (min)

Time (min)
9.5

12.1

5ppm

9.3

12

10ppm

2.2

11.7

15ppm

1.9

8.6

10ppm

9.5

20

20ppm

23

20.8

30ppm

14.2

17.3

10ppm

6.3

11.5

20ppm

5.5

30ppm

6.0

11

10ppm

9.5

11.9

30ppm

5.2

6.5

60ppm

4.8

5.4

Table 3.3: Farinograph development time and stability for each enzyme
concentration level with flour

29

14
12
10
8

Development Time
(min)

Stability (min)
4
2
0
contol

Amylase
5ppm

Amylase
10ppm

Amylase
15ppm

Graph 3.5: development time and stability of flour with amylase enzyme in
different levels
According to graph development time and stability was reduced compare to control
when increasing amylase concentration

25
20
15
10
5

Development
Time (min)

0
contol

G.O 10ppm

G.O 20ppm

G.O 30ppm

Stability (min)

Graph 3.6: development time and stability of flour with G:O enzyme in
different levels
According to graph development time and stability was increased compare to control
when increasing Glucose oxidase concentration

30

14
12
10
8
Development Time
(min)

6
4

Stability (min)

2
0

Graph 3.7: development time and stability of flour with xylanase enzyme in
different levels
According to graph development time and stability was reduced compare to control
when increasing xylanase concentration

14
12
10
8

Development Time
(min)

Stability (min)
4
2
0
contol

Lipase
10ppm

Lipase
20ppm

Lipase
30ppm

Graph 3.8: development time and stability of flour with lipase enzyme in
different levels
According to graph development time and stability was reduced compare to control
when increasing lipase concentration

31

Enzyme

Level

45
min
162

90
min
153

135min

5ppm

178

10ppm

Glucose
oxidase

Lipase

Xylanase

Resistance (BU)

149

45
min
364

90
min
512

135
min
553

169

163

358

486

574

174

171

164

394

503

528

15ppm

175

170

161

390

481

518

10ppm

157

160

136

414

568

630

20ppm

163

152

150

357

492

636

30ppm

158

152

143

438

568

716

10ppm

191

192

176

258

298

278

20ppm

193

187

195

228

299

299

30ppm

165

167

156

342

390

416

10ppm

180

165

164

362

474

489

30ppm

202

203

195

224

256

238

60ppm

217

219

218

196

204

218

Control

Amylase

Extensibility (mm)

Table 3.4: extensograph results for (45min, 90min, and 135min) Extensibility and
resistance of flour with each enzyme concentration

32

180
175
170
Extencibility (mm) 45
min

165
160

Extencibility (mm) 90
min

155
150

Extencibility (mm) 135

min

145
140
5ppm

10ppm

Control

15ppm

Amylase

Graph 3.9: Extensibility of flour with Amylase enzyme in different levels

According to graph extensibility was increased compare to control when increasing
amylase concentration

165
160
155

Extencibility (mm) 45
min

150

Extencibility (mm) 135

min

145

Extencibility (mm) 135

min

140
10ppm
Control

20ppm

30ppm

Glucose oxidase

Graph 3 10: Extensibility of flour with G: Oxidase enzyme in different levels

According to graph extensibility was decreased compare to control when increasing
glucose oxidase concentration

33

200
190
180

Extencibility (mm) 45
min

170

Extencibility (mm) 90
min

160

Extencibility (mm) 135

min

150
140
10ppm
Control

20ppm

30ppm

Lipase

Graph 3 11: Extensibility of flour with Lipase enzyme in different levels

According to graph extensibility was increased compare to control when increasing
lipase concentration up to 20 ppm, but in 30ppm it was decreased

230
220
210
200

Extencibility (mm) 45
min

190
180

Extencibility (mm) 90
min

170
160

Extencibility (mm) 135

min

150
140
10ppm
Control

30ppm

60ppm

Xylanase

Graph 3 12: Extensibility of flour with Xylanase enzyme in different levels

According to graph extensibility was increased compare to control when increasing
xylanase concentration

34

Enzyme

Level

Wet Gluten%

Dry Gluten%

5ppm
10ppm
15ppm
10ppm
20ppm
30ppm
10ppm
20ppm
30ppm
10ppm
30ppm
60ppm

31.7
31.4
31.6
31.2
31.4
31.6
30.6
31.6
31.2
31.6
31.4
31.7
31.6

10.3
10.0
10.2
10.0
10.1
10.3
10.0
10.3
10.0
10.3
10.2
10.4
10.3

Control
Amylase
Glucose
oxidase
Lipase

Xylanase

Gluten
index %
80.44
81.21
80.70
81.73
81.21
80.70
83.33
80.70
81.73
80.70
81.21
80.44
80.70

Table 3.5: Amount of wet and dry gluten for each enzyme level
According to table 3.4, amount of wet gluten and dry gluten was not showed
significant difference compare to control with each enzyme level; it was showed
enzymes not effect for gluten amount on flour

Enzyme

Level

Falling Number

5ppm
10ppm
15ppm
10ppm
20ppm
30ppm
10ppm
20ppm
30ppm
10ppm
30ppm
60ppm

630
570
530
505
594
580
585
585
569
596
540
452
431

Control
Amylase

Glucose oxidase

Lipase

Xylanase

Table 3.6: Falling number for each enzyme level and control sample

35

Falling Number
700
600
500
400
300
Falling
Number

200
100

Control

Amy:

G:O.

Lipase

60ppm

30ppm

10ppm

30ppm

20ppm

10ppm

30ppm

20ppm

10ppm

15ppm

10ppm

5ppm

Xylanase

Graph 3.13; falling number for each enzyme levels

It was showed when increasing amylase enzyme and xylanase enzyme reduce the
falling number

Sensory characteristic
According to the results, panelist found no difference (p>0.05) with respect to the
Overall appearance, crust color, symmetry of foam, mouth feel of the samples
(Appendix 02)
However panelist was found significant difference (p<0.05) with respect to texture,
grain and overall acceptability

36

CHAPTER 4
CONCLUSIONS AND RECOMMENDATION
Enzymes had positive effect on wheat flour by dough strengthening, conditioning
dough rheology and water absorption. Regarding the effect on final baked product,
enzymes improve the quality of bread by improving volume and other characteristics
Except treatment1 all other enzyme treatments were shown no significant difference
from the reference. So that according comparison cost of enzyme treatments it was
found that amylase 6ppm, glucose-oxidase 15ppm, lipase 25ppm and xylanase 40ppm
combination was the cheapest and best enzyme treatment for chosen proportions of
hard and medium flour blend
Enzymes can use for bakery products are made from composite flour or weak wheat
flour which are less suitable for bread making

37

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41