enzymes and their role in bread and chapatti … pcbs/volume/2017/issue1... · different enzymes...

Journal of Scientific Research in Pharmaceutical, Chemical &

Biological Sciences

Volume (2) Issue (1) Year (2017)

ISSN : 2455-8044

Received: 20 May 2017 Accepted: 14 June 2017 Published: 27 June 2017

Corresponding author:

ENZYMES AND THEIR ROLE IN BREAD AND CHAPATTI INDUSTRY: AMYLASES, LIPASES, AND XYLANASES

Sikander Ali*, Zobia Tabassum, Rida Fatima, Saamia Mukhtar

Institute of Industrial Biotechnology Govt. College University, Lahore.

ABSTRACT

From many years enzymes have been used that can catalyze reactions and its

chemical nature was also known. Enzymes have various applications in many fields

of science. The enzymes that are used in bread making will be added separately or in

the form of complex mixtures, that can behave collectively in the bread production.

Lipases, Xylanases, Amylases are the three major enzymes that are used in bakery

industry. Bread and Chapatti industry is very important for mankind and for economy

where main source of protein is wheat flour. Use of enzymes in bread industry is one

the greatest evolution. Three main processes involved in these industries are mixing,

fermentation and baking. Different enzymes play vital role in the production of bread

and maintaining its texture. Amylases are the enzymes which are commonly used in

bread making for the standardization of the flour and act as anti-staling agents.

Lipases are found widely in nature and are generally present in all cereal grains. The

activity of lipases in white flour is low but it is enough to avoid the rancidity caused

by the hydrolysis of the native lipids and native fats. Xylanase is a naturally

occurring enzyme which is produced by different microbes. The xylanases transform

water insoluble hemicellulose into the soluble form which binds to the water present

in the dough which results in the decrease of the dough furnace.

Keywords: Amylases, Xylanases, Lipases, bread industry and chapatti industry,

baking.

Sikander Ali et. al. 2

J. Sci. Res. Phar. Chem. Bio. Sci. Vol. 2(1),

INTRODUCTION

The enzymes have been used from decades to catalyze the reactions and their

complete chemical nature was also known. In 1960 first industrial process was developed

in which enzymes were used (Illanes, 2008).In recent years the use of enzymes in various

industries was understood in which food, laundry, feed, textiles, detergents, tanning,

cosmetics and pharmaceutical industries were included. Mostly used enzymes (almost

50%) which are marketed today are obtained from organisms that are genetically

modified. The enzymes that are used in industries accounts for over 80% (van Oort,

2010).The most widely used enzymes are that which are used in food industries.

Regarding to the use of enzymes there are present to possibilities that are either we use

enzymes just to convert the raw material in to main product or to change the functional

characteristics of products we use enzyme as additives. In the mentioned possibilities, in

first case the processes of enzyme action to increase the catalysis mechanism of enzyme

is carried out under controlled and optimize conditions. In second case one difficulty is

to control the reaction of enzyme and the other one is the optimized controlled

conditions.

The main event in mankind was the development of bread process. With the

enhancement in the fields of agriculture the prices of bread was suddenly decreased and

its quality was improved and with this white bread was easily available. In the process of

baking the industrially used enzymes led to the development of baking market. In 2010,

the market of enzymes for baked products in considerably increased from 420 million $

to 900 million $ in 2020 (Figure 1). The demand of enzymes used in world food and

beverages is basically responsible for the enzymes that are used in industries worldwide

by 40.1%.

FIG 1: Enzymes based baked product market increased from 2010 to 2020

The process of baking is basically used for making the baked products like bread;

a biscuit etc. the production of baking products is made with different ingredients which



mean the formulations vary form product to product. The production of bread is basically

done with the help of wheat flour which is used as raw material and this wheat flour is

actually a mixture of starch, lipids, non-starch polysaccharides, gluten and enzymes.

With the mixing of flour, yeast and addition of water the processes begins which are then

catalyzed the enzymes that are present in wheat flour and yeast. These complex

processes will continue in the phase of baking that will led to the development of bread.

We have to add extra enzymes that will helps in control processes of baking. The basic

component of baked product like bread is starch which when added in various foods can

help as thickening agent, as a water binder (Synowiecki, 2007).

The major components of starch are amylose and amylopectin (Goesaertet al.

2005). The amylose is actually a linear moleculeof glucose having 6000 glucose units

with α-(1,4)-glycosidic bonds. And the amylopectin is not a linear molecule it is actually

a branched polysaccharide having 10-60 glucose units which are α-1,4 linked and side

chains contain 15-45 glucose unite that are α-1,6 linked, containing approximately 2

million glucose units as shown in figure 2.

FIG 2: Amylose and Amylopectin structure

Enzymes in bread and chapatti industry

In baking the enzymes which are used are basically from three major sources that

are the use of enzymes in flour that are endogenous in nature, the enzymes related with

the organisms that are dominant, the enzymes added in dough that are exogenous in

nature. The normally used method for the regularity of flour and used in baking is the

addition of dough and flour along with enzyme improvers. The enzymes that are used in

bread making will be added separately or in the form of complex mixtures, that can

behave collectively in the bread production. Table 2 gives us the information about the

enzymes and the effect of these enzymes that are used in bread and chapatti industry.



Table 1: Enzymes used in bread and chapatti industry.

Enzyme Effect

Amylase It increases the rate of fermentation process and gives a

shredded structure

Lipase It helps in the modification of naturally present lipids in flour to

toughen the dough

Xylanases It helps in the conditioning of dough and in easy handling

Lipoxygenase It helps in the strengthening of dough and in its bleaching

properties

Maltogenic alpha-amylases It helps in the enhancement of shelf life of bread

The three main enzymes which we are going to discuss are Lipases, Xylanases

and Amylases.

Lipases

Lipases are most commonly used in many industrial products and in industrial

processes such as food, detergents and in pharmaceutical companies. In the food

industries lipases when used in one single application can have multiple roles as shown

in figure 3.

FIG 3: Industrial applications of lipases

Lipases are the third largest group of enzymes that are used commercially after

peptidases and carbohydrases. The organisms that produce the lipases are Yeast,

Archaea, Bacteria, Mold, Eucarya. The Lipases that are produced by microorganisms

extracellularly are widely used in many commercial applications. The lipid’s ester bond

hydrolysis and synthesis is basically catalyzed by the use of lipases with the help pf other

enzyme that is esterases. The most common difference between lipases and esterases is

their interfacial activation. The lipases when activated and placed at the interface



between the non-aqueous and aqueous media can be able to carry out the reaction faster.

On the other hand esterases works by acting on substrate that is dissolved in water phase.

The presence of water repellent oligopeptide surface loop that is covering the enzyme’s

active site helps in the characterization of lipases. The entrance of substrate in the active

site of enzyme is enhanced due to the conformational changes that are introduced at the

interface by this lid. Due to this the non-polarity of surface that is usually surrounding

the catalytic site is enhanced.

FIG 4: Structural view of lipases

Amylases

In biotechnology, the most common and the most important enzyme used are

amylases. These amylases occupy almost 26% of total enzyme market today. Every

enzyme is produced from one or more sources so amylases are also produced from many

different sources like plants, microbes and animals. Now days, on commercial level

many amylases are produced by microorganisms. When we produced amylases by using

plant and animal sources they are less stable than that amylases that are produced using

microorganisms and they also have wide range of applications in industries. The most

common benefit which we get when produced amylases using microbes is that we can

easily alter that microorganisms if we want the enzyme of our desire characteristics.

Alpha-amylases are produced from many yeasts, bacteria and fungi. The alpha

amylases that are produced using fungi and bacteria have wide range of applications in

industries. Alpha amylases that are produced from bacterial and fungal source also have

advantages in many industries like food, paper, fermentation and detergents. When the

field of biotechnology develops it enhances the application of amylases in many

industries like clinical, and medicinal. In many industries these alpha amylases have

many applications and the enzyme that is produced by microorganisms clearly shows

that. Amylases are of two types: Endoamylases, Exoamylases. In endoamylases, they



only drive hydrolysis in an unusual way and this is done in the inner side of the molecule

of starch. Due to this hydrolysis we get oligosaccharides that are linear and branched and

they are of variable length. In case of exoamylases, they result in a short chain length of

products by simply hydrolyzing from the ends that are non-reducing.

Xylanases:

Xylanases are actually molecules of glycoproteins that occur as single chain and

they vary from 6-80 kDa. At a temperature ranging from 40-60 °C xylanases are active

and the pH range for this is 4.5-6.5. The different range of pH and temperature is

required when xylanases are extracted from different sources. In biotechnology

xylanases have variety of applications. They have vast applications in food, paper and

pulp industries. This enzyme is most commonly produced by Bacteria, Fungi, Protozoa

and Molds.

Staphylococcus, Micrococcus, Microbacterium and Bacillus are the bacterial

genera that mostly produce xylanases. Streptomyces, Actinomadura, Nonomuraea are the

fungal genera that most commonly produce xylanases. Xylanases that are produced by

bacteria get more advantage over fungal amylases because the best suitable pH range for

bacterial amylases is alkaline or neutral but fungal amylases require acidic range of

pH.The microorganisms that produce xylanases (xylanolytic) exist in an environment

that has extreme environmental conditions. The most common polysaccharide that is

present in plants and woods that are hard in nature is Xylan.

Bakery industry

Process of bread making is very important in mankind. After 19th

century with

improvisation in agriculture price of bread was decreased but its quality was improved,

hence it became economic for every person. Use of enzymes was one of the greatest

evolutions in baking industry. Three major groups of enzymes commonly used in baking

are Enzymes that hydrolyze carbohydrate (amylases, cellulose, pentonases), Enzymes

that hydrolyze proteins (Proteases), Enzymes that effect fats and oils (Lipases,

lipoxygenases)

Baking process

Baking process is mainly divided into three operations Mixing, Fermentation

(resting and proofing), and Baking. By the process of baking the batter or fluid dough is

converted to solid baked product. Indirectly, baking interferes with sensory properties

that improve palatability, texture and aroma of food product from the raw material.

Though baking is being practiced since long time but until now complete process of

baking is unknown because of integration of several complex molecular and physical

processes. Baking includes a chain of physical, biochemical and chemical changes in the

end product. These changes include water evaporation, expansion of volume, protein



denaturation, formation of porous structure, starch gelatinization, formation of crust and

several browning reactions.

Bread is unstable, solid, elastic structure containing elastic network of cross-

linked molecules of protein and polymers of starch molecules. Mechanical mixing,

Thermal effects (time and temperature) and chemical reactions such as enzyme catalyzed

reactions affect greatly properties and nature of end product. The simplest procedure in

baking is mixing of all ingredients and developing dough. Second method is ―Dough and

sponge method‖ which includes mixing of formula ingredients in two steps. In first step,

preparation of leaving agent is carried out by mixing yeast with certain amount of flour

and water. Mixture is allowed to stay for several hours and then mixed with other

ingredients. A third procedure is named as ―Chorleywood method‖ in which ultrahigh

mixer is used for mixing of ingredients (Giannruet al. 2003).

Commonly used leaving agent in traditional bread making is yeast

Saccharomyces cerevisiae, but beside these lactic acid bacteria mainly Lactobacillus

species may be used as leaving agents for making dough sour.Baking process starts with

development of dough by mixing ingredients that are: flour, yeast, water, sugar, salt and

some other ingredients. Flour particles are sheared and hydrated during mixing when

gluten proteins form cohesive network having starch molecules dispersed in the network

by polymerization and depolymerization reactions. Air incorporation is very important

that affects final crumb, because yeast produce Carbon dioxide during fermentation may

diffuse in already existing air spaces or air bubbles (Pareytet al. 2011).After resting

stage, dough is further divided into small sized pieces, molded, placed on tray, screened

and baked. Typical solid foam like structure of bread is developed by gelatinization of

starch with gluten proteins. Partially crystallized starch molecules are transformed to

amorphous, intermediate, gelatinized network of starch. These swollen starch granules

are deformed; the polymers of starch come out of granules and then form a smooth

network in bread crumb. Along accumulation of amylase on the outer surface of

granules, an amylase-rich portion was also located in middle of starch granules after

process of baking.

While baking procedure intermediate gluten network that is formed in dough is

converted to permanent network which is predominantly formed due to modifications in

hydrophobicity of surface proteins , disulphide interchanges and formation of new cross-

links of disulphide and this leads to incorporation of α- and γ-gliadins in this network .

Further macroscopic changes that occur during baking are expansion of dough, crust

development and further browning. Yeast is continuously producing carbon dioxide, and

dough is further expanded by heating and vaporization of water and ethanol. The bread is

baked from outside to inside forming a bread crumb. The browning of crust is directly

proportional to formation of reducing sugars (fructose, maltose, glucose etc.) by

hydrolysis of starch and complex sugars of flour, while leavening and dough production.



Under heating, results in reaction of reducing sugars with free amino acid groups of

proteins produced in Millard reaction. Different flavored compounds are also produced,

which gives bread appealing texture, aroma and taste. Amylose chains are crystallized.

Amylose can also form complexes with lipids which are polar in their nature which gives

softness to fresh bread crumb. When bread loaf is removed from oven after process of

baking, a chain of unpredictable changes called as staling starts which eventually leads to

deterioration of quality. Staling refers to short shelf life of baked product. It may leads to

loss of freshness of bread and leading to undesirable aroma and non-acceptance of

market sales (Moayedallaieet al. 2010).

Chapatti industry

Chapatti are also named as flat breads, they are very popular among those

countries where major source of proteins and calories comprises of bread. There are

many types of flat breads, and variations are due to difference in ingredients,

terminology and quality. To improve quality of food products many modifications are

made in formulations. In India and Pakistan, wheat is staple, which is consumed in the

form of various flat breads such as puri, Paratha, Chapatti, Phulka and Tandoori Roti.

Various varieties of wheat are used to produce flat breads. Recently, researchers are

trying to improve ingredients, organoleptic properties, nutritional value and most

important shelf life of flat bread. Flat breads are mainly produced by mixing salt, flour

and water in definite proportion. Sometimes producers may add yeast fat, skim milk

powder or certain additives (preservatives, emulsifiers etc.) for increasing shelf life and

taste(Shalini and Laxmi, 2007).

Process of making Chapatti

Main ingredients of flat bread are water, flour, NaCl and leaving agent. Numbers

of operations are carried out to convert raw materials into final product. Such processes

are carried out in such a way that the dough formed posses special sensory and

mechanical properties which allow it to regain gas and produce loaf of well expanded

bread.

FIG 5: The flow sheet for the preparation of flat breads



Mixing is very crucial and important step in preparation of flat bread which

ensures that all ingredients are homogenized in mixture or batter. While mixing dough it

is ensured that wheat flour is hydrated and gluten protein passes are disrupted and

transformed to cohesive elastic network of gluten protein. This network is intermediate,

because of dynamic exchange of disulphide bonds between various polymers of proteins.

This protein network is primarily made up of glutenins, which are globular proteins

which don’t play any role in transient network formation. Rheology of wheat flour based

dough of bread is based on glutenin network, and some biopolymers that also participate

in dough making. An intermediate entangled network may be formed from amylase and

amylopectin from damaged starch. Mixing is partly dependent upon type of mixer used,

absorption of water, speed of mixer and type of bread which is desired.

Ability of water absorption and changes in swelling of dough leads to difference

in processing strategies during mixing. Flat breads are considered to be more tolerant to

under and over mixing process. Over mixing results in breakdown of gluten, breakage of

disulphide bonds and gluten may be partially depolymerized, which causes greater

solubility and our dough becomes sticky. This sticky dough is difficult to handle or

might become stiff, which yields poor quality bread. On the other hand under mixed

dough has under developed gluten network, which is less elastic thus resulting in poor

volume of chapatti and is not satisfactory (Slade and Levine, 1993) (Dewettinck et al.

2008).

Fermentation is one of the basic requirements of almost all the flat breads

produced worldwide. After mixing fermentation tie was varied from 0 to 3 h at room

temperature (20˚C - 35˚C). Fermentation is mostly carried by yeast or sour dough that

uptake certain substances and then produce some of the byproducts which could affect

properties of dough thus making it extensible and lighter. More the fermentation time

more will be the alteration in properties of dough. Molding is one of the important steps

in flat bread production because in thus step dough is flattened to various sizes and

shapes having uniformity in thickness. Sheeting and molding leads to subdivision of

previously existing gas cells, thus improving screening process (i.e., number and size

distribution). During fermentation gas nuclei is expanded due to release of various

fermentation gases. Temperature also increases due to release of these expansion gases.

Flat breads are immediately baked straight after flattening process (Hoseneyet al. 1978).

Baking is most important and last step in chapatti making process. Chemical and

physical properties of dough determines quality of end product .A chain of chemical and

physical changes like expansion of volume , denaturation of protein , moisture loss ,

rupturing of gas cells , crust formation etc. takes place during baking . Heating directly

affects swelling, hydration properties and extent of re-association. Solid foam like

structure of flat bread is determined by gelatinization of starch and pasting and heat



setting of gluten protein network. Starch gelatinization, heat setting and pasting starts

only when temperature reaches to 65˚C.

During this starch which was partially crystallized was converted to amorphous,

transient, gelatinized starch meshwork. Determining factor for various physiochemical

changes during baking is temperature. Parameters which affect Shelf life and quality of

bread are mainly temperature and time. Optimum temperature range is of 350˚C - 550˚C

for production of good quality chapatti. They are baked in very short time so that they

don’t lose their moisture contents and softness. Traditionally tawa is commonly used for

Chapatti preparation and Tandoor is used for preparation of Tandoor flat breads. To cope

up with daily increasing demand of flat breads, methodology to produce ready to eat flat

breads areassutting good quality product.Manohar and Sridhar made a machine for

making Chapatti which has production capacity of 160 kg /h of dough extrusion at

moisture content of about 65%-70%. (Saxenaet al. 1995)

Role of Enzymes

Amylases

Different enzymes play vital role in the production of bread and maintaining its texture.

Amylases are also one of such enzyme which are commonly used in bread making for

the standardization of the flour and also act as anti-staling agents (Goesaertet al. 2005,

2006). Amylases are the hydrolyzing enzymes which can degrade the starch to form

the

diverse products. Different types of amylases can be found but two main types are the

alpha-amylases and beta-amylases. The alpha-amylase (E.C.3.2.1.1) is a hydrolase

enzyme which catalyzes the hydrolysis of the internal glycosidic linkages of starch to

obtain the products such as glucose and maltose. It generally depends upon the presence

of a cofactor of metal for its activity. The beta-amylase is an exo-hydrolase enzyme

which acts from the non-reducing end of the polysaccharide chain by the hydrolysis of α-

1, 4-glucan linkages to yield successive maltose units. In the case of wheat flour the beta-

amylases are present in plenty amount which may cause little activity on the undamaged

and on the native starch granules so they are usually inactivated before the starch

gelatinization whereas the alpha-amylases are usually absent. Therefore, the activity of

beta-amylase in the wheat flour is minimized by the addition of alpha-amylases extracted

from fungi or malt which is also rich in alpha-amylases. Amylases play different

important roles i.e. it incredibly increases the level of fermentable and reducing sugars in

the flour and dough, it promotes the fermentation of yeast, and also helps in the

formation of the products formed by the Maillard reaction which is important to intensify

the bread flavor and its color.

Amylase also functions in the reduction of the viscosity of the dough during the

starch gelatinization which prolongs the oven rise, in delaying the crumb firming which

is a porous material with flexible elastic cell walls and also results in the increases



volume of loaf. The main cause of the significant financial loss for both the consumers

and producers are the bread and cake staling which occurs due to the changes in the

starch structure during storage. When the starch granules convert into the insoluble form

from the soluble form it loses it flexibility and become hard and brittle. And through

different mechanisms and mode of actions alpha-amylases help in the retarding or

firming processes because of it anti-staling activity which is the important property of

Amylases. The staling is basically associated with the loss of freshness in terms of

increased crumb firmness, decreased crumb elasticity and loss of the moistness. The anti-

staling products consist of the thermostable fungal or bacterial alpha-amylases (Bowles,

1996; Hebedaet al. 1991). The B. stearothermophilusmaltogenic alpha-amylase is most

effective anti-staling amylases which have expressive sequence homology and also show

starch degrading properties.

The alpha-amylase helps in the degradation of damaged starch present in the

wheat flour into the small dextrin, which enables yeast to work continuously during the

dough fermentation in the early stages of baking and produces improved crumb texture

and bread volume. The attractive baked flavor occurs due to the production of small

oligosaccharides and sugars produced by these enzymes which enhances the Maillard

reaction. Amylase enzyme in the flour has improved the quantities, taste, aroma and

porosity of the bread. It acts as the natural additive because of its ability to replace the

chemical additive i.e. Potassium bromates. The main ingredient for the dough of bread,

roll and buns consist of flour, water, yeast, salt, sugar and fats. And the flour further

consists of gluten, starch, lipids and trace amount of minerals, during the dough

processing the yeast acts on the fermentable sugars and convert them into carbon dioxide

and alcohol which makes the dough rise. Starch is the major component of wheat flour.

The amylases are able to degrade the starch and produce dextrins for the yeast to act on it

continuously during the fermentation of the dough in the early stages of baking which

results in improved bread volume and texture.

Lipases

Lipases are found widely in nature and are generally present in all cereal grains. The

activity of lipases in white flour is low but it is enough to avoid the rancidity caused by

the hydrolysis of the native lipids and native fats (Poutane, 1997). It is more useful in

baking segments as compared to the alpha-amylase and other proteases. Lipases also

plays important role in different aspects I.e. it reduces the risks for the off-flavor

formation when the baked products like butter and milk fat is stored for prolonged period

(Van Oort, 2010), lipases improve the dough rheology, it also provides the better

stability to the mechanical stress on the dough, lipases help in increasing the volume

which usually result to obtain an improved, soft and uniform crumb, along with these

functions of lipases it also increases the wall thickness and reduces the cell density.

Lipases are also used as the food additives in the bread making.



The addition of lipases helps in the retardation of the staling in the baked

products so it also exhibits anti-staling effect. As it is used as the food additive it can

develop particular flavors in the bakery products, it increases the quality of the high fiber

enriched brewer’s grain bread and it also produces beneficial effects during the bread

making. Lipases are used to produce the fiber enriched pan bread by applying the plain

dough method, by using the 50ppm dosage of lipase along with 2% MAG. The lipases

affect the rheological and thermal properties of the white and whole wheat flour doughs.

It can cause modification in the dough component i.e. gluten proteins and starch. Along

with the dough stability it provides better handling properties, maximum resistance to the

extension and also decreases the stickiness. In delaying the starch retro gradation by

lipases is indicated by the formation of amylose-lipid complexes to greater extent.

Xylanase

Xylanase is a naturally occurring enzyme which is produced by different microbes

including fungi which performs important role in the human digestion. It breaks down

hemicellulose in the digestive tract by converting one of its components into a simple

sugar called xylose. It helps to reduce the gas or intestinal discomfort. Xylanases are

used to strengthen the dough in the bakery industry, flour quality and also provides the

excellent tolerance to the dough towards the variation and processing parameters. It also

incredibly increases the volume of the baked bread, helps in greater absorption of water

as well as plays an important role in the improved resistance fermentation. The xylanases

transform water insoluble hemicellulose into the soluble form which binds to the water

present in the dough which results in the decrease of the dough furnace, increasing

volume in creating finer and more in the uniform crumps.

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