Microbial Biotechnology

Chapter Five
Application of Microbes in Food Biotechnology

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5.1 Dairy products

1. Cheese

Cheese is a highly proteinaceous food made from the milk of

some herbivores.

Cheese is originated in the warm climates of the Middle East

some thousands of years ago.

The scientific study and manipulation of milk for cheese

manufacture is however just over a hundred years old.

 Most cheese in the temperate countries of the world such as

Western Europe and the USA is made from cow’s milk. 2
The composition of cow’s milk varies according to the breed
of the cattle, the stage of lactation, the adequacy of its
nutrition, the age of the cow, and the presence or absence of
disease in the breasts (udders), known as mastitis.

 In some subtropical countries milk from sheep, goats, the

lama, yak, or ass is also used.

Sheep milk is used specifically for the production of certain

special cheese types in some parts of Europe.

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Milk from the water buffalo may be used in India and other
countries, while milk from the reindeer and the mare may be
used in northern parts of Scandinavia and in Russia,
respectively.
 Cheese made from the milk of goat and sheep has a much
stronger flavor than that made from cow’s milk. Because the
fat in goat and sheep milk contain much lower amounts of the
lower fatty acids, caproic, capryllic, and capric acids.

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These acids confer a sharp taste to cheese made from these
mammals.

 About a thousand types of cheese have been described

depending on the properties and treatment of the milk, the
method of production, conditions such as temperature, and
the properties of the coagulum, and the local preferences.

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Stages in the manufacture of cheese:

(a)Standardization of milk - The quality of the milk has a

decided effect on the nature of cheese.
Cheese made from skim milk is hard and leathery.

 The more fat a cheese contains the smoother its feel to the
palate. The fat/protein ratio is often adjusted through fat
addition in order to yield a cheese of consistent quality.

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Pasteurization (High Temperature Short Time) must be given to
milk to be in certain types of cheeses, such as cottage or cream
cheese.
For others the milk stored for at least 60 days at 2°C.

Sub-pasteurization temperatures are often the legal compromise.

Pasteurization gives a better control over the processes of cheese

production. However, the organisms present in raw milk are
important during the ripening processes.

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The milk may also be homogenized by forcing it at high speed
through small orifices to reduce the milk fat globules for use in
producing soft cheeses.

b) Inoculation of pure cultures of lactic acid bacteria as starter

cultures

Lactic acid formation is necessary in all kinds of cheese.

For cheese prepared at temperatures less than 40°C strains of
Lacto coccus lactis are used.
For those prepared at higher temp. the more thermophilic
Streptococcus thermophilus, Lactobacillus bulgaricus and Lact.
helveticus are used. 8
Lactic acid has the following effects:

i. It causes the coagulation of casein at pH 4.6

ii. It provides a favorably low pH for the action of rennin, the
enzyme which forms the curd from casein in other types of
cheeses.
iii. The low pH eliminates proteolytic and other undesirable
bacteria.
iv. It causes the curd to shrink and thus promotes the drainage
of whey.

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Metabolic products from the lactic acid bacteria such as
ketones, esters and aldehydes contribute to the flavor of the
cheese.

C) Adding of rennet for coagulum formation: The classical

material used in the formation of the coagulum is ‘rennet’
which is derived from the fourth stomach, abomasum or vell
of freshly slaughtered milk-fed calves.

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Besides those of calves, the abomasum of kids (young goats),
lamb or other young mammals have been used.

Rennet is produced by soaking and shredding air-dried vells

under acid conditions with 12-20% salt.

Extracts from young calves contain 94% rennin and 6% pepsin

and from older cows, 40% rennin and 60% pepsin.

Rennin (chymosin) is the enzyme responsible for the

coagulation of the milk.

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Pepsin is proteolytic and too high an amount of pepsin can result
in the hydrolysis of the coagulum and a resulting low yield of
cheese, and a bitter taste may result from the amino acids.

 Due to the high cost of animal rennet, other sources, mostly of

microbial origins, have been found.

 The major effect of the milk-clotting enzymes is the conversion

of casein from a colloidal to a fibrous form.

First the pH of the milk is brought down from pH 6.8 -7 to pH

5.5 by the action of lactic acid bacteria which produce lactic acid
from lactose in the milk.
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On addition of rennet, the active component, rennin, catalyses the
hydrolyses of k-casein to release para-k-casein and k-casein
macropeptide.

The latter goes into whey, while the para-k-casein remains part of
casein micelles, which now bind together to form the curd
following the removal of carbohydrates with the k-casein
macropeptide and the exposure of binding surfaces.

The events up this coagulation are aided by lowered pH and by

increasing temperatures up to 45°C.

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Most of the bacteria, fat, and other particulate matter are
entrapped in the curd.

When casein is removed the remaining liquid containing

proteins, lactalbumin, globulin, and yellow-green riboflavin
(vitamin B2) is whey.

The whey proteins may be precipitated by heat, but not acid

or rennin and they are used in making whey cheese.

 The enzymes used in cheese making are now obtained from

microorganisms, mainly fungi.

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d) Shrinkage of the curd- The removal of whey and further
shrinkage of the curd is greatly facilitated by heating it, cutting it
into smaller pieces, applying some pressure on it and lowering the
pH.

 In many types of cheeses, such as Parmesan, Emmenthal and

Gruyere, there is a stage known as scalding in which the
temperature can be as high as 56°C in the preparation.

Acid produced by the lactic starters introduce elasticity in the

curd, a property desirable in the final qualities of cheese.

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e) Salting of the curd and pressing into shape: Salt is added to
most cheese varieties at some stage in their manufacture.

Salt is important for the taste, moisture and acidity control.

Most importantly however it helps limit the growth of
proteolytic bacteria which are undesirable.
The curd is pressed into shape before being allowed to mature.

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f) Cheese ripening- The ripening or maturing of cheese is a slow
joint microbiological and biochemical process which converts
the brittle white curd or raw cheese to the final full-flavored
cheese.

 The agents responsible for the final change are enzymes in

the milk, in the rennet and those from the added starter
microorganisms as well as other micro-organisms which
confer the special character of the cheese to it.

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Yeasts, micrococci, and Brevibacterium linens impart the
characteristic flavor of Limburger cheese.
In soft cheese, such as Camembert, the protein is completely
broken down to almost amino acids, whereas in the hard
cheese, the protein remains intact.

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2. Yoghurt
Yoghurt is a fermented milk traditionally believed to be an
invention of the Turks of Central Asia, in whose language the
word yoghurt means to blend.

Although accidentally invented thousands of years ago, yoghurt

has only recently gained popularity in the United States.

Yoghurt has become popular within the last 30-40 years.

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This is due to many factors including the introduction of
fruit and other flavorings into yoghurt, the convenience of it
as a ready-made breakfast food and the image of yoghurt as
a low fat healthy food.

 In the manufacture of yoghurt, two kinds of lactic acid

bacteria, Lactococcus spp. and Lactobacillus spp., are
generally used.

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Most commonly used are Lactococcus salivarius and
thermophilus, and Lactobacillus spp., such as Lacto. acidophilus,
bulgaricus and bifidus.

Milk is heat treated at 97°C for 10 min. and cool to 45°C.

Then inoculated with starter and incubate for about 48 hr. at 4°C
with pH 4.5.

The bacteria produce lactic acid from lactose in the milk causing
the pH to drop to about 4-5 from about 7.0. This drop in pH causes
the milk to coagulate.

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A variation in pH can make the yogurt sweeter and thinner
(higher pH) or thicker and more sour (lower, more acidic
pH). More milk protein coagulates when the pH is lowered.

The lactic acid gives yoghurt its sour taste and limits the
growth of spoilage bacteria. Yoghurt is flavored usually with
fruits.

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5.2 Fermented Foods
1. Bread

Bread has been known to man for many centuries and excavations
have revealed that bakers’ ovens were in use by the Babylonians,
about 4,000 B.C.

Today, bread supplies over half of the caloric intake of the world’s
population including a high proportion of the intake of Vitamins B
and E. Bread is therefore a major food of the world.

 Bread basically consists of flour, water, yeast and a little salt.

Bread leavened with yeast is different mainly in terms of the

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texture and structure, but also has a better aroma and taste.
In modern bread making a large number of components and
additives are used.

They include yeast food, sugar, milk, eggs, shortening (fat)

emulsifiers, anti-fungal agents, anti-oxidants, enzymes, flavoring,
and enriching ingredients.

The ingredients are mixed together to form dough which is then

baked.

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Flour- chief ingredient of bread and is produced by milling the
grains of wheat. It contain starch (70%), protein (7-15%), sugar
(1%), and lipids (1%).

Flour proteins are of two types. The first type forming about
15% of the total is soluble in water and dilute salt solutions and
is non-dough forming. It consists of albumins, globulins,
peptides, amino acids, and enzymes.

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The remaining 85% are insoluble in aqueous media and are
responsible for dough formation. They are collectively known as
gluten. It also contains lipids.
 Gluten has the unique property of forming an elastic structure
when moistened with water.
 It forms the skeleton which holds the starch, yeasts, gases and
other components of dough.
Gluten can be easily extracted, by adding enough water to flour
and kneading it into dough.

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Gluten is separable into an alcohol soluble fraction which
forms one third of the total and known as gladilins and a
fraction (two thirds) that is not alcohol-soluble and known as
the glutenins.
Hard wheat with a high content of protein (over 12%) are best
for making bread because the high content of glutenins enables
a firm skeleton for holding the gases released during
fermentation.

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Yeast- Strains of Saccharomyces cerevisiae used.

The ideal properties of yeasts used in modern bakeries are:

a. Ability to grow rapidly at room temperature of about 20-25°C

b. Easy dispersability in water
c. Ability to produce large amounts of CO2 rather than alcohol in
flour dough
d. Good keeping quality
e. Ability to adapt rapidly to changing substrates
f. Ability to grow and synthesize enzymes and coenzymes under
the anaerobic conditions of the dough
g. Ability to resist the osmotic effect of salts and sugars in the dough
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The amount of yeasts used during baking depends on the flour type,
the ingredients used in the baking, and the system of baking used.
Very strong flours (with high protein levels) require more yeast than
softer ones.
High amount of components inhibitory to yeasts e.g sugar (over
2%), antifungal agents and fat) usually require high yeast additions.
Baking systems which involve short periods for dough formation,
need more yeast than others.
In general however yeast amounts vary from 2-2.75% (and
exceptionally to 3.0%) of flour weight.

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 The roles of yeasts in bread-making are leavening, flavor
development and increased nutritiveness.
Yeast food- contain a calcium salt, an ammonium salt and an
oxidizing agent. Oxidizing agents used include iodates,
bromates and peroxide.

Sugar – added to:

a) provide carbon nourishment for the yeasts.
b) sweeten the bread
c) afford more rapid browning of the crust.
Sugar is supplied by the use of sucrose, fructose corn syrups.
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Shortening (Fat) - Animal and vegetable fats are added as
shortenings in bread-making at about 3% (w/w) of flour in
order to yield:
a) increased loaf size
b) a more tender crumb and
c) enhanced slicing properties.

Butter is used only in the most expensive breads lard (fat from
pork) may be used, but vegetable fats especially soy bean oil.

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Emulsifiers (Surfactants) - used in conjunction with shortening.

Emulsifiers contain a fatty acid, palmitic, or stearic acid.

Emulsifiers are added as 0.5% flour weight. Commonly used

surfactants include: calcium stearyl- 2-lactylate, lactylic
stearate, sodium stearyl fumarate.

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Milk - Milk to be used in bread-making must be heated to high
temperatures before being dried.

 Milk is added to make the bread more nutritious, to help

improve the crust color, presumably by sugar caramelization.

The milk substitutes are added in the ratio of 1-2 parts per 100
parts of flour.

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Salt - About 2% NaCl added to bread. It serves:

a) improves taste

b) stabilizes yeast fermentation

c) As a toughening effect on gluten

d) Retard proteolytic activity, which may be related to its effect on

gluten

e) It participates in the lipid binding of dough.

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Water - needed to form gluten, to permit swelling of the starch,
and to provide a medium for the various reactions that take
place in dough formation.

Enzymes - Sufficient amylolytic enzymes must be present

during bread-making to breakdown the starch in flour into
fermentable sugars.
Fungal or bacterial amylase are used.

Bacterial amy1ase from Bacillus subtilis is particularly useful

because it is heat-stable and partly survives the baking
process.
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Systems of Bread-making

Large-scale bread-making is mechanized. The processes of yeast-

leavened bread-making may be divided into:

a) Pre-fermentation (sponge mixing): At this stage a portion of the

ingredients is mixed with yeast and with or without flour to
produce an inoculum. During this the yeast becomes adapted to
the growth conditions of the dough and rapidly multiplies.

b) Dough mixing: The balance of the ingredients is mixed together

with the inoculum to form the dough. This is the stage when
maximum gluten development is sought.

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c) Cutting and rounding: The dough formed above is cut
into specific weights and rounded by machines.

d) First (intermediate) proofing: The dough is allowed to rest

for about 15 minutes at about 27°C in equipment known as an
overhead proofer.

e) Molding: The dough is flattened to a sheet and then molded

into a spherical body and placed in a baking pan which will
confer shape to the loaf.

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f) Second proofing: This consists of holding the dough for about 1
hour at 35-43°C and in an atmosphere of high humidity (89-95%)

g) Baking: During baking the proofed dough is transferred, still in

the final pan, to the oven where it is subjected to an average
temperature of 215-225°C for 17-23 minutes. Baking is the final
of the various baking processes.

h) Cooling, slicing, and wrapping: The bread is depanned, cooled

to 4-5°C sliced and wrapped in waxed paper, or plastic bags.

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Flavor development

During baking the lower boiling point materials escape with

the oven gases; furthermore, new compounds result from the
chemical reactions taking place at the high temperature.

The flavor compound found in bread are organic acids, esters,

alcohols, aldehydes, ketones and other carbonyl compounds.

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2. Soy sauce
 Soy sauce known as shoyu in Japan is a salty pleasantly
tasting liquid with a distinct aroma.
It is made by fermenting soybeans, wheat, salt with a mixture
of molds, yeasts and bacteria.
Five different types of shoyu are recognized by the Japanese
Government, depending on the proportions of the ingredients
used and the method of preparation.

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Koikuchi-shoyu is the most produced, forming 85% of the
total produced. It is this type which is also best known in
countries outside the orient.
Koikuchi-shoyu is deep red-brown in color and is an all
purpose seasoning, with a strong aroma and myriad flavor. It
is the only type to be discussed.

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Product Country of Soy sauce: China, Japan, Philippines,
Taiwan and Indonesia
Soy sauce manufacture: The manufacture of koikuchi-shoyu
can be divided into four sections:

a. The preparation of the ingredients

b. Koji preparation
c. Brine fermentation
d. Refining process

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Preparation of the ingredients: Whole wheat is roasted and then
coarsely ground.
Roasting adds color and flavor to the resulting sauce and kill
surface organisms as well as facilitates enzymatic hydrolysis of
the grain.
Soybeans, usually defatted, are cooked under high pressure and
temperature for a short time after a previous soaking in water.

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Koji preparation: Whole wheat and soy prepared are used for
the preparation of koji.

A koji starter, or seed mold or inoculum is first prepared from the

spores of several different strains of Aspergillus oryzae by
inoculating the spores of the fungi on to a mixture of boiled rice
and wood ash or mineral salts and spreading the mixture thinly at
30°C for up to five days.

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The koji starter (also known as tane koji) is used to inoculate
equal amounts of the wheat and soy prepared. This used to
be turned manually in shallow trays, but is now also being
done mechanically.

The mixture is put into large vats and aerated by forced

aeration.

 The important requirements of koji are that it should have

high protease and amylase activities. As these are dependent
on temperature and humidity, the latter are strictly
controlled.
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After 2 to 3 days koji is harvested as a greenish-yellow
material due to the spores of Aspergillus.
Brine Fermentation: Koji is introduced into deep
fermentation tanks to which an equal volume of salt solution
20-23% is added.
The resulting mixture, known as moroni is allowed to ferment
for 6-8 months. It is frequently mixed to distribute the
material and to eliminate undesirable anaerobic organisms.

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During the period, koji enzymes hydrolyze proteins to amino
acids and low molecular weight peptides; much of the starch is
converted to simple sugars which are then ferrmented to lactic
acid, alcohol and CO2.
The pH drops from around 6.5-7.0 to 4.7-4.8.

 The effective salt concentration is about 18 % (because of the

dilution with added koji); it is never allowed to fall below 16%
otherwise putrefactive organisms might develop.

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There are three stages in the fermentation of moromi, which is
brought about by osmophilic strains of microorganisms, after
the release of simpler substances by the fungi of the koji.
 In the first stage, Pediococcus halophilus produces lactic acid,
causing a drop of the pH.
 In the second stage Saccharomyces rouxii develops and
produces alcohol.

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In the last stage, Torulopsis yeasts develop. These produce
phenolic compounds which are important components of
koichuki-shoyu flavor.

 The organisms are selected by the conditions of the

fermentation, but pure cultures as used more and more
nowadays to ensure a more consistent flavor.

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Refining: The final state consists of pressing the fermented
moromi to release the soy sauce. Hydraulic presses are used in
modern production.
The raw soy sauce is heated to 70-.80°C to pasteurize it, to
develop color and flavor and to inactivate the enzymes.
After clarification by sedimentation the sauce is bottled under
aseptic conditions, sometimes with the addition of
preservatives as well.

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3. Sauerkraut
Sauerkraut is produced by the fermentation of cabbages
(Brassica oleracea) and has been known for a long time.

Specially selected varieties which are mild-flavored are used.

The cabbage is sliced into thin pieces known as slaw and

preserved in salt water or brine containing about 2.5% salt.

The slaw must be completely immersed in brine to prevent it

from darkening.

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 Kraut fermentation is initiated by Leuconostoc
mesenteroides, a hetero-fermentative lactic acid bacterium
(it produces lactic acid as well as acetic acid and CO2.)

It grows over a wide range of pH and temperature

conditions.

 Fermentation is for about 3-10 days at room temperature

during which it should be away from direct sun light.

CO2 creates anaerobic conditions and eliminates organisms

which might produce enzymes which can cause the
softening of the slaw.
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 CO2 also encourages the growth of other lactic acid bacteria.
including L. brevis, which is hetero-fermentative, and the
homofermentative L. plantarum; sometimes Pediococcus
cerevisiae also occurs.

Compounds which contribute to the flavor of sauerkraut begin

to appear with the increasing growth of the lactics

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These compounds include lactic and acetic acids, ethanol, and
volatile compounds such as diacetyl, acetaldehyde, isoamyl
alcohol, n-hexanol, ethyl lactate, ethylbutarate, and iso amyl
acetate.

 Besides the 2.5% salt, it is important that a temperature of

about 15°C be used.

Higher temperatures cause a deterioration of the kraut.

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5.3 Single Cell Protein (SCP)

Single cell protein (SCP) refers to dead, dry cells of microorganisms,

such as yeast, fungi, bacteria and algae

These microorganisms grow on various carbon sources for their

protein content

The term, ‘single cell protein’ was firstly used by Carol Wilson in
1967 by replacing the less aesthetic terminology, ‘petro protein’ ,
‘microbial protein’.

The majority of microorganisms used are unicellular, the protein

content from them is called , ‘single cell protein’.
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 Cont..
SCP do contain contents, such as vitamins, fats and minerals etc.

 protein shortage in the Thirld World nations prompted in the

cultivation of SCP on large scale which resulted in the development
of SCP technology for livestock and human consumption

 it is estimated that SCP fermenters covering one third of a square

mile can provide 10% of the World’s protein requirement

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  Need for microbial production of scp

 it is an idea to solve global food scarcity

SCP can give relief to the agriculture sector which uses large area for
production of protein crops

production per unit area in the agriculture sector is low,

10% of the World’s protein requirement can be met by SCP technology

by using only one third of a square mile for SCP production

 climatic factor effects agriculture, whereas SCP technology is not

affected by climate

 scenario is also not so encouraging for animal protein too

 SCP has many advantages, such as 57

high protein content, contains all the essential amino acids

 some microorganisms are highly rich in vitamins

 high ratio of surface area to volume and small doubling time

high growth rate and flexibility in the use of substrate

 independence of cultivable land and climate

 works on continuous basis and eco-friendly

 cost effective , energy efficient and can also be genetically

controlled

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sources of microorganisms and the selection criteria
microorganisms, such as algae, fungi, protozoa and bacteria are used for
production of SCP
The basis for their selection are:

ability to utilize carbon and nitrogen sources

 moderate growth conditions

 tolerance to pH, temperature, and mineral concentrations

 resistance against viral infection

 non-toxicity

non-pathogenicity

 acceptable nutritive value of cell mass

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Single cell protein production from bacteria
Organism used Substrate
Bacterial species Variety of substrates
Bacteria of Methylococcaceae C1 compounds
Brevibacterium spp. C1 – C4 compounds
Cellulomonas spp . Agricultural wastes
Different species of bacteria Fruit processing wastes
Methanomonas methanica Methane
Methylophilus methanotrophus Methanol
Pseudomonas fluorescens Manure, Animal wastes
Rhodopseudomonas gelatinosus Wheat bran
Streptomyces spp . Methanol

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Production process

Technically, SCP is the manufacture of cell mass using

microorganisms by culturing on abundantly available
agricultural and industrial wastes.

The production of microbial biomass is done either by a

submerged or solid state fermentation process.

After fermentation, biomass is harvested and may be

subjected to downstream processing steps like washing, cell
disruption, protein extraction and purification.

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Production of SCP has a number of attractive features:

a) It was not subject to the vicissitudes of the weather and can

be produced every minute of the year.

b) Microorganisms have a much more rapid growth than plants

or animals.

c) waste products can be turned into food in the production of

SCP.

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Nutritional value of single cell protein

The nutritional value of SCP depends on the composition of

the microbial cells used especially their protein, amino acid,
vitamin, and mineral contents.

These to some extent also depend on the conditions of growth

of the organism.

 Fungal SCP is rich in methionine and lysine. Fungal lysine

content is higher than bacteria or algae.

Algae are rich in protein, fats and vitamins A, B, C, D and E.

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While algae are rich in all vitamins, fungi provide, to some
extent, the B-complex group of vitamins.

The amino acid composition of Aspergillus niger according

to FAO standards is well balanced.

 Bacterial SCP is high in protein and certain essential amino

acids.

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END

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