Industrially Important Yeast, Mold And Bacteria


Microorganism on one hand are responsible for causing a number of diseases,
on the other hand they are employed to produce a number of useful products.
These useful microorganisms include an array of yeasts, molds and bacteria.

Industrially Important Yeast, Mold And Bacteria

Traditionally, the man prepared wine, curd, vinegar and pickles using
fermentation. Earlier, the term ‘Fermentation’ was used for the production of
wine but at present it encompasses the foods made by the application of
microorganisms including lactic acid bacteria (LAB). Lactic acid fermentation
is one of the oldest method of preserving fruits and vegetables. 

Apart from
contributing certain desirable physical and flavour characteristics, it also
prolongs the availability and processing period of the products at
relatively low cost. Many of the fermented products are made at industrial
scale making use of microorganisms


The human food supply consists basically of plants and animals or products
derived from them, so our food supply can contain microorganisms in
interaction with the food. The interactions between microorganisms and our
food is beneficial as exemplified by many cultured products developed by
fermentation and are consumed and enjoyed by many people (Bread, bear,
wine etc). 
To produce such products microorganisms are added as pure culture
or mixed cultures. However, in some cases no cultures may be added if the
desired microorganisms are known to be present in sufficient numbers in the
original raw material. 
Starter culture, pure as well as mixed are usually employed in the manufacture
of certain fermented food and dairy products. Cultures for food fermentations
are selected primarily on the basis of their stability and their ability to produce
desired products. 
Mother culture is usually prepared daily from a previous mother culture and originally from the stock culture. The mother cultures can
be used to inoculate a large quality of culture medium to produce the mass or
bulk culture to be used in the fermentation process.
Bacterial cultures: Most of the bacterial cultures employed as starters are for
dairy products. Sausage and bread also use pure or mixed cultures of lactic
acid bacteria (LAB) e.g. Streptococcus lactis sub sp lactis, S. mesenteroides
sub-sp. cremoris etc.
Yeast cultures: Most yeasts of industrial importance are of the genus
Saccharomyces. It is used to manufacture wine, beer and other alcoholic
Bakers’ yeast: Yeast for baker’s yeast production: Strains of S.cerevisiae,
S.uvarum are used.
Wine yeast: S. cerevisiae var. ellipsoidus

Distillers yeast: High alcohol yielding strains of S.cerevisiae var ellipsoidus.
Mold cultures: Stock of cultures of molds usually are carried in slants of a
suitable agar medium and may be preserved as spore stab for a long period by
freeze drying (Penicillium roquefortii).


Enzymes: Enzymes are biological catalysts possessing extraordinary
efficiency, specificity and are mostly protein in nature. Enzyme commission
has classified various enzymes on the basis of the type of the reactions
catalysed. All the enzymes have been classified into 6 classes.
Class 1 : Oxido-reductase 
Class 2 : Transferase 
Class 3 : Hydrolase 
Class 4 : Lyase 
Class 5 : Isomerase 
Class 6 : Ligase
Properties of Enzymes 
• All enzymes are protein in nature except nucleases. 
• All enzymes are specific in their functions. 
• Enzymes are sensitive to temperature i.e. they are functional at optimal
• These are destroyed at higher temperature. 
• Enzymes are not destroyed during their use

Kinetics of Enzyme Reactions

The studies on the kinetics of enzyme reactions must be based on quantitative
measurements of the rate of the catalyzed reactions. Main factors which
influence the kinetics of enzymatic reactions are as follow: 
Enzyme concentration: The velocity of enzymatic reactions is directly
proportional to the concentration of the enzyme proteins. 
Substrate concentration: When the velocity of the reaction is plotted vs.
substrate concentration, classical enzymes give a rectangular curve. 
pH: Almost every enzyme exhibits maximum activity at a particular pH which
is called optimum pH. 
Temperature: Almost every enzyme exhibits an optional temperature at
which the enzyme exhibits maximal activity. A graph of enzyme velocity
versus temperature, is a bell shaped curve. 
Role of enzymes in food processing: The enzymes play a significant role in
food processing. Pectinase enzymes are used in juice clarification (apple juice,
guava juice), in softening of fruit (apple, tomatoes, peaches, avocadoes), and
thereby resulting in increase in yield of juices and pulps extraction of juice
from fruits. Proteases (papain) results in clarification and removal of
cloudiness in beer and wine.
Glucose oxidase enzyme is used in removal of
glucose from egg white and thereby, improve, the colour of dehydrated egg
powder. Pectinase with cellulase has been employed for extraction of oil from
oil containing fruits (olive). Enzyme diastase converts starch to sugar during
beer preparation. 


Fermentations have been classified on the basis of relationship of the formation
of product, substrate utilization or the free amount of water as given below: 

Solid state fermentation: Fermentation processes which take place in the
absence or near absence of free water in the substrate are termed as solid state
fermentation (SSF). It is imperative, however, that the substrate contain
enough moisture absorbed in the substrate particles within the substrate. SSF
have been used mostly for food fermentation and production of a few enzymes. 
Extractive fermentation: There are several industrially important products
being catalysed by enzymes which are susceptible to end product or feedback
inhibition. Hence, the increased concentration of the product inhibits the
enzymes involved in its own synthesis so that the overall rate of conversion of
substrate to the desired product is lowered. 
When the end product or anyone of the by-product of fermentation interacts with the enzyme, the synthesis of the
final product proceeds sub-optimally and in extreme case may stop altogether.
This problem has largely been overcome by using a technique called extractive
fermentation. In it there is fast removal of product, or by-product of a
metabolic pathway, so that their subsequent interference with the cellular or
medium component is not possible. 
Hence, it involves all the actions taken for
the separation of a product from its producing cell. Separation of the product
can be achieved either inside the reactor (internal) or outside the reactor
Submerged fermentation: Fermentation processes which take place in the
presence of free water in the substrate are termed as sub-merged fermentation.
Such fermentations have been used mostly to the produce fermented food and


Fermenter: The industrial usage of micro-organisms often requires that they
be grown in large vessels containing considerable quantities of nutritive media.
These vessels are commonly called fermenters. Therefore, fermenter is the
basic equipment of fermentation.
Types of fermenters: Some of the types of fermenter are listed below: 
1. Shake flasks and bottles 
2. Stirred tanks 
3. Air-lift fermenters 
4. Tower fermenter 
5. Rotating disc fermenter 
6. Fixed bed fermenter 
7. Fluidized bed fermenter 
Batch fermentation: In this fermentation, starter culture is added to the
medium and the product is withdrawn only after completion of fermentation. 
Continuous fermentation: In this fermentation, the substrate is continuously
fed to the fermenter and the product is also withdrawn continuously.


The term ‘wine’ is applied to a beverage made by alcoholic fermentation of
grape or grape juice and final production is obtained without distillation. But
now-a-days, any fleshy fruit or flower in the new world may be employed for
this purpose. 
Wine was suggested to have been made during the Neolithic
period in the near East. These are the part of food of man ever since his
settlement in Tigoris Euphorates basins and have also been used as a
therapeutic agent.
Wines are produced by the fermentation of juices/extracts of many fruits such
as apple, pear, cherries, most of berries, rhubarb, dandelion, honey, besides
bananas, pineapple, cashew nut, pomegranate, lemons, tangerines, oranges,
dates and figs. Wines from grapes are classified basically into red and white
Types of Wines

Still wines: These wines retain none of the carbon dioxide produced during the
Sparkling wines: These are the wines which have considerable amount of
carbon dioxide. Champagne in France is the sparkling wine made in
Champagne region. 
Dry wines: These wines contain little or no unfermented sugar. 
Sweet wines: Wines having either unfermented sugar or with added sugar later
on are called sweet wines. Both types of wines generally contain 11 to 14% of
Fortified wines: Wines to which distillate of wine called “Brandy” is added
and may contain 15 to 21% of alcohol. 
Table wines: It is a wine having comparatively low alcohol content (7 to
11%) and little or no sugar.
Sherry: It is produced by special processing technique from wine, containing
18 to 21% alcohol and could be sweet or dry. 
Cider: Cider is a low alcoholic beverage obtained from apple by fermentation.
Perry: It is a wine made from pear juice. 
Mead: This type of wine was prepared by the Indians from honey.
Vermouth: Wine flavoured with a characteristic mixture of herbs and spices,
some of which impart an aromatic flavour and odour while others a bitter
flavour. It can be sweet or dry with alcohol content of 15 to 21%. 
Toddy: Sweet alcoholic drink, having alcohol content of 4-6%, is made by the
fermentation of sap from coconut palm.
Pulque: National drink of Mexico, contains 6-7% alcohol and B-vitamins.

Method of Table Wine Preparation  

Grape is the most widely used fruit to make wine but it can be prepared from
any fruit having fermentable sugars, optimum acidity, nitrogenous compounds
or other growth factors to make wine of acceptable quality. The major
difference is in the extraction of sugar from the pulp of some fruits. 
grape, red and white wines are produced the world over using black/red
coloured and white varieties, respectively. The generalized flow sheets for
wine making from grapes is shown in Figure 3.1.
Preparation of must: For wine preparation, the first step is the preparation of
must which is prepared depending upon the type of fruits used and the type of
wine to be made. Must is a juice or pulp corrected for sugar, acid/pH, nitrogen
source or other requirements for the alcoholic fermentation. 
To prepare the
must, the fruits are trimmed and washed and the must Juice is extracted or fruit
is made into pulp. In the preparation of white wine only the free run juice is
used while in the red wine, the skin and seeds along with pulp/juice are
fermented together for some time to get attractive coloured wine. 
dilution of fruit pulp is required as fruits like plum and apricot are highly
acidic and effect the fermentability besides making the wine unpalatable. The
sugar content of the juice or pulp is checked with an instrument called
refractometer and is expressed as degree Brix. 
Sulphur dioxide (S02) is added
to the must to control the wild microflora and to allow the yeast to act
efficiently to conduct the alcoholic fermentation. Amelioration (or correction)
of must for better fermentability with ammonium salt and vitamins like
thiamine, biotin is necessary in some fruits. 
Preparation of active yeast culture: An active culture of wine yeast
(Saccharomyces cerevisiae var ellipsoideus) is prepared from the stock culture
in the juice to be used for wine making. 
Fermentation: After must preparation, activated yeast starter culture is added
to the must and fermentation is carried out at a temperature of 20-25o
C, till the
sugar content or the o
Brix stabilizes.
Siphoning/racking:Siphoning or racking is a simple but important process wherein the wine is
transferred through a clean pipe into another container, kept at a lower height
than the vessel with wine. It is done after completion of fermentation. Two or
three rackings are usually done at an interval of 15-20 days to separate the
yeast and other settled materials at the bottoms of container.
 As the newly made wine is harsh and has yeasty flavour maturation (from
6 months upto a year) is allowed to make the wine mellow (It is the term used
to signify the sensory quality of wine having smoothness i.e. is devoid of any
harsh taste)in taste and fruity in flavour. 
Clarification: Clarification of wine is done by using filter aids such as
bentonite, celite and tannin/gelatin using a machine called filter press. 
Blending: Blending is also practiced in some cases to make wine sweet or
better flavoured before pasteurization. 
Pasteurization: Wine is generally pasteurized at a temperature of 620
C for 15-
20 min, after bottling.
Storage: Low temperature storage is preferred for good quality wine.

Beer is an alcoholic beverage primarily prepared from barley besides other
cereals in limited quantities and is consumed in large quantities throughout the
Beer and ale the principal malt beverages made with hops, yeast, water
and malt adjuncts. Adjuncts are the malted cereals other than barley, used in minor quantities. Brewing was one of the earliest processes undertaken on a
commercial scale and became one of the first process that has developed from
an art into a technology. 
Beer can be differentiated from ale as in beer bottom
fermenting yeast is employed while in ale the top fermenting yeast is
employed. In the preparation of ale, more hops is used. It is usually pale yellow
in colour, tart in taste and have more alcohol content. 
On the basis of alcohol
content beers can be classified as light beer having 3-5% v/v and hard beer
having 5-8% alcohol content. Beer production is divided into four distinct
process as described here.
Malting: It is obtained by soaking followed by germination of barley or other
cereals and drying of the germinated cereal. Then, most of sprouts or germs are
removed and the malt remains. The malt is crushed before its use in beer
Mashing: It is the process in which extraction of the ground malted barley
with water is made. The mashing is done so as to make soluble as much as
possible of the valuable constituents of the malt and malt adjuncts. It causes
hydrolysis of starches, other polysaccharides and proteins. The insoluble
material is then filtered. The liquid so obtained is called wort.
Wort boiling: Boiling of wort with hops (Hops is the female flowers of hops
plant used in beer production to give flavour and bitter taste) is carried out to
concentate the wort, inactivate the enzymes, extract soluble substances from
the hops,coagulate and precipitate the proteins and other substances,
caramelize sugar slightly and to contribute antiseptic substances (Chiefly the
alpha resins humulone, co-humulone and adhumulone) to the wort and beer.
Fermentation: A special beer, bottom fermenting yeast strain Saccharomyces
cerevisiae var carlbergensis, is used for the inoculation or pitching of the
cooled wort. The wort temperature during the fermentation varies in different
breweries but is usually in the range from 3.3 to 14o
C. The fermentation is
usually completed within 8 to 14 days. 
During fermentation as the carbon
dioxide is evolved in increasing amounts, the foaming increases; later it
decreases to none when the fermentation is finished. At the later stage, the
bottom yeast flocculates and settles down. 

Aging or Maturation: The young, green or draft beer is stored or lagered in
vats at about Oo
C for several weeks to several months, during which period
precipitation of proteins, settling of yeast, resin and other undesirable
substances takes place and the beer becomes clear and mellowed or matured.

Finishing: After aging, the lager beer is carbonated to a CO2 content of about
0.45 to 0.52 per cent, mostly by means of gas collected during the fermentation
or by addition of CO2 from cylinders. Then, beer is cooled, clarified or filtered
and packaged in the bottles, cans or barrels.

The word vinegar is derived from two French words, vin and aigre meaning
sour wine but the term is used to denote a condiment prepared from various
sugar and starch containing materials by alcoholic and subsequent, acetic acid
fermentation. It is one of the several fermented foods prepared and consumed
by early man, even today. 
Earlier, it was used as a beverage, a condiment, a
preservative, a household cleansing and medicinal agent. Vinegar mainly
consists of a dilute solution of acetic acid in water, also contains colour flavour and extracted substances besides fruit acids, esters and inorganic salts
which vary according to its origin. The minimum legal strength for vinegar is
4% acetic acid (w/v).

Types and Composition of Vinegar

1. Synthetic vinegar: This type of vinegar is directly prepared from synthetic
acetic acid with the addition of water and finally, it is coloured by caramel.
2. Brewed vinegar: Virtually, anything having enough sugar to produce
alcohol can be used to make brewed vinegar. The vinegar usually derives
its descriptive name from the material from which it is made such as: cider
vinegar is made from apple juice, alegar from ale, malt vinegar from
malted grains spirit vinegar from alcohol etc.
Vinegar Preparation

It involves two step fermentations as detailed below:
Alcoholic fermentation: The first is alcoholic fermentation, mainly carried
out by yeast Saccharomyces cerevisiae either by pure culture inoculation or
by the natural process of fermentation. The process can be represented by a
simplified equation:
C6H12O6 → 2CO2 + 2C2H5 – OH + 55 Kcal 
Glucose Carbon dioxide Ethyl alcohol
In the process, ethyl alcohol is not the only product but small amounts of
other compounds like glycerol, succinic acid, amyl alcohol, propyl alcohol
etc. are also produced in this fermentation. The fermentation is anaerobic. 
Acetous fermentation: The second fermentation is acetic acid fermentation. It
is an oxidative fermentation carried out by acetic acid bacteria like Acetobacter
aceti. In the vinegar production, pure culture of acetic acid bacteria is not
used, due to more efficiency of mixed cultures. The oxidation reaction can
be shown as:
C2H5OH + O2 → CH3COOH + H2O + 116 Kcal 
Ethyl alcohol Acetic acid
The optimum temperature of fermentation is 26o
C which is achieved by the
heat generated in the process.

Process of Vinegar Preparation

Slow process: This process takes a long period and is generally followed in
countries like India. The juice kept in the barrels is allowed to undergo
alcoholic and acetic fermentations slowly with the passage of time. The bung
hole of the barrel is covered with a piece of cloth to screen-off the dust and
flies, and the barrel is placed in a damp but warm place. 
It takes about 5-6
months to complete the whole alcoholic and acetous fermentation to produce
the vinegar from the juice. The main drawbacks of this process are: alcoholic
fermentation is often incomplete, the acetic fermentation is very slow and the
yield is low coupled with an inferior quality vinegar.
Quick process: In the quick process like generator process alcoholic liquid is
in motion and this process is applied mostly to the production of vinegar from
spirit (alcohol). Fruit or malt liquors are well supplemented with food for the
vinegar bacteria, but to maintain active vinegar bacteria in generator methods
using alcohol denatured with ethyl acetate or vinegar, it must be supplemented
with a combination of organic and inorganic compounds known as vinegar
Combinations of substances such as dibasic ammonium phosphate, urea,
peptones, yeast extract, glucose, malt, starch, dextrin, salts etc have been made.
Materials such as pumice, branches of vines and grape stems for packing the
generators are used. Schiizenbach introduced the use of a vat instead of cask
for the acetification process and provided mechanical means for the repeated
distribution of the acidic liquid over the packing
Generator: The equipment used is known as “Upright Generator” which in its
simplest form is a cylindrical tank that comes in different sizes and is usually
made of wood. Its interior is divided into 3 parts:
i) Upper section: Here, alcoholic liquid is introduced. 
ii) Large middle section: In this section, liquid is allowed to trickle down
over beech wood shavings, corn cobs, charcoal, coke, or some other
material that will provide a large total surface area yet not settled into a
compact mass.
iii) Bottom section: This section is for the vinegar collection.
The alcoholic liquid is put at the top through an automatic feed trough or a
sprinkling device (sparger) and trickled down over the shavings or other
material on which a slimy growth of acetic acid bacteria has been developed
and the bacteria oxidize the alcohol to acetic acid and the process is called
Air enters through the false bottom of the middle section and
after becoming warm, it is exhausted out through a ventilation above. As
considerable heat is released by oxidation process, it is necessary to control
the temperature below 30o
It is usually done by using cooling coils, by
adjusting the rate of alcoholic liquid, feeding air and by cooling the
alcoholic liquid before it enters the generator or by cooling the partially
acetified liquid that is returned to the top from the bottom section of the tank
for further acetification.

Lactic Acid Bacteria (LAB) and Fermented Foods

Lactic acid bacteria (LAB) are obligate microorganisms producing lactate
from sugars as the main end product, besides producing inhibitory
substances like organic acids, bacteriocin, hydrogen peroxide which are
antagonistic towards other microorganisms. 
Fermented dairy products are
known to be inhibitory to both pathogenic and spoilage causing
microorganisms and Yoghurt is the best known fermented milk product (with
fruit pulp).Cultured milk and milk products contain lactic acid bacteria that
prevent the occurrence of stomach, colon and other cancers. 
Traditional fermentations of vegetables were depended upon growth of
naturally occurring lactic acid bacteria to metabolise sugars in the vegetables to
mainly lactic acid and improve their taste and keeping quality. However,
starter cultures are being used now to develop controlled fermentation It is established that more than one species of lactic acid bacteria are
responsible for vegetable fermentation. 
Lactic acid bacteria responsible for
natural fermentation of vegetables are within the genera of Streptococcus,
Leuconostoc, Pediococcus and Lactobacillus. Acidity, pH, salt concentration,
temperature, naturally occurring inhibitors, chemical additives, exposed
brined surface to air and sunlight, amount of fermentable carbohydrates in
the vegetables and availability of nutrients in the brine are important
factors affecting the lactic fermentation.

It is the clean, sound product of characteristic flavour, obtained by full
fermentation, chiefly lactic of properly prepared and shredded cabbage in the
presence of not less than 2% nor more than 3% of salt. It contains, upon
completion of the fermentation not less than 1.5 per cent of acid expressed as
lactic acid.
To prepare sauerkraut rough outer leaves of fully mature solid
cabbage heads are removed. Head are quartered, the cores are removed and
then, shredded the quarters into thin strips which are mixed with salt. About
2.25 to 2.5% of salt by weight should be added to the shredded cabbage to
obtain kraut of the best quality. 
Pack the cabbage loosely in a jar, place a
wooden board on the top. In order to press out juice from the cabbage, a heavy
stone is placed on the wooden board. The jar is kept at a warm place (24 to
C) for 8 to 12 days to allow fermentation to complete. The brine is
separated from the cabbage, boiled and poured hot over the cabbage shreds in
the jars. 
Sauerkraut can be packed in cans also. The cans are filled with the hot
juice, exhausted and processed till the temperature at the centre of can reaches
Prominent bacteria that attain appreciable number early in fermentation are
Enterobacter cloacea and Erwinia herbicola and contribute some flavour. 
However, Leuconostoc mesenteroides bacteria begins to outgrow all
organisms and continue acid production upto 0.7 to 1% (as lactic acid). Next,
Lactobacillus plantarum, a non-gas forming lactobacilli continues the
production of acid and can raise the acidity to 1.5 to 2.0%. 
These bacteria
produce chiefly lactic acid in their fermentation of sugars. A final acidity of
1.7% as lactic acid is most desirable and fermentation can be stopped at this
stage by canning or refrigerating the sauerkraut.

Carrots of deep purple variety are fermented in Northern India and Pakistan to
make a ready-to-serve beverage /drink called as Kanji. It is a popular beverage
and is considered to have cooling and smoothing properties besides nutritional
To prepare it, the carrots are washed, grated finally. For every Kg of
grated carrot, 7Kg of water, 200g of salt, 40 g of crushed mustard seeds and
8g of hot chillies are added followed by placing the mixture in a glazed
earthenware, leaving a tiny whole for the release of gases produced during
The mixture is fermented for 7-10 days. It is strained through a
muslin cloth. The final product is acidic in taste with an attractive purple red
colour and is usually consumed within 3-4 days.
Pickles from Vegetables
Vegetables like cucumber are pickled whole or in slices after washing in
potable water. For every one Kg of cucumber, 15g salt is added which results
in the formation of brine. It is followed by lactic acid fermentation. Depending
upon the ambient temperature it takes one to four weeks. The fermented
cucumbers are stored in clean capped jars after pasteurization.
Radish can also be pickled in a manner similar to sauerkraut as discussed
It is a fermented food of Korea with cabbage or radish as the main ingredient.
Cucumbers can also be added. Cabbages are cut and brined in 5 to 7% salt
solution for 12 hr or in 15% brine for 3 to 7 hr. Then, brined cabbage is rinsed
and mixed with 10% seasoning ingredients i.e. garlic, green onions, peppers,
ginger, mustard, parseley, sesame grains and fermented shrimp. 
This mixture is
allowed to ferment in jars which takes a few days at temperature of more than
C for a month below 10o
C. ‘Kimchi’ has a pH value of 4-4.5 and lactic acid
content of 0.4 to 0.8%. The main organisms responsible for fermentation of
‘kimchi’ are Leuconostoc mesenteroides and acidifying microorganism is
Lactobacillus plantarum.
Ethanol Production
The material rich in sugar can be converted into ethanol. The fermentation is
carried out using yeast like Saccharomyces cerevisiae. The sugars like glucose
is converted into ethyl alcohol and carbon dioxide, anaerobically. Ethanol is a
liquid fuel or liquid fuel supplement and is used as a solvent in many
The waste from fruits and vegetable processing industries being rich in
polysaccharides (cellulose, hemicellulose and lignin) has been subjected to
SSF for the production of ethanol. The cellulose and hemicellulose present
in the processing waste like apple pomace are readily fermented by anaerobic
For ethanol production, the waste from processing industries has to be
pre-treated due to presence of lignin. A SSF process has been used for
production of ethanol from apple pomace by using Saccharomyces cerevisiae.
Apple, pear, orange peel and cherry wastes have also been utilized for
production of ethanol by fermentation with Saccharomyces cerevisiae.
Enzyme Production
Both submerged fermentation (SF) and solid state fermentation (SSF) are
employed for production of enzymes. But SSF is a better method than SF for
production of enzymes. Various enzymes have been produced by fermenting
food processing waste. 
Invertase enzyme by fermenting sauerkraut waste
with the help of Canidida utilis has been produced. This enzyme is widely
used in the food processing industry. Subsequently, fungal amylase by using
baked bean waste has been produced. Enzymes like cellulase and xylanase are
produced by fermenting apple pomace, using Trichoderma viridae and
Aspergillus sp. Pectinase is another enzyme which is produced from wastes
like apple pomace

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