UNIT-4 BENEFICIAL ROLE OF MICRO-ORGANISMS

4.1 Fermentation

In the biochemical sense, the term fermentation refers to a metabolic process

in which organic compounds (particularly carbohydrates) are broken down
to release energy without the involvement of a terminal electron acceptor
such as oxygen. Partial oxidation of the substrate occurs so that only a
relatively small amount of ATP energy is released compared with the energy
generated if a terminal acceptor is involved. Partial oxidation of a carbohydrate
can give rise to a variety of organic compounds. The compounds produced by
micro-organisms vary from organism to organism and are produced via
different metabolic pathways.

The term fermentation can also be applied to any industrial process that
produces a material that is useful to humans and if the process depends on the
activity of one or more micro-organisms. These processes, known as industrial
fermentations, are usually carried out on a large scale and in vessels in which
the organism are normally grown in liquid media. Some industrial
fermentations are fermentation’s in the biochemical sense but the majority of
microorganisms are aerobic and use oxygen and metabolize carbohydrates
completely.

A vast range of material are produced by industrial fermentations. These

include:

 Organic chemicals used as fuels, food additives, antibiotics and enzymes for
use in the food and other industries. Vinegar is an example of a food
additive produced by an industrial fermentation.
 Organisms produced on a large scale for the extraction of protein (single
cell protein) that can be used as part of the human diet. Quorn is an
example of a single cell protein, produced from the fungus Fusarium
graminearum. The mycoprotein, purified from the fungus, is currently
available for use as a food and is incorporated into a range of dishes that
appear on supermarket shelves. Meatless dishes with a high protein content
made from Quorn are particularly appealing to vegetarians.

 Yeast cells produced for use in industries such as the baking industry,
which relies on the mass production of large amounts of baker’s yeast.

 Foods produced on a large scale as a result of the activities of micro-

organism, e.g. cheese, yogurt and bread.

 Production of alcoholic beverages.

 Cells extracts used as food additives, e.g. yeast extracts from yeast cells
produced as a byproduct of the brewing industry.

 Mushroom production is another process that can be considered an

industrial fermenatation.

Industrial fermentation are now often considered under the heading of

biotechnology, i.e. technology that uses living organisms and their products in
the manufacturing and service industries

4.2 Fermented food and Their importance

Fermented foods are those foods produced by the modification of a raw

material of either animal or vegetable origin by the activities of micro-
organisms. Bacteria, yeast and moulds can be used to produce a diverse range
of products that differ in flavour, texture and stability from the original raw
material. The production of many fermented foods involves organisms that are
biochemically fermentative. Lactic acid bacteria that ferment carbohydrates to
produce lactic acid are particularly important, but yeasts also play a major
role in some food fermentations, fermenting carbohydrate to produce ethanol
and other organic chemicals. Mould that do not ferment carbohydrate, also
play an essential part in some food fermentations, for example, the production
of blue cheeses and soy sauce.

Fermented foods are an extremely valuable addition to the human diet for a
whole variety of reasons:

 Increase in variety : Fermented foods increase the variety of foods that are
available, adding to our diet a group of highly nutritious products with
unique characteristics. There are, for example, about 1000 different types
of cheeses.

 Use of ingredients: Fermented foods form important ingredients for wide

variety of dishes and are often used to impart special flavours, e.g.
pepperoni in pizzas, yoghurt in curries, cheeses in a whole range of dishes,
including soups, and soy sauce in stir-fry dishes.

 Improvement in nutritional quality: The fermentation process may

improve the nutritional quality of a raw material. Here are some examples:

(a) Tempeh fermentation raises the vitamin B12 content of the original
soyabean form 0.15g / g to 5.0 g/ g.

(b) Tape fermentation doubles the protein content of cassava and increases
the level of essential amino acids.

(c) The presence of yeasts in a fermented food will increase the vitamin B
content.

(d) Antinutritional factors such as phyates, glucosinolates and lectins may

be removed by the fermentation process.
(e) Fermentation may produce an increase in the bio-availability of minerals.

These improvements in the nutritional value of raw material will have

little effect in the balanced diets of Western populations. However, of
population that subsist on diets consisting largely of polished rice, maize
or other starches, such as in Africa and Asia, the contribution that
fermented foods make to the intake of B group vitamins and proteins is
highly significant.

 Preservation : Fermentation often preserves a raw material , improving

safety with regard to food- borne pathogens and increasing shelf-life;
compare the shelf-life of raw milk (only a few days) with the shelf-life of
yoghurt (several weeks).

 Health benefits: Some fermented foods are said to have definite health
benefits, although the scientific evidence for this is limited. Reports suggest
that fermented milk products such as yoghurt can reduce serum cholesterol
levels and help avoid cancers, particularly those associated with the colon.
‘Bio’ yoghurts (AB and ABT yoghurts) are said to have a restorative effect on
a normal microflora, assisting recovery of a normal balanced flora after oral
antibiotic therapy.

 Improve digestibility: Some fermented foods are more easily digested than
the original raw material. People who cannot digest lactose properly (show
lactose intolerance) can often consume some types of fermented dairy
products (particularly yoghurts) without harmful effects. Lactose intolerance
is due to the absence of the enzyme galactosidase in digestive juices, which
converts lactose to glucose and galactose. Ingestion of dairy products leaves
unabsorbed lactose in the gut, which is fermented by the normal gut flora
giving flatulence, abdominal pain and diarrhoea. The fermentation of milk
converts the harmful lactose to the more easily digested lactate, and the
galactosidase in live starter culture organisms appears to assist in the
 digestion of any residual lactose. Legumes, e.g. soybean, contain
oligosaccharides such as stachyose which are fermented in the gut to give
gas and the associated socially embarrassing flatus. The oligosaccharides
are broken down to readily digestible monosaccharides and disacccharides
during mould fermentation of legumes, thus removing the problem.

 Detoxification of raw materials: The fermentation process may remove

toxic chemicals present in the raw material. Cassava fermentation, for
example, removes a cyanogenic (cyanide producing) glycoside; cassava is
toxic if eaten raw.

4.3 TYPES OF FOOD FERMENTATION

A number of different types of food fermentation can be recognized.

4.3.1 Acid Food Fermentations

Acid food fermentations include:

 acid fermented dairy products, e.g. cheese, butter, yoghurt and kefir;

 acid fermented vegetable products, e.g. sauerkraut, olives and various

pickles;

 acid fermented meat products, e.g. the semidry fermented meats such as
cerevelat and the dry fermented meats such as salami and pepperoni;

 sourdough breads

The common feature of all these products is the use of lactic acid bacteria to
carry out the basic fermentation process. Modern production usually involves
the use of starter cultures. The one exception is the fermentation of sauerkraut
for which the process depends on lactic acid bacteria that are natural
inhabitants of the surface of cabbage leaves. Sometimes sugar is added to raw-
material to allow the lactic acid to produce sufficient acid for a successful
fermentation. This is the case with fermented meats in which the sugar content
of the raw material is very slow. Salt may be added to suppress the growth of
the normal spoilage microflora and allow the lactic acid bacteria to dominate,
e.g. sauerkraut, pickles and fermented meats. The raw materials may be
pasteurized to eliminate pathogens and suppress natural contaminants that
compete with the lactic acid bacteria used in the starter culture.

4.3.2 Yeast Fermentation

Yeasts are important in food fermentation because of their ability to produce

carbon dioxide and ethanol. Carbon dioxide is the important metabolic product
in the manufacture of leavened bread whereas ethanol is metabolized in the
production of beers, wines and spirits. Carbon dioxide and yeast itself are
important byproducts of beer manufactutre.

4.3.3 Solid State Fermentation

Solid state fermentation involve the use of a solid substrate into which the
fermenting organism is inoculated. The organisms used are often moulds.
Examples are the ‘koji’ process and the second stage of tempeh fermentation

4.4 ORIENTAL AND INDIGENOUS FERMENTED FOODS

A large number of fermented foods can be grouped under the heading of

oriental and indigenous fermented foods. Fermented foods of this type are
produced in Asia and Africa and are often associated with specific countries or
areas. Most of the products are unknown in the West but frequently have
major nutritional role in the diets of the local population. Lactic acid bacteria
are involved in some of the fermentation but yeast and moulds are often the
main organisms responsible. Many are sold state fermentations or involve
fermentations of more than one type. Some of the products are manufactured
on large scale but many are carried out on a cottage industry or household
basis. The major groups of these products with example are given in Table 4.1.
Table 4.1 : Example of oriental and indigenous fermented foods.

Food Type Example Country of origin

Foods fermented by Soy sauce, miso Southeast Asia

fungi followed by brine
process

Meat- flavoured pastes Natto Japan, China,

produced by one-step Thailand
bacterial fermentation

Legume-based foods Tempeh Indonesia

produced by bacterial
fermentation or acid
soak followed by
fungal fermentation

Doughs fermented by Idli India

lactic acid bacteria

Alcoholic foods Tape Indonesia

produced by
fermentation with
yeasts or other fungi

4.5 ROLE OF LACTIC ACID BACTERIA

The manufacture of acid fermented foods is based on the ability of lactic acid
bacteria (LAB) to ferment carbohydrates to produce lactic acid. Mesophilic
lactic acid bacteria Lactococcus, Leuconostoc, Lactobacillus and Pedicococcus
are used in fermentation that take place between 20and 30° C., e.g. most
cheeses, fermented meats, sourdough breads and fermented vegetable
production, whereas theremophilic Lactobacillus spp and Streptococcus spp are
used in fermentation carried out at higher temperature up to 45°C.e.g. in the
manufacture of yoghurt.

4.6 METABOLISM OF LACTIC ACID BACTERIA

Two types of lactic acid bacteria are recognized in terms of the metabolic
pathways used to break down carbohydrates to release energy, with the
fermentation products formed as biproducts: the homolactic bacteria that
produce mainly lactic acid and the heterolactic bacteria that produce a mixture
of lactic acid, acetic acid, carbon dioxide and ethanol. The metabolic pathways
involved are illustrated in Fig. 4.1.

Lactic acid bacteria are also important in the production of organic molecules
that impart flavour to fermented foods in addition to the typical flavour of lactic
acid. Although these substances are produced in very small quantities, they
impart flavours to fermented diary products that are often responsible for their
unique flavour characteristics, for example, Lactococcus var diacetylactis and
Leuconostoc spp can covert citrate to diacetyl which is the main flavouring
constituent of cottage cheese and butter. The characteristics flavour of yoghurt
is associated with the production of acetaldehyde by Lactobacillus delbreukii
subsp. bulgraicus.
Bacteria used in starter
cultures for the specific
production of aromas
and flavours are known
as aroma bacteria. Some
lactic acid bacteria

produce extracellular
polymers (slimes) that
contribute to the texture
of the final product also.
Fig. 4.1 : Metabolic pathways associated with lactic acid fermentations

4.7 EXAMPLES OF FOOD FERMENTATIONS

4.7.1 Fermented baked preparations

In baked products such as bread and bun, the yeast Saccharomyces cerevisiae
which is popularly known as “baker’s yeast”, helps by raising the dough, giving
it the texture and also adding flavours. The different ingredients further added
contribute distinct different tastes to each of these products. The naan which is
popular in India is made from maida (refined wheat flour) to which salt, yeast
or curd is added. It is kneaded vigorusly for 15 minutes adding vegetable oil for
softening. It is allowed to ferment for 30 minutes to 1 hour. It is then baked
rapidly on metal trays for 5 to 10 minutes. Intense heat causes centre of the
dough to expand rapidly and create a central pouch. Saccharomyces cerevisiae
is mainly responsible for leavening by carbondioxide production.

4.7.2 Fermented vegetable foods

Sauerkraut
Sauerkraut is fermented fresh cabbage product. It is popular in USA and
Europe. The main organism involved in the fermentation of this pickle is lactic
acid bacteria. Leuconostoc mesenteroides followed by Lactobacillus plantarum.

Cucumber pickle
Cucumber pickle is a fermentation product of fresh cucumbers. Several lactic
acid bacteria are involved in preparation of this pickle. Lactobacillus plantarum
is the most important organism required for fermentation of cucumber pickle.

4.7.3 Fermented soyabean products

Tempeh
Tempeh is a highly popular soyabean preparation in Indonesia. The chief
organism in this preparation is the mould species of Rhizopus oligosporus. The
soybean mash is wrapped in a banana leaves or kept in boxes and the mash is
inoculated with tempeh fungus by addition of a portion of previous batch and
allowed to ferment for about 24-48 hours at a temperature 30-40oC until there
is a good mycelium growth which is then sliced and prepared as per the taste
such as roasting or frying. The taste of the tempeh is considered to be bland
but it is highly nutritious.

Soya sauce
Soya sauce is a very popular preparation of Japan which has received wide
acceptance world over. This is prepared by inoculating an aspergillus species
mostly Aspergillus oryzae in a mixture of soaked and steamed soya bean with
roasted wheat in the ratio of 2:1. The mixture is allowed to be incubated for 3
to 5 days. Subsequently, it is subjected to various processing steps using
Lactobacillus bacteria and the yeast Saccharomyces rouxii. After 3 months, the
final product is filtered, pasteurized and bottled for use.

4.7.4 Fermented Dairy Products

The fermented dairy products assume greater importance in the human diets
as invariably most of the diets consist of milk byproducts especially the cheese,
butter, yoghurt, curd etc.

Cheese
There are several varieties of cheese which manufactured all over the world. All
types of cheese are the byproducts of lactic acid fermentation of milk. There
are several varities of cheese which are classified as hard, semihard and soft
cheese. These are prepared with culturing of the milk either with bacteria or
mould species. Among the several varieties, the popular ones are Cheddar
cheese and Swiss cheese which are known as hard cheese whereas Roquefort
cheese (blue cheese) is a semi-soft cheese and the soft variety is the
Camembert cheese. The Cheddar cheese originated from England and the
colour of which ranges from white to orange-yellow depending upon the colour
added. The curing is done with the help of Streptococcus and Lactobacillus.
This cheese is without the gas holes (the eyes) which characterizes the swiss
cheese. The swiss cheese is cultured with the help of mixed culture.
L.bulgaricus and Streptococus-thermophilus and Propionbacterium shermanii
which imparts the characteristics eye formation. The Roquefort cheese is
prepared by the use of Penicillium roqueforti where as the camembert cheese is
produced by Penicillum camemberti.

Dahi / curd
Dahi is an Indian fermented product which resembles yoghurt and is widely
used in the daily menu of an average Indian. The dahi / curd is obtained from
the milk by use of mixed culture of heterolactic bacteria

Yoghurt
Yoghurt is the preparation which uses the action of two lactic acid bacteria
Streptococcus thermophilus and Lactobacillus bulgaricus. The ideal ratio of
these two organisms is 1:1 for the best result.

Butter
The microorganisms which are involved in the preparation of butter are
Streptococus lactis and Streptococcus cremoris which convert the lactose in the
milk to lactic acid. Then the organisms like Streptococcus diacetylactis,
Leuconostoc dextranicum, Laeuconostoc citrovorum are involved in imparting the
aromatic flavours to the butter. The preparation of commercial butter involves
the ageing of cream overnight at 5-10°C and subculturing for 15-16 hours with
lactic acid bacteria followed by churning.

4.7.5 Other Fermented Foods

Idli
Idli is prepared from rice and urad lentil (dhal). They are cleaned, washed and
grounded in equal proportions and left overnight for fermentation. The
microorganism involved in this preparation are Leuconostoc mesenteroides
which grows first in the batter which is followed by Streptococcus faecalis and
Pediococus cerevisiae. Once the batter raises sufficiently, it is steam cooked
and served.

Vada
Vada is prepared by soaking urad lentil (dhal) in water for sometime, then
ground to paste. It is then left to ferment at 23-32°C for 12-48 hours, usually
overnight. It is made into balls and deep fried in vegetable oil. The dal paste is
fermented by lactic acid bacteria. Leuconostoc mesenteroides being the major
organism. The organism produces carbondioxide which aerates the product
and contributing to its texture..

Dosa
Dosa is a light, shallowed-fried thin pan-cake. It is prepared from fermenting
rice and urad dhal batter (ratio 2:1) followed by overnight fermentation. The
aeration of batter is caused by lactic acid bacteria fermentation by
Streptococcus faecalis with carbondioxide production by Leuconostoc
mesenteroides.

Bhatura
Bhatura is prepared from wheat flour, salt and sometimes with pepper or
cumin and made into dough with water and curd. Curd is used as a starter
and the dough is then fermented at 20-30°C overnight. It is rolled and
flattened into discs and deep fried in vegetable oil. The major organism
involved in fermentation are Streptococcus and Lactobacillus species
introduced by curd.

Dhokla
Dhokla is similar to idli except that rice and bengal gram are used in this
preparation. It is steamed into large sheets, then cut into pieces and seasoned.
It is yellow in colour unlike idli.

4.7.6. Economically important fermentation products.

Apart from helping man in preparing his foods, the microrganisms have
contributed to a great extent in perpetuating man’s desire for alcoholic
beverages which are usually made up of plant products.

Beer
Beer is an alcoholic product produced by brewing. It is a principal malt
beverage where the fermentation of carbohydrate to alcohol takes place. Barley
is used in the preparation of beer. Yeasts play a major role in the preparation of
beer. Lagerbeer is produced by Saccharomyces uvarum which settles at the
bottom of the fermenting vat and is known as bottom yeast.

Ale
The ale is produced by strains of S.cerevisiae which is collected at the top and
is called top yeast.

Rice Beer
Rice beer is a low-alcohol beverage made from rice which is more popular in
North Eastern India. The rice is milled, water is added and cooked. It is cooled
and then starter is added and fermented for 18 hours at 20-28oC. Rice beer is
then decanted from solid residue, which often is used as a breakfast cereal.

The starter introduces a mixture of moulds and yeasts. The rice starch is
broken down to sugars by amylase enzymes of moulds of Rhizopus, Mucor and
Aspergillus species. The yeasts like Endomycopsis and Hansenula then converts
the sugars to ethanol and carbondioxide.

Wine

Wine is the oldest and well known fermented alcoholic beverage produced by
the fermentation of good and sound grapes which is further processed, known
as ageing, before consumption. Although there are other fruits wines, they are
not as popular as grape wine. The grape fruits are crushed to give ‘must’. The
fermentation of ‘must’ is initiated by yeasts Kloeckera apiculatus and
Metschnikowia pulcherrima together with yeasts of Torulopis, Candida etc. The
main fermentation is by the yeasts, Saccharomyces cerevisiae and S. uvarum or
S. bayanus which converts sugars to ethanol and carbondioxide.

Champagne
Champagne, is a product of secondary fermentation. Fresh must, yeast and
sugar are added to the wine selected for champagne preparation.

4.7.7 Distilled liquor products

The distilled alcoholic products of interest are rum, whisky and brandy. These
products are manufactured from the distillation of yeast fermentation of sugar
cane juice, molasses, grains and grain products.

Rum
The rum is an alcoholic distillate of fermented sugarcane juice or molasses.

Brandy
The brandy is produced by distilling grapes or other fruit wines.

Whisky
Whisky is produced by distilling the fermented mash of wheat, barley malt and
other grains.

4.7.8 Other uses of microbes in industry

Vinegar
If the alcohol produced by the fermentation process is further oxidised to acetic
acid producing bacteria, the product is vinegar. Vinegar is made by different
processes. It can be made from fruit juices, starchy vegetables, malted cereals,
sugars and alcohol. Vinegar is widely used as preservative in food preparation,
wherein, the minimum percentage of acetic acid should be 3.75% as per PFA
Act.

Enzymes
Enzymes, which are known as biocatalysts are very useful in the
manufacturing of several products of commercial value. The enzymes are used
widely for manufacture of alcoholic beverage etc. The enzyme alpha-amylase
used in bread making is commercially prepared from Aspergillus oryzae. The
amyloglucosidase used as a substitute for malt in the production of beer and
spirits is commercially prepared from Aspergillus niger. Pectolytic enzymes are
produced from a number of fungi for use in fruit processing. Cellulase used for
removing cellulose cloud and clarify the citrus juices is produced from the
mould Trichoderma viride and proteases used in cheese making form
Aspergillus niger.

Amino acids and Vitamins

The importance of amino acid and vitamins in human health is welll
recognized. Several microbes have been used for their production which are
biologically suitable. Yeast is one of the best sources of the vitamin B-complex.
A number of preparations of high potency vitamin B-complex made from dried
yeast, yeast extracts are available in the market. Ribolfavin one of the B group
vitamins is produced from the yeast Eremothecium ashbyii. Ergosterol, the
prescursor of vitamin D is synthesized by a number of moulds and yeasts.
Betacarotene is produced commercially by fermentation using the yeasts
Rhodotorula.

Citric acid
Citric acid is one of the widely used chemical which finds applications in
several divergent industries such as pharmaceuticals, flavouring extractions in
food preparations, dyeing etc. Citric acid is produced by mould, Aspergillus
niger which converts sugars to citric acid where molasses is generally the raw
material.

Antibiotics
Apart from giving man several food products, certain microroganisms especially
moulds have given products which are life saving. The antibiotics are the
products of living organisms which in small proportions could be acting as
inhibitory agent for the growth of other microbes. The discovery of Penicillin
produced by Penicillium notatum by Sir Alexander Fleming in 1929 has
triggered off the manufacturing of modern antibiotics. There are over 600
antibiotics derived from bacteria and over 150 from fungi. The genus
Streptomyces has yielded a wide range of useful antibiotics such as strept,
streptomycin, aureomycin , chloromycetin and terramycin.