Basic of Fermentation Technology

About the Author

Rajan Sharma*

Department of Molecular Biology, Dehradun & Uttaranchal Institute of

management, India
*Corresponding author: Department of Molecular Biology, Dehradun &
Uttaranchal Institute of management, India, Email:

Published By:
MedCrave Group LLC
Date: January 09, 2017
 Contents
Origin and Evolution of Fermentation Process and Fermented Foods 1
Development of Fermentation Process and Industry 1
Fermented Foods 2
Products of Fermentation 3
Probiotic 4
How Temperature and pH Affect the Growth of Microorganisms 8
Lactic Acid 9
Medium 9
Microorganisms for Commercial Production of Lactic acid 10
Medium 10
Production of Lactic Acid 10
Product Recovery and Grades 11
Uses of Lactic Acid 11
Single Cell Protein (SCP) 14
Ethanol as Fuel 15
Different Types of Substrates for Industrial fermentations 17
Yogurt 22
Kefir 24
Bulgaricus Butter Milk or Bulgarian Milk 24
Acidophilus Milk or Reform Yogurt 24
Miso 30
Dosa 35
Fermented Sausages 36
Sauerkraut 38
Cucumber Pickles 41
Fermented Dill Pickles 42
Production of Industrial Enzymes 43
Production of Vitamins 46
Carotenoid 47
Recovery and Purification of β-Carotene 47
General Calculations 56
 Basic of Fermentation Technology

Origin and Evolution of Fermentation Process the production of liquid, fermented mashes from cereals
1
and Fermented Foods are closely related processes. It is likely that liquid from a
fermented mash was drunk as a slightly alcoholic beverage,
In earliest times, man was plagued with either feast while semisolid mash was needed into dough and baked.
or famine, so any means he could discover to conserve Even today yeast strain used in the production of ale
food when it was plenty was a great step forwarded in and bread is that from single species of Saccharomyces
his survival and his conquest of earth. Since man was of cerevisiae. Until into the middle of the 19th century bakers
necessity a wanderer and a hunter, he learned about the obtain their yeast from breweries. At that time lager beer
drying and smoking of meat. Certainly these methods not strains of Saccharomyces uvarum (S. carlsbergensis)
only conserved his supplies, but they also reduced weight, were introduced into central Europe and later in the United
enabling him to carry more food with him. At the times of States. These strains tolerate high osmotic pressure
discovery of North America by Europeans, most of the in the dough and bakers were forced to look for another
Indians were in this stage of development. The discovery of source of yeast. Distiller’s yeast that are also strains of
two methods Drying and Smoking just like the invention of Saccharomyces cerevisiae perform reasonably well in
wheel perhaps took place by serendipity. Early man might bread making, but they were difficult to separate from the
not know why foods spoil he knew they did. Later we can distilling mash. This led to the establishment of a separate
speculate that he discovered the use of salt with drying and industry that produced baker’s yeast on commercial scale
smoking. for sale to bakers and for home baking. The production of
baker’s yeast was increased many folds with the advent of
Man’s next discovery for preserving food was the
Fed-batch culture.
fermentation of foods, although he had no idea what
happened when microbial growth occurred, he learned that The discovery of fruit fermentation was made so
plant materials and meat could be kept for long periods long ago that the ancient Greek believed wine had been
of time when they have undergone fermentation. It was invented by one of their gods, Dionysus. The manufacture
also essential that he knew how to use salt (a necessary of wine have been recorded about 3500 B.C. as wine
agent that inhibits toxin production from microorganisms industry of Fertile Crescent that spread west (around the
in a successful fermentation). Undoubtedly many an early Mediterranean), North (to Hungry, Germany and France)
ancestors of man died from botulism or was made ill by and in the post Columbus period to America, South Africa
Staphylococcus aureus. After the addition of salt in natural and Oceania. Romans advanced the art of wine making,
fermentation there he knew definite changes in color, odor but it was an industry of large risks due to spoilage until
appearance and taste, which helped the product to be the mid nineteenth century. The research of Louis Pasteur
wholesome. Probably the first fermentation was discovered revolutionized the wine industry. A Mesopotamian clay
accidentally when salt might have selected certain tablet written in Sumerian-Akkadian about 500 B.C. tells
harmless microorganisms that fermented the product to that brewing was an established profession 1500 years
give nutritious and acceptable food. If we speculate along earlier. An Assyrian tablet of 2500 B.C. lists beer among
these lines, we might expect the first fermented food have commodities that Noah took abroad his ark. Egyptian
been fish. With the advent of certain religions in which meat documents dating back to 4th dynasty about 2500 B.C.
was excluded from diet, the use of salt and fermentation describes malting of barley and the fermentation of beer.
was adapted to certain plant products. For instance Bush Kui- a Chinese rice beer has been traced back to 2300 B.C.
(1959) states that Buddhism was well established religion in When Columbus landed America, he found that Indians
China and Korea by 4th century. It was introduced to Japan drank beer made from Corn. According to Weeks (1949)
between 500-600 AD. It may very well be the cultivation of the etymology of word “beer”, as we know today indicates it
Soya beans and their use in food including fermented foods originated from Latin verb ‘bibre’ (means to drink). Similarly,
were then introduced in Japan. For centuries Balkan people the Spanish word for beer ‘Cervaza’ apparently originated
have enjoyed fermented milk or yogurt and central Asian from cervisia, which combines Latin word ‘Ceres’ (goddess
tribesmen have found equal pleasures in sour camel’s milk of grain) and ‘Vis’ (vigor). The art of brewing was spread
or Koumiss. The ancient Sanskrit scriptures of India, the to England by Teutons that settled in Rhine area became
Vedas, documented the food value of Dahi- a fermented Germanic tribe. The major brewing centers were eventually
milk product similar to yogurt. Further evidences for the established in Pilsen, Czechoslovakia, Munich, Dortmund,
existence of soured milk as a food in the early times can Germany, Burton-O-Trent, England, Dublin and Ireland.
be found in Bible. The historical, geographical, ecological American Indians were already making beer from maize but
and dietetic patterns in various regions of the world are Mayflower Company brought English type beer to America.
reflected in diversity, variety and types of fermented milks in English Ale beer was used till 1840s, but German Lager
vogue today. These products are generally produced by the beer became more accepted type of beer because of its
intense activity of the Lactic acid bacterial cultures. superior keeping quality.
Bread that has been known almost as long as agriculture The process of fermenting sausages was probably
itself, its preparation involves a yeast fermentation. Loaves one of the earliest forms of meat processing and its
of bread have been found in Egyptian pyramids built in six manufacturing probably began before written history. The
thousand years ago. The art of fermented doughs from first mention of written history was in 9th century B.C. when
cereals was practiced before recorded history. This and it was mentioned in “Homer’s Odyssey”. The sausage was
 Basic of Fermentation Technology

2
called as “Oryae”. The word “Salami” was coined from the subsequent growth of yeast cells. This technique now is
product made in Salamis- a Cyprus city destroyed in 449 called as “Fed-batch Culture. Further studies also showed
BC (Pederson, 1979). Sausages eaten by Babylonians, that growth of yeast cells could be improved by sparging
Greeks and Romans were no doubt fermented and dried air in the fermentation broth. During the First World War
meat products. Brested (1938) stated that “Caeser’s Weizmann introduced a concept of Aseptic fermentation
legions” in Gaul consumed dry sausages. The descriptions the development of Acetone-Butanol fermentation. Steam
of the process of making sausages confirm that sterilized hemispherical topped and bottomed vertical steel
Babylonians, Greeks and Romans ate many types of dry cylinders were used as fermentors. These fermentors
sausages. The various regions of Mediterranean developed had problems of inoculum development and maintenance
characteristics sausages e.g. Salamis developed Genoa, of aseptic conditions. In spite of all the hindrances these
Milano and Lambardi types of sausages (Anon, 1938) The organic fermentations paved a way for the introduction of
Mediterranean countries consumed a highly seasoned non- aseptic aerobic fermentation technology.
smoked products classified as Latin type. Non-Europeans
In the Third Stage Penicillin fermentation process was
countries developed a Roman product, but slightly spiced,
developed, which was a wartime need. This fermentation
heavily smoked, moist and higher in salt content. This
was very vulnerable to contamination. All the knowledge
product is often referred to as Germanic type. In colder
gained in the previous year’s regarding process control, air
areas sausages are made in the winter months, stored
sparging, isolation and propagation etc. were applied for
and aged until summer, hence they are called as Summer
the synthesis of Penicillin at a large scale. The development
Sausages. The aging occurs by indigenous flora prompting
of large-scale extraction process and initiation of strain
the growth of Lactic acid bacteria, yeasts and molds in and
improvement programme was advancement at that time.
on the surface of sausages. Early in 20th century bacteria
Many other fermentation processes were developed at that
were discovered to be responsible for Lactic acid production
time like antibiotics, vitamins, gibberellins, amino acids,
and nitrate reduction in sausages. Further research in
enzymes and steroid transformations. The fourth Stage
the microbiology has led to the production of very safe
(early 1960s) is marked by the production of microbial
processed meat products and newer products are under
biomass as a source of feed proteins. Many waste products
the stage of development. Many fermented products have
were considered as a carbon source for the development
been proved to possess some medicinal values.
of microbial biomass. Hydrocarbons as another potential
Development of Fermentation Process and source for carbon for microorganisms were discovered.
In this period Jet and Pressure cycle fermentors were
Industry
developed that eliminated the need of mechanical stirring.
Development of fermentation process may be Other advantages of these processes were that they can be
represented by five overlapping stages. Stage I represents run continuous and were economic. At this time Batch and
the pre-1900 development that is confined to potable Fed batch culture techniques were common in the industry,
alcohol and vinegar. Wooden vats and even fitted with some but their application became short lived because of the
process control like thermometers (1757) and primitive heat development of Continuous Culture. The high standards of
exchangers (1801) replaced the ancient traditional Beer the aseptic operation and process controls were achieved
production by Egyptians. In mid 1800s Cagniard-Latour, by the introduction of computer systems in the fermentation
Schwann and Kutzing demonstrated role of yeast in alcoholic process to minimize the possibility of human error. The
fermentation independently. Pasteur later convinced that fifth Stage in the progress of fermentation process is the
pure culture of these microorganisms produces more introduction of Genetic Engineering and Recombinant
alcohol than the mixed culture. Methods for isolating and DNA technology in the strain improvement programme.
propagating pure yeast cultures were developed in the These Techniques not only allowed the transfer of genes
late 1800s. By the late 1800s and early 1900 generator between unrelated organisms but also enable the extremely
for the production of vinegar was developed, which was precise alteration of genome of a particular organism. The
considered as the first aerobic fermentor to be developed. development of further stages in fermentation will depend
In this method 10% good vinegar was added to the medium upon the new advances in this area.
as an inoculum that also makes the medium acidic to make
it contamination free. Thus in the beginning of 20th century Fermented Foods
concepts of process control for the fermentation process Fermented foods form an important part of human diet.
were developed. Fermented legume and cereal products are especially
Between the years 1900-1940 the main thrust areas popular in South East Asia including India, Middle East and
of research were baker’s yeast and organic solvent Africa. Traditional fermented foods are important elements
fermentations. Newer products developed were yeast in the diets of millions of people of particularly in developing
biomass, glycerol, citric acid, lactic acid and acetone- countries and the methods for their preparation are simple
butanol. Studies indicated that growth of yeast in the and inexpensive. Indigenous fermented foods are so
fermentation broth leads to oxygen depletion, which results prepared that they utilize cheap sources, supply proteins,
in the ethanol production at the expense of cell formation. and enrich starchy diets with vitamins and other nutrients.
Adding more broth in the previous broth can regulate the The exact origin of fermented foods is not known and their
 Basic of Fermentation Technology

3
discovery is considered to be purely by chance. The Asians
centuries ago knew the art to produce meat like flavors dp / dt = Yp / x .µ . x -----------(iii)
from vegetable proteins. The Indonesians had various Combining equations (i) and (iii) we have
methods to introduce meat like texture into the vegetable
products. Such foods have a particular place in their diets. Q = Yp / x.m
p
Koreans introduced acid fermented vegetables and People
of Egypt developed Bread leavened with yeasts while It may be seen that when product formation is growth
Indians discovered methods for souring and leavening associated the specific rate of product formation increases
cereal-legume batters. Nearly every nation in the world has with specific growth rate. When product is Non-growth
one or more fermented milks. Fermented milks are used associated the specific rate of product formation may
to restore the natural flora of intestine impaired by disease remain constant over a wide range of growth rates or
or antibiotic activity. In many countries cultured milks are it may vary in a complex manner. Garden relates the
widely promoted and credited with health giving properties. formation of products to substrate utilization or in other
Yogurt, Kefir, Acidophilus milk, Bulgarian milk, and Koumiss way this classification assesses the extent to which the
are a few names that are very popular in many parts of the energy producing reactions are coupled to the product
world and even in western countries. Much interest over the forming reactions. This approach is now very much used
years has been generated in the fermented foods of Asian in studying the continuous process. According to Gaden’s
and African countries because such foods in these countries Classification product forming reactions fall into following
are prepared traditionally, using simpler technology and three categories:
equipments. In India preparation of fermented foods Type -I, Type -II and Type III
has gained a status of small-scale cottage industry that
manufactures such foods utilizing natural microflora from Type I arise as a result of primary energy metabolism.
staples and surroundings. The desired product results from a carbohydrate substrate
e.g. glucose to ethanol, glucose to lactic acid etc. The
Products of Fermentation metabolic roots are serial with µF negative. The kinetic
A variety of products can be obtained by fermenting approximation of alcohol fermentation is given by:
different substrates with the help of microorganisms.
Fermentation products are the primary or secondary
metabolic products of microorganisms that are produced
at certain stage of their life cycle. Primary products are
Growth Associated products, their concentration in the
medium increases, as microorganism grows in the medium
i.e. concentration increases gradually in the exponential Negative sign indicates that substrate is decreasing.
growth phase of microorganism. Secondary products
are Non-Growth Associated products and are produced In Type II the main product arises from energy metabolism
in the stationary phase of growth of microorganism. The but indirectly e.g. Citric acid fermentation, some amino
concentration of Non-growth associated products may acid fermentation. The reaction patterns are complex and
sometimes become toxic to the microorganism itself e.g. restricted or abnormal metabolism is involved. The overall
Antibiotics. free energy change is negative. Such types of products are
also called as Intermediate metabolites. Gaden suggests
Kinetics of Growth Linked Product Formation following prototype reaction for the complex dissimilation of
The formation of growth Associated product may be Type II metabolites: (Figure 1)
described by the equation:
dp / dt = qp. x (1)
Where p is the concentration of product and qp is the
specific rate of product formation. Also the product formation
is related to biomass production by the equation as:
dp / dx = Yp / x (2)
Where Yp/x is the yield of product in terms of substrate
consumed.
Multiplying equation (2) on both sides by dx/dt we get:
dp / dt = Yp / x -----------(i)
dp / dx = Yp / x ---------(ii)
Figure 1
Therefore:
 Basic of Fermentation Technology

4 are prepared by fermentation process. However some of

them now are produced by synthetic process.
Polymers: Dextran is the only polymer that is being
produced on large scale by fermentation process. It is used
as a blood extender and blood thickener.

Type III, Secondary, or Non-growth Associated products Miscellaneous: Sorbose (an intermediate in ascorbic
acid manufacture), fructose (a liquid sweetener),
Secondary products result from biosynthetic reactions dihydroxyacetone (a sun-tanning agent) and
where the main product does not result from energy phenylacetylcarbinol (an intermediate in L-ephedrine
metabolism. Cell and metabolic activities reach maximum synthesis) are a few miscellaneous compounds produced
in the early stages of the life cycle and product formation by fermentation process. Spores of Bacillus thuringiensis
takes place at the later stages of the life cycle. Oxidative used as an insecticide for chickens, is also prepared by
metabolism is low at the time of maximum product formation fermentation process.
e.g. Antibiotic fermentation and biosynthesis of Vitamins.
In Non-growth associated product formation a period of Probiotic
negative specific growth rate occurs where the terms
A bacterial supplement of a single or a mixed culture
This shows that the population has moved beyond the of selected non-pathogenic bacterial strains is termed as
stationary phase for the latter part of the fermentation. Probiotic. The term ‘Probiotic’ was firstly coined by Parker
Under such conditions dead cell lysis may provide a (1974) and originated from two Greek words ‘pro’ and
second nutrient source for the other cells. Fermentation ‘bios’ which means ‘for life’. Probiotic generally includes
processes are the prime sources of over hundred products bacteria, cyanobacteria, fungi etc. They may be called as
for food, chemical and pharmaceutical industries. Various normal micro biota or “Effective micro biota”. Probiotic,
fermentation products are as follows: Probiotic, Probiotic bacteria, beneficial bacteria, or friendly
bacteria are the synonymously used for probiotic bacteria.
Antibiotics: Fermentation industry once dominated by
According to some recent publications, the mechanisms of
alcohol and solvent making now derives its primary income
action of probiotic bacteria have several aspects: 1) they
from antibiotics like Penicillin, Streptomycin, Vancomycin,
competitively exclude the pathogenic bacteria or produce
Neomycin, Chloramphenicol, and Erythromycin etc.
substances that inhibit the growth of pathogenic bacteria
Steroids: Transformation of steroids i.e. induction of (e.g. bacitracins and polymyxins produced by Bacillus spp.)
hydroxyl in 11β 11β and 16β position, dehydrogenation 2) provide the essential nutrients to enhance the nutrition of
of 1,2 position and hydrolysis of esters of the 3 hydroxyl the cultured organisms 3) they may directly uptake the or
group can be achieved by fermentation process. Key decompose the organic matter or toxic materials in water
steroids in microbial transformation process are Cortisone, improving the quality of water in the mediums or in the
Hydrocortisone, and Prednisolone etc. treatment of water. Beneficial effects of the Probiotic may be
mediated by: 1) Neutralization of the toxins 2) suppression
Enzymes: Many enzymes can be produced by
of the viable count 3) production of antibacterial substances
fermentation process. These find application in production
4) competition of adhesion sites 5) Alternation of microbial
of food, chemical and medicines e.g. amylases, pectinases,
metabolism 6) Stimulation of immunity of the host 7)
proteases, cellulases, catalases, invertases, lipases,
Accelerate the sediment decomposition by producing
streptokinases glucose oxidases and collagenases.
organic acids in water treatment 8) production of hydrogen
Organic Acids: Organic acids find their use in food, peroxide 9) production of enzymes.
chemical and medicines as an acidulant, sequestrate,
a. Types of Probiotic
plasticizer, flavouring and reducing agents. Commonly
used organic acids are citric acid, lactic acid, gluconic acid i. Non-viable Probiotic –these are dead.
and itaconic acid. Amino acids like lysine is used as food
ii. Freeze-dried Probiotic –these will die rapidly upon
supplement glutamic acid as a flavouring agent and are
leaving refrigeration.
ascorbic acid as a reducing agent.
iii. Fermentation Probiotic –these are produced through
Vitamins and Growth factors: Fermentations have been
fermentation.
used to produce growth factors for many years. These are
used in pharmaceuticals and as food and feed supplement iv. Viable Probiotic–these live with guaranteed shelf life.
e.g. Riboflavin, vitamin B12. Gibberellin is used in germinating Guaranteed number of organisms has a protocol for
barley and ripening fruits, Xanthophylls produced by algal counting and to be very stable and efficacious. Produce
cultures is added to chicken feed to give color to egg yolks many benefits.
and chicken meat. Torula yeast is added in animal feed as
a source of B vitamins is derived by fermentation of waste b. Bioreactor or fermentor
liquors from paper industry. Bioreactor is a device in which biochemical
Solvents: Many solvents like alcohol, acetone and butanol transformations take place. It is here a less expensive
material is converted into a more valuable product or
 Basic of Fermentation Technology

5
service is rendered. Even though the term is new but sterilization and downstream processing. Temperature
the concept is old. The terms like bioreactors, microbial is usually measured with the help of Resistance
reactors, fermentors, and biochemical reactors all have the thermometers, Thermocouples and liquid expansion
same meaning. Until about four decades ago, fermentation thermometers. Risk of contamination is minimal with
had been practiced as an art with a little engineering input, all these methods. Resistance thermometry prevails
but with the realization of the potential of this process, the because of its accuracy and reliability. Sensors
need of its instrumentation and control was felt. With proper usually are the encased platinum wires. The use of
instrumentation and control of a bioreactor it is possible thermocouples is less frequent. Electric measuring
to increase the conversion yield and the productivity of a signals from both the resistance thermometers and
biological product manifolds. The first step in understanding, thermocouples can be transferred to control boards.
controlling and optimizing a process is the precise, accurate This is not possible with liquid expansion thermometers
and timely monitoring of important parameters. The state of like mercury or ethanol in glass, which occasionally
the art in automated monitoring is very advanced in mature are employed for direct on the spot measurements.
industrial sectors but even today it is adapting sensors Another method of on the spot temperature indicator
developed for other applications or designing new sensors is by means of thermo colors that change their colors
to satisfy its needs. at certain temperature. They can be applied in the form
of thermo foils attached at critical spots. Some details
c. Measurement system
of the devices used to control the temperature are as
From system’s point of view, a measurement is achieved follows:
through the use of a meter or sensor expanding the human
b. Mercury in glass thermometers: They may be used
senses ability to detect measure and quantify. To control a
in small bench fermentors and are very fragile in
fermentation process we need to know 1) state of the process
nature. That is why their use is restricted. They usually
within a small time increment i.e. continuous monitoring of
are used enclosed in a pocket which protects the glass.
the state variables 2) The microorganism’s response to any
They are used only as an indicator.
set of measurable environmental conditions i.e. a control
model for fermentation. Before describing the various c. Bimetallic thermometer: It consists of bimetallic
sensors available for a bioreactor, it needs to emphasize coil surrounded by protective tubing. The coil Winds
the requirements of an ideal sensor. The requirements and or unwinds with changes in temperature causing
characteristics must be met within reasonable limits; it may movement of fixed pointer onto it. They are more
vary from one case to another. expansive and less accurate.
These are: d. Pressure bulb thermometer: It is basically a pressure
gauge connected by a small bore tubing which may be
i. Reliability-Long term reliability is of great importance
up to 60m in length to the detecting bulb. The whole
and a time of about 2000-3000 hours continuous
system is gas tight and filled with an appropriate gas or
operation should be attainable for most instruments
liquid under pressure. The movement of the free end of
ii. Repeatability or Reproducibility -Measurements made the receiving element can be used to operate a pen on
under standard conditions should be repeatable from a chart recorder or an electrical or pneumatic control.
day to day and from laboratory to laboratory (Figure 2)

iii. Accuracy- Accuracy is the measure of how close

the empirical measurement is to the true value or its
conformity to an accepted standard value
iv. Rough and Tough- Sensor should be rugged and
repeatedly withstand the conditions of steam
sterilization, variety of chemicals besides acidity,
basicity, salinity, and water etc.
All the fermentation sensors can be categorized as 1)
Physical environmental sensors-to measure the physical
process variables and 2) Chemical environmental sensors-
to measure the chemical process variables.
Figure 2
d. Methods of Measurement of Process variables
e. Thermocouples: In 1821 Seebak discovered that if
Physical process variables a circuit consisting of wires of two dissimilar metals
a. Temperature: It is an important parameter in the had the junction of the wires maintained at different
biochemical process. This is not true only further temperatures, a current flowed through the circuit. This
reaction itself, but also for auxillary operations such as current produced can be measured on a calibrated
 Basic of Fermentation Technology

6
instrument or recorder and is a measure of point which is very often kept constant. It is usually measured
temperature. At a point therefore by holding the by monitoring the number of revolutions per unit of
temperature constant at all junctions except one, which time. Outside the aseptic area, the impeller speed is
is a given circuit, it is possible to measure temperature measured with the help of a device called Tachometer.
with reference to the old junction temperature. (Figure 2)
Thermocouples have limited use because they are
j. Power Input: The power consumption of agitators
normally operated at ambient temperatures.
depend on stirrer speed and physical properties of
f. Electrical resistance thermometers: Electrical the stirred fluid especially on its viscosity, which may
resistances of metals changes with temperature change drastically during batch fermentations e.g. in
variations. The bulb of electrical thermometers some processes for the production of antibiotics like
contains a resistance element, a mica framework penicillin viscosity changes take place. In large-scale
(for accurate measurement) or a ceramic framework, fermentors, the consumption of electric energy as
around which the sensing element is wound. Platinum determined by a Wattmeter, yields useful information
wires of 100Ω resistance are normally used. The on the input of agitating power when friction losses in
wires are then connected to the measuring element. the stuffing box, seals, and motor are accounted for.
Reading is normally obtained by a wheat-Stone bridge A direct measurement of agitation power is possible
circuit and is the measure of the average temperature by using Torsion dynamometer or Strain gauges. The
of the sensing element. The electrical resistance latter method is an accurate method.
thermometers are very accurate, more sensitive to
k. Foam: It is a nuisance occurring in most fermentation
small temperature changes and are very fast.
broths. It may be caused by surface-active metabolites
g. Thermistors: These are semiconductors made from (proteins, polysaccharides etc.), components of the
specific mixtures of pure oxides of iron, nickel and other medium, or by cells. Two types of foams have been
metals. Their main characteristic is a large change distinguished 1) Soft foam 2) Hard foam Soft foam
in resistance as a function of absolute temperature. is unstable while hard foam is stable. Foam of either
Temperature reading is obtained with a wheat-Stone form must be suppressed in order to prevent the
bridge. They are relatively cheap and stable. contamination, clogging of the exhaust system including
its measuring devices and loss of culture broth. Foam
h. Pressure: It is measured by means of conventional
destruction can be achieved by mechanical or by
pressure gauge. Since the manometer is not in the
chemical methods (antifoaming agents). Often both
direct contact with the fermenter contents therefore
the methods are used in combination. Foam control
no sterility problem arises. Often measurement of
necessitates its detection. This can be achieved by
pressure is not included in the standard equipment,
employing sensors mounted inside the fermenter
though it may yield valuable information especially with
above the liquid level. Examples of the various probes
laboratory glass vessels. Here any clogging of exhaust
used are: Electric conducting probe, Capacitance
pipe may cause a buildup of a pressure head, and
probe and Heat conducting probe.
thereby apart from the danger of cracking the glass.
Other parameters, such as solubility of gases will be l. Volume: Information on liquid volume is essential
affected. In fermentors containing cultures, which tend in liquid flow control in the filling of the vessel with
to form wall growth, deposits of microbial mass on medium or in continuous culture feed in the continuous
membranes may lead to errors in the monitoring of the culture, which ultimately affects the metabolic activity
pressure. of the inoculum culture? There are two systems
generally used for the on-line volume determinations:
i. Flow Rate: Gas flow rate is important in aerobic
1) liquid level sensors and 2) weight or mass
fermentations. Likewise the rate of gas production is of
measurement devices. Capacitance probes measures
interest for cultures producing biogas. Liquid flow rates
the change in capacitance when liquid level changes
must be known for continuous and fed batch processes,
in small-scale fermentors. In large-scale fermentors
where the rate of nutrient feed is an essential variable
∆P measurement method is generally used, where a
for efficient operation of the process by means of
flush mounted diaphragm pressure cell is located in
mass balancing control. Furthermore, knowing the
the base of the vessel for liquid height and hence the
liquid flow rate is necessary to control the addition
volume measurement.
of corrective liquid feed streams, such as amount of
base or acid consumed for pH control or the amount m. Weight/Mass: Scales of various types determine
of antifoam input. Flow rates are mainly measured weight or mass of the liquid. In this method the vessel
by the following devices: 1) Floating body flow meter is suspended on a scale and the combined weight
2) Differential pressure flow meter 3) Rotating flow of the vessel and strain type gauges electronically
meter 4) Electromagnetic flow meter Floating Body measure liquid. This method cannot be applied to the
Flow Meter Impeller Speed For stirred tank fermenter, fixed existing installations.
impeller speed is an important operating variable,
 Basic of Fermentation Technology

7
Chemical process variables measurement, which in turn is correlated to oxygen flux
reaching the cathode surface. Fluorescence Quenching-
a. Exhaust gas analysis: The concentration of carbon For medical investigations so called Optodes has been
dioxide in the exhaust gas from cell reaction is indicative developed for oxygen and carbon dioxide determinations.
of the respiratory activity and fermentative activity In this, the sensitive element is the membrane into which
of the inoculum culture and hence is one of the most a fluorescence indicator (Pyrene butyric acid or β- methyl
useful and widely applied measurement methods in belliferon purine) has been incorporated. This membrane
the monitoring and controlling a cell bioreactor. Using is brought in contact with the broth. The fluorescence
an Infrared spectrophotometer, gas chromatography quenching in indicator is indicative of the presence of
and mass spectrometer most commonly controls oxygen or carbon dioxide. This method does not consume
carbon dioxide content in a bioreactor. Gas stream oxygen or carbon dioxide.
oxygen partial pressure is usually measured using a
paramagnetic analyzer. Care should be taken in both c. pH: pH value is an important indicator of the state of the
the cases that water vapors should be eliminated from state of biochemical process. The automatic addition of
the exhaust before feeding them into the analyzer. The alkali or acid to fermentation broth can be achieved by
paramagnetic analyzers are quite sensitive to small techniques already in use in other chemical industries
changes in total atmospheric pressure so they require but special electrodes have been developed for use
simultaneous monitoring of barometric pressure for in fermentation industry. The half-cell of the glass
compensation in oxygen analysis. Apart from water electrode was composed of Ag/AgCl saturated with KCl.
vapors the samples should also be free from dust The solid KCl increases the mechanical resistance of
particles, aerosols and oil droplets. the glass particles of KCl on the glass surface during
heat sterilization and cooling of the electrode. Other half
b. Dissolved gases and volatiles: Dissolved oxygen of the electrode is composed of the same material as
and carbon dioxide are both important variables in the glass electrode, asbestos or porcelain cylinder being
fermentations. These are normally determined by 1) used as the junction material. To ensure good insulation,
Electrochemical methods 2) Fluorescence quenching both the glass and reference electrode were mounted in
and 3) Mass Spectrometry Electrochemical Methods- Teflon gaskets and silicone rubber washer were fixed.
Electrochemical determination of oxygen and carbon The internal resistance of the electrode is 300-500meg
dioxide in fermentation media is performed by means of Ohm. Steel sleeves provided with several holes to allow
special sterilizable electrode. Analysis by this electrode free passage of broth protect both of these electrodes.
is based on detecting the amount of oxygen diffusing
from the liquid membrane into an Amperometric or d. Redox potential: Another method obtained with
Polarographic measuring cell. Amperometric principles electrochemical method is the Redox potential
are most frequently used. In Polarographic type oxygen measurement method. Every redox system consists of
electrode a constant voltage is applied between cathode two components, one is oxidized by electron donation
and anode. At cathode the oxygen that has diffused into and the other is reduced by electron acceptance.
the cell is reduced to hydroxyl ion as shown below: In such a system, an electrochemical potential can
be measured by means of an unprotected electrode
Cathode: - O2 + 2H2O + 4 ē → 4 OH ˉ consisting of a noble metal (Au/Pt), the composition
Anode: 4 Ag + 4 Cl ˉ → 4 AgCl + 4 ē of which is chosen on the basis of relation of donor to
the acceptor. In a fermentation system/culture, a great
The response of the probe is proportional to the oxygen number of redox systems are present simultaneously.
activity in liquid. Since at equilibrium i.e. for a saturated Accordingly an exact interpretation of signals from the
liquid the activity of a solute is directly related to its partial redox potential measurements cannot be given. It is for
pressure (fugacity), the readings of electrochemical that reason that some scientists suggested to rather
process are commonly given as percent partial pressure or name this potential as Platinum-electrode potential
saturation. instead of redox. The competing donors i.e. oxygen
In Potentiometer probe same principle of oxygen and glucose that are present in a fermentation system
diffusivity is used and this diffused oxygen is reduced at may serve as a typical example. In spite of the difficulty
cathode surface according to the same above equation: of interpreting the results, measurements of redox
potentials permit an important insight into the course
Pt of fermentations. Sterilizable Platinum electrodes are
½ O2 + H2O + 2 ē → 2OH ‾ commercially available. They either contains built in
reference electrode (Ag/AgCl) similar to pH electrode
The reaction at anode in galvanic electrode is as follows: or they are used in combination with pH measurement
making use of the same reference electrode. The
Pb → Pb2+ + 2 ē
amplifier for redox measurements is of the same type
This reaction competes with the cell from which a as in conventional pH meters. As in the latter case,
small amount of current is drawn to provide a voltage the built in electrode is sterilized together with the
fermenter. Since no membrane is required so any
 Basic of Fermentation Technology

8
special sterilization problem exists in these electrodes. fold increase in growth rate per 10 °C rise in temperature.
The signal of the redox meter is influenced by pH. This Growth rate approaches zero at 10 to 25 °C below the
can be tolerated because fermentations are usually run optimum temperature. Chemical reaction rates are related
at constant pH. to temperature by Arrhenius equation:
e. Enzymatic analysis of substrate: Enzymatic analysis – E / RT
K = Ae
allows very specific determination of many organic
compounds. This group of methods takes advantage Where K = reaction rate; A= Arrhenius constant; R = Gas

1/ T
of the ability of enzymes to react selectively with well- constant; T = Absolute temperature; E = Activation energy
defined compounds or rather chemical structures; or temperature characteristics.
organic compounds present in fermentation media
Taking log on both sides we have:
(substrates and metabolites) can be analyzed.
Usually this is performed offline with samples taken Log µ
from fermentors but methods for online enzymatic
analysis have also been developed. The procedure
consists of determining the conversion of the enzyme- 1/ T
catalyzed reaction with the respective substrate for
analyzing one of the products either calorimetrically or Plot of Log K against 1/T should be a straight line with
electrochemically. In applying online enzymatic analyses slope of E/2.3 RT. If we substitute specific growth rate ‘µ’
to sterile fermentations, special difficulties arise because for reaction rate K in the above equation then converting
conventional sterilization techniques apply steam at it to straight line equation and plotting log µ against 1/T,
120˚C that will destroy the enzymes. One solution to this keeping the value of E constant we find a straight line with
is to employ dialysis through which a recycled sample slope of E/2.3RT. The Q10 value also varies inversely as
stream of broth is conducted. Components diffusing the temperature varies from normal as
through the dialysis membrane can then be monitored
continuously by means of enzymatic analysis. Such Q10 = E . 10 / 2.3RT (T+ 10)
systems have been developed for measuring glucose,
saccharine and lactose.
f. Ion specific electrodes: Not only carbon source and The activation energy is a valuable constant as it can be
other organic compounds but also inorganic salts (N, P, used to predict the effect of temperature on growth rate over
S, K, Mg, Ca, Na, and Fe) are essential constituents the normal temperature range. Changing in the activation
of fermentation broths. Ion specific electrodes have energy indicates that differences in rate controlling reactions
been proposed for a number of these ions. Some or in the metabolic regulations can occur. Temperature
electrodes of this type are commercially available for range for growth of individual bacteria extends over about
offline measurements. There is little known about the 35°C. Extreme psychrophiles grow between -5 to 30°C
online measurement using ion selective electrodes. Ion and extreme thermophiles 55 to 90°C. Decrease in growth
selective electrodes are Infact potentiometer electrodes rate at high temperature is due to disruption of metabolic
applying different principles e.g. for measurement of regulations or death of cells by protein denaturation. When
Na+ glass electrodes with glass membrane especially death of cells occur the growth rate of the viable biomass
sensitive to Na+ are employed. In some electrodes (X) is given by:
organic membranes are employed while in others
dX/dt = (µ -K) X where µ = specific growth rate; K =
enzymes may be incorporated. For these types of
specific death rate and X = biomass
electrodes sterilization may be the problem.
Death rate becomes dominant if at high temperature
How Temperature and pH Affect the Growth activation energy for death exceeds that for growth. Increase
of Microorganisms in temperature causes breakdown of protein structure so
the affinity for substrate and enzyme regulators will be
Effect of temperature on the growth of affected. Thermophiles possess proteins with exceptional
microorganisms heat resistance. Temperature also affects the nutrient
Bioprocesses of microorganisms are heavily affected by requirements, lowering of the growth temperature causes
the temperature. The cell temperature must become equal small increase in the growth yield from carbon and energy
to the culture temperature. Temperature affects the rate source. The pathways of metabolism of carbon and energy
of cell reactions, the nature of metabolism, the nutritional source can be temperature sensitive e.g. Lactobacillus
requirements and biomass composition. The temperature brevis ferments glucose by heterolactic pathway at 24°C but
coefficient of growth rate is denoted by Q10 value (It at 32oC requires fructose as hydrogen acceptor for glucose
is defined as increase in growth rate per 10°C rise in fermentation. Growth factor requirements also change
temperature e.g. If Q10 is equal to 2 that means there is two with temperature e.g. Yersinia pestis requires different
amino acids and vitamins at growth from 37 °C to that at
 Basic of Fermentation Technology

28°C. Temperature affects the product formation e.g. over industrially by the fermentation process. The first commercial
9
production of riboflavin by Ashbya gossypii requires growth production of lactic acid in USA by microbiological process
of the microorganism at 28°C than at its normal temperature took place in 1881 as its calcium salt Calcium lactate.
because growth at low temperature causes breakdown of The plant for its manufacture was built in Littleton,
normal regulation of synthesis of enzyme system which Massachusetts, but little is known about its process. The
produces the riboflavin. Similarly the optimum temperature Clinton processing Company, Clinton Iowa is the only
of production of Penicillin is lower than that of normal manufacturer using fermentation process for the production
growth temperature. Temperature affects the microbial of lactic acid in United States.
composition as RNA content of bacteria or yeasts increase
i. Isomers: Lactic acid of commerce is the L (+) isomer,
several folds on decreasing the temperature. Yeast lipids
D (-) isomer or any possible mixture of the two. The
increase their unsaturated fatty acids when temperature
entire range of isomers has been found. The mixture
is lowered. Antigenic composition of bacteria varies both
of the two forms of isomers is called as DL mixture or
qualitatively as well as quantitatively with temperature e.g.
racemic mixture. DL mixture is optically inactive form.
virulent Yersinia pestis is produced at 37°C but not at 25°C.
Microorganisms differ in their ability to produce either D
Mechanism of temperature effect (-) lactic acid or L (+) lactic acid or racemic mixture and the
particular acid formed are the characteristic of individual
The effect of temperature can be explained as: 1) microorganism. From industrial standpoint the lactic
Dependence of structure of cell components on temperature, acid recovered from fermentation broth usually is the
2) Activation energy required for the reactions to occur racemic mixture because fermenting microorganisms
inside the cell which in other term affects the regulatory or contaminants like Lactobacillus plantarum produce
mechanisms of the cell, cell composition and permeability an enzyme known as Racemase that converts either of
functions. the optically active isomer to optically inactive racemic
Effect of pH on the growth of microorganisms mixture. Some trace impurities in the medium also
have been reported to bring about racemization. The
The influence of [H+] on biological activities is related to Racemase are known to be lactic dehydrogenises that
either hydrogen ion concentration or hydrogen ion activity maintain equilibrium between lactic acid isomers and
(ah). These two parameters are proportional as: pyruvic acid. When both dehydrogenase enzymes are
present, racemization occurs.
ah = f [H+] where f is the activity coefficient which may
vary with the ionic strength and other factors. The glass ii. Microorganisms: Many types of microorganisms
electrodes respond to hydrogen ion activity so that strictly have been isolated that accumulate lactic acid or
pH = -log (ah) and [H+] can be substituted for hydrogen lactates in the culture solutions e.g. Lactic acid
ion activity. It can only be possible when activity coefficient bacteria, algae, molds, yeasts and phycomycetous
is one. In dilute media solutions f approximately is one. fungi. Apart from many Lactic acid bacteria, mold
But this may be far from true when media are strong Rhizopus oryzae has been found to produce lactic acid
salt solutions. As far as cell properties are concerned comparable to homofermentative lactic acid bacteria
hydrogen ion activity is more meaningful parameter so from glucose. Lactic acid bacteria fall under two main
that it is appropriate to express the effect of hydrogen ion groups: (1) Homofermentative Lactic acid Bacteria (2)
concentration in terms of pH. Plasma membrane is not Heterofermentative Lactic acid Bacteria
freely permeable to hydrogen ions or OH- ions. So that
intracellular and extracellular hydrogen ion concentrations
do not necessarily equilibrate and a gradient of hydrogen
ion across the plasma membrane is established. According
to chemiosmotic theory this gradient of hydrogen ions
together with membrane electric potential makes a proton
motive force that derives the membrane reactions.

Lactic Acid
Scheele (1789) first isolated lactic acid from sour milk.
The studies on the physical and chemical properties have
shown that the compound occurs in two isomeric forms and
as a mixture of the two isomeric forms. Lactic acid is also
produced in muscles. Many microorganisms produce lactic
acid by the fermentation of sugars. The structural formula of
Lactic acid is CH3 -CH (OH)–COOH.
iii. Homofermentative lactic acid bacteria: These
Technological development bacteria produce lactic acid as the major or sole
Lactic acid was the first organic acid to be manufactured product of glucose fermentation. The homofermentative
 Basic of Fermentation Technology

10
pattern is observed when glucose is metabolized but Medium
not necessarily when pentoses are metabolized, for
some homolactics produces acetic acid and lactic acid The fermentation solutions usually contain hydrolyzed
when utilizing pentoses. Also, the homofermentative starches or dextrose syrup, although D-glucose, maltose,
character of homolactics may be shifted for some lactose or sucrose can also be fermented. It is advantageous
strains by altering cultural conditions such as to start with a relatively simple medium or mash in order
glucose concentration, pH and nutrient limitation. The to facilitate recovery of the product. Crude carbon sources
homolactics are able to extract about two times as are generally avoided because the impurities interfere
much energy from a given quantity of glucose as are with the recovery and purification procedure. The sugar
the heterolactics. They are less important in producing concentration in the medium should not be more than
flavor and aroma components e.g. acetaldehyde 12-15% because Calcium lactate produced at high sugar
and diacetyls in food products. They possess the concentration tends to crystallize from the medium late in
enzymes aldoses and hexose isomerase but lack the fermentation, thereby slowing the fermentation process.
phosphoketolase. They use EMP pathway to produce Nitrogen sources are added in small amounts and are
two molecules of lactate per glucose molecule. usually inorganic in nature e.g. Ammonium phosphate.
Examples of homofermentative lactic acid bacteria are: This is because impurities in the nitrogen sources might
All members of genera Streptococcus, Pediococcus interfere in the recovery and purification procedure.
and some genera of Lactobacilli like L. delbrueckii Calcium carbonate (10%) is added to neutralize the
Homofermentative lactic acid bacteria are very lactic acid produced because lactic acid bacteria cannot
important for the production of Lactic acid industrially. tolerate high acid concentration. Lactic acid bacteria have
complex requirements of B-vitamins. This ordinarily is met
iv. Heterofermentative lactic acid bacteria: They by enrichment of the culture medium with crude vegetable
produce some lactic acid, but at the same time, they materials. Malt sprouts are commonly used vegetable
also produce carbon dioxide, ethanol, and acetic acid materials, but if they have been overheated in drying, they
and trace amounts of a few other products. These lose some of their value as a nutrient for Lactobacilli.
organisms are of little use for industrial lactic acid
fermentations because too much of the substrate Production of Lactic Acid
carbon is directed towards products other than lactic
Today much of the lactic acid is produced by the
acid. The end product differences between homo and
hydrolysis of lactonitrile, a byproduct of another process.
Heterofermentative lactics when glucose is attacked are
Only a few companies in the world are producing lactic acid
a result of basic genetic and physiological differences.
by fermentative process.
The heterolactics have phosphoketolase pathway
but do not possess aldolase and hexose isomerase. Equipment
Instead of EMP pathway such organisms use
Hexose monophosphate shunt as energy pathways. Lactic acid is very corrosive to metals therefore
Heterofermentative bacteria are very important for wooden fermentors are used in most of the plants. These
the production of flavor and aroma components like fermentors may be uncovered or covered with loosely
diacetyls in food products. Examples of Heterolactics fitting wooden lids. They are to be steamed empty before
are: All species of genus Leuconostoc, and some charging. Fermentation solutions are pasteurized or
species of Lactobacilli. sterilized by passing it through a steam jacketed heat
exchanger. Contamination of culture solutions sometimes
Microorganisms for Commercial Production by thermophilic Clostridia results in the production of some
of Lactic acid butanol and butyric acid. Such contaminated lactic acid
could only be sold to leather tanners for delining of hides. A
The microorganisms used for commercial production of very pure lactic acid is required for manufacturing of plastic.
lactic acid depends upon the raw material to be fermented, Such grade of lactic acid is called Plastic grade lactic acid.
but the most common bacterium used for this purpose is
Lactobacillus delbrueckii; it is employed in fermentations Inoculum
utilizing corn dextrose medium. Although increasing Cultures of L. delbrueckii are transferred from test tubes
use is being made of a flat sour Bacillus coagulans yet through successively larger culture vessels, held at 45-55
Lactobacillus bulgaricus is used for the production of lactic °C. Each stage of the culture build up requires 16-18hours
acid from whey because it utilizes lactose as a carbon and slight excess of Calcium carbonate is required at each
source. L. pentosus (L.plantarum) is recommended for use stage. Inoculum volume should be 5% of the volume of
in spent sulfite liquor, as it is able to utilize pentoses. L. fermentation solution.
brevis is used when medium contains hydrolyzed corncobs,
cotton seed hulls etc. Other homofermentative species of pH
potential industrial importance are L.casei, L.leichmannii,
An excess of Calcium carbonate keeps the pH in the range
and Streptococcus lactis. These are facultative anaerobes
of 5.5-6.5. The pH necessary varies with the composition
and can withstand some oxygen. Streptococcus lactis is
of culture solution but it is controlled by continuous
particularly useful under such conditions.
 Basic of Fermentation Technology

11
neutralization with the slurry of Calcium hydroxide between confectionary, sherbets, soft drinks, extracts and other
6.3-6.5. Fermentations utilizing grain may resist increase in products. It is added to brines for pickles and olives and
pH with the buffering capacity of mash. to fish to aid preservation. Its addition makes milk more
digestible to infants. Calcium lactate is an ingredient of
Aeration and agitation some baking powders. In tannery, it is used for washing of
The medium is not aerated but agitation is done to hides. In textile industry it is employed for fibre washing.
keep the Calcium carbonate in suspension. Fermentation Lactic acid is used in the preparation of medicines. It is also
Time and Fermentation Temperature: The fermentation used as a laboratory reagent and a research tool.
temperature is adjusted to 45-50 °C and varies the type of
Citric Acid
organism used. Same is the case with fermentation time.
It is usually completed in six days or less depending on Scheele first isolated Citric acid in 1784 from Lemon
the time required by the organism to deplete the sugar in juice by crystallization process. Members of citrus family
the medium. Fermentation time is usually 5-10 days. It is of fruits especially are rich in this organic acid, but citric
important that residual sugars be reduced to 0.1% or less acid is also found as a natural constituent of a variety of
during the fermentation because residual sugars make fruits. Citric acid extracted from fruits is designated as
recovery of better quality lactic acid difficult. Natural Citric acid in contrast to the Citric acid produced
by Microbial Fermentation process. Citric acid can also be
Yield prepared synthetically but no equivalent synthetic process
Commercial yields are 93-95% by weight of glucose has been invented to the microbial fermentation.
supplied. Recovery yields vary with the various recovery
Microorganisms
processes and product grades.
Many microorganisms like molds (Aspergillus niger,
Product Recovery and Grades A. awamori, Penicillium janthinallum, Trichoderma
viridae, Mucor piriformis, etc.), yeasts (Candida lipolytica,
Technical grade lactic acid
C.tropicalis, C.citrica, Hansenula, Rodotorula, Pichia,
Calcium from the fermentation solution is precipitated as Torulopsis etc.) and bacteria (Bacillus licheniformis, Bacillus
Calcium Sulfate, filtered, and filtrate is evaporated to 35- subtilis, Brevibacterium flavus, Corynebacterium species)
40% lactic acid. Now more Calcium sulfate is precipitated, have the capability to produce citric acid. Commercially
filtered, and filtrate is evaporated to 44-55% total acidity. It spores of Aspergillus niger are employed to produce citric
is then crystallized. acid.

Food grade lactic acid Methods of fermentation

It is a pale yellow, straw colored solution of about 50% Fermentation of Citric acid is carried out by any of the
total acidity. Calcium in this case is precipitated as Calcium following methods 1) Stationary or Surface culture, 2)
sulfate, precipitates are washed, washed water is combined Submerged Culture, 3) Solid State Culture, 4) Continuous
with the filtrate, filtrate is bleached with activated carbon Culture, 5) Multistage Culture process, 6) Semi-Continuous
and it is then subjected to evaporation firstly to 25% solids, Culture process.
again bleached and then secondly evaporated to 50%
In stationary or surface culture, sterile nutrient medium
solids, bleached finally to produce an off colored product.
with sugar is added into stainless steel or high-grade
Impurities like Iron and Copper metals are removed by
aluminum trays sterilized with formaldehyde or sulfur
adding Potassium ferrocyanide in the filtrate of first filtration.
dioxide. Spores of Aspergillus niger are inoculated and
Plastic grade lactic acid incubated at 28-30 °C for 8-12 days. Submerged process
consists of two phases i.e. growth phase and productive
It is a colorless product. Product is recovered by phase. In Growth phase medium is inoculated with spores of
etherification with methanol after concentration or by solvent A. niger, after 3-4 days mycelium is separated from solution
extraction with isopropyl ether followed by re-extraction of and added to the fermentation medium and fermentation is
isopropyl ether with water. allowed to occur for 3-4 days. During the period production
Pharmaceutical grade lactic acid (Lactic Acid of the Citric acid takes place. This phase now is called
as Production phase. In Solid state Culture as described
USP)
by Calm (1935), fermentation medium is impregnated in
It is a colorless product with 85% total acidity and 76- porous solid materials like sugarcane baggase, potato or
78% concentration. It contains 2-3.5% volatile acids 0.5- beet pulp, or pineapple pulp in an appropriate ratio, sterilized
1.0% ash content. and then inoculated with a suspension of fungal spores
and incubated at 25-30 °C for 6-7 days. In Continuous or
Uses of Lactic Acid multistage culture, the medium is replaced after 24 hours
In the foods, lactic acid is added to acidify jams, jellies, and that medium goes to second fermenter. It is then
 Basic of Fermentation Technology

12
aerated by gradually increasing the amount of air. In Semi- from the culture medium various methods adopted are: 1)
Continuous Culture, the whole medium is not replaced but Pretreatment of the raw materials. 2) Ion exchange resins.
a part is replaced. 3) Development of resistant varieties of fungus. The raw
materials are treated with Potassium ferrocyanide to reduce
Medium the concentration of iron ions in molasses. The chemical
Components of the medium varies with the type of the is either added directly in the medium or the molasses
process used because it has been seen that the strains that is treated with its high concentration. Raw materials are
give good result in surface culture do not perform better also sometimes treated with chelating agents like EDTA,
in submerged culture. Fungi have been seen to give much activated charcoal or polythene amine. Chemicals of
better results in simple synthetic media. A variety of carbon quaternary ammonium compounds category like Diiosbutyl
sources are being used these days, which include Sucrose, phenoxy ethyl dimethyl benzyl ammonium chloride and
Citrus molasses, Cane juice, Starch from various sources, Triton-B are also used.
Cane or Beet molasses. The initial sugar content determines
pH
the amount of citric acid produced by A. Niger. Normally
15-18% sugars are added into the medium. Concentration Fungus can tolerate high concentration of acid therefore
of sugar more than 15-18% leads to greater amounts of calcium carbonate is not added in the medium. The initial
residual sugars that make the process uneconomical. required pH of the medium depends upon the carbon
When the sugar concentration is lower than the above source used as follows: Glucose or clarified molasses pH =
percentage, it leads to lesser yield of citric acid as well as 3.0, Crude molasses pH = 5-6, Decationized molasses pH
accumulation of oxalic acid. Molasses contains 50-60% = 1.4-2.8, Sucrose pH = 2.0-3.0. pH is adjusted with HCl,
sucrose and is of three types: 1) Black Strap molasses- it H2SO4 or NaOH.
is obtained from the last stages of crystallization of sugar.
2) Refinery molasses- it is obtained at the second stage of Additive and stimulants
refining sugar. It contains 48-50% sucrose and has high ash Methanol is usually used to increase the yield at a
content. 3) Invert or High-test molasses- it is partly inverted concentration of 3-4%. At this level it retards growth, delays
cane juice syrup from which no sugar has been extracted. sporulation and increases citric acid yield. This is added
Molasses is diluted to sugar concentration of 15-20% and before inoculation. It has some role in the conditioning the
pH adjusted to 5.5-6.5 with sulfuric acid. Molasses is added mycelium without impairing their metabolism. It is thought
with other required nutrients and mixture is sterilized for that it increases the tolerance of the fungus towards the
30 minutes. For the growth stage of Aspergillus niger, the harmful effects of the trace elements. Other additives like
organism needs major elements (C, N, P, S) and trace hydrogen peroxide, Methylene blue, Naphthaquinone,
elements. The type of inorganic source and its concentration aromatic amides, esters of dichloroacetic acid, sodium
affects the performance of the fungus. Inorganic sources sulfite, crysyllic acid, glycerol, vegetable oils (corn oil,
like Ammonium sulfate prolong vegetative growth. almond oil and peanut oil) and cAMP are added as stimulant
Ammonium nitrate shortens the growth phase. Ammonium to increase the yield.
nitrate in concentration greater than 0.25% accumulates
more of oxalic acid whereas sodium nitrate at concentration Antifoaming agents
0.4% delays the onset of production phase and vegetative
Antifoaming agents like Octadecanol (0.75% solution) or
growth also increases. Phosphate also affects the citric
Antifoam AE (silicone oil) are added in the medium. To control
acid yield. High phosphate concentration promotes growth
contamination by bacteria Pentachlorophenol ate, formic
and there is less acid production. Therefore phosphate
acid, tetracycline’s, and 3-furaldehyde semicarbazone are
concentration in the medium should be kept at 0.1%-0. 2
added in the medium.
%. Trace elements (Fe2+, Cu2+, Zn2+, Mn2+ and Mg2+)
are necessary for A. niger. Mg2+ is necessary for a variety Biochemistry of Citric Acid Formation
of enzyme reactions in the cell. It is required for growth
Citric acid is formed via following pathways: (Flow Chart 1)
and production phase. Its optimum concentration in the
medium should be 0.02-0.025%. Fe2+ and Zn2+ have Citric acid accumulates in culture solutions of pH 1.8-
critical role to play in the growth and production of citric 2.0. In fungi different metabolic pathways are involved in the
acid. Both are essential for the production of citric acid in production of citric acid but 78% of the citric acid is produced
low concentration because their high concentration allows by the involvement of glycolysis and Tricarboxylic acid cycle.
vegetative growth. Iron also has deleterious effects on the The acetyl COA derived by EMP pathway condenses with
fungus but its action is counteracted by copper ions. Zn2+ Oxaloacetate of Kreb’s cycle to produce citric acid. Since it
at concentration 1-2 µM allows growth phase but its less has been observed that, during accumulation of citric acid,
concentration restricts growth. Its excess in the citric acid A.niger demonstrates decreased activity of condensing
producing culture reverses the production phase. It also has enzyme and almost no activity of Isocitrate dehydrogenase
its indirect role in the functioning of cAMP that enhances the and aconitase therefore another theory of its production
production phase. To remove the excess of trace elements came into light. In this pathway, glucose first splits up into
 Basic of Fermentation Technology

13
two 3-Carbon fragments followed by decarboxylation of heating the mixture to 80-90°C. Calcium citrate is filtered off
other fragment to yield a 4-Carbon compound. The 2-C on conventional filter and the filter cake is transferred to a
and 4-C compounds then combine to yield citric acid. In tank called acidulator where it is treated with Sulfuric acid to
methyl citrate pathway Propionyl COA formed through the precipitate calcium sulfate, which is then filtered. The dilute
β-oxidation of n-alkanes condenses with Oxaloacetate to filtrate containing citric acid is purified by treatment with
yield methyl citrate. activated charcoal and demineralized by passing it through
ion exchange resins. The purified solution is evaporated
Product recovery in a circulated granulator or circulating evaporator called
The crude fermented liquor is subjected to filtration to crystallizer. Crystals are removed by centrifugation. Citric
remove mycelia of fungi. Calcium citrate is precipitated acid so prepared is subjected to recrystallization. The
from the clear liquid by adding slurry of calcium hydroxide remaining mother liquor is added into the main stream prior
(add hydrated lime 1 part for every 2 parts of liquor added to limming and decolorization (Flow Chart 2).
over one hour period and temperature raised to 95°C) and

Flow Chart 1

Flow Chart 2
 Basic of Fermentation Technology

14
Single Cell Protein (SCP) the source of carbon for photosynthetic-autotrophically
growing cultures. Air contains only 0.03% carbon dioxide.
Definition -The dried cells of various groups of Accordingly, additional carbon dioxide must be supplied to
microorganisms like Bacteria, Yeasts, Molds, Higher fungi the cultures. Some algae can also be grown in lakes rich
and algae that have been considered for food or feed are in sodium carbonate. In sewage ponds, the algal growth
collectively referred to as Single Cell Protein. C.L. Wilson is limited by the extent of liberation of carbon dioxide and
in Massachusetts institute of technology coined the term ammonia by bacterial action. Slow and uniform liberation
SCP in 1966. People have eaten certain microorganisms of carbon dioxide is necessary to provide a uniform
as a portion of their diet e.g. Top fermenting yeasts supply of carbon source to the growing culture. Nitrogen
(Saccharomyces spp.) was recovered as a leavening sources suitable for algal growth include ammonium salts
agent for bread as early as 2500 B.C. Fermented milks or nitrates. The N-sources together with phosphorous and
and Cheeses produced by Lactic acid bacteria of genera mineral nutrients may be readily available in sewage ponds.
Lactobacilli and Streptococci were consumed by early However on other water supplies where synthetic media
Egyptians and Greeks and reached a higher state of are used in culturing algae, these nutrients may have to
development during the Roman era (100-50 B.C.). Pharaohs be supplied. Aseptic conditions are not maintained during
of Egypt prized wild mushrooms as a delicacy. Romans the large-scale growth of algae in ponds but the potential
regulated the grading and selling of mushrooms. People contamination by pathogenic bacteria may be given
of Chad regions of Africa and Aztec in Mexico have eaten serious consideration. The key factors in a large-scale algal
Spirulina spp. (Blue green Algae) for many generations. cultivation are agitation and flow rates. Agitation prevents
The purposeful cultivation of microorganisms for direct use sedimentation and keeps the cells in suspension while flow
in human food or animal feed is a fairly recent development. rate adjustments allows the detention period of algal culture
Considerable efforts have been expanded since World War- to exceed the generation time. This helps in maximum
II to develop mass cultivation of microbial cells. population development.
Nutritive value and use of SCP Cell recovery
Nutritive value of SCP varies with the microorganism Cells must be recovered by concentration, dewatering
used. Protein digestibility of SCP is expressed as and drying. The inorganic compounds like Aluminum
percentage that ranges from 65-96 for various cultures. sulfate, calcium hydroxide and cationic polymers have
Protein efficiency ratio ranges from 0.6-2.6. Food yeasts been investigated as flocculating agents but these do
are high in proteins and vitamins of B-12 category but not separate from algae. Algae may auto flocculate in
may be deficient in some amino acids such as cysteine shallow ponds at pH 9.5 or above without flocculants. Ion
and methionine. The UN protein advisory group has major exchange resins at pH 2.8-3.5 can also recover algae but
concern over the use of SCP for human beings because this method is expensive. Spirulina maxima float on the
of the following reasons: 1) The high nucleic content of the surface in clumps when maximum growth is attained. It can
SCP may elevate serum uric acid level, which may result be harvested by skimming at much lower cost than would
in kidney stone formation or gout. 2) Certain skin reactions be incurred by centrifugation.
may occur by consuming foreign proteins 3) Possibility
of carrying over of carcinogenic factors 4) possibility of SCP production by bacteria and actinomycetes
gastrointestinal reactions.
Bacteria are of interest for use in SCP production
Production of SCP because of following reasons: 1) High growth rate (20-
30 min. in bacteria, 2-3 hours in yeasts and 16 hours in
a. Production of SCP by algae: Algae can be grown algae, molds and higher fungi). 2) Bacteria can utilize a
photo synthetically or autotrophically in the Presence of variety of carbon and energy sources, renewable sources
sunlight, artificial light or heterotrophically in dark with as carbohydrates (starch, sugars and cellulose) and non-
organic carbon and energy sources. renewable sources as hydrocarbons and petrochemicals.
Microorganisms Actinomycetes are of interest because their growth patterns
and substrate utilization patterns are similar to bacteria.
The most important algal strains used for SCP production
include: Microorganisms
Chlorella sorokiniana, Scenedesmus acutus, S. Methylococcus capsulatus, Methylomonas methylovora,
quardicanda, S. obliqus, Spirulina maxima, S. platensis. Methylophilus methylotrophus, Pseudomonas spp.

Growth conditions Growth conditions

In an open circulating system particularly sewage Some important considerations for selecting bacterial
oxidation ponds, mixed culture of algae tend to predominate cultures suitable for use in SCP production are: High Specific
rather than a single strain. The limiting factor in the algae growth rate, yield on a given substrate, pH and temperature
growth on a large scale is illumination. Carbon dioxide is tolerance, less aeration requirement, genetic stability and
 Basic of Fermentation Technology

freedom from associated bacteriophage. Criterion for using having hydrocarbons and alcohols; lower concentrations
15
bacteria for SCP production is as follows: 1) Strains should are to be used. Ammonium salts or anhydrous Ammonia
not be pathogenic to plants, animals or human beings. 2) is suitable as nitrogen source. Phosphoric acid is used
Strains should not have potential for mating with known to adjust pH. Yeasts when grow on substrates like
pathogenic bacteria to yield pathogenic hybrids. Growth carbohydrates, hydrocarbons or alcohols, a lot of heat
condition requirements are: 1) Carbon and Nitrogen ration is liberated therefore heat tolerant varieties should be
in the medium should be 10:1 or less to favor high protein employed for production (e.g. Hansenula polymorpha can
content in cells and to prevent accumulation of lipids or grow at 37-42°C) and cooling water or refrigeration facilities
storage substances such as Poly-β-hydroxy butyrate. should be employed in the fermentation. The growth rate
Nitrogen is added as anhydrous ammonia or ammonium of yeasts under aerobic conditions depends upon the rate
salts and phosphates as phosphoric acid (feed grade) to of mass transfer of oxygen and substrate to and across the
avoid contamination with arsenic or fluorides found in crude cell surface. Yeast SCP production may or may not take
industrial phosphoric acid. Minerals are added as Magnesium place under sterile conditions. In either batch or long-term
and Manganese in water. Other minerals are added as continuous yeast production one must balance the need for
sulfates and hydroxides but not chlorides. pH is controlled contamination control by maintaining sterile conditions, with
in the range of 5-7. Temperature tolerance is important for the capital and operating costs of the equipment required.
bacteria grown on alcohol hydrocarbons. Maintenance of The temperature of fermentation is maintained at 36-37°C
sterile conditions is important to avoid contamination at and pH 4.5-6.0. Yields are 45% or more of the dry yeast on
pH 5-7. Different carbon sources may be used for bacteria the basis of the sugar fed.
these may be carbohydrates sugar, starch, cellulose, and
baggase, wheat bran, and wood, petroleum products diesel Cell recovery
oil, gas oil, and n-hexane, proteins (Collagen, meat packing Yeast cells range in size from 5-8µm and have a density
waste), methane, Alcohols (methanol) and n-alkanes. 1.04-1.09 gm/cm3. They can be recovered readily from the
medium by centrifugation. The cells are centrifuged in 2
Cell recovery
stages. In the first stage it is dewatered and yeast cream
A number of problems come in way in bacterial cell is separated. This is followed by two subsequent washing
recovery process because large volume of water is to be centrifugations. The final washed yeast cream usually
handled and bacteria have very small size in addition to contains 15-20% solids. In SCP by Kluyveromyces fragilis
that the bacterial cell densities are very close to water. grown on cheese whey, the entire growth medium is passed
Therefore they are separated by centrifugation, filtration through a three-stage evaporation to concentrate the solids
and electrochemical coagulation methods. After separation from 8-27% to give a feed grade product. The separated
they are spray dried. cells can either be drum or spray dried.

Production of SCP by yeasts Ethanol as Fuel

Yeasts are probably the most widely acceptable and Ethanol can be used as such or in blends with petrol as
used microorganism for SCP production. Yeasts have the a motorcar fuel. About 10% ethanol can be used without
capacity to grow on a variety of substrate including waste modifications in the engine but the only disadvantage it has
products that contain pentose sugars. Yeasts in general is that it increases the octane rating. Combustion engines
have several advantages over bacteria and algae these are: can be built to run straight on ethanol or on ethanol of
1) better public acceptance 2) lower nucleic acid content 3) lower concentration (80% ethanol and 20% water). Current
easier harvesting because of size and concentration 4) high interest in various parts of the world centers to use a blend of
protein content 5) production of vitamins of B-12 category ethanol called ‘Gasohol’, which is a blend of 90% unleaded
6) growth in substrates of low pH. petrol and 10% ethanol. Anhydrous ethanol is preferred for
this application but 96.5% ethanol can be used if mutually
Growth conditions miscible solvent such as isopropyl alcohol is added.
a. Raw materials used as substrates: Materials that
are employed to produce SCP by yeasts
Raw materials
Forty-five kilograms of fermentable sugars (glucose) is
include: 1) molasses from sugar industry 2) starch after
assumed to yield 18-23 kg or 23-28 litres of ethanol. The
hydrolysis 3) spent sulfite liquor from paper industry 4) acid
yield is same for starchy raw materials i.e. between 40-50%
hydrolysates from wood industry 5) whey from dairy industry
based on dry weight of carbohydrates. Complete hydrolysis
6) hydrolyzed starchy foods (grains and cull potatoes, fruit
of starch yields about 50 kg of glucose but conversion is
wastes etc.) 7) methane 8) methanol and ethanol 9) alkanes
never complete, therefore with 90% conversion the yield is
and paraffins 10) gas oil 11) combustion gas Substrates and
40-50%. For cellulosic raw materials yield is less because
nitrogen concentration for yeast growth should be adjusted
α-cellulose is quite resistant to enzymatic attack.
to provide C  N in the range of 7: 1 to 10: 1 to favor high
protein content. Concentration of carbohydrates in batch Sugar containing raw materials
culture should range from 1-5% while in continuous culture
 Basic of Fermentation Technology

16
Any sugar containing fruits, fruit juices or extracts, sugar- and succinate), which may consume up to 4-5% of the total
containing effluents from canneries, sugar beet, sugar cane, substrate.
sugar cane molasses, cheese whey or sweet sorghum may
C6H12O6 → 2 C2H5OH + 2 CO2 + 2 ATP (Gay
be used as raw materials.
Lusac’s Equation)
Starchy raw materials Glucose Ethanol carbon dioxide Energy
Cereal grains (wheat, rice, corn etc.), root crops
(cassava), tubers (potatoes) are gelatinized by heating and
Modes of fermentation
then hydrolyzed to fermentable sugars by enzymes. Batch fermentation: Most of the production of ethanol
is carried out on the same lines as Established hundreds
Cellulosic raw materials of years ago. These methods are based on the simple
Saw-mill residues, paper-mill residues, newsprints, batch fermentation of substrates inoculated with culture
potato peelings, rice straw, corn stover, peanut shells, cocoa microorganism at temperature 20-30 °C, initial pH adjusted
and coffee husk, tobacco stalks and wheat straw contain to 4.5 and the time varies between 36-72 hours. Final
α-cellulose, hemicellulose and lignin. These materials when ethanol concentration is usually 10-16%. Batch technology
used are delignified, hydrolyzed and then enzymatically has been preferred because of its ease of operation, low
treated with cellulases to convert them into fermentable requirement of sterilization, use of unskilled labor, low risk
sugars. of financial loss and easy management of feedstocks. It has
its disadvantages also such as low productivity due to long
Microorganisms turnaround times, expenditure of cooling as fermentation by
Most of the commercial production is carried out with this mode raises the temperature of the fermentation broth
Saccharomyces cerevisiae. For production of ethanol and initial lag in growth.
from pentose solutions from corn stover hydrolysis and Continuous fermentation: In continuous fermentation,
from spent sulfite liquor of wood industry Candida utilis is fresh medium along with inoculum (yeast)is fed to the
used and glucose is fermented with immobilized cells of S. fermenter and an equal volume of the fermented liquor is
cerevisiae. Many strains of S. cerevisiae readily produce withdrawn from the fermenter for the recovery of ethanol
10-13% ethanol by volume in batch fermentation of and yeast. Continuous ethanol production eliminates much
molasses solutions with initial fermentable sugars 20-25%. of the unproductive down time associated with the batch
Higher concentration of ethanol can be achieved in Fed- fermentation process. The microorganism inoculated in
batch culture. The fermentation of whey requires the use of the continuous mode should always be in its exponential
dairy yeast Kluyveromyces fragilis and K. lactis. growth phase because this increases the time dependent
productivity of ethanol. The rate at which medium in the
Nitrogen sources
fermenter is added or withdrawn is usually expressed as
Assailable Nitrogen sources such as ammonia, ‘Dilution rate’. It is denoted by a letter ‘D’. The time for which
ammonium salts, urea, or amino acids are added in addition the fermentation solution remains inside the fermentor for
to phosphorus in the form of ortho-phosphate. Minor fermentation is referred as ‘Residence Time’. The dilution
quantities of potassium, magnesium, calcium and trace rate is the ratio of withdrawn liquid to the volume of total
elements are also added. The addition of vitamins is rarely liquid in the fermentor. Hence, a dilution rate D = 0.1
required but thiamine has been often seen to accelerate the indicates that one-tenth of the fermentor liquid is withdrawn
rate of fermentation. S. cerevisiae requires the presence of per hour (or the residence time in the fermentor is 10 hours).
biotin. The best source of all required nutrients including the In continuous fermentation residence time and dilution rate
trace elements and vitamins is yeast extract. should be so adjusted to get higher ethanol production.

Fermentation Cell recycle: In Cell recycle, yeast cells are separated

from the withdrawn fermented solution by means of
Biochemical basis of fermentation: Yeasts, under centrifugation or by any comparable process or settling and
anaerobic conditions, metabolize glucose to ethanol returned to the fermentor. This is done to overcome low cell
primarily by way of the EMP pathway. The overall net density limitations and to maintain high cell concentration in
reaction involves the production of 2 moles each of ethanol, the fermentor.
carbon dioxide and ATP per mole of glucose fermented.
Therefore on a weight basis, each gram of glucose can Vacuum fermentation: This is a novel fermentation process
theoretically give rise to 0.51g of alcohol. The yield attained in which alcohol formed by fermentation is continuously
in practical fermentations however does not exceed 90- removed from the system so that it does not become toxic
95% of theoretical. This is due to the requirement for to the microorganism. This process was described by
some nutrient for to be utilized in the synthesis of new Cysewski and Wilke in 1977, 1978 and by Ramalingham
biomass and other cell maintenance related reactions. Side and Finn in 1977. The former took the advantage of high
reactions also occur in the fermentation (usually glycerol volatility of alcohol and conducted the fermentation at
 Basic of Fermentation Technology

17
temperature conducive for the growth of yeast and sufficient by the use of adsorbents, such as cellulose, or other dry
vacuum to boil off the alcohol while latter used vacuum plant materials (cracked grains). Inorganic desiccants have
lesser than what former had used and temperature 30 °C. also been suggested for this purpose. An endless loop of
yarn fibers is also used to remove water from ethanol water
Aeration azeotrope.
Aeration is done to keep the cells under suspension and
By-Products of ethanol fermentation
to maintain viability in cells. Therefore sufficient amount of
air should be sparged into the fermentor so that dissolved During the fermentation of ethanol many by-products
oxygen concentration (expressed as Oxygen Tension) may are also formed which must be removed effectively by
not exceed its toxic levels. the distillation system. The by-products more volatile
than the ethanol are mainly aldehydes with acetaldehyde
Distillation the principal compound. Methanol in small amounts is
Distillation process may be divided into two stages i.e. also formed by the hydrolysis of pectins present in the
Distillation proper and Rectification. In distillation proper, fermentation broth. The less volatile compounds formed
volatile components of the fermentor are separated from the as a by-product are Fusel oils which are a mixture of
insoluble solids and ethanol is concentrated to a distillate isomers of amyl alcohol 60-80%, isobutyl alcohol 15-30%
containing 30-96% ethanol by weight. In rectification, and n-propyl alcohol 0-10%. Separation of by-products:
ethanol is separated from other volatile components such The aldehydes are removed relatively easily due to the low
as aldehydes (acetaldehyde) and fuel oils (amyl alcohol 60- boiling point of acetaldehyde as a distillation head product;
80%, isobutyl alcohol 15-30%, and n-propyl alcohol 0-10%) however the separation is not complete. The residual
about 1 litre of acetaldehyde and 5 litre of fuel oil is produced aldehyde content in alcohol is typically 5-100 mg/L. Fuel
for every 1000 litre of ethanol produced. The amount of the oil, owing to its complex composition and limited solubility
volatile component depends upon the starting material used in water is more difficult to separate. Dry fuel oils boil in
for fermentation and fermentation process itself. the range from 128-137°C and normally, being even less
volatile than water, they remain in the bottom product. In the
Ethanol has a lower boiling point than water. presence of water however, the boiling point of the mixture
Concentration of ethanol is therefore always more in the falls below that of the water alone as a consequence of their
vapor phase of ethanol solution in equilibrium with the immiscibility. Fuel oil, therefore rises from the bottom of an
water. When distillation of the water and ethanol mixture alcohol-purifying column and concentrate in the middle of
is carried out, ethanol concentration of ethanol is always the column, from they are removed. In a continuous process
more in the distillate. Water and ethanol form an azeotrope. fuel oil is removed by bleeding from 6th to 15th plate of the
(Azetrope is a mixture of volatile substances that at a given rectification section and then fed to the fuel oil separator.
concentration has identical liquid and vapor compositions).
For ethanol and water this concentration is 96% by weight of Then specification of the industrial alcohol depends upon
ethanol and the boiling point of ethanol is 78.2°C. Therefore the intended use. Premium grade alcohol has low water
initially easy separation of ethanol from the broth becomes content, low odor ratings and high permanganate time test
increasingly difficult as concentration 96% by weight of values (presence of aldehydes).
ethanol is reached because volatilities of both water and a. Definitions
ethanol become identical. Boiling at this point gives a vapor
of same composition as that of boiling liquid and no further A. Denatured Spirit- Alcohol that is denatured by adding
concentration of ethanol can be achieved. 10% methanol is called denatured alcohol or denatured
spirit.
Anhydrous ethanol may be obtained from 96% ethanol
solution by forming a ternary azeotrope with benzene. B. Rectified Spirit- Alcohol from which fuel oil has been
This ternary azeotrope contains 74% benzene, 18.5% removed is called rectified spirit or rectified alcohol.
ethanol and 7.5% water and its boiling point is 68oC.
C. Anhydrous Alcohol- Alcohol that has very less water
Since this azeotrope contains more water than the
content is called anhydrous alcohol.
ethanol water azeotrope, it can be distilled overhead and
leaves anhydrous ethanol behind. Such a process usually D. Potable Alcohol- Alcohol that can be used for drinking
consists of two steps simple fractionation to produce near purposes is called potable alcohol.
azeotrope mixture followed by ternary distillation with an
added chemical agent yielding anhydrous ethanol. If the Different Types of Substrates for Industrial
extraneous material is less volatile than the feed, it is called fermentations
a solvent and the operation is called extractive distillation.
Substrates are used as components of media suitable
When the extraneous material is more volatile than the feed
for the growth of microorganisms or the production of
it is withdrawn with the overhead product and the operation
desirable microbial metabolites. Nutrient medium form
is called azeotrope distillation. In addition to these methods
the environment, in which microorganisms grow and from
water may also be removed from ethanol water azeotrope
 Basic of Fermentation Technology

18 which they draw substances necessary for the synthesis Water is an indispensable component of a medium. It is
of cellular components and to produce energy that runs much needed by microorganisms for fermentation process.
the biochemical machinery of the cell. Bio-chemically It is also needed in other processes like isolation of the cells
substrates may be classified as: 1) Simple-that contain the and products. In fermentation, we usually use tap water for
lowest amount of energy, e.g. water, carbon dioxide, oxygen, the preparation of culture media, separation and washing
nitrogen and inorganic ions, 2) High-energy-contain highest of microorganisms. The quality of water used depends
amount of energy and 3) Intermediary substrates-that are upon the process in which is used e.g. A water of low grade
formed during the transformation of simple substrates to the quality can be used for cooling purposes, whereas for the
high-energy substrates. The function of the nutrient medium preparation of media and isolation of cells and products etc.
is to ensure the growth of the microorganism and has to a water of high quality i.e. Potable water or treated water is
contain individual substrates in a form best accessible to the needed. The treated water can be produced by four ways:
microorganism, which usually means in solution. However, 1) By adjusting the chemical composition i.e. by removing
in some cases solid media are used and cells from gaseous salts or ions like iron, free chloride or chlorine, salts of
phase may sometimes utilize some substrates. Besides carbonic acid and adjusting the hardness. 2) By removing
major components medium also contains many other the extraneous materials by sedimentation, filtration etc.
components necessary for microbial growth. Physico- 3) by removing microorganisms from the water i.e. by the
chemical properties and medium composition adjustment processes like filtration, sterilization and disinfection. 4) By
help in the maintenance of the maximum rate and desirable removing the colloids i.e. by the process of osmosis. The
direction of the microbial process. quality of water always fluctuates and can be controlled by
various chemical and biological methods e.g. when levels
Complex media are prepared from natural raw materials
of ammonia in water increases, the water is said to be
of animal or plant origin; the exact chemical composition of
polluted and has to be treated by various chemical methods
these media is usually not known and individual components
or by growing certain organisms like ammonifying bacteria.
can be determined only with difficulty. A complex medium
Similarly when levels of salts of nitrous acid or nitric acid
is prepared by dissolving appropriate natural substances
rise above the normal limits, growing denitrifying bacteria
in water and is often further processed by hydrolysis,
also controls them. When iron levels in water increases,
clarification complemented by additional nutrients, growth
it causes discoloration of microorganisms such as yeasts
factors, and missing trace elements. Synthetic media are
and inhibit the biosynthesis of various substances e.g.
prepared by dissolving defined chemicals in distilled water to
antibiotics. It may also produce corrosion like effect in water.
eliminate completely all minerals and trace elements usually
The norms for high quality water are 20-100 germs per ml.
present in un-distilled water. Complex media are most often
The hardness of water is due to the presence of various
used for technological purposes while synthetic media are
salts of calcium and magnesium, which when rise above
employed only in special cases e.g. in the processing of
their normal limits, they lead to instability in product yields.
substrates from the chemical industry or utilization of some
In that case, water is deionized and appropriate portions of
waste materials. In laboratories usually synthetic media are
other salts are added. Such types of treatments are only
used despite their numerous shortcomings because they
possible in laboratory and at commercial scale; the cost of
are defined therefore it is possible to draw exact material
applicability becomes very high. The quality of ground water
and energy balances from changes in the levels of individual
now days is further deteriorating due to the increases use of
components. As a qualitative and quantitative combination
chemicals in agriculture and utilization of nuclear materials
of substrates, the medium must meet the microbiologist’s
in the warfare. So the demand of water for the commercial
demand. The consumption of substrates in the laboratory is
purpose has further increases and further prospects of
very small and it is quite within the range of microbiologist’s
research in this regard are needed.
to use more expansive substrates that cannot be utilized
on commercial scale due to certain reasons. According to Carbon sources
state of the medium, media can be classified as: 1) Solid
media. 2) Liquid media. 3) Gaseous media. The solid media They represent the main component of nutrient media
are present in the solid state and they are used immediately quantitatively. They are utilized for the biosynthesis of
on dissolving in distilled water. The liquid media are readily cell materials and are also used as main energy source.
utilized as they contain more water. These can be converted Carbon is also used to build carbohydrates, lipids, fats
onto solid ones or gels by the addition of certain thickening and proteins etc. in the cell and it is derived from various
agents like agar-agar, gums, and gelatin etc. The gases organic compounds added in the medium. Some new types
also play an important role in the growth and multiplication of substrates have also come into light such as synthetic
of the organisms and they also have a profound effect on alcohols, alkanes and many hydrocarbons. The nature of
the production of metabolites. The main components of microbial process, product purity and metabolism of the
a medium are as follows: 1) Water. 2) Carbon source. 3) inoculum microorganism may dictate the use of chemically
Nitrogen source. 4) Accessory substances. 5) Antifoam defined carbon sources such as glucose, sucrose, lactose
agents. or starch as well as complex raw materials like molasses,
sulfite liquors, wood hydrolysates, cellulose and raw
Water materials of petrochemical origin.
 Basic of Fermentation Technology

Carbohydrates temperatures while at high temperatures, they swell and

19
change into starch gel. The gelation is usually performed
They form a distinct group of organic compounds that at 100 °C in water. At 120-130 °C, the gel liquefies and
are divided into three categories as monosaccharides, may be easily degraded by enzymes. Starch is a mixture
disaccharides and polysaccharides. of two related polysaccharides, amylose (linearly arranged
Monosaccharides chain of α-glucosidically linked molecules of D-glucose with
1-4 bonds) and amylopectin (branched chain of D-glucose
Glucose-In fermentation process, glucose is the most molecules bonded also -glucosidically by 1-4 and less
frequently used monosaccharide (Grapes contain 25- often 1-6 bonds); one molecule contains more than one
30% of glucose) but it has its own disadvantages like thousand glucose residue. Acid hydrolysis of amylose
Catabolite repression and Pasteur effect. For industrial yields first maltose and then glucose. Enzymic degradation
purposes glucose is obtained by the hydrolysis of starch by amylases proceeds via amylodextrin and low molecular
both by chemical and enzymatic methods. Since it is weight dextrins to maltose.
rapidly assimilated therefore a continuous inflow of glucose
is required in growing cultures for the biosynthesis of Cellulose: It is a structural polysaccharide, forms a
complex compounds. It is sterilized separately from the substantial part of the plant cells and is found abundantly in
other components of the medium because at neutral pH nature. Isolation of pure cellulose is difficult because other
and at high temperature it undergoes Maillard’s reaction natural substances like lignin, hemicellulose and waxes
with amino acids to give dark brown colored solution. It accompany it. For industrial purposes, cellulose is obtained
is usually sterilized at pH 3.0 as a 30-50% solution (w/v); from wood and straw of various plants. Recently an intensive
under these conditions the solution remains colorless. In research has been initiated concerning cellulose-degrading
microbial processes, the so-called “Hydrol”, a waste product enzymes like cellulases. The cellulase is a multienzyme
in the manufacture of pure glucose, can also be used as a complex of three enzymes namely endoglucanase,
substrate. exoglucanase and α-glucosidase that act synergistically to
form glucose. A compound known as “Cadoxen” (25-30%
Disaccharides and oligosaccharides ethylene diammine in water and 4.5-5.2% cadmium) has
the capacity to disrupt the crystalline structure of cellulose,
1. Sucrose- It is obtained from sugar cane or sugar beet
which renders it susceptible to rapid and total hydrolysis
and is available in different levels of purity. It is much
to form soluble products. Other agricultural wastes, which
needed for the fermentation by fungi, yeast and in the
can be used as carbon sources are: Molasses from sugar
fermentation of microorganisms those biosynthesize
industry, Barley water and its products from brewing
antibiotics, amino acids and organic acids.
industry, sulfite waste liquor from paper and pulp industry,
2. Lactose- Lactose is present in the milk of mammals at acid-wood hydrolysates from wood and paper industry,
the levels of 3-8% and is prepared by the evaporation of vegetable oils and animal fat from oil industry and alcohol
whey that remains after the processing of milk to butter. and hydrocarbons from petrochemical industry.
Only a few species of microorganisms can assimilate
Molasses: It is a thick, syrup-like, viscous and dark colored
lactose; yeasts with a few exceptions do not assimilate
liquid obtained as a byproduct in the production of raw and
lactose. Penicillium chrysogenum can assimilate lactose
refined sugar from sugar cane or sugar beet (remains after
in low but in efficient levels. The main disadvantage of
the crystallization of main fraction). It is named after its
the lactose is its varying quality, which can be attributed
source as Cane or Beet molasses. Cane molasses contains
to different milk processing operations.
a high level of sugars
Polysaccharides Barley and its products: Barley and its products such
These substances represent a suitable source for as malt, wort and malt extract. Malt is prepared by the
bacteria, yeast and fungi and are used in the biosynthesis of germination of barley. During malting amylases and
many products. They are macromolecular, forming colloidal pectinases are only partially activated since the process
solutions or exhibiting negligible solubility in water, for this takes place at low temperatures and in a medium containing
reason they must often be converted into a soluble form only vegetable liquor. In the brewery malt is crushed,
before being used in nutrient media. mixed with water and kept at a higher temperature; under
these conditions the enzyme reactions proceed rapidly
Starch: It is one of the most important polysaccharide and completely as far as the degradation of malt starch is
both biologically and industrially. It is accumulated as a concerned. Wort is obtained from malt residue by filtration. It
reserve substance in fruits, seeds, and bulbs of higher is used as a raw material for Beer brewing. After the addition
plants in the form of granule of different shapes and sizes. of hops it is boiled and filtered. The resulting solution is
It is usually denoted according to its origin i.e. according to hopped wort; after cooling it is inoculated by a culture of
the plant from which it is manufactured i.e. Potato starch brewer’s yeast and fermented. Malt extract is prepared from
from potatoes, Maize starch from maize, Wheat starch from wort by filtration and evaporation; it is thick syrup containing
wheat, Rye starch from rye and Barley starch from barley. 80% dry weight that may be vacuum dried to a powder.
Starch granules are hygroscopic and do not changes at low
 Basic of Fermentation Technology

20
Sulfite waste liquor-It is a waste product in the production and therefore are used for the production of fodder yeasts.
of pulp from wood by the sulfite method. It is a brown-yellow Vegetable oils and Animal Fats-Majority of the vegetable
liquid with a density of 5 to 90 B at 20°C. Its specific weight is oils are easily metabolized by microorganisms therefore
1.045 to 1.060. It contains 8 to 14% dry weight that consists they are used as a carbon sources in many microbial
of 18 to 20% minerals and 80 to 90% organic substances. processes especially in the production of antibiotics. They
The inorganic part is composed chiefly of sulfur dioxide, are also often used as an antifoam agent. Fats on the other
sulfite and calcium sulfate. A part of the calcium is bound to hand are solid at room temperature and their liquefaction
lignin as a calcium lignosulfate salt, which is water soluble requires high temperature. For this reason they are not so
in acidic media. Other inorganic compounds present in the often used as substrates.
liquor are salts of heavy metals such as copper, arsenic
Alcohols-Synthetic alcohols, especially ethanol and
and lead. These heavy metals ions in their minute amount
methanol have importance as potential raw materials
also affect the growth of microorganisms. The organic
for microbial processes. Ethanol is available in sufficient
compounds found in the liquor i.e. lignin and carbohydrates,
quantities, has a reasonable price, standard quality and
are released from wood during treatment with the sulfite
homogeneity. Its physico-chemical properties place it
cooking acid. A short cooking at low temperature yields solid
among surface-active substances; hence it increases the
pulp containing higher levels of hemicelluloses and lignin;
foaming of nutrient media. However, the foam is thin and
therefore the resulting liquor contains lower amount of these
facilitates a better distribution of air into liquid. Synthetic
substances. On the other hand long and intensive cooking
alcohol is easy to produce in pure form and is highly water-
at higher temperature produces soft pulp having very
soluble. Its disadvantage is the presence microorganism
less lignin and hemicelluloses resulting in liquor of higher
inhibiting substances like crotonaldehyde. Methanol is
contents of these substances. The liquor further contains
also water-soluble and has similar properties like that
small amounts of volatile acids like acetic and formic acid.
of ethanol. In comparison with ethanol it has a higher
Further fermentation of sulfite liquor depends upon how
volatility and toxicity. Hydrocarbons-Microorganisms can
much carbohydrates are released during cooking and
utilize almost all hydrocarbons to larger or some smaller
pulping. The amount of carbohydrates varies according to
extent. Bacteria can assimilate all hydrocarbons while
the species of the pressed wood and technology of pulping.
yeasts can assimilate only some of them. The best carbon
The composition differs in liquors from conifers and from
sources among hydrocarbons are n-alkanes. Alkanes can
deciduous trees. The total carbohydrates in the coniferous
be divided in to several groups according to the number of
wood liquor usually include 30-40% mannose, 30-35%
carbon atoms they contain. These are: C1- C9 n-alkanes,
glucose, 10% galactose, 15% xylose, and 5-10% arabinose.
C10-C20 n-alkanes and C20 and higher n-alkanes. C1-C9
In deciduous trees, the liquor contains 50% xylose, 15-
n-alkanes-Methane is the most widely used alkane in this
20% arabinose, 10% methylpentoses, 10% mannose, and
group because it is readily available. It is assimilated by
about 10% uronic acids. In addition they contain higher
most of the bacteria but not readily by yeasts. An amount of
levels of furfural, which originates from pentoses during
1 Kg of methane yields 0.8 kg of dry cell matter. Due to their
cooking at high temperature and pressure. The furfural
yield and narrow range of possible sources the alkanes of
affects negatively on the growth of microorganisms. The
this group are not suitable for microbial industry. C10-C20
sulfite waste liquor cannot be used directly for fermentation
n-alkanes -The alkanes of this group are readily assimilated
in the form obtained from the cooker since 1) it contains
by almost all microorganisms including bacteria, yeasts,
a considerable amount of sulfur dioxide, which has to
actinomycetes and molds. The yield of cell mass per
be removed. 2) Has fluctuating pH values, which can be
consumed alkane of this group is 90-100%. C20 and higher
adjusted with calcium carbonate or calcium hydroxide. 3) it
alkanes-Assimilation of this group of alkanes is slower
may contain insignificant levels of assimilable nitrogen and
because alkanes of this group do not exist in liquid state at
phosphorus and these nutrients must therefore be added.
normal cultural temperatures.
Sulfite liquors are used especially for the production of
ethanol and production of fodder yeasts. Nitrogen sources
Acid wood hydrolysates: The resulting pulp after the Substances containing nitrogen in various forms may
sulfite waste liquor has been removed still contains a serve as sources of nitrogen for microbial growth. The
considerable amount of carbohydrates other than cellulose. range of nitrogen assimilation is much wider than the
These are removed by low concentrations of acids, which carbon assimilation. Many microorganisms, especially
hydrolyse hemicelluloses to soluble sugars; this process lower fungi, utilize nitrogen from inorganic nitrogen sources
is called as prehydrolysis. Prehydrolysis removes only like inorganic ammonium salts. Ammonium sulphate, the
hemicelluloses and not cellulose in contrast to the complete cheapest of these compounds is often used in combination
hydrolysis that cleaves all the polysaccharides including with ammonium hydroxide and gaseous ammonia for
cellulose. The prehydrolysis is carried out on deciduous continuous regulation of pH. Another very good source of
wood, cereals, rape or rice straw, and reed grass. The nitrogen is urea; since it decomposes at high temperature
straw prehydrolysates contain higher amount of pentoses is should be sterilized by filtration. Quality wise nitrogen
 Basic of Fermentation Technology

21
sources cannot match with the pure laboratory chemicals either dry or in the form of paste. The extract is prepared
because of the economic reasons. Although many by autolysis or plasmolysis of Baker’s or Brewer’s yeast. To
microorganisms assimilate inorganic nitrogen, they often remove bitter hop substances, Brewer’s yeast is washed at
grow faster in the presence of organic nitrogen sources. At pH 6.5 to 7.0 prior to treatment. Yeast cells must be kept
present many suitable nitrogen sources of plant and animal in a viable state during washing and filtration in order to
origin are available as waste products. prevent losses of cell components. Baker’s yeast does
not need washings. The autolysis is performed in a large
Inorganic and synthetic organic nitrogen sources
vessel under continuous stirring at a temperature above
Ammonium sulphate is readily soluble in water; the purest 75°C. The yeast is then transferred to a vessel containing
form used in microbial industry is technical ammonium sodium chloride solution, which induces cell plasmolysis.
sulphate that contains a minimum of 20.7% nitrogen. It is then filtered or centrifuged to separate cell walls and
The so-called coke and gas sulphates are considerably membranes. The liquid is rapidly concentrated at 37oC
contaminated with tar substances and are used only to prevent degradation of thermolabile substances and
exceptionally as substrates. Diammonium hydrogen also to prevent contamination. Drying the liquid form in an
phosphate, which is used especially in yeast production, is atomizer or spontaneously in vacuum makes the powered
a source of both nitrogen and phosphorus. Ammonia is used form of the yeast extract. Yeast extract is a mixture of amino
either in the form of 25% aqueous solution or in gaseous acids, peptides, water, soluble vitamins and carbohydrates.
form. Urea is used only exceptionally in microbial industry Soybean and Peanut flour-Soybean flour is an important
i.e. in the production of fine and high-grade products, due to source of nitrogen. Its most important constituents are
its relatively high price. proteins 44-47%, fat 5.5-6.0% and phosphorus 0.62-0.65%.
It is obtained by the extraction of oil from soybeans. Peanut
Natural sources of nitrogen flour is also obtained after the extraction of oil from peanuts.
Corn Steep Liquor-It is obtained as byproduct in the It’s important constituents are nitrogen, fat and phosphorus.
production of maize starch. It contains biologically active The composition of proteins is variable and depends on the
substances that differ qualitatively and quantitatively source of raw material. Both peanut and soybean flours
according to the quality of the corn used and its are used as substrates in the biosynthesis of antibiotics.
technological processing. These substances are released Peanut flour is suitable for the biosynthesis of Penicillin and
into the corn steep liquor during corn steeping and some Lysine while Soybean flour is suitable for the biosynthesis
microbial processes, especially lactic acid fermentation of Chlortetracycline. Peanut flour is not suitable for the
participating in the release, occur during steeping process. biosynthesis of Tetracyclines.
Moyer and Coghil first used corn steep liquor in the Antifoam agents
Penicillin fermentation in 1946 and that enhanced the yield
of antibiotic many folds. Corn steep liquor contains nitrogen Media when are rich in natural substances; foaming
substances, lactic acid, reducing substances and ashes occurs in the microbial processes. This foam poses
with many trace elements. An important criterion of the difficulties when it is uncontrolled. The outflow of a large
quality of corn steep liquor is the ratio of amino nitrogen to amount of foam from the fermentor may reduce the volume
total nitrogen, which should be about 0.4 i.e. approximately of the culture and may lead to contamination. Various
2% of amino nitrogen dry matter. This ratio indicates the antifoam devices and certain natural or synthetic antifoam
intensity of lactic fermentation during steeping. Another agents are employed to control foam in a fermentation
criterion is the content of amino acids, which should be system. The effect of these substances is based on their
higher than 20%. ability to reduce the surface tension of the liquids. The term
‘Antifoam’ is used for the substances that are added before
Potato liquor and fermented extracts from bran and oil the foaming actually occurs in fermentation while the term
seeds-Sometimes liquor from potato starch industry and ‘Defoam’ applies to a substance added afterwards to knock
fermented extracts from bran and oil seeds are used as down the foam. The natural antifoam agents used are soya,
substrates in microbial industry. Potato liquor is obtained rape, coconut, sunflower and mustard oils. Antifoam agents
as follows: The liquid formed by pressing potato pulp in of animal origin are tallow and deodorized fish fat. Mineral
starch production is subjected to sulphuriation and lactic oils may also be used. Another group of substances used
acid fermentation by Lactobacillus delbrueckii and carefully are alcohols. The most common compound of this group
dried. Though it contains many reducing substances yet is Octadecanol either pure or in combination with lard oil
it lacks amino acids like arginine, glutamic acid, histidine, or mineral oil. Specially designed antifoam agents are also
leucine, isoleucine, lysine, methionine, phenylalanine, available; among them are silicon oils. The silicon oils are
proline, threonine and valine. The fermented extract of usually used in water based emulsions containing 10%
bran and oil seeds is prepared as follows: Wheat bran and silicon oil. They are most effective in bacterial or yeast
ground tobacco seeds are mixed with water and a small cultures but are less effective in cultures of filamentous
amount of lactic acid and superphosphate is added. After six microorganisms. Since the microorganism does not
days of lactic acid fermentation at 50oC the liquid is filtered assimilate silicon oils, that is why silicon oils are very efficient
and evaporated in vacuum. Yeast extract-It is available
 Basic of Fermentation Technology

22
and a single dose at the start of the fermentation is usually or on top of the yogurt to be mixed by the consumer). 4) Soft
sufficient to prevent the formation of the foam. A similar frozen yogurt (Flavored and Unflavored). 5) Hard frozen
effect is exhibited by polyalcohol’s with molecular weight of yogurt (Flavored and Unflavored). 5) Frozen yogurt sticks.
2000 and alkylated glycols. Phosphorus and magnesium 6) Fluid yogurt drinks. 7) Fruit topped with yogurt 8) Spray
are important compounds of nutrient agar medium since dried yogurt products for confectionary, bakery and soups.
they participate in energy transduction, especially in
reactions mediated by ATP. Microorganisms need calcium, Yogurt ingredients
potassium, sulfur and sodium; therefore are added to the a. Dairy ingredients: The dairy ingredients for the
medium for microorganisms. Though trace elements such preparation of yogurt include whole, partially defatted milk,
as iron, cobalt, copper and zinc are indispensable, they are condensed skim milk, cream, and non-fat dry milk. All the
usually present as impurities in other components and in dairy ingredients should be of very high bacteriological
water. Enrichment by these trace elements is necessary only quality. Mastitis milk, rancid milk, partially fermented milk;
when analysis of synthetic media or raw materials indicate milk containing antibiotics and sanitizing chemical residues
their shortage. A major element in media is phosphorus, cannot be used for yogurt production. Since yogurt is a
which can be added both in organic and inorganic form. The manufactured product, it may have variations from the quality
compound most frequently used in industrial microbiology standards established by the marketing considerations.
is Diammonium hydrogen phosphate that also serves as Therefore it becomes necessary to standardize the milk
nitrogen source. Superphosphate is used in the production used in the yogurt production. The milk fat levels in yogurt
yeast; it is a mixture of calcium hydrogen phosphate and range from 1.0 -3.25%. Yogurt can be classified on the basis
calcium sulphate with variable content. Growth Factors and of fat present as: 1) Yogurt -contains minimum of 3.25% fat,
Vitamins Growth factors and vitamins are usually present 2) Low-fat yogurt -containing not less than 0.5% and not
in natural sources of carbon or nitrogen compounds. When more than 2.0% milk fat, 3) Non-fat yogurt -contains less
these natural substrates are used as the only source of than 0.5% milk fat. In all the categories of yogurt, a minimum
vitamins, it becomes sometimes necessary to add additional milk solid non-fat and minimum titrable acidity stipulated is
growth factors and vitamins in a pure form. Vitamins of the 8.25% and 0.5% respectively. In order to standardize the
B-complex category are necessary as growth factor in the milk solids non-fat, cream, partially skimmed milk, and skim
production of Baker’s yeast. The production of amino acids milk alone or in combination, concentrated skim milk, non-
by auxotrophic mutants requires the presence of certain fat dry milk, or other milk derived ingredients may be used.
vitamins; shortage or excess of these substances in the The milk-derived ingredients include casein, sodium and
medium affects the yield. Production of certain secondary calcium caseinates, whey; whey protein concentrates alone
substances is greatly increased when precursors of or in combination.
these substances are added into the medium e.g. when
phenylacetic acid or phenylacetamide is added into the Sweeteners
medium; biosynthesis of Penicillin G increases but when
Sucrose is the major sweetener used in yogurt
phenoxyacetic acid is added into the medium; biosynthesis
production. Sometimes corn sweeteners and honey may
of Penicillin V is enhanced.
also be used. The level of sucrose in yogurt mix appears to
Yogurt affect the production of lactic acid and flavor in the yogurt.
Sucrose concentrations above 4% inhibit the growth of S.
Yogurt is defined as the end product of a controlled thermophilus. Sucrose may be added in a dry, granulated,
fermentation of high solids whole milk with a symbiotic free-flowing, crystalline form or as a liquid of 670Brix.
mixture of Streptococcus thermophilus and Lactobacillus Commercial yogurt has an average of 4.06% lactose, 1.85%
bulgaricus in the ratio of 1:1 for the manufacture of high- galactose, 0.05% glucose and pH of 4.15. In frozen yogurt
grade yogurt. Among the various cultured dairy products it is 6% corn syrup solids are added. Non-nutritive sweeteners
unique because it is the only product in which acetaldehyde like calcium-saccharine, maltol, and sorbitol alone or in
in relatively high concentration is desired as an essential combination may be used for diabetics. Sometimes lactase
flavor component. Similar sour milk products are called as is added to break the lactose of milk.
‘Matzoon’ in Armenia, Matsoni in Georgia, Leben in Syria,
Egypt and Algeria and Matsurad or Jodda in Sycilly. Stabilizers

Forms of yogurt They produce smoothness, body texture, gel structure,

reduce wheying off or syneresis, and increase shelf life.
At present yogurt is sold and used in various forms in Stabilizers function through their ability to form gel structure
different parts of the world. These forms are as follows: 1) in water, thereby leaving less free water for syneresis. In
Plain Yogurt (Set type and Stirred type). 2) Flavored yogurt addition, some stabilizers perform their action by forming
without fruits (lemon or Vanilla). 3) Yogurt with fruit puree a complex with casein. A good stabilizer should not impart
(French style or Swiss style or Continental style -Whole or any flavor, should be effective at low pH values, and should
sliced fruits are mixed uniformly throughout the yogurt and be easily dispersed in the normal working temperatures in
Sundae style -Fruits may be filled in the bottom of the cup a dairy plant. The stabilizers generally used are gelatin,
 Basic of Fermentation Technology

23
vegetable gums like carboxy methylcellulose, locust of the system is depressed. Depletion of oxygen and the
bean, carob, guar and seaweed gums like alginates and availability of the formate ion stimulate the growth of rods.
carrageen an. Sometimes agar-agar or pectin is also used. Until the pH value reaches 4.2 Streptococci grow very
Calcium chloride is added to control whey separation. slowly. Below pH 4.2 Lactobacilli grow rapidly and dominate
the fermentation. The major portion of the lactic acid and
Fruit preparations for flavoring yogurt acetaldehyde necessary for the characteristic flavor of
Fruit preparations are present at the level of 15-20% yogurt is contributed by the lactobacilli but the second part
in the final product. Generally fruits are added to meet of the fermentation is greatly aided by the initial metabolic
the market demand. In every type of flavored yogurt, the activity of the coccus component. Yogurt should be cooled
composition of basic fruit preserve remains the same while at pH value of 4.2-4.3 to obtain a high-grade product.
its pouring styles are different. Fruit preserve consists of
Flavors of yogurt
55% sugar and a minimum of 45% fruit which is cooked
until the final soluble solid content is 68% or higher (65% Typical flavor of yogurt can only be detected in plain
in the case of certain fruits). Frozen fruits and juices are yogurt because it is a product of bacterial symbiosis. The
the usual raw materials. Commercial pectin, 150grade, is production of flavor depends upon the proportion of rods
normally utilized at a level of 0.5% in preserves and the pH and cocci and their combined metabolic activity. The
is adjusted to 3.0-3.5 with a food grade acid, viz. citric acid distinctive flavor is contributed by lactic acid (odorless) and
during manufacturing of the preserve. Calcium chloride and by acetic acid, which are volatile and have strong odors.
certain food grade phosphates are also used in several fruit Milk components and other ingredients in the mix also play
preparations. In Fruit on Bottom or Eastern Sundae style a role in providing a background flavor. To obtain a good
yogurt, 59ml fruit preserve on bottom is followed by 177ml flavor one should keep in mind that 1) starter strain should
of inoculated yogurt mix on the top. No flavor or sweetener be selected with a view to obtain a good symbiosis 2)
is added. It is incubated till pH becomes 4.2 and then proportion of rods to cocci should be equal 3) rapid cooling
refrigerated. In western Sundae style yogurt, the top yogurt should be done at pH 4.2. If the pH value falls below 4.2,
layer contains flavors and colors while in Swiss style yogurt, in large tanks that require a long time to cool down to 10oC
yogurt and mixed fruit layers are mixed together. then the desired cocci to rods ratio and flavor balance is
lost. The product so obtained will be too sour and coarse.
Flavors and colors
Characteristics of a good yogurt
Only certified flavors and colors should be used for
the preparation of yogurt. It should meet the following 1) The body of yogurt should possess a relatively high
requirements. It should 1) exhibit true color and flavor of viscosity and should be firm and cohesive enough to be
the fruit when blended with yogurt, 2) be easily dispersible removed and eaten with a spoon. 2) There should be very
in yogurt without causing texture defects, phase separation little wheying off during normal handling for mixing, cooling,
or syneresis. pumping and packaging. 3) It should have a smooth, rich
texture free from lumps, granules or graininess. 4) There
Microorganisms should be no gas packets, fissures or gassy effervescence.
The culture is specified as a mixture of Lactobacillus Factors affecting body characteristics of yogurt
bulgaricus and Streptococcus thermophilus in 1:1 ratio.
The inoculum culture can be regenerated from a lyophilized The factors are: 1) Fat and SNF concentration in the
culture tubes on media containing milk as a component. mix. 2) Stabilizers used. 3) Control over weighing and
blending of ingredients in the mix. 4) Heat treatment of the
Microbiology of yogurt mix 5) Concentration of protein by new process such as
ultrafiltration. 6) Concentration of calcium and magnesium
Streptococcus thermophilus is commonly referred as
ions. 7) Type of starter culture used 8) Incubation conditions
coccus and Lactobacillus bulgaricus as rod. Equal numbers
used. 9) Initial pH value of the mix. 10) Handling during pre
of both of these bacteria are desirable for the production
and post incubation operation. 11) Sucrose concentration
of tartness and green flavor of yogurt. The fermentation is
in the mix.
carried out within a temperature range of 35 -45°C, optimum
being 40°C. During fermentation, both rods and cocci work a. Defects and their causes in yogurt
together to produce yogurt. Rod shaped Lactobacillus
bulgaricus grows first slowly but their weak proteolytic activity 1) Graininess -If coagulum becomes exceedingly
liberates sufficient amount of the amino acids (glycine and firm before stirring, the finished yogurt tends to grainy. In
histidine) and peptides to stimulate cocci. Streptococcus addition, the use of rennet to obtain a good body invariably
thermophilus now grows vigorously to produce moderate leads to graininess. 2) Coarse Texture -Disturbance of the
amounts of lactic acid, acetic acid, acetaldehyde, diacetyl yogurt mix just before the gelling stage gives rise to coarse
and formic acid. When the pH of the yogurt mix (initial 6.3- texture. 3) Granular feeling in Mouth -Inadequate mixing
6.5) drops below 5.5, the rapid growth of cocci is arrested of the powdered products causes a granular feeling in the
and that of rods is favored. Oxidation-Reduction potential mouth. 4) Gas packets and Fissures -These are caused by
 Basic of Fermentation Technology

24
the trapped carbon dioxide or hydrogen gas produced by heating at such high temperature denatures the proteins of
contaminant flora like E. coli, Bacillus spp. and yeasts. 5) the milk that releases some peptides required for the growth
Slimy feeling in Mouth -Slime producing strains when used of bacteria. It is then cooled to 37-38°C and inoculated with
in starter culture would result in a mouth feeling similar to 5% milk starter culture. The incubation at 37-38°C for 18-
the white of eggs. 6) Off Taste and Off Odor -These develop 24 hours yields Acidophilus milk, which is quickly cooled to
due to faulty fermentation. 5-7°C and bottled when final acidity of the finished product
reaches 1.0%.
Kefir
Bulgaricus Butter Milk Or Bulgarian Milk
It is a historic and old product from Caucasus region of
Russia. Kefir grains are used as starter culture that can be It is prepared from cow or mare’s milk, which is fermented
reused several times. The kefir grains are gelatinous white by a pure culture starter of Lactobacillus bulgaricus. This
or cream-colored irregular granules from the size of wheat is particularly consumed in various parts of the world
grain to walnut. These are made up of a polysaccharide particularly Balkan countries. The starter produces the
called ‘Kefiran’, which is associated with denatured milk required acidity and flavor. It can also be prepared from cow
protein. These are insoluble in water and swell up when or buffalo’s skimmed milk.
soaked in water to form a jelly like product. Bacteria and
yeasts are present within the folds of the grains and there Method of preparation
may be some symbiotic relationship between the grains The milk is heated at 95°C for 30 minutes and cooled
and microorganisms. Various kinds of microorganisms to 37-38°C. It is then inoculated with 2% milk starter
are involved in the kefir fermentation these include: culture prepared with Lactobacillus bulgaricus. The milk
Saccharomyces kefir, Torula kefir, Lactobacilli, Streptococci, is incubated until the acidity reaches 1.4%. The product is
and Leuconostoc spp. Coliforms, micrococci and spore cooled to 7°C. After incubation a curd like smooth mass is
forming rods contaminate the fermentation. formed that is diluted and churned. The butter is removed.
Buttermilk left has its acidity in terms of lactic acid (0.25%).
Method of preparation
The final product lacks aroma but has a pleasant flavor. It
Fresh cow’s milk is pasteurized at 85 °C for 30 minutes. can be used as a substituent for milk but it has very less
It is inoculated with kefir grains taken from the previous sugar content. It has diuretic effect when taken in large
batch after cooling to 25 °C. The incubation at 23 °C yields a quantities (Flow Chart 3).
soft curd, which after being agitated forms a beer like foam
and fuzziness from which kefir grains come upward with Role of heat treatment at high temperature in
the evolution of carbon dioxide. Kefir grains are separated culture dairy products
and washed in cold water. They are stored in cold water Heat treatment at 85-95 °C for 30 minutes or equivalent
at 4 °C or are dried in a warm oven. The kefir grains have is an important step in the manufacture of cultured dairy
alcoholic, yeasty sour with tangy effervescent flavor. Fresh products. The heat treatment 1) produces a relatively
kefir grains have activity up to 8-10 days while dried grains sterile medium for the exclusive growth of starter culture. 2)
may remain active for 8-12 months. They contain lactic acid Removes air from the medium to produce more conducive
0.8%, alcohol 1%, carbon dioxide and flavor compounds medium for microaerophilic lactic cultures to grow. 3)
(acetaldehydes, diacetyl and acetone). Effects thermal breakdown of milk constituents, especially
proteins, releasing peptones, sulfhydryl groups which
Acidophilus Milk or Reform Yogurt
provide nutrition and anaerobic conditions for the starter. 4)
It is a product formed by fermenting milk with an authentic denatures and coagulates milk albumins and globulins that
culture of Lactobacillus acidophilus. The organism has its enhance the viscosity and produce custard like consistency
unique features that it can survive in the severe conditions of in the product.
intestinal tract of human beings, animals and birds. Its ability
to initiate growth in, or form colonies on media containing Flavor production in cultured dairy products
bile salts should be used as a distinguishing characteristic. The characteristic flavor of cultured dairy products
The strains of man and animals have certain differences is produced by the activity of lactic culture by metabolic
like DNA of human isolates generally had lower G+C ratio transformation of milk constituents. An important
than that of pig and chicken biotypes. The acidophilus milk transformation of milk components into an essential flavor
has its therapeutic value in controlling intestinal disorders compound in cultured dairy products involves citrate
but its mode of action has not yet been elucidated. metabolism. Milk contains an average of 0.2% citrate
and it exhibits the greatest seasonal fluctuation (0.07-
Method of preparation
0.4%). Citrate is converted into ‘Diacetyl’ by flavor bacteria
The acidophilus milk is an extremely sour product. Leuconostoc cremoris and Streptococcus lactis sub sp.
The finished product contains very little, if any metabolic Diacetylactis. Leuconostocs ferment citrate only when there
byproduct other than lactic acid. It is prepared from partially is sufficient acid development during the fermentation.
skimmed milk, which is heated at 120°C for 15 minutes. The Because leuconostocs do not produce much acid from the
 Basic of Fermentation Technology

25
lactose of the milk, therefore require an associative growth acetic acid and propionic acid. In koumiss and Kefir alcohol
with lactic acid producing strain. Streptococcus lactis sub is an important component characteristic of the product.
sp. diacetylactis on the other hand produces sufficient Torula kefir yeast and Saccharomyces kefir are responsible
lactic acid, which required for the fermentation of citrate to for the production of alcohol. One very important compound
produce diacetyls. The literature contains conflicting reports causing flavor problems in cultured milks is 3-methylbutanal.
on the biosynthetic pathways involved for the production of This imparts a malty flavor to cultured milks and ripened
diacetyls in aroma bacteria. Diacetyl synthesized by flavor cream butter. Carbon dioxide plays an essential role in the
bacteria in dairy products does not accumulate indefinitely. flavor impact of cultured buttermilk, kefir and koumiss. The
Once the concentration of diacetyl precursor, citrate falls gas entrapped in the thickened milk provides lift, fizz, or
below a critical value, the diketone is rapidly converted into effervescence to these cultured products. Carbon dioxide is
a flavorless compound, Acetone. An enzyme called diacetyl derived from lactose by heterolactic bacteria. Fermentation
reductase is widely distributed among flavor bacteria and of citrate by aroma bacteria also produces considerable
other contaminating psychotropic bacteria commonly found amount of carbon dioxide. The minor metabolic products,
in dairy environment, catalyzes conversion of diacetyl although found in small or even trace amounts, may be
into acetone. Acetaldehyde is another important flavor important in maintaining a desirable flavor balance in
compound but in cultured creamy butter, cultured buttermilk cultured dairy products. Any shift in the flavor balance in
and cultured sour cream it is undesirable because it imparts cultured dairy products may result in the organoleptic
a flavor defect referred to as green or yogurt flavor. It is a very perception of off-flavor. Rancid flavor present in milk is
important flavor component in yogurt and related products. It derived from straight chain free fatty acids up to 18-carbons
is primarily derived from lactose although other mechanisms by the action of lipase enzyme. Cream flavor is derived
for production of carbonyl residue are found among lactic from isomeric 18-carbon unsaturated fatty acids by auto
acid bacteria. For example, metabolism of threonine and oxidation, 4-cis-Heptenol is produced. Butter flavor is due to
deoxyribonucleic acid results in acetaldehyde formation by β -lactones derived from hydroxy fatty acids by ring closure.
bacteria. Many bacteria produce acetaldehyde; major ones Sharp sour flavor is produced by the fermentation of lactose
are streptococcus lactis subsp. diacetylactis, Streptococcus to lactic acid by starter culture. Aldehydes, ketones, alcohols
thermophilus, Lactobacillus bulgaricus. Lactic Streptococci and esters combined to give milk a cowy flavor. Acetic acid
also produce a variety of carbonyl compounds that impart contributes significant culture flavors. (Flow chart 4, 5)
flavor important ones are: volatile fatty acids, formic acid,

Flow Chart 3
 Basic of Fermentation Technology

26

Flow Chart 4
 Basic of Fermentation Technology

27

Flow Chart 5

Starter cultures acid from lactose, citric acid from various compounds
or are involved in the fermentation of sugars to produce
Cultures used to start the fermentation in the preparation alcohols, diacetyls, or other flavor and aroma compounds.
of butter, ripening of cream, souring of milk in cheese The microorganisms of the starters may grow in association
manufacture; preparation of various fermented milks and or in succession to produce the desired product. Various
preparation of fermented cereal products are called “Starter purposes for which they may be used are:
Cultures”. They contain microorganisms that produce lactic
 Basic of Fermentation Technology

28
Aroma production milk product. The samples of dahi from south India
showed predominance of Lactobacilli. But samples of dahi
Aroma producing microorganism is Streptococcus lactis from North India showed predominance of Streptococci. The
that usually grows in fermented milk products. It produces species of lactic acid bacteria occurring most commonly in
lactic acid from lactose and also ferments citric acid to dahi include L. bulgaricus, Streptococcus thermophilus, S.
produce acetic acid, carbon dioxide, acetyl methyl carbinol, faecalis, S. lactis, L. casei and L. plantarum, in order of their
diacetyls and 2,3-butylene glycol. frequency.
Butter production Soy sauce: Soy sauce is a dark brown liquid with a salty
During butter ripening Streptococcus lactis produces taste and distinct aroma, which is made by
lactic acid, volatile acids as acetic acid, propionic acid and fermenting soybeans, wheat, and salt with a mixture
carbon dioxide. Some neutral 4-carbon compounds like of mold, yeast and bacteria. It is a seasoning agent used
diacetyls, acetyl methyl carbinols and 2,3-butylene glycol as a substitute for salt in the preparation of food as well
add to the flavor and aroma. as a table condiment. It enhances the flavor of the food
Batter fermentation and adds color into meats, sea foods, vegetables etc.
The fermentation of soy sauce is essentially a process of
In case of idli, dosa, jalebi, vada and bhatura, culture of enzymatic hydrolysis of proteins, carbohydrates and other
the previous batch is added as starter culture that starts the constituents of soybeans and wheat to peptides, amino
fermentation. The starter causes leavening and acidification acids, sugars, alcohols acids and other low-molecular
of the batter that produces a pleasant flavor in the batter. compounds by the enzymes of microorganisms. The
brewing of soy sauce originated in China many centuries
Preparation of fermented milk products ago and later was introduced in Japan and other oriental
In fermented milk products preparation, the flavor and countries. Soy sauce is consumed in almost every oriental
body is so related with the starter that without good starter country and has different names in different countries. It
culture, the product is sure to become unsatisfactory. is known as Chiang-yu in China, Shoyu in Japan, Kecap
in Indonesia, Kangjang in Korea, Toyo in Philippines and
Dahi or curd See-iew in Thailand. Japan leads the soy sauce industry
Dahi is the most important sour milk product used in the world. It has not only the largest fermentation plants
throughout India. Dahi is the common term used in India for but also has the most advanced technology. Japanese
any kind of sour milk. But sour milk from different parts of shoyu is produced primarily in the areas near Tokyo and in
India varies very much in quality, consistency and microflora. Chiba prefecture. Japanese shoyu is mainly of three types:
Dahi of Northern India is much thicker than in Southern India Koikuchi, Usukuchi and Tamari. Nearly 90% of the Japanese
where dahi is generally used in diluted form. In some parts shoyu is of Koikuchi type, with dark reddish brown color
of India Cane sugar is added to milk to prepare sweet dahi. and strong flavor and made from a nearly equal mixture
Sweet dahi is liked very much in Northern parts of India. of soybeans and wheat using Aspergillus oryzae hydrolytic
Dahi sold in the market is prepared from fresh raw milk, enzymes followed by vigorous lactic acid and alcoholic
but it is advisable to prepare dahi from boiled milk. Dahi yeast fermentations. It is pasteurized at a relatively high
prepared from fresh raw milk is fermented by natural flora temperature. Ten percent of shoyu is Usukuchi type, light in
i.e. no starter is added. When it is prepared from boiled milk, color with maximum total nitrogen content 1.2%. Third type
a little portion of dahi from the previous day is added and of shoyu is Tamari in which major portion is soybean and
milk is allowed to curdle at room temperature. As dahi is smaller portion is wheat. The highest quality shoyu is made
prepared without special care regarding its mother starter, by fermentation but second and third grades contain some
its quality varies from place to place and is never uniform. portion of chemical hydrolysates.

Method of preparation of dahi: Dahi of good quality Preparation of soy sauce: Soy sauce is prepared by
should be prepared in the following way: Good fermentation of a mixture of soybeans and

quality whole milk or skim milk may be used for the Cereal usually wheat and salt. However, in recent years,
purpose. Milk should be heated to 85 to 95 °C for half an defatted soybean meals and flakes have taken place. Today,
hour or boiled for 10 minutes. It is then cooled to 45 °C. A more than 90% of soy sauce production in Japan is from
small portion of the starter from good quality dahi is then defatted soybean products. In addition to the fermentation
inoculated into the milk. The amount of starter added will process, a chemical process in which acid hydrolyzes
vary with the type of the starter, condition of the milk and the proteins and carbohydrates is also being used in
incubation period. The milk is then incubated at temperature some western countries. In this method acid hydrolysis
near about 40 0C. After 24 hours of incubation dahi of good usually results in a complete breakdown of the substrate
quality may be obtained. than enzyme hydrolysis. However, acid hydrolysis cannot
perform many other specific reactions or interactions of
Microflora of dahi: Very little information is available hydrolyzed products as carried out by the multiple enzyme
regarding the microflora of this important system produced by molds, yeasts and bacteria. That is
 Basic of Fermentation Technology

29
why chemically hydrolyzed product does not possess the adding a koji starter culture called “Tane Koji” in Japan in
flavor and odor of the soy sauce prepared by fermentation cooked cereals and /or soybean substrate. Different types
process. of tane koji (soy sauce koji, miso koji) are available for
commercial use in making soy sauce, miso and others.
Treatments of the raw materials
Preparation of tane koji: Tane koji is prepared by using
Soaking soybeans: In preparation for fermentation, naturally selected or mutant strains of Aspergillus oryzae
selected soybeans are cleaned by ashing and soaked for or A. soyae to give desirable starter for a particular
10-15 hours at room temperature. The water is changed fermentation. Although strains used for preparation of koji
every few hours to prevent acidification by bacteria. Weight starter are different, the method for preparation is similar.
of beans should increase 2.1-2.5 times during soaking. Polished rice is soaked in water overnight, drained, steamed
Cooking soybeans: Hydrated soybeans are cooked for 1 for 1 hour and mixed thoroughly with 2% wood ash as a
hour in steam at 10-14 lb/sq in. ina rotary cooker (capacity 1 source of trace elements. The mixture is then inoculated
ton). They are then cooled rapidly. When defatted soybean with spores of selected strains of A. oryzae and spread out
meals or flakes are used, they are first moistened by on trays in layers approximately 1.5 cm deep. And covered
spraying with water amounting to about 130% of soybean with a moistened cloth to favor the growth of mold mycelium.
weight and then are steamed at 13lb/sq in. for 45 minutes. After incubation at 30 °C for 5 days, the rice is well covered
with mycelium and with green to yellowish green spores of
Roasting wheat: Whole wheat or wheat flour is essential A. oryzae. The spores are harvested, dried at 50 °C and
for production of typical Japanese stored at 15 °C. A koji starter is usually composed of a blend
of spores of different strains in a definite proportion, so that
shoyu flavor. Usually low protein flour is used. Wheat is
various enzymes are produced in proper amounts during
roasted in sand for several minutes at 170-180 °C and then
the preparation of koji.
the grains are crushed into 4-5 pieces in a roller mill. The
roasting process adds flavor and color to the resulting soy Preparation of soy sauce koji: Soy sauce koji is prepared
sauce and in addition destroys surface microorganisms and from a mixture of roasted wheat and steamed soybeans
facilitates enzymatic hydrolysis. inoculated with a koji starter (soy sauce Tane Koji) consisting
of selected strains of Aspergillus oryzae grown on polished
Effect of addition of wheat to the fermentation mixture:
rice. Inoculated soybean and wheat mixture is placed in
According to Yokotsuka (1964),
wooden or stainless steel porous trays of depth 30-40 cm
the addition of wheat to the fermentation mixture and several meters in length and width. The trays are then
serves several functions. Firstly, the mold grows better incubated at 25-35 °C. Aeration and moisture is carefully
and produces more enzymes on a mixture of wheat and controlled. This step is completed in 45 hours because this
soybeans than on wheat or soybeans alone. Secondly, the prevents the development of Mucor spp. and bacteria and
addition of roasted, crushed wheat to the cooked soybeans enhances the development of proteolytic enzymes. The end
would minimize the growth of undesirable bacteria i.e. product is called Soy sauce koji, which is a mixture of fungal
moisture of cooked soybeans is 60%, which is ideal for hydrolytic enzymes on soybean wheat mixture substrate.
bacterial growth whereas moisture of soybeans and Soy sauce koji of superior quality has a dark green color,
wheat mixture (1:1) is about 45%, which is adequate for pleasant aroma, sweet but bitter taste and high protease
mold growth but not for bacteria. Thirdly, wheat serves as and amylase activity.
a precursor of sugars, alcohols, organic acids, and flavor
Fermentation
compounds. Lastly, wheat is rich in glutamic acid.
The soy sauce koji is mixed with 1.2-1.5 volumes of
Addition of salt: Commercial salt is generally preferred
salt brine (23% w/v salt) to make mash called “Moromi”
for making soy sauce because it may carry an inoculum
in Japan. Moromi is fermented in large concrete tanks or
of halophilic and halotolerant bacteria and yeasts. Salt is
wooden vats for 8-12 months. Since koji is not prepared
added in such quantities that it prevents spoilage and/or
under aseptic conditions, one would expect the presence
food poisoning bacteria and permits the development of
of yeasts and bacteria in moromi. However pure cultures of
flavor and aroma forming bacteria and yeasts.
Pediococcus soyae, Saccharomyces rouxii and Torulopsis
Role of salt: In addition to giving a salty taste, sodium spp. are added to the mash at the start of fermentation and
chloride acts as a preservative and also at one month after the start of fermentation to accelerate
has a selective action on microorganisms that grow in the the fermentation and to improve the flavor of the final
fermentation substrate. product. High salt content ensures the development of
flavor enhancing yeasts while lower brine to koji ration
Koji and tane koji result in decreased utilization of total nitrogen. Traditional
Koji is a Japanese name given to a preparation consisting fermentation starts in April and takes a year to complete.
of mold growth on cooked cereals and/or soybeans. Koji In general low temperature fermentation gives better
serves as an enzyme source for converting complex plant results, because the rate of enzyme inactivation is slow and
constituents to simpler compounds. Koji is prepared by enzymes remain active for a longer time.
 Basic of Fermentation Technology

30
Pressing: The matured moromi is pressed in a hydraulic used for the fermentation of oriental foods, have shown
press at 100kg/sq cm. (1379lb) for 2-3days into a liquid that they do not produce Toxins. But, on the other hand
part, known as raw soy sauce, and a solid cake. When they resist accumulation of certain toxins which otherwise
whole soybeans are used as raw materials, soy sauce will be produced by other microorganisms in the food. This
oil, consisting chiefly of ethyl esters of higher fatty acids, could be considered as a protection of the product against
is produced during fermentation and appears at the upper other harmful microorganisms. A good example of such a
layer of raw soy sauce. This oily layer must be removed protective role is demonstrated by Rhizopus oligosporus,
and has no potential use. This is one of the reasons that the mold species used for the production of Tempeh. This
defatted soybean meal is used instead of whole soybeans mold species does not produce aflatoxins. On the contrary,
as raw materials in soy sauce fermentation. if aflatoxins are already present in the growth substrate,
R.oligosporus could lower its contents to about 40% of its
Pasteurization and bottling: The raw soy sauce liquid
original content. In addition it was found that R. oligosporus
is pasteurized at 70-80 °C in a kettle or heat exchanger,
inhibits growth, sporulation, and aflatoxin production by
cooled, filtered to remove precipitates and stored. The
Aspergillus flavus.
final product is bottled for market. The bottles are usually
made of plastic or glass and sometimes benzoic acid or Mold starters
propyl or butyl ester of p-hydroxy benzoic acid is added as
preservative. The mold species that are traditionally used for
fermentation of foods in different parts of the world
belong to different genera. Species of Rhizopus, Mucor,
Benefits of pasteurization
and Aspergillus are used for the fermentation of foods
1) Flavor, color and clarity is produced by the removal throughout the orient, with the exception of Japan. In
of oil particles mixed with heat coaguable substances 2) Japan, it is restricted to species of Aspergillus including A.
Inactivation of enzymes occur, which gives a stable product oryzae and A. soyae. These species are used in Tane koji,
3) Concentration of compounds like aldehydes, acetals, which is used as starter culture for the preparation of soy
phenolic compounds, mercaptans, organic acids, pyrazines, sauce, miso etc. Tane koji is prepared by growing the mold
furfurals, and β-diketones is increased 4) Because of on steamed rice. In other Asian countries, Ragi type starter
the increase in phenolic compounds and organic acids cultures are in common use. Cultivating molds on cakes
resistance to spoilage by film forming yeasts occur. made of rice or wheat flour, which has not been steamed or
cooked, makes these starters. The difference in preparing
Role of Mold in foods fermented by molds growth substrates for manufacture of two types of inocula
Synthesis of enzymes: Molds, in these food fermentations is thought to be the cause of a natural selection of mold
synthesize enzymes that decompose complex compounds, species which are developing in each of the type starters
including proteins, carbohydrates and fats into smaller over many centuries, when they were produced with non-
molecules. At the same time, other compounds may be aseptic, traditional methods.
synthesized from the food substrates. These complex
Yeast fermentations
changes are accompanied by changes in the original
properties of the raw materials. Taste, flavor, texture, When yeasts are involved in the fermentation process,
color, palatability and other properties of the raw materials production of alcohols improves the aroma of the product.
are usually modified in such a way that product becomes In addition, alcohol at a certain concentration makes the
more attractive to the consumer. In addition to this general substrate unsuitable for microorganisms, which may create
function of producing enzyme, in certain products the mold undesirable properties in the product. Combined with the
has a special role to play. organic acids that are produced by lactic acid bacteria, the
inhibitory effect of alcohol on undesirable microorganisms is
Mold growth: Mold growth on certain products contributes
increased. (Flow Chart 6)
to the appearance of the food, which is desired by the
consumer. Neurospora spp. provides oncom (fermented Miso
food) cake with a coating of its pink orange colored and
powdery conidia. Rhizopus oligosporus covers tempeh cake Miso is a paste like product made by fermenting cereals,
with a clean white mycelium surface layer and additionally soybeans and salt with molds, yeasts and bacteria in
has the function of binding together the soybean into a Japan. This product is generally known as Bean Paste. It
solid, compact cake. has the consistency of peanut butter, some smooth and
some chunky and its color varies from light yellow to reddish
Synthesis of coloring compounds: The function of brown. It has distinctive pleasant aroma resembling that of
Monascus purpureus during fermentation of Angkak soy sauce, and it is typically salty (degree of saltiness may
(fermented food) is the production of red colored compound vary) and may sometimes has sweet taste. It is like soy
monascorubin and a yellow pigment monascoflavin in sauce is used as a flavoring agent in cooking as well as
soaked rice. table condiment and can be used in place of soy sauce. The
Protection of the product: Molds, which are traditionally product is known as Chiang in China, Doenjang in Korea,
 Basic of Fermentation Technology

31
Tao-Chico in Thailand, and Tauco in Indonesia. All mean also be made according to the length of fermentation and
bean paste. In Japan, there are many types of miso and fermentation temperature. This can be understood from the
they can be prepared by varying the ratios of soybeans to following table: (Table 1)
cereals, salt content, length of fermentation and addition of
other ingredients such as hot pepper, which is very popular General method of preparation of Japanese rice
in China and Korea. miso
Cooking Soybeans: Whole soybeans are generally
used for the preparation of miso, but sometimes dehulled
soybeans or full fat soybean grits are also used for making
white or yellow rice miso. Soybeans used should be of
large size because the ratio of hull to cotyledons is lower in
large sized beans. They should have high water absorbing
capacity and when cooked under described conditions,
the beans should be homogeneously soft with fine texture
and bright color. To prepare the whole soybeans for
fermentations, they are washed, soaked in water for about
20 hours at 16 °C and drained. The soaked beans are then
cooked in water (white miso) or steamed at a temperature
11 °C for about 20 minutes in a closed cooker and slightly
mashed Table 2.

Preparation of rice miso koji

For Rice miso koji preparation, polished rice is used.
Since it is essential that the mold mycelium quickly
penetrates the rice kernels, the rice is soaked in water
(15°C) overnight or until moisture content is about 35%.
Excess water is drained off, and the soaked rice is steamed
at atmospheric pressure for 40 minutes. After cooling to
35°C, tane koji (koji starter culture), which is a blended
mixture of several different strains of A. oryzae, prepared as
described in the soy sauce preparation, is sprayed over the
rice and mixed well. The inoculated rice is then incubated
in a temperature and humidity controlled room. In about
40 -48 hours at 30 -35°C. The rice is completely covered
with white mycelium of the inoculate culture. Harvesting
is done while koji is white and before any sporulation has
occurred. At this time, koji has a pleasant odor, lacks any
musty or moldy odors, and is quite sweet in taste. The koji
is removed from the fermentor and mixed well with salt to
stop any further development of the mold.

Yeast and bacterial fermentation

Next fermentation is carried out under anaerobic
conditions by yeast and bacteria. Cooked and slightly
mashed soybeans are mixed with the salted koji and
inoculated with a starter culture containing pure culture of
Yeasts (Saccharomyces rouxii, Torulopsis spp.) and bacteria
(Pediococcus halophilus, Sterptococcus faecalis). Miso
Flow Chart 6 from the previous batch can also be used as an inoculum
Japanese miso can be categorized into three major of yeast and bacteria. Sufficient water is added to bring the
groups as follows: moisture content equal to 48%. The mixture, now known
as Green Miso, is thoroughly blended and tightly packed
1) Rice miso -it is prepared from rice, soybeans, and salt into a vat or tank for fermentation at 25 – 30 °C. During
2) Barley miso -it is prepared from barley, soybeans and the fermentation period, the green miso is transferred from
salt 3) Soybean miso -it is prepared from soybeans and one vat to another at least twice to improve fermentation
salt. Each group can further be subdivided into white, light conditions. Fermentation time varies widely depending
yellow, red according to color, and sweet, medium salty, upon the type of miso. It is one week for white miso, 1 -3
and salty according to taste. Similar classifications can months for salty miso and one year for soybean miso.
 Basic of Fermentation Technology

32
Table 1

Soybean : Rice : Salt Type Color Taste Fermentation Time Fermentation Temperature

100 : 200 : 35 White miso Bright light yellow Sweet 2-4 days 50 °C

100 : 60 : 45 Salty miso Light yellow Salty 30 days 30 –35 °C

100 : 50 : 48 Red salty miso Yellow red Salty 60days 30-35 °C

Table 2

Protein Isoelectric point Protein Isoelectric point

Egg albumin ~ 4.6 γ1-Globulin 6.6

Haemoglobin 6.8 Lysozyme 11.0

Pepsin ~ 1.0 Myoglobin 7.0

Serum Albumin 4.9 Chymotrypsinogen 9.5

β- Lactoglobulin 5.2 -- --

Aging and packaging soybeans that most people find unpleasant. Because of its
high protein content and universally acceptable taste and
At the end of fermentation, the fermented mass is kept texture, it can be a potential source of low -cost proteins.
at room temperature for about 2 weeks to ripen. The aged
product is then blended mashed, pasteurized (60 – 70°C Preparation of tempeh
for 30 minutes), and packaged. Traditionally, miso is sold in
wooden kegs of various sizes. Presently, it is sold in sealed Microorganism: The mold used for tempeh fermentation
polythene bags or tubes. For packaging into the plastic was reported earlier to be Rhizopus oryzaebut in Indonesia,
bags, miso must first be pasteurized at 60 –70°C for 30 a strain identified as Rhizopus oligosporus saito NRRL 2710
minutes to prevent swelling. Sorbic acid or its potassium is considered better producer of a good product. This strain
salt is also added at a level of less than 1 gm/kg of miso is characterized by sporangiospores showing no striations
(Flow Chart 7). and being very irregular in shape under any condition of
growth. The sporangiospores are short unbranched and
Dehydrated miso powder arise opposite to rhizoids that are much reduced in length
and branching. It’s all isolates also show Chlamydospores.
This product has become increasingly popular. The Rhizopus oligosporus is highly proteolytic, which is important
dehydration is carried out by freeze-drying process. in tempeh fermentation because of the high protein content
Dehydration process does not affect the flavor of the of the substrate. Two proteolytic enzyme systems were
product. It has its potential use as an ingredient in the observed in the fungus; one has an optimum pH at 3.0 while
instant mix products. (Flow chart 7) the other at 5.5. Both the enzyme systems have maximum
activities at 50 – 55 °C and are fairly stable at pH 3 -6.
Tempeh They rapidly denature at pH below 2 or above 7. In addition
Tempeh or Tempe kedelee is a cake -like product to high protease activity, the mold possesses strong lipase
that originated in Indonesia and is widely consumed in activity, low amylase and no detectable pectinase activity.
the regions of Malaysia and Indonesia. It is prepared by Preparation of soybeans for fermentation: The Full
fermenting dehulled and briefly cooked soybeans with a -fat soybeans are soaked in water overnight at room
mold, Rhizopus; the mycelia bind the soybean cotyledons temperature. The soybean cotyledons can be mechanically
together in a firm cake. The raw tempeh has a clean, fresh, cracked into 4-5 pieces, so that they absorb water easily
and yeasty odor. When sliced and deep -fat fried, it has a and this also reduces the soaking time from 20 hours to 30
nutty flavor, pleasant aroma, and texture that are familiar minutes. They are dehulled by hand or by a simple roller
and highly acceptable to almost all people around the mill. The grits of beans are boiled in water for 30 minutes.
world. Unlike most of other fermented soybean products They are then drained and spread to cool and surface
that are used as flavoring agents or relishes, tempeh is drying.
used as a main dish and meat substitute in Indonesia. It
is easy to cook and does not possess the beany flavor of
Basic of Fermentation Technology

33
Fermentation
A pure culture of R. oligosporus spore suspension
(prepared by adding a few milliliters of sterilized distilled
water to the slant culture) already grown on potato dextrose
agar and incubated at 28 °C for 5-7 days is mixed with
the cooled and surface dried soybeans at the rate 106
spores/100gm of cooked soybeans. The inoculated
soybeans are tightly packed into an appropriate container
(petri dish) for incubation to obtain a final product in which
a white mycelium developed abundantly but black spores
are minimal. Rhizopus oligosporus like many molds require
air to grow, but it does not require as much aeration as
many other molds. In fact, too much aeration causes
spore formation and may dry the beans resulting in poor
growth. Therefore, it is important to properly pack the
beans for fermentation. Many workers have successfully
tried different types of packing materials. These are petri
plates, aluminum foils or metal trays, with perforated or
woven mesh bottoms and covers or perforated plastic
film covers or perforated plastic bags and tubings. When
the fermentation is complete, the beans are covered with
and bound together by white mycelia. Thus raw tempeh
looks like a firm white cake and has an attractive and
slightly yeasty odor. Prolonged fermentation often causes
the product to become obnoxious due to the enzymatic
breakdown of proteins (Flow Chart 8).

Flow Chart 8

Flow Chart 7
 Basic of Fermentation Technology

34
Idli Soaking
Idli is a breakfast food in most parts of India especially Generally, the ingredients are soaked separately in water
popular in southern India. It is acidified and leavened at room temperature for 5 to 10 hours before grinding to
through fermentation by hetero fermentative acid bacteria prepare the batter. Parboiled rice semolina can frequently
rather than by the activity of the yeast. It is closely related be used while dry black gram dhal flour has been found
to sourdough bread of the western world, but it does not unsuitable for the preparation of idli. If the idli batter is to
depend upon wheat or rye as a source of protein to retain be made without inoculation, it is essential that cereal and
the carbon dioxide gas during leavening. The importance legume be soaked, ground with water and incubated at
of idli lies in 1) its high degree of acceptability as a food room temperature. Hot soak or hot grind will destroy the
2) its protection against food poisoning and transmission organisms essential for fermentation and, unless they are
of pathogenic microorganisms, because of acidity and 3) replaced by an inoculum, the fermentation will not proceed
the fact that idli fermentation can be used in many parts properly.
of the world using various combinations of cereal grains
and legumes to produce acid, leavened bread, or pancake
Proportion of water and salt to other ingredients
like products 4) No wheat or rye flour is needed. Idli is a The amount of water added to the rice and dhal batter
small, white, acid leavened, and steamed cake made by the has varied from 1.5 to 2.2 times the dry weight of the
bacterial fermentation of a thick batter made from carefully ingredients. Batter should be rather thick for idli. Generally
washed rice (Oryza sativa) and dehulled black gram dhal salt 0.8 to 1% is added to the batter as a seasoning before
Phaseolus mungo. Idli cakes are soft, moist and spongy fermentation.
and have a desirable sour flavor. It is served like a pancake
with butter, honey, and jam or with other sauces. It can also Fermentation time
be consumed directly “out-of-hand” following steaming or Fermentation time varies from 14-24 hours, with overnight
the cake may be deliciously flavored with fried mustard being the traditional time interval for the preparation of idli.
seeds and chopped coriander leaves. The unflavored cakes The fermentation time must be sufficient to allow a definite
are eaten with chutney and /or samber, a thin-spiced soup leavening of the batter and allow for the development of
of dhal and vegetables. pleasant acid flavor.
Details of manufacture of Idli Inoculum
Ingredients: Idli preparation contains a number of different Ordinarily, the microorganisms developing during initial
ingredients these are 1) Rice 2) Black gram dhal 3) Salt 4) soak and then during the overnight fermentation are
water. Dehulled soybeans or Bengal gram can be used as a sufficient to leaven idli. The Central food technological
substitute for black gram dhal and a number of cereal grains Research Institute recommends adding one tablespoonful
can replace rice. However, there may be marked change of buttermilk to each pound of its dry idli mix. The
in the texture and flavor when using substituted materials. microorganisms that develop during overnight soaking
It has been reported that rice variety and its physical are: Leuconostoc mesenteroides, Streptococcus faecalis,
characteristics are very important to produce a good quality Pediococcus cerevisiae, Lactobacillus fermentum,
idli. White Kar and IR20 varieties of rice have given much Torulopsis candida, Trichosporon pullutans, Torulopsis
better performance in the production of idli, especially the holmii etc.
White Kar variety because of its high amylose content, low
amylopectin content better gelatinization, and better water Incubation temperature
uptake ability.
Ordinarily, the idli fermentation is carried out at room
Proportion of cereal to legume temperature. It generally means that temperature of 25 °C
to 30 °C is probably an optimum temperature.
Ordinary idli consists of three parts rice and one part
black gram dhal plus salt to taste. Kancheepuram idli is Steaming
prepared from one part rice and one part black gram dhal
The fermented batter is steamed as soon as the product
plus cashew nuts, ghee, salt, pepper, ginger and cumin
has become leavened and acidified for 15-30 minutes at
added to taste. Normally proportions of rice to black gram
atmospheric pressure to get a soft, spongy and sour tasted
dhal varies from 4:1 to 1:4, the 2:1 being the best. It has
and flavored idli. The fermented batter is poured into the
been seen that when black gram dhal proportion is less
cups of an idli steamer, which is placed in a covered pan
than 25%, the steamed idli was hard and organoleptically
and steamed until the starch is gelatinized and idli cakes
unacceptable whereas when it is more than 50%, the
are soft and spongy. They are generally consumed on the
product obtained is too sticky to be acceptable. Thus, not
same day and there is no effort to preserve the product.
only can the ingredients be varied, but the proportions can
The acid content of the product retards the growth of food
also be varied within a wide range and still an acceptable
poisoning and food spoilage causing microorganisms.
product is obtained.
 Basic of Fermentation Technology

35
Microbiology of fermentation Method of dosa preparation
In a study of sequence of microorganisms that Ingredients: The ingredients used for dosa preparation are
developed during soaking of ingredients and subsequently similar to as that of idli preparation i.e.rice, black gram dhal,
during fermentation of the batter at 30 °C, it has been salt and water. Rice may be substituted by wheat, bajra
found that Leuconostoc mesenteroides and Streptococcus (Pennisetum typhoideum), maize, or kodri and black gram
faecalis developed concomitantly and then continued dhal may be substituted by sprouted peas, cowpeas (Vigna
to multiply following grinding. The number of these two catjang), field beans (Dolichos lablab) or soybeans. Fresh
species remained very high until 23 hours while in the later groundnut oilcakes may also be substituted for black gram
stages Pediococcus cerevisiae was also found. During this dhal.
stage idli is being steamed and consumed. Thus, these
Soaking and batter formation: Generally, equal quantities
two species are responsible for the acid production and
of rice and dehulled black gram dhal are soaked in water at
leavening of the batter. The usual aerobic contaminants
room temperature separately for 5-10 hours. It is common
present on the ingredients are eliminated partly by careful
practice that finely ground powders are used to prepare
washing and partly by acidic conditions produced by the
batter. The finely ground powders are mixed with water at
fermentation. The fermenting microorganisms appear to be
temperatures ranging from 480 to 980 °C, best at 80 °C for
present on the ingredients and if the product has to be made
the preparation of batter. Water is added in the range of 2.0
daily, there might be some advantage in adding a bit of the
to 2.2 times the initial dry weight of ingredients to prepare a
freshly fermented batter to the newly ground batter. Yeasts
batter of viscosity desired for dosa. Salt is added from 0.8
like Torulopsis and Trichosporon can possibly leaven the
to 1.0% as a seasoning before fermentation.
batter if present in sufficient number, but it is highly unlikely
that they produce the acid characteristics of idli. Fermentation Time: Traditionally, dosa batter is kept
overnight for fermentation. The fermentation time should be
Jalebi sufficient to allow a definite leavening and acidification of
To make jalebi, refined wheat flour (Maida), dahi and the batter and to allow for the development of a pleasant
water are mixed into a thick batter and fermented for 14- acid flavor.
16 hours. The fermented batter is deep fat fried in spiral
Inoculum: The natural microflora developed during the
shapes and immediately immersed in sugar syrup (600B to
soaking operation and then at the overnight fermentation
750B) for a minute or two and eaten.
is sufficient to leave the dosa batter. However, fermented
Microbiology of jalebi fermentation batter of the previous batch may also be used as a
starter culture for fermentation. The microorganisms that
Inoculum in jalebi ads from the natural microflora develop during the overnight soaking are: Leuconostoc
includes Lactobacillus fermentum, Streptococcus lactis, mesenteroides, Streptococcus faecalis, lactobacillus
Streptococcus faecalis, Lactobacillus buchneri and fermentum, Pediococcus cerevisiae, Trichosporon
Saccharomyces spp. pollutants, Trichosporon beigelii, and Candida kefyr etc.
Changes during jalebi fermentation Incubation temperature: Ordinarily, the dosa fermentation
is carried out at room temperature. In the tropics, this
During fermentation, pH decreases from 4.4 to 3.3,
generally means a temperature of 25-30 °C.
volume of the batter increases about 9%; both amino
nitrogen and free sugar decrease. Fermentation containers: In Indian homes, fermentation
is customarily carried out in utensils that have sufficient
Dosa capacity to hold the batter and clean to avoid excessive
It is a thin crisp, fried, pancakes like staple food of contamination. The containers are covered with a clean
southern India and is also gaining much popularity in other damp cloth to prevent the entry of insects.
parts of the country. Like most of the fermented foods Grinding of the Ingredients: Stone mortars are used for
consumed in India and other Asiatic countries, dosa is grinding the ingredients in Indian homes. These provide
prepared by natural fermentation. Dosa batter is very similar excellent control of the particle size in batter. The ingredients
to idli batter, except that both the rice and black gram dhal are very finely ground to a thin paste and then salt is added
are finely ground. The batter of dosa is thinner than the idli in it.
batter. Following fermentation, the dosa is quickly fried as a
thin, fairly crisp pancake and eaten directly. The unflavored Harvesting and preservation: As soon as the batter
crisp pancake may be rolled onto a cooked mixed vegetable becomes leavened and acidified, it is spread onto a hot
mixture and eaten with samber, which is a thin richly spiced and greasy griddle where it assumes the shape of a crisp
soup of dhal and vegetables. It is an important source of pancake. The spreading of dosa on the griddle is an art
protein and calories in the diet and nutrition of south Indians. that matures with practice. Sometimes a mixture of cooked
Since is easily digested therefore it is often used as food for different vegetable is poured onto the crisp dosa and the
infants and invalids. sides are rolled, which now becomes ready to be eaten with
a spiced soup of dhal and vegetables popularly known as
Samber.
 Basic of Fermentation Technology

36
Microbiology of dosa fermentation: Traditional dosa Fermented sausages
batter fermentation has revealed the Occurrence and
role of several bacteria alone or in combination with Sausages are cylindrically shaped, fermented, solidified,
yeasts in bringing about various biochemical changes but and heat stabilized emulsions formed by mixing together
Leuconostoc mesenteroides appears in the fermentation proteins, fats, water, salt and flavorings. Color, flavor and
early and brings about the leavening in batter. It is also texture of sausages depend on the type of meat, which
along with Streptococcus faecalis involved in the acid is mixed and comminuted together with ice, salt, spice
production. These organisms appear to be present on the flavorings, curing agents and selected meat trimmings, to
ingredients and develop during fermentation. Therefore it form a sausage emulsion. The word “sausage” is derived
becomes an advantage that previous batch inoculum is from the Latin, salus; meaning salted or literally, preserved
used as a starter for fermentation. Yeasts, if are present in meat. The term “salus” undoubtedly was used by the
sufficient number can leave the batter but they have no role Romans to denote meat preserved through the use of salt.
to play in acid production, which is characteristic of dosa Basically all sausages are comminuted meats. Products
batter fermentation. Pediococcus cerevisiae appear very differ primarily because they are spiced in varied fashion
late in the fermentation when the batter becomes ready to and because of differences in methods of processing.
be fried on hot and greasy griddle. (Flow Chart 9) Classification of sausages
Sausages may be loosely classified into three general
categories: 1) Fresh sausages. 2) Cooked or Smoked
sausages and. 3) Dry Sausages. Some sausages,
especially the dry and semi-dry types depend on bacterial
fermentation of the production of their characteristic
flavors, while in manufacture of some of the more common
sausages such as frankfurters bacteria play no role. No
distinct classification can be made based on spice formulae
because basic spices are used in virtually all products.
Fresh sausages: Fresh sausages are always kept under
refrigeration and are fried, boiled, or Cooke thoroughly
before serving. They are prepared from selected cuts of
fresh meats, principally pork, or beef that has not been
previously cured. Examples of fresh sausages are fresh
Pork sausages that are prepared selectively from pork meat
and fresh sausages that may also contain a percentage
of beef and other meats in addition to pork meat. Pork
trimmings are put through a grinder, seasoning is added
and mixed, and the product is stuffed into natural casings
or sold in bulk.
Cooked or smoked sausages: These can be divided
into two categories -1) smoked and un cooked and 2)
smoked and cooked. Both these categories of sausages
are prepared from cured meats and must be kept under
refrigeration prior to preparation for serving. Smoked and
uncooked sausages -include Smoked country-style Pork
sausages that are subjected to mild curing and then placed
in casings and smoked and cooked before serving. Other
varieties like mettwurst, smoked country-style sausage and
polish sausage, all contain beef and pork mixed in various
proportions. Smoked and cooked sausages -these are
prepared from both uncured and cured meats. They are
processed by cooking and smoked after cooking. Examples
of these are Frankfurters (60% beef and 40% pork), Bolonga
(cured beef and pork), Berliner sausages (cured, coarsely
ground pork and finely chopped beef), and German type
Mortadella (Cubes of fat pork and pistachio nuts), Liver
sausages (pork liver and gelatin), Blood sausages or
Bluwurst diced, cooked fat pork plus finely ground cooked
Flow Chart 9 meat, blood and gelatin.
 Basic of Fermentation Technology

37
Dry sausages: Dry sausages are prepared by curing proportion of fillers in sausages should not exceed 3.5%.
freshly comminuted meats added with Curing agents and When corn syrup and milk solids are used their proportion
spices for 2 to 3 days. They are then placed in casings and should not exceed 2%.
are processed by carefully controlled air-drying. When pork
Curing agents: The common curing agents for sausage
is used in such sausages, it is subjected to a light smoke in
preparation are salt, sodium nitrite and/or nitrate and sugar.
order to destroy live trichinae. The principal dry sausages
Three percent of salt on the basis of the meat ingredients
are either salamis or cervelats. Salamis are generally more
is frequently used in sausages. Sodium nitrite and/or nitrate
highly seasoned than cervelats.
are usually added along with the salt to the beef portion of
Meat Ingredients for sausages: The meat ingredients the sausage emulsion. The proportion of sodium nitrite may
used chiefly in preparing sausages are those parts of the not exceed ¼ oz per 100 lb of meat; if sodium nitrate is used
animal that do not have a ready sale as such. These are with nitrite, not more than 2 oz should be employed per 100
classified according to their “water binding” properties i.e. lb of meat. Sugar is added in many sausage products at a
their ability to retain moisture during thermal processing. level of 0.5-1.0%. Other curing agents such as potassium
Meats considered to have best binding properties are nitrate (saltpeter), potassium nitrite, vinegar and flavorings
skeletal tissue from the beef animal, and include bull meat, are also added.
shank meat, chucks and boneless cow meat. Medium
Seasonings: Variation in seasoning is one factor that
binders are head meat, cheek meat, and lean pork
is responsible for the large number of sausage varieties.
trimmings. The meat with low binding properties usually
These seasonings may be added as ground natural spices,
contains large proportions of fat or are non-skeletal or
extracted oils, and oleoresins, or a mixture of the two.
smooth muscles. Examples are regular pork trimmings,
Spices that are usually employed include allspice, black
beef brisklets, heart, giblets and tongue trimmings. Meat
pepper, cardamom, cinnamon, coriander, garlic, mace,
tissues classified as “Filler Meats” and considered to have
nutmeg, paprika and sage. Most seasonings for pork
little or no binding properties include ox lips and tripe, pork
sausage contain 1.75-2.0 lb of salt, 6-8 oz dextrose, 4-5
stomachs, skin, snout, lips, and particularly defatted pork
oz pepper, 2-3 oz sage and 0.25 oz of ginger, Per 100 lb
tissue; their use in sausages must be severely limited if the
of meat.
quality of the product is to be maintained. The muscles of
beef in sausages enhances flavor as well as contributes to Other additives: The other condiments used in meat
color and texture. The beef muscles contain water-soluble products are pistachio nuts, mono sodium glutamate,
nitrogenous extractives that act favorably on flavor. Fat in ascorbates and isoascorbates. Pistachio nuts are used
the sausages enhances flavor and changes the texture of in headcheese, meat loaves and Braunschweiger.
the sausages. They become tender and juicy, but total fat in Monosodium glutamate is occasionally employed in pork
sausage must not exceed 50%. sausage at a level of 0.1% to enhance flavor. Ascorbic acid
or d-isoascorbic acid (erythrobic acid) is used at a level of ¾
Ice and salt: The addition of ice (moisture) assists in
oz or ⅞ oz of their sodium salts, in each 100 lb of sausage
controlling the temperature of the emulsion while it is
product. These are commonly used in smoked sausage to
comminuted. Otherwise the chopping temperature will
assure minimum color development and retention. Coloring
exceed 60oF, which will lead to instability of emulsion and
matter and dyes, which are approved by the regulatory
promote bacterial growth. The moisture in the final cooked
bodies may be mixed with the rendered fat, applied to
sausage should not exceed four times the meat proteins
animal and artificial casings, and applied to such casing
plus 10%. For fresh sausages that have not been heat-
enclosing products. The following coloring matter and
processed the limit is four times plus 3%. Water and salt
dyes are acceptable 1) Natural coloring matters -alkanet,
make meat proteins soluble, which then stabilizes the fat
annatto, carotene, cochineal, green chlorophyll, saffron and
globules in the sausage emulsion. Salt also adds to flavor.
turmeric 2) Coal tar dyes -all food certified dyes.
Binders or fillers: Cereal flour, potato flour, soy flour,
Sausage casings: Sausage casings may be natural
bread or cracker crumbs, milk powder, casein are some
casings prepared from some part of alimentary tract of
of the binders that are used both to hold together and to
cattle, sheep or hogs, or they may be cellulosic casings.
extend the meat ingredients in the sausages. The flours
These latter are frequently used on frankfurters and may be
that are made from cereals as corn, durum wheat, and rye
clear or colored.
and from potato starch absorb water highly; however, they
must not readily ferment when mixed with it. The soy flour a. Method of preparation of frankfurter sausage
with its low fat content enables making sausage flour high
in protein content. Rice and cracker flours are also high in Frankfurters are the most popular of all the sausage
protein content. Binders or fillers improve color, provide products. More frankfurters are consumed than any other
better binding properties, improve slicing characteristics, type of smoked and cooked category of sausage; they
change or improve flavor and reduce cost of the product. represent 25% of all sausage sold. The meat formulation of
Their value depends on their ability to absorb moisture in ordinary frankfurters is 40-60% beef and 60-40% pork. Beef
the emulsion and retain it throughout heat processing. The content includes Bull meat, boneless chuck, plates, hearts
and trimmings while pork ingredients are filler meats such
 Basic of Fermentation Technology

38
as tongue, snout, lips and other byproducts in proportion sausage makers developed products that would keep
not exceeding 20%; more will result unsatisfactory product. under the climatic conditions of their particular area. Since
Fillers employed are dry skim milk, corn syrup solids and artificial refrigeration and canning processes were unknown
sometimes cereals. Total filler content should not exceed therefore sausage makers of Italy and southern France,
3.5%. Meat used for preparation was pre-cured, but at developed dry sausage products. German and Hungarians
the present time the curing agents are normally added produced definite types of dry sausages.
at the time of chopping. Salt sugar and curing salts are
c. Action of microorganisms in sausages
added at the level of 3 lb of salt, ½ lb of dextrose, ¼ lb
oz of sodium nitrite and 2 oz of sodium nitrate, for each Paulo and Smith (1977) have shown that Micrococci
100 lb of meat. The more common spices and seasonings dominate the surface of stuffed sausage during the early
used are pepper, nutmeg, mace, cinnamon, mustard and stages of fermentation. These are killed at pH 5.5 and
garlic. After chopping and mixing, frankfurters are stuffed are not found in the sausage after heat treatment of
into casings and linked. They then held at refrigeration or drying. The results corroborated the research carried out
ambient temperatures for varying periods of time prior to by Sison (1967) in Philippines on the native chorizo-type
heat processing to permit completion of curing process. sausages. The major functions of micrococci during the
Ascorbic acid may be used to obviate the need for this fermentation are the reduction of nitrate to nitrite and the
holding period. The sausages are then heated and smoked. production of catalase. Lactic acid bacteria rarely reduce
Immediately after smoking, they are cooked with a spray the nitrates to nitrites. Lactic acid bacteria were also found
of hot water at a temperature of 77-82 °C. Simply raising in the fermentation. The activity of the lactic acid bacteria
the smoke house temperature finishes some frankfurters; is the conversion of sugars to lactic acid by EMP pathway.
such dry-processed products are usually given a brief hot Streptococcus diacetylactis produces diacetyls and acetoin
water shower to plump the sausages and provide better that imparts nutty flavor and aroma to some sausage.
peeling characteristics. The frankfurters are then cooled Staphylococci also actively reduce nitrates to nitrites.
by cold water showering to an internal temperature slightly Excessive nitrite level in sausage has been noted when
above ambient; the remaining heat is usually sufficient to using high nitrate concentrations with micrococci, producing
dry the product prior to its placement in the holding cooler a defect in sausage called “nitrite burn”. The micrococci are
at 2-7 °C. The total processing time may be as short as 65 also lipolytic, and they produce lipase during early stages of
minutes (when the cure proceeds rapidly) or as long as 2.5 fermentation. This results in an increase in free fatty acids,
hours. Colored cellulosic casings may be used to add color, volatile fatty acids and carbonyl compounds after 28 days
or dye may be mixed with the hot shower water, which must of drying. Lactic acid bacteria produce varying amounts
then be recirculated; occasionally frankfurters are colored of hydrogen peroxide, which is destroyed by the catalase
by dipping. Certified coal tar colors can also be used. of micrococci. Another important function of lactic acid
Frankfurters processed in cellulosic casings may have their bacteria is the inhibition of Staphylococci. This inhibition
casings peeled from them after processing to produce the or suppression also suppresses the enterotoxin production
product sold as skinless variety. by Staphylococci. This inhibition is more pronounced as
the ratio of lactic acid bacteria to Staphylococci increases
b. History of sausage
and as temperature of the fermentation decreases. The
Sausage, one of the oldest forms of processed food, was beneficial effects of lactic acid bacterial starter cultures
developed some thousand years before the birth of Christ. in inhibiting Staphylococci and enterotoxin production in
It started by slow stages from simple process of salting fermented sausages have also been demonstrated.
and drying meats by the aborigines and was originated in
part as a means of preserving meat that could not once be Sauerkraut
consumed. The American Indians combined chopped, dried These are German terms for sour cabbage, which is
meat with dried berries and pressed these ingredients into a generally prepared from shredded cabbage. The yellow
cake for use when food was scarce. Similar drying of meat -white shreds are approximately 2-5 mm in width and as
was a common place along the shores of the Mediterranean long as 20 cm.
centuries before the rise of Roman Empire. The ancient
Romans were extremely fond of a sausage made of fresh Standards for sauer kraut
pork and white pine nuts, chopped fine and seasoned
It must contain at least 0.75% lactic acid and less than
with cumin seed, bay leaves and black pepper. Salami is
10% of the total acid can be volatile. The pH must not
mentioned in Grecian literature of about 5th century B.C.
exceed 4.1. The strainable brine should amount to about
Sausage was made and eaten by Babylonians (1500 B.C.).
10% of the total weight of sauerkraut and should contain
The fact that a city of Salamis existed on the east coast of
from 0.7 to 3.0% NaCl.
Cyprus in Aegean Sea about 449 B.C. provides foundation
for a supposition that salami sausage may have originated Preparation for fermentation
in this ancient Grecian city. The various types of sausages
as we know today were developed in certain European Properly matured sound heads of cabbage are trimmed
localities because of local climate conditions. European to remove the damaged parts and outer green or dirty
 Basic of Fermentation Technology

39
leaves. The cabbage is then sliced to shreds of size 0.16 to Influence of Temperature
0.08 cm in thickness. The shredded cabbage is conveyed
to vats or tanks for salting and fermentation. At low temperature (7.5 °C), fermentation is very slow.
Leuconostoc mesenteroides grows slowly attaining an
Role of salt acidity of 0.8-0.9% in terms of lactic acid in a month. Acidity
is important for its preservative effect. Other lactobacilli
Salt plays a primary role in the preparation of sauerkraut; and pediococci cannot grow at this low temperature. The
therefore its concentration is carefully controlled. According sauerkraut may not be completely fermented for 6 months
to legal standards salt must not be less than 2% and must or more or until the temperature rises to a temperature
not be more than 3%. Most of the producers of sauerkraut suitable for the growth of higher lactic acid producing
add salt in the concentration of 2.25 to 2.5%. Salt extracts lactics. At a temperature of 18 °C with a salt concentration
water from shredded cabbage by the process of osmosis, of 2.25%, a total acidity of 1.7-2.3% as lactic acid will be
thus forming fermentation brine. It suppresses the growth of attained, with an acetic to lactic acid ratio of about 1:4 in
some undesirable bacteria that might cause deterioration of about 20 days. At higher temperature i.e. 23 °C, the rate
the product and at the same time makes conditions favorable of fermentation will be greater so that a brine acidity of
for the growth of lactic acid bacteria. Salt also contributes 1.0-1.5% (lactic acid) may be attained in 8 to 10 days.
to the flavor of finished product by yielding a proper salt Active growth of Lactobacillus plantarum and Lactobacillus
acid ratio. The use of too little salt causes softening of brevis may be initiated in 3-5 days and the kraut may be
tissues and produces a product lacking in flavor. Too much completely fermented in approximately one month. At still
fermentation and over salting may produce a product with higher temperature of 32 °C, the rate of fermentation may
a sharp, bitter taste. It may also cause darkening of the be very rapid and an acidity of 1.8-2.0 may be attained in
color of product and may favor the growth of pink yeasts. 8 to 10 days. The major share of the acid produced will
Brine begins to form once the shreds are salted and tank is result from the growth of homofermentative bacteria L.
closed when it is filled to the proper level. Then a weight is plantarum and P. cerevisiae. The flavor of the sauerkraut
placed over these shreds so that it squeezes the water out will be inferior, similar to an acidified cabbage. At the higher
of the shreds. The weight may be of wood (old method) or temperature sauerkraut will darken rapidly unless canned
some plastic bag filled with water may be placed (modern immediately. It will have a poorer shelf life than sauerkraut
method). fermented at lower temperature. This sauerkraut also has
Microbiology of sauerkraut fermentation low percentage of acetic acid and will not attain as high a
total acidity, even though the pH is lower. It will also subject
Pederson first described the lactic acid bacteria that to yeast spoilage, partly because of its low content of carbon
he observed in fermenting sauerkraut. He found that the dioxide. It is also low in ascorbic acid content.
fermentation was initiated by the species of Leuconostoc
mesenteroides. This species was followed by gas forming Influence of salt
rods and finally by non-gas forming rods and cocci. Salt plays a primary role in the preparation of sauerkraut;
Leuconostoc mesenteroides is a heterofermentative therefore its concentration is carefully controlled. According
bacteria and it grows more rapidly than other lactic acid to legal standards salt must not be less than 2% and must
bacteria. It is active over a wide range of temperature and not be more than 3%. Most of the producers of sauerkraut
salt concentrations. It produces acid and carbon dioxide add salt in the concentration of 2.25 to 2.5%. Salt extracts
that rapidly lowers the pH, thus inhibiting the activity of water from shredded cabbage by the process of osmosis,
undesirable microorganisms and enzymes that may soften thus forming fermentation brine. It suppresses the growth of
the shredded cabbage. The carbon dioxide replaces some undesirable bacteria that might cause deterioration of
the air and creates an anaerobic condition favorable to the product and at the same time makes conditions favorable
prevent oxidation of ascorbic acid and natural color of for the growth of lactic acid bacteria. Salt also contributes
the cabbage. It also stimulates the growth of many lactic to the flavor of finished product by yielding a proper salt
acid bacteria. While this initial fermentation is developing, acid ratio. The use of too little salt causes softening of
the heterofermentative species of lactobacillus brevis and tissues and produces a product lacking in flavor. Too much
homofermentative species of lactobacillus plantarum and fermentation and over salting may produce a product with
sometimes Pediococcus cerevisiae begin to grow rapidly a sharp, bitter taste. It may also cause darkening of the
and contribute to the major end products like lactic acid, color of product and may favor the growth of pink yeasts.
carbon dioxide, ethanol, acetic acid. Minor products also Brine begins to form once the shreds are salted and tank is
appear in the fermentation. The minor products are a closed when it is filled to the proper level. Then a weight is
variety of volatile compounds e.g. diacetyls, acetaldehyde placed over these shreds so that it squeezes the water out
and primary carbonyls. of the shreds. The weight may be of wood (old method) or
Control of fermentation some plastic bag filled with water may be placed (modern
method).
Temperature, salt concentration and sanitary conditions
are the primary environmental factors controlling the
sauerkraut fermentation.
 Basic of Fermentation Technology

40
Acetylcholine content in sauerkraut at household level and consumed directly while limited
amounts of cabbage-based kimchi are canned in factories
Sauerkraut is known to provide certain laxative and sold in the market. Kimchi is available throughout the
properties; both sauerkraut and its juice have been used as year and, served three times a day, is a staple in the diet
purgative. The strain of l. plantarum produces acetylcholine along with cooked rice and accessory side dishes. It is a
in the presence of choline, while simultaneously fermenting favorite food unique in its complex of sour, sweet and hot
carbohydrates. This acetylcholine is of significance in nerve pepper flavors accompanied by carbonation derived from
activity. fermentation with natural microflora. Kimchi differs from
Defects and spoilage of sauerkraut sauerkraut in two respects: 1) It has, optimally much less
acid and 2) It is carbonated.
Pink-Kraut: Pink-kraut was observed first by Butjagin
(1904) Wchmer (1905) and Henneberg
1916. Brunkow et. al (1925) and Fred and Peterson 1922
noted that this cause was the growth of pigmented yeast
i.e. asporogenous yeasts presumably members of genus
Rodotorula. Pederson and Kelly (1938) observed that Pink-
kraut usually contained a salt content greater than 2.5%.
They associated the growth of yeasts with any factor that
would inhibit a normal fermentation or that would suppress or
adversely affect the heterofermenting bacteria. Sometimes
pink-kraut was observed in vats of sauerkraut only a few
feet away from an area of soft-kraut. The latter condition
arises due to insufficient salt concentration. Stamer (1975)
reported that L. brevis produces a red pigment under certain Flow Chart 10
condition that can be related to discoloration or darkening
of sauerkraut. The red color occurs between pH 4.4 and Methods of preparation for typical korean kimchi
5.2 and is most readily generated under aerobic conditions. (Tongbaechu-Kimchi or Kakduggi-kimchi)
Chemical reducing agents like ascorbic acid, cystein or
glutathione inhibit this color formation. Materials for kimchi preparation include 1) fresh
vegetables (major vegetables are Korean cabbage, and
Slimy or Ropy Kraut: This is generally caused by dextran radish; minor vegetables are garlic, green onion, ginger, leaf
formation induced by the L.mesenteroide and is transitory mustard, hot pepper, parsley pear, chest nut and carrot),
in nature. This species prefers to ferment fructose rather 2) Jeotkal (Korean pickled fish) 3) fresh fish 4) seasoning
than glucose, therefore in the fermentation of sucrose; the agents (table salt, sesame seeds, sugar, monosodium
fructose is fermented leaving the glucose which interacts to glutamate, chenggak (type of seaweed), pear etc. additional
form slimy, ropy water insoluble dextrans. These vary from minor ingredients may also be added depending on the
an almost solid, gelatinous mass to ropy slime surrounding household maker these are: saeujeot (pickled shrimp),
the bacterial cell. meolchijeot (pickled anchovy), whangsegijeot, frozen
Other Defects: Discoloration caused by autochemical Pollack, oyster, shrimp and small octopus. The ratio of major
oxidation. Loss of acidity caused bgrowth of molds and to minor ingredients varies depending upon the household
yeasts. Spoilage caused by molds and yeasts cause off- maker; the range generally is 70-90 to 30-10. Although the
flavors and off-odors (yeasty and rancid). Slimy, softened proper combination of minor ingredients is reported to be
kraut caused by aerobic growth of asporogenous yeasts. the key to good-tasting kimchi, the most important factor
seems to be the salt concentration. Salting of cabbage can
Advantages of the acid food fermentations: 1) They be done at 5 to 7% for 12 hours or in 15% saline solution
render foods resistant to microbial spoilage and development for 3-7 hours followed by rinsing and draining. Optimum salt
of food toxins 2) They generally preserve the food between concentration during kimchi fermentation is approximately
the time of harvest and consumption 3) they make the 3% and is adjusted by experience at the household level.
food less likely to transfer pathogenic microorganisms 4) Fermentation of kimchi in the homes is usually carried out
they modify the flavor of the original ingredients and often at ambient temperature. Using 3% salt concentration, the
improve the nutritional value. (Flow Chart 10) optimum fermentation period is one day at 30 °C, 2 to 3
days at 20 °C, 12-15 days at 10oC and 30-60 days at 5 °C.
Kimchi Optimum acidity of kimchi is 0.4 to 0.8% (as lactic acid).
Kimchi is the general name given to a group of Higher acidity makes the product unacceptable.
fermented acid vegetable foods with a long tradition in
Microorganisms
Korea. More specific names are used for these pickled
vegetables depending on the raw materials, processing Kimchi is fermented by the microflora of the region.
methods, seasons and localities. Most kimchi is prepared Organisms isolated include lactic acid bacteria, such as
 Basic of Fermentation Technology

41
Leuconostoc mesenteroides, Streptococcus faecalis, harvested while still immature. Fully grown (ripe) ones
Lactobacillus brevis, Pediococcus cerevisiae, Lactobacillus are undesirable for pickling because they are too large,
plantarum, and aerobic bacteria, such as Achromobacter, change color and shape, are full of mature seeds, and
flavobacterium and pseudomonas spp. In the later stages are too soft for most commercial uses. After harvesting
of fermentation yeasts and molds appear that are reportedly the cucumbers are immediately transferred to the salting
causes of softening. station to avoid sweating i.e. growth of undesirable
softening microorganisms. Unsound, decomposed, broken
Preservation of kimchi or crushed, distorted (wilt, rots, crooks, nubbins, etc.) are
Kimchi is preserved at low temperature (below 5 °C) sorted out. They are then graded in a mechanical grader
for a very short period of time. During storage at elevated into 4 or more sizes. Three types of cucumber pickles
temperature, rancidity and soft rot are accelerated by the are made: 1) Fresh pack -These are held in salt brine
microbial action. Thus shelf life is very short in summer for as long as 2 days, then packed into jars or cans and
months. pasteurized. They undergo marginal fermentation. 2) Salt
Stock Pickles -From these pickles a variety of processed
Biochemical changes in kimchi products are prepared. They undergo complete lactic acid
fermentation. 3) Fermented Dill Pickles -They also undergo
The initial pH 5.5-5.8 falls to an optimum of 4.5-
complete lactic acid fermentation. Dill herb is also added in
4.0. Optimum acidity (as lactic acid) is 0.4-0.8%. Salt
these types of pickles.
concentration remains constant during fermentation. Kimchi
fermented at low temperature (6-7 °C) contains more lactic, Brining techniques for salt stock
succinic, oxalic, tartaric, malonic, maleic, and glycolic acid
than that fermented at 22-23 °C. Vitamins B1, B2, B12 and There are two general methods for preparing salt stock
niacin reach the highest levels (twice the initial level) when pickles for fermentation these are Dry Salting and Brining
kimchi possesses the most palatable taste and decrease Dry Salting
when kimchi becomes sour. Vitamin C and carotene content
decreases upon ripening. (Flow Chart 11) For cucumbers, dry salting is done after first adding
salt brine to cover the bottom of the tank (at least 12 in.)
to form a cushion. This prevents bruising, breaking, or
crushing the fresh cucumbers. Dry salt is then added at the
rate of about 22.5 kg for every 450 kg of small cucumbers
and 29.25 kg for every 450 kg large cucumbers. When full,
the tank is covered with a wooden lid very tightly. Brine
forms by osmosis. If the brine formed by osmosis does not
cover cucumbers then 400 salometer brine is added to the
desired level. The brine should be recirculated a day or two
after tank is filled in order to equalize the concentration of
salt throughout the brine. For long storage 60o salometer
brine is used. In industry 100 salometer is equal to 2.64%
NaCl by weight or 100o salometer is equal to 26.359 g NaCl
at 15.5°C (saturated solution of salt).
Brine salting: Brine salting process is preferred over
dry salting because dry salting yields soft,
Flow Chart 11
flabby, shriveled pickles that do not fill out properly when
Cucumber Pickles processed. Therefore most picklers mostly use brine-salting
The cucumber (Cucumis sativus) is popular both as technique for fermenting cucumbers. For brine salting ‘low’
a fresh and as a pickled vegetable. It is grown widely in or ‘high’ brine process may be used. In low brine salting, a
temperate climates although originally of semitropical salt brine of 25-30°C salometer is added into cucumbers
origin. Cucumbers for pickling must be grown from whereas in high brine technique, 40°C salometer salt is
varieties known to have regular form, firm texture, and added into cucumbers and tank is closed to air tight first
good pickling characteristics. Earlier varieties used by covering it with polythene sheet and then by lid. The
for pickling were monoecious plants but new varieties cucumbers are handled by the same procedure as described
developed by hybridization method have preponderance in dry salting except brine is used to cover the cucumbers.
of female flowers and are called Gynoecious. These new Now a days, molded plastic and fiberglass tanks are being
cultivars often have greater vigor and uniformity than the used in place of wood or concrete tanks. These plastic
open pollinated ones formerly grown. In addition, several and fiberglass tanks have several advantages. These are
of the hybrids are early maturing so they can be used to 1) They are not subject to biological degradation or metal
advantage in harvest scheduling. Pickling cucumbers are corrosion 2) They do not have to be maintained during the
 Basic of Fermentation Technology

42
off season, as do wooden tanks 3) As all the valves and Post fermentation
piping are made of plastic, the problem of metal corrosion
and contamination is eliminated 4) They are properly When fermentable carbohydrates are exhausted,
designed, closures are nearly airtight so problems of loss microbial growth is restricted to the surface of brines
of acidity is reduced. exposed to air; the spoilage bacteria may become
established on the surface of improperly managed tanks.
Microbiology of the cucumber fermentation At the end of fermentation, total acidity is 0.9% and pH is
equal to 3.3.
A rapid development of the microorganism causes a
spontaneous fermentation as soon as the lid of the tank Fermented dill pickles
is closed after adding brine. The rapidity of fermentation is
directly related to the temperature of the brine, concentration Cucumbers when are subjected to bacterial fermentation
of the salt in brine, availability of fermenting materials and in dill flavored, spiced, salt brine; the product obtained is
relative number of microorganisms available on cucumbers. called Dill pickles. They have their distinctive flavor and
Fresh cucumbers contain numerous and varied microflora aroma due to the products of fermentation of lactic acid
including many potential spoilage microorganisms and a bacteria and to the blending of flavor and aroma of dill herb
small number of lactic acid bacteria (5-103 acid forming and spices that were added to the brine.
bacteria per gram of cucumber). When cucumbers are Method of preparation: The larger size cucumbers are
brined at 5-8% NaCl range and allowed to undergo natural washed and placed in suitable containers, together with the
fermentation, the salt solution supports the fermentation requisite amount of dill weed (which was earlier cured in
by a sequence of various types of microorganisms. This vinegar, salt and brine) and dill spice and brine solution.
sequence is categorized into four stages 1) Initiation 2) Dill pickles are generally fermented in low salt (5% NaCl)
Primary fermentation 3) Secondary fermentation and 4) brine solution. Vinegar is added to retard the growth of
Post-fermentation undesirable microorganisms (by decreasing the pH value).
Initiation The fermentation is carried out at a temperature between
21 °C and 26.7 °C for 3-4 weeks. A curing period of 3-4
This stage may include growth of many facultative and weeks further is also necessary. During this period, the flesh
strictly anaerobic microorganisms originally present on the of pickles becomes entirely translucent and acidity about
fresh material, the growth of undesirable microorganisms 0.5-1.2% (lactic acid). In addition, there is small amount of
such as Gram-negative and spore-forming bacteria is volatile acid (acetic acid); lactic acid bacteria and yeasts
inhibited as the pH gets lowered and lactic acid bacteria produce ethanol and other minor products.
become established. The quality of the final product
depends largely of the rapidity with which lactic acid bacteria Microbiology
are established and undesirable bacteria are excluded. In the beginning, a wide variety of unrelated
Primary fermentation microorganisms start growing in the fermentation but soon
lactic acid bacteria predominate them. At low temperatures
In this stage lactic acid bacteria (Leuconostoc, Leuconostoc mesenteroides play an important role in the
Lactobacilli and Pediococci) and both fermentative and fermentation. Once Leuconostoc starts predominating other
oxidizing yeasts are the predominant active microflora. species like Lactobacillus brevis, Lactobacillus plantarum
They grow in brine until the fermentable carbohydrates begin to grow in the fermentation and ultimately complete
are exhausted or until there is production of lactic and the fermentation.
acetic acids. In normal fermentation the undesirable
microorganisms are excluded within 10-14 days. Buffering Packaging
capacity and the fermentable carbohydrate content present Fermented dill pickles are marketed in bulk plastic
in the medium are the important factors that govern the containers and glass containers covered with acidified
extent of fermentation by lactic acid bacteria and the extent brine, closed and pasteurized at 74 °C for 15 minutes.
of subsequent fermentation by yeasts.
Spoilage of cucumber pickles
Secondary fermentation
In Cucumber pickles, most of the deterioration is caused
Pediococci, Lactobacillus brevis, and Lactobacillus by the microorganisms; chemical defects are generally
plantarum and fermentative yeasts are responsible for the caused by metallic contamination, auto-chemical and
completion of lactic acid build up in this final stage of the physico-chemical reactions. Microorganisms damage the
fermentation. The acid tolerant yeasts still remain in the tissues by their cellulolytic or pectinolytic enzymes that
medium after the lactic acid bacteria are inhibited by low pH result in loss of texture of firmness. Gaseous deterioration
values and continue to grow till fermentable carbohydrates caused by microorganisms, resulting in the production of
are exhausted. internal cavities or distorted stock caused by excessive gas
pressure is another common spoilage. This defect is known
as bloater or floater spoilage.
 Basic of Fermentation Technology

43
Softening of cucumber pickles Production of Industrial Enzymes
Softening occurs most frequently after brining of the Introduction
cucumbers for dill or salt stock pickles. The entire skin of the
cucumbers become slippery and can be removed easily. Enzymes are biocatalysts produced within the living
Such condition of pickles is sometimes referred to as ‘Slip cells to bring about specific chemical changes. They
or Slippery pickles’ in industry. When softening progresses are present in all living cells and the metabolic reactions
into the deeper layers of cells and more and more pectic common to all cells are catalyzed by these enzymes. The
materials, present in the middle lamella separating the use of enzymes in industry dates back to some centuries
individual cells of the cucumber are attacked, the condition before the discovery of enzymes. For example use of barley
is known as ‘Mushy Pickles’. A variety of bacteria of Gram- malt for starch conversion in brewing is a notable example.
positive type (Bacillus subtilis, B.pumilis, B.polymyxa, The field of enzymology was opened by Buchner brothers
B.stearothermophilus) and yeasts such as Saccharomyces who showed that cell free extracts from yeasts fermented
fragilis, and Rhodotorula that produce pectinolytic and sugar to produce alcohol and carbon dioxide. In oriental
cellulolytic enzymes cause mushy deterioration. countries microorganisms are directly employed as enzyme
source for the preparation of products such as shoyu,
Gaseous Spoilage of Cucumber Pickles miso, natto, and sake. Other important processes in which
enzymes are primarily used are cheese making, leavening
A number of genera of bacteria and yeasts cause
of bread, manufacture of vinegar, tanning of leather etc.
gaseous spoilage in cucumber pickles. Undesirable yeasts
It was Takamine who laid the foundation for the industrial
produce gas because they utilize lactic acid and cause a
production of microbial enzymes by developing process for
rise in the pH. The fermenting yeasts have been identified
producing diastase from fungi. Boidin and Effront of France
as belonging to genera Brettanomyces, Hansenula,
were the first to produce industrial enzymes from bacteria.
Saccharomyces and Torulopsis. Among bacteria,
So far more than 1300 enzymes have been identified, out
Lactobacillus brevis, Lactobacillus plantarum cause
of which nearly 100 have been obtained in crystalline form.
serious bloater spoilage. The major gases formed during
spoilage are generally hydrogen and/or carbon dioxide. Commercial production of industrial enzymes
Nitrogen purging of the fermenting brine is used to reduce
undesirable levels of carbon dioxide that otherwise might Selection of microorganisms: The potential
result in bloater formation. (Flow Chart 12) microorganisms are isolated from soil, decaying Organic
matter or air and are tested individually for their capability to
produce the desired product; this process is called primary
screening. A few members of this potential natural isolates
will possess the desired characteristics. It is also customary
to grow the selected organisms on their substrates.
In certain cases such as in the case of pectinases the
organisms is induced to secrete the desired enzyme.
The selected strains are maintained in pure form by
lyophilization, on agar slant, or soil culture. The isolates are
periodically checked for purity and for the retention of their
original activity. Secondary screening is conducted in flasks
or small fermentors. This evaluates the true potential of the
organism to produce the desired product both qualitatively
as well as quantitatively. Once potentiality of the organism
is established, investigations are undertaken to work out a
suitable medium and optimization of other conditions like
pH, temperature, aeration etc. for the maximal enzyme
production are carried out. Continuous maintenance of
strains may cause degeneration and ability to produce
the desired enzyme. Therefore, periodic re-isolation and
reevaluation is necessary. The strains are also continually
improved to enhance their capabilities by various physical
agents such as UV treatment or by chemical methods.
Recombinant DNA technology has also playean important
role in the strain improvement programme.
Flow Chart 12
 Basic of Fermentation Technology

44
Methods of cultivating microorganisms: Several the bran can be stirred. The chamber is also provided with
methods of culturing the microorganisms are being cooling coils and an inlet for aeration. Bran is loaded into
employed industry that can be classified as follows: 1) the drum and moistened with dilute acid and sterilized with
Solid Culture -i) Conventional koji culture ii) Bulk koji culture steam. After cooling, the inoculum prepared in bran culture
iii) Rotary drum culture 2) Liquid culture -i) Stationary ii) is added and mixed. After charging, the air is passed slowly
Submerged These methods are explained in the coming into the drum which is maintained at a temperature of 28-
section. 30 °C. The spores germinate within 5-6 hours during which
period drum is rotated slowly for 15-20 minutes after every
Preparation of starter culture: Working cultures are first
2 hours. The drum is rotated continuously for 5-6 hours
prepared from stock culture (i.elyophilized or soil culture).
at a speed of 1 rpm or less. The growth of the fungus is
These cultures are tested for their potency from time to
completed within 60 hours. The moldy bran is then spread
time. In certain countries like Japan, there are firms that
in the form of layers on paper for drying. After drying, the
specialize in supplying pure fungal spores called Tane koji
moldy bran is ground and utilized as such, as an enzyme
of desired strains of fungi to large manufacturing units. In
source or to extract the enzyme.
such cases spores are directly inoculated into the growth
medium. Starter inoculums for large fermentations (both Extraction process: Powdered moldy bran prepared
for solid and liquid fermentations) have to be progressively from the foregoing procedures is utilized for extraction and
built up. The quantity of inoculum required depends upon purification of the enzyme. The moldy mass is extracted
the batch size and it varies between 0.01 to 0.001 of the with cold water (1-2 °C) or with solvents like ethanol. The
volume of the medium and is expressed as number of cells, common procedure is to employ counter current extraction
weight of cell mass or just on the volume basis. Generally system which gives a better and clear extract of enzyme.
the amount of inoculum is kept as low as possible. Large The quantity of water utilized generally is 5-10 times the
Scale Mold Fermentation using Fungi weight of the moldy bran. The clear extract thus obtained
is utilized for concentration and purification of the enzyme.
Bran process: Wheat bran is moistened with 0.2 to 0.3N
HCl and autoclaved at 15lb pressure for1 hour. Addition of Submerged fermentation: After selecting a suitable
acid improves sterilization and inhibits growth of undesirable microorganism capable of producing the desired enzyme
microorganisms. Sterilization can also be carried out by and standardizing the conditions for its maximum output,
direct injection technique and continuously stirring the mass large scale fermentation is taken up. This involves various
SO that bran particles come in direct contact with steam. operations that are discussed below: The substrate
When dilute acid is used for moistening bran, it is sufficient for growing microorganism is designed, based on the
to hold the medium for 15-30 minutes in live steam to obtain availability of cheap raw materials and types of enzyme
practical sterility. The cooked bran is then cooled to room to be produced. Composition of media recommended for
temperature and inoculated with inoculum grown earlier, at different enzymes consists of components selected from
1% level. It has been reported that 0.4% dry spore inoculum starch hydrolysates, wheat bran extract, milled cereal
is sufficient for good growth of fungus. The inoculated bran products, soybean meal, peanut meal, corn steep liquor,
is mixed well and transferred to trays having false bottoms. distiller’s solubles, yeast extracts and other organic and
Layers of 5 centimeter are considered good for uniformly inorganic nitrogenous compounds and mineral salts. The
good growth. The trays are placed one above the other liquid nutrient medium is charged into cleaned fermentors
8 to 10 cm apart. Spores germinate within 3-4 hours and and sterilized by means of steam. The medium is constantly
temperature begins to rise after 5-6 hour. Aeration is started kept stirred during sterilization. The sterilization is carried
at this stage and continued till growth is completed. It takes out for 2-3 hours depending on the size of the batch.
48-120 hours for the fermentation to complete depending Generally the holding temperature is 121 °C for 20 minutes.
upon the microorganism. After the completion of growth, the For large fermentors a continuous high temperature and
trays are shifted to drying tunnels with a central exhaust. short time regime is adopted. Direct heating with steam
The hot air is blown into the tunnel and air temperature is is preferred when medium is thick and viscous. After
not allowed to rise above 40 °C. Different modification of the sterilization medium is cooled to the desired temperature
above mentioned process are adopted by different enzyme and pH is adjusted to optimum, the inoculum is introduced
producing companies. under aseptic conditions. In commercial production of
microbial enzymes generally aerobic microorganisms
Bulk-koji process: In this method the fungus is cultivated
are used for fermentation. In such cases aeration and
on thick layers of bran up to 25cm t50cm high. The chamber
agitation is started soon after inoculation. The fermentor is
has a false bottom and air is circulated under pressure from
kept under constant pressure to avoid contamination. The
the bottom of the chamber. The chambers have a floor
agitator speed and quantity of air depends on the size of the
area of 2 to 30 meters by 6 to 10 meters. Temperature
batch, medium composition, and the requirements of the
and humidity of the air in the chamber are automatically
selected microorganism. After inoculation microorganisms
controlled.
establish themselves and passing through a lag phase,
Rotary drum method: In this method, the fermentation begin to grow exponentially. The standard fermentors are
vessel consists of a rotating drum fitted with baffles so that provided with aseptic sampling devices that allow samples
 Basic of Fermentation Technology

45
to be withdrawn for routine checking of contaminants, cell sulphite, magnesium sulphite, sodium sulphite, or by other
population, morphological observations and enzyme yield. substances added to bring the pH of the solution to its
Fermentation is carried out for 1-7 days. Most of the enzymes isoelectric point to separate the protein. The former process
are secreted into the medium by the microorganism and is called as ‘Salting Out’ and latter referred to as ‘Isoelectric
extracellular enzymes appear in the medium during early Precipitation’. Water soluble solvents such as ethanol,
part of the logarithmic phase. In case of intracellular methanol, isopropyl alcohol, acetone, dioxane etc. are also
enzymes cell disruption techniques are employed to recover used for precipitating the enzyme. The precipitated enzyme
the enzymes. The enzyme rich material obtained in any of is separated by process of centrifugation or filtration. The
the three procedures i.e. moldy bran, cultural extract, or cell organic solvents are removed by low temperature drying
autolysates is concentration and purified after solution is and the inorganic salts by dialysis. Enzyme now gets
stabilized by the addition of chemical s such as ammonium concentrated manifolds. To obtain an enzyme in pure form,
phosphate, ascorbic acid, calcium salts, hydrochloric acid, the enzyme solution is absorbed on ion exchange resins,
phosphoric acid, sodium citrate, sodium phosphate, sodium inert earth, calcium phosphate, aluminum hydroxide,
sulphite or organic substances such as gelatin, gums, colloidal iron etc. after adjusting the pH to the optimum
Arabic gum etc. level. Highly purified enzyme is freeze-dried or crystallized.
Purification of enzymes: The fermentation solution is Enzyme activity
subjected to centrifugation or filtration to obtain a clear
liquid free of suspended particles. Concentration of the Commercial enzymes are evaluated according to their
enzyme is brought by employing techniques such as specific activity per unit volume or weight. The activity
vacuum evaporation and fractionation. In fractionation, all is usually measured in ‘Enzyme Units’. An enzyme unit
substances in the crude enzyme solution except the desired indicates an amount of chemical change catalyzed by a
enzyme are separated out. Enzyme is then purified by definite quantity of enzyme. The activity can be standardized
precipitation, absorption, and crystallization. Precipitation by blending the enzymes with inert materials such as
of enzyme is carried out with salts such as ammonium diatomous earth, glucose, sucrose etc (Flow Chart 13).

Flow Chart 13
 Basic of Fermentation Technology

46
Production of Vitamins 26-28 °C for 4-5 days. The fermentation is submerged,
aerated but high levels of aeration may inhibit mycelial
Riboflavin production and reduces the product yield. When Candida
spp. are used for riboflavin production, the vessels made
Microbiologically produced Riboflavin has long been
of steel cannot be used because the organism is very
available in yeast and related preparations in association
sensitive to traces of iron, therefore the vessels may be
with many other vitamins of the B-complex category.
lined with plastic. Cobalt at proper concentration (stimulates
Riboflavin is essential for the growth and reproduction of
the ascomycete fermentation) can be added into the
both humans and animals. Thus it is often incorporated
fermentation broth as it partially counteracts the iron toxicity.
into the feed of the animals. By fermentation process the
Candida fermentation can be carried out at low pH, which
riboflavin content of the medium can be raised up to 7
eliminates the bacterial contamination and less sterilization
gm/L. Various microorganisms involved in the fermentation
is required.
of Riboflavin are as follows: a) Ascomycetes- Ermothecium
ashbyii and Ashbya gossypii are the commercial strains. b)
Bacteria- recovered from the acetone butanol fermentation
e.g. Clostridium butylicum, C. acetobutylicum and
Mycobacterium smegmatis also produce riboflavin. c)
Yeasts- Candida guilliermondia, C. flareris and Mycocandida
riboflava are the non-commercial strains. (Figure 3)

Fermentation process for ascomycetes

Ascomycetes for the production of Riboflavin require
semi purified sugars (glucose) and crude organic nutrients
like corn steep liquor, animal stick liquor and meat scraps.
Glucose may be totally replaced by corn oil, however low
levels of corn oil may be added to the glucose to stimulate
riboflavin yields. pH is adjusted to 6.5-7.5 and temperature Figure 3

Mechanism of riboflavin accumulation

Kaprálek (1962) and Stárka (1957) demonstrated that fermentation of riboflavin progresses through three phases.
First phase is a rapid growing phase with no riboflavin production. The substrate is utilized and oxidized; pH decreases as
pyruvic acid accumulates. In the second phase glucose gets exhausted, sporulation begins, pyruvate decreases, ammonia
accumulates because of deaminase activity, pH becomes alkaline, Rapid synthesis of cell bound riboflavin occurs in the
form of FAD and rapid catalase activity causes disappearance of cytochromes. In the third phase autolysis of the cells
occurs with the release of riboflavin into the medium (Flow Chart 14).

Flow Chart 14
 Basic of Fermentation Technology

47
Recovery of riboflavin
On completion of fermentation, the solids were dried to a
crude product for feed supplement. For a crystalline product,
broth is heated for 1 hour at 15lb pressure to solubilize the
riboflavin. Insoluble matter was removed by centrifugation
and riboflavin is recovered by conversion to the less
soluble form by chemical and microbiological methods. The
precipitated riboflavin was then dissolved in water or polar
solvents or in an alkaline solution, oxidized by aeration and
recovered by recrystallization from aqueous or polar solvent
solution or by acidification of the alkaline solution.

Carotenoid
Carotenes
Carotenes are precursors of vitamin A. Some carotenes
are normally present in foods and have an essential
biological function to perform. They are used as food
supplement to prevent or cure vitamin deficiency diseases.
In addition other pigmented carotenoids are used both
as food additives for intensifying or modifying the color in
fats, oils, cheese, and beverages and also as animal feed
supplement to enhance the color of such foods as egg yolks
and chicken flesh. Though carotenoids are widely found in
plants and animals, only microorganisms and plants have
the necessary systems to synthesize a wide range of these
products.

Microorganisms
Many species of algae and fungi (e.g. Neurospora
crassa, Penicillium sclerotium, Phycomyces blakesleeanus)
and also yeasts (Rhodotorula) were considered for use in
β-Carotene production but were found unsuitable. Some Flow Chart 15
particular fungi in mucorales group and choanophoraceae
family of Phycomyces concentrated the interest on the Recovery and Purification of β-Carotene
development of industrial fermentation. Particularly
Crystallization
Blakeslea trispora have received extensive study for their
ability to produce β-Carotene. This microorganism is It is the formation of solid particles within a homogenous
heterothallic in nature. High concentrations of β-Carotene phase. It may occur as the formation of solid particles in a
are produced only when both the mating types are present vapor, as solidification from a liquid melt or as crystallization
in the medium. from liquid solution. Crystallization from solution is important
industrially because of the variety of materials that are
Growth conditions marketed are in crystalline form. In industrial crystallization
The medium should be viscous and rich in vegetable from solution, the two-phase mixture of mother liquor
oils, kerosene and surface-active agents, β or β- ionones and crystals of cell sizes that occupy the crystallizer and
are added during the incubation. withdrawn as product is called Magma. Crystals have been
classified into seven classes these are: cubic, hexagonal,
Activators of β-carotene production trigonal, tetragonal, orthorhombic, monoclinic, and triclinic.
β-Ionone, a precursor of β-Carotene is not directly A given material may crystallize in two or more different
incorporated into the β-Carotene but it activates the classes depending upon the conditions or crystallization e.g.
enzymes required for carotene production. β-Carotene calcium carbonate occur commonly in nature in hexagonal
production can also be enhanced by the presence of form (as calcite) but also it occurs in the orthorhombic form
dimethyl formamide, α-pyrrolidone and succinimide in (as aragonite). Under ideal conditions, a growing crystal
addition to β-ionones. (Flow Chart 15) maintains geometric similarity during growth; such a crystal
is called invariant. Unless the crystal is a regular polyhedron,
 Basic of Fermentation Technology

48
the rates of growth of various faces of an invariant crystal Organic solvent + Erythromycin + water β Erythromycin
are not equal. hydrate

Principles of crystallization Biopolymer recovery is also obtainable by salt addition

e.g. Xanthan gum is a polyanion and calcium ion can
Crystallization may be analyzed from the standpoint of be used to form gel precipitate. Alginate biopolymer is
purity, yield, energy requirements, rates of nucleation and recoverable from algal biomass by cell removal (filtration),
growth. Purity- crystals are purified from the mother liquor followed by calcium chloride precipitation of the biopolymer.
by filtration, centrifugation and then washing the crystals
with fresh solvent. The effectiveness of these purification 2) Solvent driven precipitations are useful in the
steps depends upon the size and uniformity of the crystals. production of microbial biopolysaccharides including
Equilibrium- Equilibrium in crystallization process is reached dextrans and xanthan gums. The biogum fermentations are
when the solution is saturated and equilibrium relationship typically aerobic and produce a highly viscous final broth
for bulk crystals is the solubility curve. Yields- In many with xanthan production, final broth pasteurization kills
industrial crystallization processes, the crystals and mother Xanthomonas cells. After adding KCl and then methanol
liquor are in contact long enough to reach equilibrium, and or isopropyl alcohol the gum polysaccharide directly
the mother liquor is saturated at the final temperature of the precipitates out. Dextran recovery is achieved by alcohol
process. The yield of the process can then be calculated or acetone precipitation. In solvent driven precipitation for
from the concentration of the original solution and the the production of bulk polysaccharide, the modest product
solubility at the final temperature. Supersaturation- In the value requires efficient recovery and reuse of solvent as
formation of a crystal two steps are required 1) the birth of a well as good solvent removal for food or pharmaceutical
new particle 2) its growth to macroscopic size. The first step grade product. 3) Protein precipitation techniques- The
is called Nucleation. techniques result in a phase change to form a precipitate,
require some alteration of protein solution conditions to
Types of crystallizers render the original, thermodynamically stable one phase
Commercial Crystallizers are different in several ways. system unstable with respect to precipitation. The various
The difference lies in how the crystals are brought into methods for causing the needed reduction in solubility of
contact with supersaturated liquid. The first technique protein include: 1) Added high salt concentration to give
called the ‘Circulated liquid method’, a stream of precipitates by salting out 2) pH adjustments to protein’s
supersaturated solution is passed through a fluidized bed of pH of neutral charge, the isoelectric point, at which point
growing crystals, within which supersaturation is released the protein has minimum solubility 3) Reduction of medium
by nucleation and growth. The saturated liquid then is dielectric constant to enhance electrostatic interaction by
pumped through a cooling or evaporating zone, in which e.g. addition of miscible organic solvent 4) Addition of non-
supersaturation is generated and finally the supersaturated ionic polymers that reduce the amount of water available
solution is recycled through crystallizing zone. In the for protein solvation 5) Addition of polyvalent metal ion to
second technique called ‘Circulating Magma method’, the form reversibly a protein precipitate. The method of choice
entire magma is circulated through both crystallization and includes considerations not only of the protein concentration
supersaturation steps without separating the liquid from needed and cost of separation technique, but also the purity
solid. Supersaturation as well as crystallization occurs in of the final product compared to precipitating agent (Table
the presence of crystals. In both the methods feed solution 2).
is added to the circulating stream between crystallizing and Recovery and purification of microbial products
supersaturating zone.
When biosynthesis of products in a fermentor takes
Precipitation place this becomes necessary to isolate the product and
Precipitation phenomenon is used to obtain products convert it into a form suitable for the required purpose. The
from the broth or some times, to remove impurities from isolation procedures differ considerably depending upon
the ongoing fermentation process. There are a number of the location of the product i.e. intracellular or extracellular
processes where insoluble precipitates are isolated. Since and also depend upon the concentration and stability of the
organic solutes have solubilities dependent on the solution product. Sometimes microorganisms also produce many
temperature, pH, composition, ionic strength and dielectric other organic products apart from the main product that may
constant therefore precipitation can be brought about in complicate the process of isolation. The simplest situation
many ways as follows: is the isolation of microbial cells when they represent the
desired end product. The basic process to isolate the
1) By adding precipitant to react with solute and microbial products is shown in the following figure: (Flow
producing an insoluble product, often a salt e.g. procaine Chart 15)
hydrochloride + penicillin β Procaine-penicillin.
After cultivation, the culture fluid is usually processed
Organic solvent + streptomycin + sulfuric acid β in order to facilitate the separation of microorganisms. The
Dihydrostreptomycin sulfate treatment depends upon the composition of the fluid, the
 Basic of Fermentation Technology

49
type of cultured microorganisms and the product; it may the diminishing distance between particles inside the pores.
include an adjustment of pH, heating and /or addition of For rigid or non-compressible particles the filter cake may
substances that coagulate the microbial cells. After such be considered as a system comprising a large number of
treatments the microorganisms are separated by filtration or capillary channels through which the fluid flows according
centrifugation. Further treatments depend on whether the to Poiseuille’s law. Then
product is intracellular or extracellular in the supernatant.
The microorganisms themselves, or the filtrate, may, after
suitable processing by pressing, evaporation, and etc.
constitute the end product. If the product is contained
in the cells, it is necessary first to disintegrate them; the
method of disintegration again depends upon the type of
microorganism, physiological state and composition of the Where u = linear flow rate; V= filtrate volume; T= time;
cell wall. Following disintegration, the cell walls are separated P= pressure drop through filter cake; L= thickness of filter
and product is isolated by the methods shown in the above cake; A= filter area; and = viscosity of fluid
figure. Products contained in the supernatant are isolated-
depending on their chemical properties-by precipitation,
extraction, adsorption, dialysis, ultrafiltration, evaporation
etc.; these methods are often used in combination. After
isolation, the products are further processed to a form
they are to be used for the said purpose (in medicine, food
industry, agriculture, etc.) (Table 3)
Table 3

Grape Fermenting Wine

Parameter Juice
(mls) 7 days 14 days 21 days Food Fermentation Practical (1) -To study the wine
% Tartaric acid production by the fermentative activity of Yeast cells.
% Acetic acid I. Principle
Alcohol Wine is a product of the natural fermentation of the
juices of grapes and other fruits such as peaches, pears,
Taste
plums, and apples by the action of yeast cells. This
Aroma biochemical conversion of juice to wine occurs when the
Clarity yeast cells enzymatically degrade the fruit sugars, fructose
and glucose firstly into acetaldehyde and then into alcohol.
Mechanical separation of microorganisms Grapes containing 20-30% sugar contents will yield
wines with an alcohol content of 10-15%. Also present in
The choice of the method of mechanical separation grapes are acids and minerals whose concentration are
and the appropriate equipment depend on the type increased in the finished product and are responsible for
of microorganism (Bacteria, yeast, actinomycetes or the characteristic taste and bouquet of different wines. For
filamentous fungi), composition of the medium (synthetic red wines crushed grapes must be fermented with their
or complex) and the absence or presence of suspended skins to allow extraction of their color into juices while white
particles. Basically, the separation of microorganisms is wines are produced from the juice of white grapes without
carried out using one or two main methods, filtration and skins. The commercial production of wine is a long and
centrifugation. exacting process. First the grapes are crushed to express
Filtration juice called “must”. Potassium metabisulfite is added to the
must to retard the growth of acetic acid bacteria, molds, and
It may be defined as the separation of suspended wild yeasts that are endogenous to grapes in the vineyard.
particles from a liquid by means of a pressure difference A wine producing yeast Saccharomyces cerevisiae var
through a permeable partition. The diameters of the ellipsoideus is used to inoculate the must that is used to
particles may be smaller than the openings in the filter inoculate the must, which is then incubated for 3-5 days
so that initially they pass through these openings. When, under aerobic conditions at 21-32oC. This is followed by
however, the filter pores become clogged with the particles, an anaerobic incubation period. The wine is then aged for a
the filter begins to retain further particles almost completely. period of one to 5 years in aging tanks or wooden barrels.
As filtration proceeds the thickness of the particle cake on During this time the wine is clarified of any turbidity and
the filter increases and the flow rate of the filtrate decreases. formation of esters responsible for characteristic flavor are
The gradual decrease in flow rate is caused partly by produced. The clarified product is then filtered, pasteurized
clogging of the pores on the filter surface and partly also by at 60oC for 30 minutes and bottled.
 Basic of Fermentation Technology

50 II. Materials required 40-60 minutes; and incubated at 21-28oC for 12-18 hours
till the pH reaches 4.9-5.0. The starter culture now is cooled
Fifty mls of white grape juice broth culture of
to 5-10 °C and is kept at the same temperature until used.
Saccharomyces cerevisiae incubated for 48 hours at 25oC.
It is not a good idea to hold ripe starter for more than 24
Five hundred mls of pasteurized Welch commercial white
hours.
grape juice. Phenolphthalein solution 1%. Sodium hydroxide
0.1N and sucrose. One litre Erlenmeyer flask, One holed VI. Fermentation
rubber stopper containing a 2” glass tube plugged with
Weigh desired quantity of milk and adjust its SNF to
cotton plug, Pan balance, Spatula, Glassine paper, 10 ml
10-12% by adding whole dry milk. Add sugar at the rate of
graduated cylinder, Ebulliometer, Burette and Pipettes for
10%. Heat it to 80-90 °C for 20 minutes. Cool the milk to
titration.
45-48 °C (for S. thermophilus) and inoculate with 5% yogurt
III. Procedure starter culture. Mix well. Keep the milk in clean and sterile
container for setting. Incubate the milk containers at 45 °C
Pour 500 ml of white grape juice into one litre Erlenmeyer
for 3-4 hours till a firm coagulum is obtained. Remove the
flask. Add 20gm sucrose and 50 ml of Saccharomyces
product from incubator and keep it at 5 °C till it is consumed.
cerevisiae containing grape juice broth culture (10% starting
culture). Close the flask with the stopper containing cotton Calculation of Milk Solid Non Fat (MSNF)
plugged air vent. Incubate the wine at 25 °C. After 2nd
Milk at a temperature of 60oF is added up to the brim
and 4th day of incubation, add 20gm more sucrose to the
of the cylinder and lactometer is gently dropped into it.
fermenting wine. Now again incubate at 25oC for 21 days.
Reading on the lactometer is noted down. This reading is
IV. Total acidity (expressed as % Tartaric acid) corrected as follows:
To 10 ml of aliquot of fermenting wine, add 10ml distilled  Add one for every 10oF rise in temperature
water and 5 drops of phenolphthalein indicator. Mix and
 Add 0.5 for upper meniscus of the milk
titrate it with 0.1N NaOH solution. (Table 2)
This now is called as Corrected lactometer reading
(CLR). The MSNF is calculated by the formula as: MSNF =
CLR/4 + 0.2 × Fat + 0.14
E.g. If lactometer reading observed at 70oF comes out
to be 27 then CLR is equal to 27+1+0.5 (for meniscus) =
28.5 and MSNF = 28.5/4 + 0.2× Fat + 0.14
Calculation of Acidity in Yogurt
Take one gram of yogurt in titration flask and dilute
it with 5 ml of distilled water. Add to it a few drops of
phenolphthalein indicator. The solution is colorless. Now
add from the burette 0.1N NaOH solution drop wise till
Theory the color of solution changes to light pink. This is the end
Milk of high SNF (10-12 or 15%) is used for the point. Repeat the experiment till a concordant set of three
preparation of yogurt. It is different from curd in the sense readings is obtained.
that, in yogurt milk of high SNF is inoculated with pure culture General Calculations
of Lactobacillus bulgaricus and Streptococcus cremoris or
Streptococcus thermophilus in the ratio of 1:1 while in curds Take one gram of yogurt in titration flask and dilute
natural flora acts as inoculum. After fermentation, the acidity it with 5 ml of distilled water. Add to it a few drops of
of the final product is measured in terms of lactic acid (gm phenolphthalein indicator. The solution is colorless. Now
per 100ml of yogurt). add from the burette 0.1N NaOH solution drop wise till
the color of solution changes to light pink. This is the end
V. Procedure point. Repeat the experiment till a concordant set of three
Preparation of starter culture: The inoculum of readings is obtained.
Lactobacillus bulgaricus is maintained in Micro-inoculum VII. Theory
broth of composition: Yeast extract 20g, Peptone 5g,
Dextrose 10g, Potassium dihydrogen phosphate 2g, Natural microflora present on cabbage produces lactic
Sorbitan monooleate complex 0.1g or Tween-80 few drops, acid from carbohydrates. In due course of time, after the
and distilled water one litre. The inoculum of Streptococci is accumulation of lactic acid to certain extent, all proteolytic
maintained in Nutrient broth of composition: Beef extract 3g, and other microorganisms are eliminated from the product
Peptone 5g, NaCl 5g, and distilled water one litre. Cultures except lactic acid tolerant Lactobacilli. These bacteria
of both the strains are to be mixed in 1:1 ratio in Peptonized further produce more lactic acid resulting in lowering of the
milk of composition: Skim milk powder 10g, Peptone 5g, pH of the product significantly after 3-4 weeks. Because of
and distilled water 100 ml; pH 6.5 sterilized at 85-95oC for this lowering in pH other organisms do not find any access
to grow in the same product.
 Basic of Fermentation Technology

51

VIII. Procedure Weigh the salt (2.25%) i.e. 11.25 gm for ½ kg of cabbage
shredding. Put the outer leaves that were kept aside, at the
Wash cabbage with clean water. Remove the outer
bottom of a glass jar. Take one lot of shredded cabbage
leaves. These leaves are kept aside for their further use.
and layer onto the leaves inside the glass jar. Sprinkle ¼th
Remove the case and other undesirable area. Prepare
of the salt on the cabbage and wait for its absorption in the
lots of cabbage weighing ½ kg each and slice them into
shredded cabbage. Similarly layer the rest of the shredded
shredding or small pieces of 0.16-0.08 cm in thickness.
 Basic of Fermentation Technology

52
cabbage and sprinkle the salt onto it till all the cabbage and therapeutic value and has been successfully tried in cases of
salt finishes for the preparation of sauerkraut. The addition chronic colitis and gastro-intestinal disorders in general. It is
of salt serves two main functions. Firstly, it draws moisture prepared by the inoculation of pure culture of Lactobacillus
out of cabbage that dissolves the salt forming a brine acidophilus. It is a very acidic product. The acidophilus
solution, which acts as a fermenting medium for Lactobacilli milk has not gained popularity as that of other fermented
and also equally distributes them in the medium. Secondly, milk products because of its taste and off-flavor. When it is
it inhibits the growth of proteolytic bacteria. Now place sweetened with sugar, it is called as sweet acidophilus milk.
the plastic bags filled with water as a weight to press the Sweet acidophilus milk is gaining popularity now a day.
cabbage and close the lid of the glass jar. Fermentation was
carried out at 25oC for 3-4 weeks. Note down the change in Medium for maintenance for lactobacillus
pH and color after every week. acidophilus
Microbiology of Sauerkraut Fermentation Pederson The organism is maintained in Tomato Juice Agar
first described the lactic acid bacteria that he observed in of Composition: Tomato juice 400ml equivalent to 20g
fermenting sauerkraut. He found that the fermentation was tomatoes, Peptone 15g, Skimmed milk powder 10g, Agar-
initiated by the species of Leuconostoc mesenteroides. This Agar 1.5-2.0%; pH 5.0; sterilize at 15lb pressure for 15
species was followed by gas forming rods and finally by non- minutes.
gas forming rods and cocci. Leuconostoc mesenteroides is
Procedure for ordinary acidophilus milk
a hetero fermentative bacteria and it grows more rapidly
than other lactic acid bacteria. It is active over a wide Take adequate quantity of low fat milk. Boil or steam it
range of temperature and salt concentrations. It produces for 20 minutes. Cool it to a temperature of 28-300C. Pass
acid and carbon dioxide that rapidly lowers the pH, thus carbon dioxide gas through it for 1-2 minutes. Inoculate it
inhibiting the activity of undesirable microorganisms and with 1-2% inoculum of Lactobacillus acidophilus. Incubate
enzymes that may soften the shredded cabbage. The at 37-40 °C for 30-40 hours.
carbon dioxide replaces the air and creates an anaerobic
condition favorable to prevent oxidation of ascorbic acid and Procedure for sweetened acidophilus milk
natural color of the cabbage. It also stimulates the growth Inoculate cold pasteurized sweetened low fat milk with
of many lactic acid bacteria. While this initial fermentation is 1-2% of pure culture of Lactobacillus acidophilus. No
developing, the hetero fermentative species of lactobacillus incubation is done. Inoculated milk is held under refrigeration
brevis and homofermentative species of lactobacillus at 7°C or below for 30-40 hours or till it is consumed. It
plantarum and sometimes Pediococcus cerevisiae begin tastes exactly like low fat milk.
to grow rapidly and contribute to the major end products
like lactic acid, carbon dioxide, ethanol, acetic acid. Minor Food Fermentation Practical (5) -Production of Lactic
products also appear in the fermentation. The minor acid
products are a variety of volatile compounds e.g. diacetyls,
Materials required
acetaldehyde and primary carbonyls.
Pure culture of Lactobacillus delbrueckii B-70 and
Role of temperature in sauerkraut fermentation Production medium of composition: Sucrose 100g/L, Yeast
Lower the initial temperature better is the product extract 20g/L, Potassium dihydrogen orthophosphate
formation. It is considered that the initial temperature of 2.5g/L, Calcium carbonate 10%, Agar-Agar 2% (for solid
18.3oC produces superior quality sauerkraut because at medium); pH 7.0-7.2; Sterilize at 15 lb pressure for 15
lower temperature hetero fermentative lactic acid bacteria minutes. Vitamin B-complex+ Aspartic acid +Folic acid
exert a greater effect. combination may be used in place of yeast extract.

Spoilage of Sauerkraut Medium for inoculum preparation

Common spoilage signs found in sauerkraut are Inoculum is prepared in Glucose Yeast Extract medium
discoloration, off-flavor, off-odor caused by yeast and mold of composition: Glucose 10%, Yeast extract 2% and
growth, loss of acidity and slimy product due to the dextran Potassium dihydrogen orthophosphate 0.25%.
formation by Leuconostoc mesenteroides. The proteolytic I. Procedure
activity of molds and yeasts and also by asporogenous
yeasts produces product pink in color. Such type of spoilage Take two litres of production medium in laboratory
is known as “Pink kraut”. fermenter and inoculate it with 10% of inoculum of L.
delbrueckii B-70 prepared in Glucose Yeast extract medium.
Food Fermentation practical (4) -Preparation of Sweet Incubate it at 37 °C for 5-7 days. The medium is gently
Acidophilus milk stirred during the incubation period to keep the calcium
I. Theory carbonate in suspension.

The fermented acidophilus milk is known for its

 Basic of Fermentation Technology

Recovery of lactic acid II); add solution-I into solution-II and adjust pH to 5.2, make
53
volume 100ml and heat it to 60oC for 10-15 minutes.
Filtration -The suspension is filtered with conventional
laboratory filters. Acidification -To the filtrate add I. Procedure
concentrated sulfuric acid to form precipitates of calcium
Dispense 100ml of production medium in two 250ml
sulfate. Filter and wash the precipitates with water. The
conical flasks and sterilize it at 15lb pressure for 15 minutes.
washings are added into the filtrate. Removal of Impurities
Inoculate the flasks with Streptomyces spp. culture that
-The filtrate is treated with activated charcoal to remove
was preserved in nutrient broth medium at the rate of 5%.
the impurities. Filter again. Concentration –The filtrate
Incubate the flaks on shaker at 37 °C for 96 hours.
is evaporated on a steam bath to concentrate it to 25%
solids and then again subjected to evaporation till 50% Standard curve for starch
solids are obtained. Removal of Heavy metals -The heavy
metals like lead is removed by adding sodium or potassium Prepare dilutions of starch in acetate buffer from 0.1% to
ferrocyanide into the concentrated mass and filtering it. The 0.01%. To each of these dilutions, add 0.2ml Iodine solution
filtrate contains lactic acid, which can be purified by passing and add water to dilute it to 10ml. Measure the optical
it through ion exchange resin column. The lactic acid so density at 520nm. For control take 1ml of distilled water
obtained may have 93-95% purity. in place of starch dilution and to this add iodine solution;
observe optical density in a similar manner.
Food Fermentation Practical (6) –Production of
Amylase enzyme and its estimation Estimation of amylase
Materials required Prepare Control, Blank and Digest. Control -To 6ml of
0.1% starch solution, add 1ml of 1% calcium chloride, 2ml
Inoculum -An Amylase producing strain of Streptomyces of hydrochloric acid, and 1ml of distilled water and 0.2ml of
spp. preserved in Nutrient medium of composition: Beef iodine solution. Measure optical density at 520nm. Blank
extract 3g, Peptone 5g, NaCl 5g, and distilled water one litre. -To 6ml of 0.1% starch solution, add 1ml of 1% calcium
Calcium chloride solution 1% in water, Starch solution 0.1% chloride solution, 1ml of enzyme extract, 2ml of hydrochloric
in 0.05M acetate buffer pH 5.2 (dissolve 1mg starch in 1ml acid and 2ml of iodine solution. Measure optical density at
of acetate buffer), Production medium of composition: Beef 520nm. Digest -To 6ml of 0.1% starch solution, add 1ml of
extract 3g, Peptone 5g, NaCl 5g, and distilled water one 1% calcium chloride solution and 1ml of enzyme extract.
litre. Add starch 10g per litre into the nutrient broth medium. This is incubated at 30 °C for 10 minutes. Now add 2ml of
Sodium acetate-Acetic acid buffer solution (0.05M) -Sodium HCl and 0.2ml of iodine solution. Measure optical density
acetate 2.72g dissolve in 100ml distilled water (solution-I), at 52 nm.
Acetic acid 1.15g dissolve in 100ml distilled water (solution-

Calculation of Enzyme activity

I. Theory Materials required

The yeast Saccharomyces cerevisiae converts Molasses; fermentation jar (10 L); micro distillation
fermentable sugars (glucose, fructose and sucrose) into unit; Test tubes; conical flasks; Standard volumetric flasks
ethanol and carbon dioxide. In large-scale production of (25ml); Urea; DNS reagent (3,5-Dinitosalicylic acid 1%,
alcohol, molasses is used as substrate. Blackstrap molasses Phenol 0.2%, Sodium carbonate 0.05%, Sodium hydroxide
contains 45-55% w/v fermentable sugar as sucrose, which 1% and Sodium potassium tartarate 20%); Dichromate
is metabolized by yeast through Embden-Meyerhoff-Parnas reagent (Dissolve 34 gm of Potassium dichromate in 500
Pathway to produce ethanol and carbon dioxide as the end ml of distilled water, add 325 ml of concentrated Sulfuric
products. acid slowly keeping the flask in ice bucket); YPD medium
of composition: Yeast extract 10gm, Peptone 20 gm,
 Basic of Fermentation Technology

54 Dextrose 20gm and Agar-Agar 20gm; Slant culture of yeast Black gram dhal 3) Salt 4) water. Dehulled soybeans or
S. cerevisiae. Bengal gram can be used as a substitute for black gram dhal
and a number of cereal grains can replace rice. However,
I. Procedure
there may be marked change in the texture and flavor
Preparation of Inoculum: Prepare 250ml GYE / YPD when using substituted materials. It has been reported
broth medium. Add 50ml in separate flasks and autoclave that rice variety and its physical characteristics are very
at 15lb pressure for 15 min. Inoculate slant culture of yeast important to produce a good quality idli. White Kar and IR20
aseptically in each 50ml flask. Incubate the flasks at 28 °C varieties of rice have given much better performance in the
for 16-18h. production of idli, especially the White Kar variety because
of its high amylose content, low amylopectin content better
II. Inoculum for fermentation gelatinization, and better water uptake ability.
Dilute molasses to 12 oBrix (1.1Kg molasses in 8 litre of Proportion of cereal to legume: Ordinary idli consists of
water). Adjust pH to 5.5 with 10N Sulphuric acid. Sterilize at three parts rice and one part Black gram dhal plus salt to
10lb pressure for 30min and then cool. Inoculate this 250 ml taste. Kancheepuram idli is prepared from one part rice
molasses medium (2 flasks) with 10% of the culture grown and one part black gram dhal plus cashew nuts, ghee,
in YPD. Incubate the culture in shaker for 12h at 28 °C. salt, pepper, ginger and cumin added to taste. Normally a
III. Fermentation medium proportion of rice to black gram dhal varies from 4:1 to 1:4,
the 2:1 being the best. It has been seen that when black
Dilute the Black strap molasses with tap water to 22 0Brix gram dhal proportion is less than 25%, the steamed idli was
(2.1Kg molasses in 8 litre water). Adjust pH to 5.5 with 10N hard and organoleptically unacceptable whereas when it is
Sulphuric acid. Add 200mg Sodium dihydrogen phosphate more than 50%, the product obtained is too sticky to be
and 200mg Urea per litre respectively. Maintain pH 5.5. acceptable. Thus, not only can the ingredients be varied,
Sterilize medium at 10lb pressure for 30min. Inoculate with but the proportions can also be varied within a wide range
10% v/v inoculum and incubate at 28 °C for 48-72h for and still an acceptable product is obtained.
fermentation. After the fermentation has ceased close the
mouth of the flask with an airtight bung to provide anaerobic I. Procedure
conditions so that alcohol production may take place. White polished rice is carefully washed and soaked for
Withdraw 10ml sample at every 12h interval and estimate 5-10 hours. Black gram dhal is carefully washed and soaked
alcohol and sugar concentration. Plot a graph with time on for 5-10 hours. The rice is then drained and coarsely ground
X axis and alcohol and sugar concentration on Y- axis. in a stone mortar or other grinder. The black gram dhal is
Alcohol estimation drained and finely ground in a stone mortar. The rice and
black gram dhal slurries are combined to form a rather thick
Preparation of standard curve for alcohol concentration: batter, which is stirred with hands. Salt is added to taste.
Prepare 1-10% v/v alcohol in Other seasonings, such as chilies are occasionally added.
The batter is placed in a warm place to ferment overnight.
Various test tubes. Take 1ml of the various concentration
In the morning, the batter is poured into the cups of an idli
of alcohol into 25ml of distilled water in 100ml distillation
steamer, which is placed in a covered pan or cooker and
flask fitted with Liebig condenser. Distill and collect 15ml
steamed until the starch is gelatinized and the idli cakes are
of the distillate in a 50ml volumetric flask containing 25ml
soft and spongy.
Pot. Dichromate solution. Make up the volume to 50ml.
Keep the alcohol-Pot. dichromate complex at 60 °C for 30 Food Fermentation Practical (9) -Preparation of
min. Measure OD at 600nm. Plot a standard curve with Dosa
concentration of alcohol on X-axis and OD at 600nm on
Y-axis. II. Ingredients

Determination of Alcohol Concentration from The ingredients used for dosa preparation are similar to
fermentation Medium as that of idli preparation i.e. rice, black gram dhal, salt and
water. Rice may be substituted by wheat, bajra Pennisetum
Take 1ml sample and mix it with 25ml of distilled water. typhoideum, maize, or kodri and black gram dhal may be
Distill as above and measure OD at 600nm. Find the alcohol substituted by sprouted peas, cowpeas (Vigna catjang),
concentration from the standard graph. field beans (Dolichos lablab) or soybeans. Fresh groundnut
oilcakes may also be substituted for black gram dhal.
A. If molasses could not be obtained for laboratory
exercise, use YPD medium with 50g/L sucrose for inoculum III. Soaking and batter formation
development and with 150g/L sucrose for preparation of
fermentation broth. Generally, equal quantities of rice and dehulled black
gram dhal are soaked in water at room temperature
Food Fermentation Practical (8) -Preparation of Idli separately for 5-10 hours. It is common practice that finely
ground powders are used to prepare batter. The finely
Ingredients: Idli preparation contains a number of different
ground powders are mixed with water at temperatures
ingredients these are 1) Rice 2)
 Basic of Fermentation Technology

55
ranging from 48o to 98 °C, best at 80 °C for the preparation the crisp dosa and the sides are rolled over it, which now
of batter. Water is added in the range of 2.0 to 2.2 times becomes ready to be eaten with a spiced soup of dhal and
the initial dry weight of ingredients to prepare a batter of vegetables popularly known as Samber. Food Fermentation
viscosity desired for dosa. Salt is added from 0.8 to 1.0% as Practical (10) -Production of Citric acid by Aspergillus niger
a seasoning before fermentation. in media containing molasses and sucrose.
IV. Fermentation time VII. Theory
Traditionally, dosa batter is kept overnight for Many microorganisms like molds (Aspergillus niger,
fermentation. The fermentation time should be sufficient to A. awamori, Penicillium janthinallum, Trichoderma
allow a definite leavening and acidification of the batter and viridae, Mucor piriformis, etc.), yeasts (Candida lipolytica,
to allow for the development of a pleasant acid flavor. C.tropicalis, C.citrica, Hansenula, Rodotorula, Pichia,
Torulopsis etc.) and bacteria (Bacillus licheniformis, Bacillus
V. Incubation temperature
subtilis, Brevibacterium flavus, Corynebacterium species)
Ordinarily, the dosa fermentation is carried out at have the capability to produce citric acid. Commercially
room temperature. In the tropics, this generally means a spores of Aspergillus niger are employed to produce citric
temperature of 25-30 °C. acid. It accumulates in culture solutions of pH 1.8-2.0. In
fungi different metabolic pathways are involved in the
VI. Harvesting and preservation production of citric acid but 78% of the citric acid is produced
As soon as the batter becomes leavened and acidified, by the involvement of glycolysis and Tricarboxylic acid cycle.
it is spread onto a hot and greasy griddle where it assumes The acetyl COA derived by EMP pathway condenses with
the shape of a crisp pancake. The spreading of dosa on Oxaloacetate of Kreb’s cycle to produce citric acid. Citric
the griddle is an art that matures with practice. Sometimes acid is an important chemical used in medicines, flavoring
a mixture of cooked different vegetable is poured onto extracts, food and candies and in dyeing industry.

Flow Chart 16
 Basic of Fermentation Technology

56
VIII. Materials required sucrose) of 250ml flasks. Each set having three flasks
containing 100ml production medium. One set having
Molasses; Sucrose; Flasks; Test tubes; Sodium
molasses and sucrose in the second set. Label the flasks
hydroxide; Ammonium dihydrogen orthophosphate; Trace
of each set as control flask, Methanol containing flask, and
element solution of composition: Zinc sulfate 3mg/100ml,
flask without methanol. Add 3ml of methanol in the flask
Copper sulfate 680mg/100ml, Magnesium sulfate
labeled as methanol flask. Sterilize both the sets at 15 lb
2.0g/100ml and EDTA 2.0g/100ml; Dissolve in 100ml
pressure for 15 minutes. Cool the flasks and inoculate all
distilled water and incubate at 25oC for 3 days.
the flasks of both the sets with spores of Aspergillus niger
Composition of production medium of citric acid except the control flask. Incubate the flasks at 25 °C for
4-5 days on shaker. After the fermentation is over, filter the
Molasses or Sucrose 45gm/300ml, Ammonium contents. Compare the acidity of the filtrate with the control
dihydrogen orthophosphate 0.75gm/300ml, Potassium flask.
dihydrogen orthophosphate 0.3gm/300ml, Tween-80
0.6gm/300ml; pH for molasses 5.0-6.0 and for sucrose 2.0- II. Acidity
3.0 Add trace element solution before sterilization. Sterilize Titrate the filtrate against 0.1N NaOH using
at 15lb pressure for 15 minutes. phenolphthalein as an indicator. Appearance of pink color
I. Procedure is the end point. Note down the volume of filtrate used in
titration and calculate the strength of citric acid in filtrate.
Prepare two sets (one for molasses and second for

General calculations
 Basic of Fermentation Technology

About the Author

Rajan Sharma*

Department of Molecular Biology, Dehradun & Uttaranchal Institute of

management, India
*Corresponding author: Department of Molecular Biology, Dehradun &
Uttaranchal Institute of management, India, Email:

Published By:
MedCrave Group LLC
Date: January 09, 2017
 Contents
Origin and Evolution of Fermentation Process and Fermented Foods 1
Development of Fermentation Process and Industry 1
Fermented Foods 2
Products of Fermentation 3
Probiotic 4
How Temperature and pH Affect the Growth of Microorganisms 8
Lactic Acid 9
Medium 9
Microorganisms for Commercial Production of Lactic acid 10
Medium 10
Production of Lactic Acid 10
Product Recovery and Grades 11
Uses of Lactic Acid 11
Single Cell Protein (SCP) 14
Ethanol as Fuel 15
Different Types of Substrates for Industrial fermentations 17
Yogurt 22
Kefir 24
Bulgaricus Butter Milk or Bulgarian Milk 24
Acidophilus Milk or Reform Yogurt 24
Miso 30
Dosa 35
Fermented Sausages 36
Sauerkraut 38
Cucumber Pickles 41
Fermented Dill Pickles 42
Production of Industrial Enzymes 43
Production of Vitamins 46
Carotenoid 47
Recovery and Purification of β-Carotene 47
General Calculations 56
 Basic of Fermentation Technology

Origin and Evolution of Fermentation Process the production of liquid, fermented mashes from cereals
1
and Fermented Foods are closely related processes. It is likely that liquid from a
fermented mash was drunk as a slightly alcoholic beverage,
In earliest times, man was plagued with either feast while semisolid mash was needed into dough and baked.
or famine, so any means he could discover to conserve Even today yeast strain used in the production of ale
food when it was plenty was a great step forwarded in and bread is that from single species of Saccharomyces
his survival and his conquest of earth. Since man was of cerevisiae. Until into the middle of the 19th century bakers
necessity a wanderer and a hunter, he learned about the obtain their yeast from breweries. At that time lager beer
drying and smoking of meat. Certainly these methods not strains of Saccharomyces uvarum (S. carlsbergensis)
only conserved his supplies, but they also reduced weight, were introduced into central Europe and later in the United
enabling him to carry more food with him. At the times of States. These strains tolerate high osmotic pressure
discovery of North America by Europeans, most of the in the dough and bakers were forced to look for another
Indians were in this stage of development. The discovery of source of yeast. Distiller’s yeast that are also strains of
two methods Drying and Smoking just like the invention of Saccharomyces cerevisiae perform reasonably well in
wheel perhaps took place by serendipity. Early man might bread making, but they were difficult to separate from the
not know why foods spoil he knew they did. Later we can distilling mash. This led to the establishment of a separate
speculate that he discovered the use of salt with drying and industry that produced baker’s yeast on commercial scale
smoking. for sale to bakers and for home baking. The production of
baker’s yeast was increased many folds with the advent of
Man’s next discovery for preserving food was the
Fed-batch culture.
fermentation of foods, although he had no idea what
happened when microbial growth occurred, he learned that The discovery of fruit fermentation was made so
plant materials and meat could be kept for long periods long ago that the ancient Greek believed wine had been
of time when they have undergone fermentation. It was invented by one of their gods, Dionysus. The manufacture
also essential that he knew how to use salt (a necessary of wine have been recorded about 3500 B.C. as wine
agent that inhibits toxin production from microorganisms industry of Fertile Crescent that spread west (around the
in a successful fermentation). Undoubtedly many an early Mediterranean), North (to Hungry, Germany and France)
ancestors of man died from botulism or was made ill by and in the post Columbus period to America, South Africa
Staphylococcus aureus. After the addition of salt in natural and Oceania. Romans advanced the art of wine making,
fermentation there he knew definite changes in color, odor but it was an industry of large risks due to spoilage until
appearance and taste, which helped the product to be the mid nineteenth century. The research of Louis Pasteur
wholesome. Probably the first fermentation was discovered revolutionized the wine industry. A Mesopotamian clay
accidentally when salt might have selected certain tablet written in Sumerian-Akkadian about 500 B.C. tells
harmless microorganisms that fermented the product to that brewing was an established profession 1500 years
give nutritious and acceptable food. If we speculate along earlier. An Assyrian tablet of 2500 B.C. lists beer among
these lines, we might expect the first fermented food have commodities that Noah took abroad his ark. Egyptian
been fish. With the advent of certain religions in which meat documents dating back to 4th dynasty about 2500 B.C.
was excluded from diet, the use of salt and fermentation describes malting of barley and the fermentation of beer.
was adapted to certain plant products. For instance Bush Kui- a Chinese rice beer has been traced back to 2300 B.C.
(1959) states that Buddhism was well established religion in When Columbus landed America, he found that Indians
China and Korea by 4th century. It was introduced to Japan drank beer made from Corn. According to Weeks (1949)
between 500-600 AD. It may very well be the cultivation of the etymology of word “beer”, as we know today indicates it
Soya beans and their use in food including fermented foods originated from Latin verb ‘bibre’ (means to drink). Similarly,
were then introduced in Japan. For centuries Balkan people the Spanish word for beer ‘Cervaza’ apparently originated
have enjoyed fermented milk or yogurt and central Asian from cervisia, which combines Latin word ‘Ceres’ (goddess
tribesmen have found equal pleasures in sour camel’s milk of grain) and ‘Vis’ (vigor). The art of brewing was spread
or Koumiss. The ancient Sanskrit scriptures of India, the to England by Teutons that settled in Rhine area became
Vedas, documented the food value of Dahi- a fermented Germanic tribe. The major brewing centers were eventually
milk product similar to yogurt. Further evidences for the established in Pilsen, Czechoslovakia, Munich, Dortmund,
existence of soured milk as a food in the early times can Germany, Burton-O-Trent, England, Dublin and Ireland.
be found in Bible. The historical, geographical, ecological American Indians were already making beer from maize but
and dietetic patterns in various regions of the world are Mayflower Company brought English type beer to America.
reflected in diversity, variety and types of fermented milks in English Ale beer was used till 1840s, but German Lager
vogue today. These products are generally produced by the beer became more accepted type of beer because of its
intense activity of the Lactic acid bacterial cultures. superior keeping quality.
Bread that has been known almost as long as agriculture The process of fermenting sausages was probably
itself, its preparation involves a yeast fermentation. Loaves one of the earliest forms of meat processing and its
of bread have been found in Egyptian pyramids built in six manufacturing probably began before written history. The
thousand years ago. The art of fermented doughs from first mention of written history was in 9th century B.C. when
cereals was practiced before recorded history. This and it was mentioned in “Homer’s Odyssey”. The sausage was
 Basic of Fermentation Technology

2
called as “Oryae”. The word “Salami” was coined from the subsequent growth of yeast cells. This technique now is
product made in Salamis- a Cyprus city destroyed in 449 called as “Fed-batch Culture. Further studies also showed
BC (Pederson, 1979). Sausages eaten by Babylonians, that growth of yeast cells could be improved by sparging
Greeks and Romans were no doubt fermented and dried air in the fermentation broth. During the First World War
meat products. Brested (1938) stated that “Caeser’s Weizmann introduced a concept of Aseptic fermentation
legions” in Gaul consumed dry sausages. The descriptions the development of Acetone-Butanol fermentation. Steam
of the process of making sausages confirm that sterilized hemispherical topped and bottomed vertical steel
Babylonians, Greeks and Romans ate many types of dry cylinders were used as fermentors. These fermentors
sausages. The various regions of Mediterranean developed had problems of inoculum development and maintenance
characteristics sausages e.g. Salamis developed Genoa, of aseptic conditions. In spite of all the hindrances these
Milano and Lambardi types of sausages (Anon, 1938) The organic fermentations paved a way for the introduction of
Mediterranean countries consumed a highly seasoned non- aseptic aerobic fermentation technology.
smoked products classified as Latin type. Non-Europeans
In the Third Stage Penicillin fermentation process was
countries developed a Roman product, but slightly spiced,
developed, which was a wartime need. This fermentation
heavily smoked, moist and higher in salt content. This
was very vulnerable to contamination. All the knowledge
product is often referred to as Germanic type. In colder
gained in the previous year’s regarding process control, air
areas sausages are made in the winter months, stored
sparging, isolation and propagation etc. were applied for
and aged until summer, hence they are called as Summer
the synthesis of Penicillin at a large scale. The development
Sausages. The aging occurs by indigenous flora prompting
of large-scale extraction process and initiation of strain
the growth of Lactic acid bacteria, yeasts and molds in and
improvement programme was advancement at that time.
on the surface of sausages. Early in 20th century bacteria
Many other fermentation processes were developed at that
were discovered to be responsible for Lactic acid production
time like antibiotics, vitamins, gibberellins, amino acids,
and nitrate reduction in sausages. Further research in
enzymes and steroid transformations. The fourth Stage
the microbiology has led to the production of very safe
(early 1960s) is marked by the production of microbial
processed meat products and newer products are under
biomass as a source of feed proteins. Many waste products
the stage of development. Many fermented products have
were considered as a carbon source for the development
been proved to possess some medicinal values.
of microbial biomass. Hydrocarbons as another potential
Development of Fermentation Process and source for carbon for microorganisms were discovered.
In this period Jet and Pressure cycle fermentors were
Industry
developed that eliminated the need of mechanical stirring.
Development of fermentation process may be Other advantages of these processes were that they can be
represented by five overlapping stages. Stage I represents run continuous and were economic. At this time Batch and
the pre-1900 development that is confined to potable Fed batch culture techniques were common in the industry,
alcohol and vinegar. Wooden vats and even fitted with some but their application became short lived because of the
process control like thermometers (1757) and primitive heat development of Continuous Culture. The high standards of
exchangers (1801) replaced the ancient traditional Beer the aseptic operation and process controls were achieved
production by Egyptians. In mid 1800s Cagniard-Latour, by the introduction of computer systems in the fermentation
Schwann and Kutzing demonstrated role of yeast in alcoholic process to minimize the possibility of human error. The
fermentation independently. Pasteur later convinced that fifth Stage in the progress of fermentation process is the
pure culture of these microorganisms produces more introduction of Genetic Engineering and Recombinant
alcohol than the mixed culture. Methods for isolating and DNA technology in the strain improvement programme.
propagating pure yeast cultures were developed in the These Techniques not only allowed the transfer of genes
late 1800s. By the late 1800s and early 1900 generator between unrelated organisms but also enable the extremely
for the production of vinegar was developed, which was precise alteration of genome of a particular organism. The
considered as the first aerobic fermentor to be developed. development of further stages in fermentation will depend
In this method 10% good vinegar was added to the medium upon the new advances in this area.
as an inoculum that also makes the medium acidic to make
it contamination free. Thus in the beginning of 20th century Fermented Foods
concepts of process control for the fermentation process Fermented foods form an important part of human diet.
were developed. Fermented legume and cereal products are especially
Between the years 1900-1940 the main thrust areas popular in South East Asia including India, Middle East and
of research were baker’s yeast and organic solvent Africa. Traditional fermented foods are important elements
fermentations. Newer products developed were yeast in the diets of millions of people of particularly in developing
biomass, glycerol, citric acid, lactic acid and acetone- countries and the methods for their preparation are simple
butanol. Studies indicated that growth of yeast in the and inexpensive. Indigenous fermented foods are so
fermentation broth leads to oxygen depletion, which results prepared that they utilize cheap sources, supply proteins,
in the ethanol production at the expense of cell formation. and enrich starchy diets with vitamins and other nutrients.
Adding more broth in the previous broth can regulate the The exact origin of fermented foods is not known and their
 Basic of Fermentation Technology

3
discovery is considered to be purely by chance. The Asians
centuries ago knew the art to produce meat like flavors dp / dt = Yp / x .µ . x -----------(iii)
from vegetable proteins. The Indonesians had various Combining equations (i) and (iii) we have
methods to introduce meat like texture into the vegetable
products. Such foods have a particular place in their diets. Q = Yp / x.m
p
Koreans introduced acid fermented vegetables and People
of Egypt developed Bread leavened with yeasts while It may be seen that when product formation is growth
Indians discovered methods for souring and leavening associated the specific rate of product formation increases
cereal-legume batters. Nearly every nation in the world has with specific growth rate. When product is Non-growth
one or more fermented milks. Fermented milks are used associated the specific rate of product formation may
to restore the natural flora of intestine impaired by disease remain constant over a wide range of growth rates or
or antibiotic activity. In many countries cultured milks are it may vary in a complex manner. Garden relates the
widely promoted and credited with health giving properties. formation of products to substrate utilization or in other
Yogurt, Kefir, Acidophilus milk, Bulgarian milk, and Koumiss way this classification assesses the extent to which the
are a few names that are very popular in many parts of the energy producing reactions are coupled to the product
world and even in western countries. Much interest over the forming reactions. This approach is now very much used
years has been generated in the fermented foods of Asian in studying the continuous process. According to Gaden’s
and African countries because such foods in these countries Classification product forming reactions fall into following
are prepared traditionally, using simpler technology and three categories:
equipments. In India preparation of fermented foods Type -I, Type -II and Type III
has gained a status of small-scale cottage industry that
manufactures such foods utilizing natural microflora from Type I arise as a result of primary energy metabolism.
staples and surroundings. The desired product results from a carbohydrate substrate
e.g. glucose to ethanol, glucose to lactic acid etc. The
Products of Fermentation metabolic roots are serial with µF negative. The kinetic
A variety of products can be obtained by fermenting approximation of alcohol fermentation is given by:
different substrates with the help of microorganisms.
Fermentation products are the primary or secondary
metabolic products of microorganisms that are produced
at certain stage of their life cycle. Primary products are
Growth Associated products, their concentration in the
medium increases, as microorganism grows in the medium
i.e. concentration increases gradually in the exponential Negative sign indicates that substrate is decreasing.
growth phase of microorganism. Secondary products
are Non-Growth Associated products and are produced In Type II the main product arises from energy metabolism
in the stationary phase of growth of microorganism. The but indirectly e.g. Citric acid fermentation, some amino
concentration of Non-growth associated products may acid fermentation. The reaction patterns are complex and
sometimes become toxic to the microorganism itself e.g. restricted or abnormal metabolism is involved. The overall
Antibiotics. free energy change is negative. Such types of products are
also called as Intermediate metabolites. Gaden suggests
Kinetics of Growth Linked Product Formation following prototype reaction for the complex dissimilation of
The formation of growth Associated product may be Type II metabolites: (Figure 1)
described by the equation:
dp / dt = qp. x (1)
Where p is the concentration of product and qp is the
specific rate of product formation. Also the product formation
is related to biomass production by the equation as:
dp / dx = Yp / x (2)
Where Yp/x is the yield of product in terms of substrate
consumed.
Multiplying equation (2) on both sides by dx/dt we get:
dp / dt = Yp / x -----------(i)
dp / dx = Yp / x ---------(ii)
Figure 1
Therefore:
 Basic of Fermentation Technology

4 are prepared by fermentation process. However some of

them now are produced by synthetic process.
Polymers: Dextran is the only polymer that is being
produced on large scale by fermentation process. It is used
as a blood extender and blood thickener.

Type III, Secondary, or Non-growth Associated products Miscellaneous: Sorbose (an intermediate in ascorbic
acid manufacture), fructose (a liquid sweetener),
Secondary products result from biosynthetic reactions dihydroxyacetone (a sun-tanning agent) and
where the main product does not result from energy phenylacetylcarbinol (an intermediate in L-ephedrine
metabolism. Cell and metabolic activities reach maximum synthesis) are a few miscellaneous compounds produced
in the early stages of the life cycle and product formation by fermentation process. Spores of Bacillus thuringiensis
takes place at the later stages of the life cycle. Oxidative used as an insecticide for chickens, is also prepared by
metabolism is low at the time of maximum product formation fermentation process.
e.g. Antibiotic fermentation and biosynthesis of Vitamins.
In Non-growth associated product formation a period of Probiotic
negative specific growth rate occurs where the terms
A bacterial supplement of a single or a mixed culture
This shows that the population has moved beyond the of selected non-pathogenic bacterial strains is termed as
stationary phase for the latter part of the fermentation. Probiotic. The term ‘Probiotic’ was firstly coined by Parker
Under such conditions dead cell lysis may provide a (1974) and originated from two Greek words ‘pro’ and
second nutrient source for the other cells. Fermentation ‘bios’ which means ‘for life’. Probiotic generally includes
processes are the prime sources of over hundred products bacteria, cyanobacteria, fungi etc. They may be called as
for food, chemical and pharmaceutical industries. Various normal micro biota or “Effective micro biota”. Probiotic,
fermentation products are as follows: Probiotic, Probiotic bacteria, beneficial bacteria, or friendly
bacteria are the synonymously used for probiotic bacteria.
Antibiotics: Fermentation industry once dominated by
According to some recent publications, the mechanisms of
alcohol and solvent making now derives its primary income
action of probiotic bacteria have several aspects: 1) they
from antibiotics like Penicillin, Streptomycin, Vancomycin,
competitively exclude the pathogenic bacteria or produce
Neomycin, Chloramphenicol, and Erythromycin etc.
substances that inhibit the growth of pathogenic bacteria
Steroids: Transformation of steroids i.e. induction of (e.g. bacitracins and polymyxins produced by Bacillus spp.)
hydroxyl in 11β 11β and 16β position, dehydrogenation 2) provide the essential nutrients to enhance the nutrition of
of 1,2 position and hydrolysis of esters of the 3 hydroxyl the cultured organisms 3) they may directly uptake the or
group can be achieved by fermentation process. Key decompose the organic matter or toxic materials in water
steroids in microbial transformation process are Cortisone, improving the quality of water in the mediums or in the
Hydrocortisone, and Prednisolone etc. treatment of water. Beneficial effects of the Probiotic may be
mediated by: 1) Neutralization of the toxins 2) suppression
Enzymes: Many enzymes can be produced by
of the viable count 3) production of antibacterial substances
fermentation process. These find application in production
4) competition of adhesion sites 5) Alternation of microbial
of food, chemical and medicines e.g. amylases, pectinases,
metabolism 6) Stimulation of immunity of the host 7)
proteases, cellulases, catalases, invertases, lipases,
Accelerate the sediment decomposition by producing
streptokinases glucose oxidases and collagenases.
organic acids in water treatment 8) production of hydrogen
Organic Acids: Organic acids find their use in food, peroxide 9) production of enzymes.
chemical and medicines as an acidulant, sequestrate,
a. Types of Probiotic
plasticizer, flavouring and reducing agents. Commonly
used organic acids are citric acid, lactic acid, gluconic acid i. Non-viable Probiotic –these are dead.
and itaconic acid. Amino acids like lysine is used as food
ii. Freeze-dried Probiotic –these will die rapidly upon
supplement glutamic acid as a flavouring agent and are
leaving refrigeration.
ascorbic acid as a reducing agent.
iii. Fermentation Probiotic –these are produced through
Vitamins and Growth factors: Fermentations have been
fermentation.
used to produce growth factors for many years. These are
used in pharmaceuticals and as food and feed supplement iv. Viable Probiotic–these live with guaranteed shelf life.
e.g. Riboflavin, vitamin B12. Gibberellin is used in germinating Guaranteed number of organisms has a protocol for
barley and ripening fruits, Xanthophylls produced by algal counting and to be very stable and efficacious. Produce
cultures is added to chicken feed to give color to egg yolks many benefits.
and chicken meat. Torula yeast is added in animal feed as
a source of B vitamins is derived by fermentation of waste b. Bioreactor or fermentor
liquors from paper industry. Bioreactor is a device in which biochemical
Solvents: Many solvents like alcohol, acetone and butanol transformations take place. It is here a less expensive
material is converted into a more valuable product or
 Basic of Fermentation Technology

5
service is rendered. Even though the term is new but sterilization and downstream processing. Temperature
the concept is old. The terms like bioreactors, microbial is usually measured with the help of Resistance
reactors, fermentors, and biochemical reactors all have the thermometers, Thermocouples and liquid expansion
same meaning. Until about four decades ago, fermentation thermometers. Risk of contamination is minimal with
had been practiced as an art with a little engineering input, all these methods. Resistance thermometry prevails
but with the realization of the potential of this process, the because of its accuracy and reliability. Sensors
need of its instrumentation and control was felt. With proper usually are the encased platinum wires. The use of
instrumentation and control of a bioreactor it is possible thermocouples is less frequent. Electric measuring
to increase the conversion yield and the productivity of a signals from both the resistance thermometers and
biological product manifolds. The first step in understanding, thermocouples can be transferred to control boards.
controlling and optimizing a process is the precise, accurate This is not possible with liquid expansion thermometers
and timely monitoring of important parameters. The state of like mercury or ethanol in glass, which occasionally
the art in automated monitoring is very advanced in mature are employed for direct on the spot measurements.
industrial sectors but even today it is adapting sensors Another method of on the spot temperature indicator
developed for other applications or designing new sensors is by means of thermo colors that change their colors
to satisfy its needs. at certain temperature. They can be applied in the form
of thermo foils attached at critical spots. Some details
c. Measurement system
of the devices used to control the temperature are as
From system’s point of view, a measurement is achieved follows:
through the use of a meter or sensor expanding the human
b. Mercury in glass thermometers: They may be used
senses ability to detect measure and quantify. To control a
in small bench fermentors and are very fragile in
fermentation process we need to know 1) state of the process
nature. That is why their use is restricted. They usually
within a small time increment i.e. continuous monitoring of
are used enclosed in a pocket which protects the glass.
the state variables 2) The microorganism’s response to any
They are used only as an indicator.
set of measurable environmental conditions i.e. a control
model for fermentation. Before describing the various c. Bimetallic thermometer: It consists of bimetallic
sensors available for a bioreactor, it needs to emphasize coil surrounded by protective tubing. The coil Winds
the requirements of an ideal sensor. The requirements and or unwinds with changes in temperature causing
characteristics must be met within reasonable limits; it may movement of fixed pointer onto it. They are more
vary from one case to another. expansive and less accurate.
These are: d. Pressure bulb thermometer: It is basically a pressure
gauge connected by a small bore tubing which may be
i. Reliability-Long term reliability is of great importance
up to 60m in length to the detecting bulb. The whole
and a time of about 2000-3000 hours continuous
system is gas tight and filled with an appropriate gas or
operation should be attainable for most instruments
liquid under pressure. The movement of the free end of
ii. Repeatability or Reproducibility -Measurements made the receiving element can be used to operate a pen on
under standard conditions should be repeatable from a chart recorder or an electrical or pneumatic control.
day to day and from laboratory to laboratory (Figure 2)

iii. Accuracy- Accuracy is the measure of how close

the empirical measurement is to the true value or its
conformity to an accepted standard value
iv. Rough and Tough- Sensor should be rugged and
repeatedly withstand the conditions of steam
sterilization, variety of chemicals besides acidity,
basicity, salinity, and water etc.
All the fermentation sensors can be categorized as 1)
Physical environmental sensors-to measure the physical
process variables and 2) Chemical environmental sensors-
to measure the chemical process variables.
Figure 2
d. Methods of Measurement of Process variables
e. Thermocouples: In 1821 Seebak discovered that if
Physical process variables a circuit consisting of wires of two dissimilar metals
a. Temperature: It is an important parameter in the had the junction of the wires maintained at different
biochemical process. This is not true only further temperatures, a current flowed through the circuit. This
reaction itself, but also for auxillary operations such as current produced can be measured on a calibrated
 Basic of Fermentation Technology

6
instrument or recorder and is a measure of point which is very often kept constant. It is usually measured
temperature. At a point therefore by holding the by monitoring the number of revolutions per unit of
temperature constant at all junctions except one, which time. Outside the aseptic area, the impeller speed is
is a given circuit, it is possible to measure temperature measured with the help of a device called Tachometer.
with reference to the old junction temperature. (Figure 2)
Thermocouples have limited use because they are
j. Power Input: The power consumption of agitators
normally operated at ambient temperatures.
depend on stirrer speed and physical properties of
f. Electrical resistance thermometers: Electrical the stirred fluid especially on its viscosity, which may
resistances of metals changes with temperature change drastically during batch fermentations e.g. in
variations. The bulb of electrical thermometers some processes for the production of antibiotics like
contains a resistance element, a mica framework penicillin viscosity changes take place. In large-scale
(for accurate measurement) or a ceramic framework, fermentors, the consumption of electric energy as
around which the sensing element is wound. Platinum determined by a Wattmeter, yields useful information
wires of 100Ω resistance are normally used. The on the input of agitating power when friction losses in
wires are then connected to the measuring element. the stuffing box, seals, and motor are accounted for.
Reading is normally obtained by a wheat-Stone bridge A direct measurement of agitation power is possible
circuit and is the measure of the average temperature by using Torsion dynamometer or Strain gauges. The
of the sensing element. The electrical resistance latter method is an accurate method.
thermometers are very accurate, more sensitive to
k. Foam: It is a nuisance occurring in most fermentation
small temperature changes and are very fast.
broths. It may be caused by surface-active metabolites
g. Thermistors: These are semiconductors made from (proteins, polysaccharides etc.), components of the
specific mixtures of pure oxides of iron, nickel and other medium, or by cells. Two types of foams have been
metals. Their main characteristic is a large change distinguished 1) Soft foam 2) Hard foam Soft foam
in resistance as a function of absolute temperature. is unstable while hard foam is stable. Foam of either
Temperature reading is obtained with a wheat-Stone form must be suppressed in order to prevent the
bridge. They are relatively cheap and stable. contamination, clogging of the exhaust system including
its measuring devices and loss of culture broth. Foam
h. Pressure: It is measured by means of conventional
destruction can be achieved by mechanical or by
pressure gauge. Since the manometer is not in the
chemical methods (antifoaming agents). Often both
direct contact with the fermenter contents therefore
the methods are used in combination. Foam control
no sterility problem arises. Often measurement of
necessitates its detection. This can be achieved by
pressure is not included in the standard equipment,
employing sensors mounted inside the fermenter
though it may yield valuable information especially with
above the liquid level. Examples of the various probes
laboratory glass vessels. Here any clogging of exhaust
used are: Electric conducting probe, Capacitance
pipe may cause a buildup of a pressure head, and
probe and Heat conducting probe.
thereby apart from the danger of cracking the glass.
Other parameters, such as solubility of gases will be l. Volume: Information on liquid volume is essential
affected. In fermentors containing cultures, which tend in liquid flow control in the filling of the vessel with
to form wall growth, deposits of microbial mass on medium or in continuous culture feed in the continuous
membranes may lead to errors in the monitoring of the culture, which ultimately affects the metabolic activity
pressure. of the inoculum culture? There are two systems
generally used for the on-line volume determinations:
i. Flow Rate: Gas flow rate is important in aerobic
1) liquid level sensors and 2) weight or mass
fermentations. Likewise the rate of gas production is of
measurement devices. Capacitance probes measures
interest for cultures producing biogas. Liquid flow rates
the change in capacitance when liquid level changes
must be known for continuous and fed batch processes,
in small-scale fermentors. In large-scale fermentors
where the rate of nutrient feed is an essential variable
∆P measurement method is generally used, where a
for efficient operation of the process by means of
flush mounted diaphragm pressure cell is located in
mass balancing control. Furthermore, knowing the
the base of the vessel for liquid height and hence the
liquid flow rate is necessary to control the addition
volume measurement.
of corrective liquid feed streams, such as amount of
base or acid consumed for pH control or the amount m. Weight/Mass: Scales of various types determine
of antifoam input. Flow rates are mainly measured weight or mass of the liquid. In this method the vessel
by the following devices: 1) Floating body flow meter is suspended on a scale and the combined weight
2) Differential pressure flow meter 3) Rotating flow of the vessel and strain type gauges electronically
meter 4) Electromagnetic flow meter Floating Body measure liquid. This method cannot be applied to the
Flow Meter Impeller Speed For stirred tank fermenter, fixed existing installations.
impeller speed is an important operating variable,
 Basic of Fermentation Technology

7
Chemical process variables measurement, which in turn is correlated to oxygen flux
reaching the cathode surface. Fluorescence Quenching-
a. Exhaust gas analysis: The concentration of carbon For medical investigations so called Optodes has been
dioxide in the exhaust gas from cell reaction is indicative developed for oxygen and carbon dioxide determinations.
of the respiratory activity and fermentative activity In this, the sensitive element is the membrane into which
of the inoculum culture and hence is one of the most a fluorescence indicator (Pyrene butyric acid or β- methyl
useful and widely applied measurement methods in belliferon purine) has been incorporated. This membrane
the monitoring and controlling a cell bioreactor. Using is brought in contact with the broth. The fluorescence
an Infrared spectrophotometer, gas chromatography quenching in indicator is indicative of the presence of
and mass spectrometer most commonly controls oxygen or carbon dioxide. This method does not consume
carbon dioxide content in a bioreactor. Gas stream oxygen or carbon dioxide.
oxygen partial pressure is usually measured using a
paramagnetic analyzer. Care should be taken in both c. pH: pH value is an important indicator of the state of the
the cases that water vapors should be eliminated from state of biochemical process. The automatic addition of
the exhaust before feeding them into the analyzer. The alkali or acid to fermentation broth can be achieved by
paramagnetic analyzers are quite sensitive to small techniques already in use in other chemical industries
changes in total atmospheric pressure so they require but special electrodes have been developed for use
simultaneous monitoring of barometric pressure for in fermentation industry. The half-cell of the glass
compensation in oxygen analysis. Apart from water electrode was composed of Ag/AgCl saturated with KCl.
vapors the samples should also be free from dust The solid KCl increases the mechanical resistance of
particles, aerosols and oil droplets. the glass particles of KCl on the glass surface during
heat sterilization and cooling of the electrode. Other half
b. Dissolved gases and volatiles: Dissolved oxygen of the electrode is composed of the same material as
and carbon dioxide are both important variables in the glass electrode, asbestos or porcelain cylinder being
fermentations. These are normally determined by 1) used as the junction material. To ensure good insulation,
Electrochemical methods 2) Fluorescence quenching both the glass and reference electrode were mounted in
and 3) Mass Spectrometry Electrochemical Methods- Teflon gaskets and silicone rubber washer were fixed.
Electrochemical determination of oxygen and carbon The internal resistance of the electrode is 300-500meg
dioxide in fermentation media is performed by means of Ohm. Steel sleeves provided with several holes to allow
special sterilizable electrode. Analysis by this electrode free passage of broth protect both of these electrodes.
is based on detecting the amount of oxygen diffusing
from the liquid membrane into an Amperometric or d. Redox potential: Another method obtained with
Polarographic measuring cell. Amperometric principles electrochemical method is the Redox potential
are most frequently used. In Polarographic type oxygen measurement method. Every redox system consists of
electrode a constant voltage is applied between cathode two components, one is oxidized by electron donation
and anode. At cathode the oxygen that has diffused into and the other is reduced by electron acceptance.
the cell is reduced to hydroxyl ion as shown below: In such a system, an electrochemical potential can
be measured by means of an unprotected electrode
Cathode: - O2 + 2H2O + 4 ē → 4 OH ˉ consisting of a noble metal (Au/Pt), the composition
Anode: 4 Ag + 4 Cl ˉ → 4 AgCl + 4 ē of which is chosen on the basis of relation of donor to
the acceptor. In a fermentation system/culture, a great
The response of the probe is proportional to the oxygen number of redox systems are present simultaneously.
activity in liquid. Since at equilibrium i.e. for a saturated Accordingly an exact interpretation of signals from the
liquid the activity of a solute is directly related to its partial redox potential measurements cannot be given. It is for
pressure (fugacity), the readings of electrochemical that reason that some scientists suggested to rather
process are commonly given as percent partial pressure or name this potential as Platinum-electrode potential
saturation. instead of redox. The competing donors i.e. oxygen
In Potentiometer probe same principle of oxygen and glucose that are present in a fermentation system
diffusivity is used and this diffused oxygen is reduced at may serve as a typical example. In spite of the difficulty
cathode surface according to the same above equation: of interpreting the results, measurements of redox
potentials permit an important insight into the course
Pt of fermentations. Sterilizable Platinum electrodes are
½ O2 + H2O + 2 ē → 2OH ‾ commercially available. They either contains built in
reference electrode (Ag/AgCl) similar to pH electrode
The reaction at anode in galvanic electrode is as follows: or they are used in combination with pH measurement
making use of the same reference electrode. The
Pb → Pb2+ + 2 ē
amplifier for redox measurements is of the same type
This reaction competes with the cell from which a as in conventional pH meters. As in the latter case,
small amount of current is drawn to provide a voltage the built in electrode is sterilized together with the
fermenter. Since no membrane is required so any
 Basic of Fermentation Technology

8
special sterilization problem exists in these electrodes. fold increase in growth rate per 10 °C rise in temperature.
The signal of the redox meter is influenced by pH. This Growth rate approaches zero at 10 to 25 °C below the
can be tolerated because fermentations are usually run optimum temperature. Chemical reaction rates are related
at constant pH. to temperature by Arrhenius equation:
e. Enzymatic analysis of substrate: Enzymatic analysis – E / RT
K = Ae
allows very specific determination of many organic
compounds. This group of methods takes advantage Where K = reaction rate; A= Arrhenius constant; R = Gas

1/ T
of the ability of enzymes to react selectively with well- constant; T = Absolute temperature; E = Activation energy
defined compounds or rather chemical structures; or temperature characteristics.
organic compounds present in fermentation media
Taking log on both sides we have:
(substrates and metabolites) can be analyzed.
Usually this is performed offline with samples taken Log µ
from fermentors but methods for online enzymatic
analysis have also been developed. The procedure
consists of determining the conversion of the enzyme- 1/ T
catalyzed reaction with the respective substrate for
analyzing one of the products either calorimetrically or Plot of Log K against 1/T should be a straight line with
electrochemically. In applying online enzymatic analyses slope of E/2.3 RT. If we substitute specific growth rate ‘µ’
to sterile fermentations, special difficulties arise because for reaction rate K in the above equation then converting
conventional sterilization techniques apply steam at it to straight line equation and plotting log µ against 1/T,
120˚C that will destroy the enzymes. One solution to this keeping the value of E constant we find a straight line with
is to employ dialysis through which a recycled sample slope of E/2.3RT. The Q10 value also varies inversely as
stream of broth is conducted. Components diffusing the temperature varies from normal as
through the dialysis membrane can then be monitored
continuously by means of enzymatic analysis. Such Q10 = E . 10 / 2.3RT (T+ 10)
systems have been developed for measuring glucose,
saccharine and lactose.
f. Ion specific electrodes: Not only carbon source and The activation energy is a valuable constant as it can be
other organic compounds but also inorganic salts (N, P, used to predict the effect of temperature on growth rate over
S, K, Mg, Ca, Na, and Fe) are essential constituents the normal temperature range. Changing in the activation
of fermentation broths. Ion specific electrodes have energy indicates that differences in rate controlling reactions
been proposed for a number of these ions. Some or in the metabolic regulations can occur. Temperature
electrodes of this type are commercially available for range for growth of individual bacteria extends over about
offline measurements. There is little known about the 35°C. Extreme psychrophiles grow between -5 to 30°C
online measurement using ion selective electrodes. Ion and extreme thermophiles 55 to 90°C. Decrease in growth
selective electrodes are Infact potentiometer electrodes rate at high temperature is due to disruption of metabolic
applying different principles e.g. for measurement of regulations or death of cells by protein denaturation. When
Na+ glass electrodes with glass membrane especially death of cells occur the growth rate of the viable biomass
sensitive to Na+ are employed. In some electrodes (X) is given by:
organic membranes are employed while in others
dX/dt = (µ -K) X where µ = specific growth rate; K =
enzymes may be incorporated. For these types of
specific death rate and X = biomass
electrodes sterilization may be the problem.
Death rate becomes dominant if at high temperature
How Temperature and pH Affect the Growth activation energy for death exceeds that for growth. Increase
of Microorganisms in temperature causes breakdown of protein structure so
the affinity for substrate and enzyme regulators will be
Effect of temperature on the growth of affected. Thermophiles possess proteins with exceptional
microorganisms heat resistance. Temperature also affects the nutrient
Bioprocesses of microorganisms are heavily affected by requirements, lowering of the growth temperature causes
the temperature. The cell temperature must become equal small increase in the growth yield from carbon and energy
to the culture temperature. Temperature affects the rate source. The pathways of metabolism of carbon and energy
of cell reactions, the nature of metabolism, the nutritional source can be temperature sensitive e.g. Lactobacillus
requirements and biomass composition. The temperature brevis ferments glucose by heterolactic pathway at 24°C but
coefficient of growth rate is denoted by Q10 value (It at 32oC requires fructose as hydrogen acceptor for glucose
is defined as increase in growth rate per 10°C rise in fermentation. Growth factor requirements also change
temperature e.g. If Q10 is equal to 2 that means there is two with temperature e.g. Yersinia pestis requires different
amino acids and vitamins at growth from 37 °C to that at
 Basic of Fermentation Technology

28°C. Temperature affects the product formation e.g. over industrially by the fermentation process. The first commercial
9
production of riboflavin by Ashbya gossypii requires growth production of lactic acid in USA by microbiological process
of the microorganism at 28°C than at its normal temperature took place in 1881 as its calcium salt Calcium lactate.
because growth at low temperature causes breakdown of The plant for its manufacture was built in Littleton,
normal regulation of synthesis of enzyme system which Massachusetts, but little is known about its process. The
produces the riboflavin. Similarly the optimum temperature Clinton processing Company, Clinton Iowa is the only
of production of Penicillin is lower than that of normal manufacturer using fermentation process for the production
growth temperature. Temperature affects the microbial of lactic acid in United States.
composition as RNA content of bacteria or yeasts increase
i. Isomers: Lactic acid of commerce is the L (+) isomer,
several folds on decreasing the temperature. Yeast lipids
D (-) isomer or any possible mixture of the two. The
increase their unsaturated fatty acids when temperature
entire range of isomers has been found. The mixture
is lowered. Antigenic composition of bacteria varies both
of the two forms of isomers is called as DL mixture or
qualitatively as well as quantitatively with temperature e.g.
racemic mixture. DL mixture is optically inactive form.
virulent Yersinia pestis is produced at 37°C but not at 25°C.
Microorganisms differ in their ability to produce either D
Mechanism of temperature effect (-) lactic acid or L (+) lactic acid or racemic mixture and the
particular acid formed are the characteristic of individual
The effect of temperature can be explained as: 1) microorganism. From industrial standpoint the lactic
Dependence of structure of cell components on temperature, acid recovered from fermentation broth usually is the
2) Activation energy required for the reactions to occur racemic mixture because fermenting microorganisms
inside the cell which in other term affects the regulatory or contaminants like Lactobacillus plantarum produce
mechanisms of the cell, cell composition and permeability an enzyme known as Racemase that converts either of
functions. the optically active isomer to optically inactive racemic
Effect of pH on the growth of microorganisms mixture. Some trace impurities in the medium also
have been reported to bring about racemization. The
The influence of [H+] on biological activities is related to Racemase are known to be lactic dehydrogenises that
either hydrogen ion concentration or hydrogen ion activity maintain equilibrium between lactic acid isomers and
(ah). These two parameters are proportional as: pyruvic acid. When both dehydrogenase enzymes are
present, racemization occurs.
ah = f [H+] where f is the activity coefficient which may
vary with the ionic strength and other factors. The glass ii. Microorganisms: Many types of microorganisms
electrodes respond to hydrogen ion activity so that strictly have been isolated that accumulate lactic acid or
pH = -log (ah) and [H+] can be substituted for hydrogen lactates in the culture solutions e.g. Lactic acid
ion activity. It can only be possible when activity coefficient bacteria, algae, molds, yeasts and phycomycetous
is one. In dilute media solutions f approximately is one. fungi. Apart from many Lactic acid bacteria, mold
But this may be far from true when media are strong Rhizopus oryzae has been found to produce lactic acid
salt solutions. As far as cell properties are concerned comparable to homofermentative lactic acid bacteria
hydrogen ion activity is more meaningful parameter so from glucose. Lactic acid bacteria fall under two main
that it is appropriate to express the effect of hydrogen ion groups: (1) Homofermentative Lactic acid Bacteria (2)
concentration in terms of pH. Plasma membrane is not Heterofermentative Lactic acid Bacteria
freely permeable to hydrogen ions or OH- ions. So that
intracellular and extracellular hydrogen ion concentrations
do not necessarily equilibrate and a gradient of hydrogen
ion across the plasma membrane is established. According
to chemiosmotic theory this gradient of hydrogen ions
together with membrane electric potential makes a proton
motive force that derives the membrane reactions.

Lactic Acid
Scheele (1789) first isolated lactic acid from sour milk.
The studies on the physical and chemical properties have
shown that the compound occurs in two isomeric forms and
as a mixture of the two isomeric forms. Lactic acid is also
produced in muscles. Many microorganisms produce lactic
acid by the fermentation of sugars. The structural formula of
Lactic acid is CH3 -CH (OH)–COOH.
iii. Homofermentative lactic acid bacteria: These
Technological development bacteria produce lactic acid as the major or sole
Lactic acid was the first organic acid to be manufactured product of glucose fermentation. The homofermentative
 Basic of Fermentation Technology

10
pattern is observed when glucose is metabolized but Medium
not necessarily when pentoses are metabolized, for
some homolactics produces acetic acid and lactic acid The fermentation solutions usually contain hydrolyzed
when utilizing pentoses. Also, the homofermentative starches or dextrose syrup, although D-glucose, maltose,
character of homolactics may be shifted for some lactose or sucrose can also be fermented. It is advantageous
strains by altering cultural conditions such as to start with a relatively simple medium or mash in order
glucose concentration, pH and nutrient limitation. The to facilitate recovery of the product. Crude carbon sources
homolactics are able to extract about two times as are generally avoided because the impurities interfere
much energy from a given quantity of glucose as are with the recovery and purification procedure. The sugar
the heterolactics. They are less important in producing concentration in the medium should not be more than
flavor and aroma components e.g. acetaldehyde 12-15% because Calcium lactate produced at high sugar
and diacetyls in food products. They possess the concentration tends to crystallize from the medium late in
enzymes aldoses and hexose isomerase but lack the fermentation, thereby slowing the fermentation process.
phosphoketolase. They use EMP pathway to produce Nitrogen sources are added in small amounts and are
two molecules of lactate per glucose molecule. usually inorganic in nature e.g. Ammonium phosphate.
Examples of homofermentative lactic acid bacteria are: This is because impurities in the nitrogen sources might
All members of genera Streptococcus, Pediococcus interfere in the recovery and purification procedure.
and some genera of Lactobacilli like L. delbrueckii Calcium carbonate (10%) is added to neutralize the
Homofermentative lactic acid bacteria are very lactic acid produced because lactic acid bacteria cannot
important for the production of Lactic acid industrially. tolerate high acid concentration. Lactic acid bacteria have
complex requirements of B-vitamins. This ordinarily is met
iv. Heterofermentative lactic acid bacteria: They by enrichment of the culture medium with crude vegetable
produce some lactic acid, but at the same time, they materials. Malt sprouts are commonly used vegetable
also produce carbon dioxide, ethanol, and acetic acid materials, but if they have been overheated in drying, they
and trace amounts of a few other products. These lose some of their value as a nutrient for Lactobacilli.
organisms are of little use for industrial lactic acid
fermentations because too much of the substrate Production of Lactic Acid
carbon is directed towards products other than lactic
Today much of the lactic acid is produced by the
acid. The end product differences between homo and
hydrolysis of lactonitrile, a byproduct of another process.
Heterofermentative lactics when glucose is attacked are
Only a few companies in the world are producing lactic acid
a result of basic genetic and physiological differences.
by fermentative process.
The heterolactics have phosphoketolase pathway
but do not possess aldolase and hexose isomerase. Equipment
Instead of EMP pathway such organisms use
Hexose monophosphate shunt as energy pathways. Lactic acid is very corrosive to metals therefore
Heterofermentative bacteria are very important for wooden fermentors are used in most of the plants. These
the production of flavor and aroma components like fermentors may be uncovered or covered with loosely
diacetyls in food products. Examples of Heterolactics fitting wooden lids. They are to be steamed empty before
are: All species of genus Leuconostoc, and some charging. Fermentation solutions are pasteurized or
species of Lactobacilli. sterilized by passing it through a steam jacketed heat
exchanger. Contamination of culture solutions sometimes
Microorganisms for Commercial Production by thermophilic Clostridia results in the production of some
of Lactic acid butanol and butyric acid. Such contaminated lactic acid
could only be sold to leather tanners for delining of hides. A
The microorganisms used for commercial production of very pure lactic acid is required for manufacturing of plastic.
lactic acid depends upon the raw material to be fermented, Such grade of lactic acid is called Plastic grade lactic acid.
but the most common bacterium used for this purpose is
Lactobacillus delbrueckii; it is employed in fermentations Inoculum
utilizing corn dextrose medium. Although increasing Cultures of L. delbrueckii are transferred from test tubes
use is being made of a flat sour Bacillus coagulans yet through successively larger culture vessels, held at 45-55
Lactobacillus bulgaricus is used for the production of lactic °C. Each stage of the culture build up requires 16-18hours
acid from whey because it utilizes lactose as a carbon and slight excess of Calcium carbonate is required at each
source. L. pentosus (L.plantarum) is recommended for use stage. Inoculum volume should be 5% of the volume of
in spent sulfite liquor, as it is able to utilize pentoses. L. fermentation solution.
brevis is used when medium contains hydrolyzed corncobs,
cotton seed hulls etc. Other homofermentative species of pH
potential industrial importance are L.casei, L.leichmannii,
An excess of Calcium carbonate keeps the pH in the range
and Streptococcus lactis. These are facultative anaerobes
of 5.5-6.5. The pH necessary varies with the composition
and can withstand some oxygen. Streptococcus lactis is
of culture solution but it is controlled by continuous
particularly useful under such conditions.
 Basic of Fermentation Technology

11
neutralization with the slurry of Calcium hydroxide between confectionary, sherbets, soft drinks, extracts and other
6.3-6.5. Fermentations utilizing grain may resist increase in products. It is added to brines for pickles and olives and
pH with the buffering capacity of mash. to fish to aid preservation. Its addition makes milk more
digestible to infants. Calcium lactate is an ingredient of
Aeration and agitation some baking powders. In tannery, it is used for washing of
The medium is not aerated but agitation is done to hides. In textile industry it is employed for fibre washing.
keep the Calcium carbonate in suspension. Fermentation Lactic acid is used in the preparation of medicines. It is also
Time and Fermentation Temperature: The fermentation used as a laboratory reagent and a research tool.
temperature is adjusted to 45-50 °C and varies the type of
Citric Acid
organism used. Same is the case with fermentation time.
It is usually completed in six days or less depending on Scheele first isolated Citric acid in 1784 from Lemon
the time required by the organism to deplete the sugar in juice by crystallization process. Members of citrus family
the medium. Fermentation time is usually 5-10 days. It is of fruits especially are rich in this organic acid, but citric
important that residual sugars be reduced to 0.1% or less acid is also found as a natural constituent of a variety of
during the fermentation because residual sugars make fruits. Citric acid extracted from fruits is designated as
recovery of better quality lactic acid difficult. Natural Citric acid in contrast to the Citric acid produced
by Microbial Fermentation process. Citric acid can also be
Yield prepared synthetically but no equivalent synthetic process
Commercial yields are 93-95% by weight of glucose has been invented to the microbial fermentation.
supplied. Recovery yields vary with the various recovery
Microorganisms
processes and product grades.
Many microorganisms like molds (Aspergillus niger,
Product Recovery and Grades A. awamori, Penicillium janthinallum, Trichoderma
viridae, Mucor piriformis, etc.), yeasts (Candida lipolytica,
Technical grade lactic acid
C.tropicalis, C.citrica, Hansenula, Rodotorula, Pichia,
Calcium from the fermentation solution is precipitated as Torulopsis etc.) and bacteria (Bacillus licheniformis, Bacillus
Calcium Sulfate, filtered, and filtrate is evaporated to 35- subtilis, Brevibacterium flavus, Corynebacterium species)
40% lactic acid. Now more Calcium sulfate is precipitated, have the capability to produce citric acid. Commercially
filtered, and filtrate is evaporated to 44-55% total acidity. It spores of Aspergillus niger are employed to produce citric
is then crystallized. acid.

Food grade lactic acid Methods of fermentation

It is a pale yellow, straw colored solution of about 50% Fermentation of Citric acid is carried out by any of the
total acidity. Calcium in this case is precipitated as Calcium following methods 1) Stationary or Surface culture, 2)
sulfate, precipitates are washed, washed water is combined Submerged Culture, 3) Solid State Culture, 4) Continuous
with the filtrate, filtrate is bleached with activated carbon Culture, 5) Multistage Culture process, 6) Semi-Continuous
and it is then subjected to evaporation firstly to 25% solids, Culture process.
again bleached and then secondly evaporated to 50%
In stationary or surface culture, sterile nutrient medium
solids, bleached finally to produce an off colored product.
with sugar is added into stainless steel or high-grade
Impurities like Iron and Copper metals are removed by
aluminum trays sterilized with formaldehyde or sulfur
adding Potassium ferrocyanide in the filtrate of first filtration.
dioxide. Spores of Aspergillus niger are inoculated and
Plastic grade lactic acid incubated at 28-30 °C for 8-12 days. Submerged process
consists of two phases i.e. growth phase and productive
It is a colorless product. Product is recovered by phase. In Growth phase medium is inoculated with spores of
etherification with methanol after concentration or by solvent A. niger, after 3-4 days mycelium is separated from solution
extraction with isopropyl ether followed by re-extraction of and added to the fermentation medium and fermentation is
isopropyl ether with water. allowed to occur for 3-4 days. During the period production
Pharmaceutical grade lactic acid (Lactic Acid of the Citric acid takes place. This phase now is called
as Production phase. In Solid state Culture as described
USP)
by Calm (1935), fermentation medium is impregnated in
It is a colorless product with 85% total acidity and 76- porous solid materials like sugarcane baggase, potato or
78% concentration. It contains 2-3.5% volatile acids 0.5- beet pulp, or pineapple pulp in an appropriate ratio, sterilized
1.0% ash content. and then inoculated with a suspension of fungal spores
and incubated at 25-30 °C for 6-7 days. In Continuous or
Uses of Lactic Acid multistage culture, the medium is replaced after 24 hours
In the foods, lactic acid is added to acidify jams, jellies, and that medium goes to second fermenter. It is then
 Basic of Fermentation Technology

12
aerated by gradually increasing the amount of air. In Semi- from the culture medium various methods adopted are: 1)
Continuous Culture, the whole medium is not replaced but Pretreatment of the raw materials. 2) Ion exchange resins.
a part is replaced. 3) Development of resistant varieties of fungus. The raw
materials are treated with Potassium ferrocyanide to reduce
Medium the concentration of iron ions in molasses. The chemical
Components of the medium varies with the type of the is either added directly in the medium or the molasses
process used because it has been seen that the strains that is treated with its high concentration. Raw materials are
give good result in surface culture do not perform better also sometimes treated with chelating agents like EDTA,
in submerged culture. Fungi have been seen to give much activated charcoal or polythene amine. Chemicals of
better results in simple synthetic media. A variety of carbon quaternary ammonium compounds category like Diiosbutyl
sources are being used these days, which include Sucrose, phenoxy ethyl dimethyl benzyl ammonium chloride and
Citrus molasses, Cane juice, Starch from various sources, Triton-B are also used.
Cane or Beet molasses. The initial sugar content determines
pH
the amount of citric acid produced by A. Niger. Normally
15-18% sugars are added into the medium. Concentration Fungus can tolerate high concentration of acid therefore
of sugar more than 15-18% leads to greater amounts of calcium carbonate is not added in the medium. The initial
residual sugars that make the process uneconomical. required pH of the medium depends upon the carbon
When the sugar concentration is lower than the above source used as follows: Glucose or clarified molasses pH =
percentage, it leads to lesser yield of citric acid as well as 3.0, Crude molasses pH = 5-6, Decationized molasses pH
accumulation of oxalic acid. Molasses contains 50-60% = 1.4-2.8, Sucrose pH = 2.0-3.0. pH is adjusted with HCl,
sucrose and is of three types: 1) Black Strap molasses- it H2SO4 or NaOH.
is obtained from the last stages of crystallization of sugar.
2) Refinery molasses- it is obtained at the second stage of Additive and stimulants
refining sugar. It contains 48-50% sucrose and has high ash Methanol is usually used to increase the yield at a
content. 3) Invert or High-test molasses- it is partly inverted concentration of 3-4%. At this level it retards growth, delays
cane juice syrup from which no sugar has been extracted. sporulation and increases citric acid yield. This is added
Molasses is diluted to sugar concentration of 15-20% and before inoculation. It has some role in the conditioning the
pH adjusted to 5.5-6.5 with sulfuric acid. Molasses is added mycelium without impairing their metabolism. It is thought
with other required nutrients and mixture is sterilized for that it increases the tolerance of the fungus towards the
30 minutes. For the growth stage of Aspergillus niger, the harmful effects of the trace elements. Other additives like
organism needs major elements (C, N, P, S) and trace hydrogen peroxide, Methylene blue, Naphthaquinone,
elements. The type of inorganic source and its concentration aromatic amides, esters of dichloroacetic acid, sodium
affects the performance of the fungus. Inorganic sources sulfite, crysyllic acid, glycerol, vegetable oils (corn oil,
like Ammonium sulfate prolong vegetative growth. almond oil and peanut oil) and cAMP are added as stimulant
Ammonium nitrate shortens the growth phase. Ammonium to increase the yield.
nitrate in concentration greater than 0.25% accumulates
more of oxalic acid whereas sodium nitrate at concentration Antifoaming agents
0.4% delays the onset of production phase and vegetative
Antifoaming agents like Octadecanol (0.75% solution) or
growth also increases. Phosphate also affects the citric
Antifoam AE (silicone oil) are added in the medium. To control
acid yield. High phosphate concentration promotes growth
contamination by bacteria Pentachlorophenol ate, formic
and there is less acid production. Therefore phosphate
acid, tetracycline’s, and 3-furaldehyde semicarbazone are
concentration in the medium should be kept at 0.1%-0. 2
added in the medium.
%. Trace elements (Fe2+, Cu2+, Zn2+, Mn2+ and Mg2+)
are necessary for A. niger. Mg2+ is necessary for a variety Biochemistry of Citric Acid Formation
of enzyme reactions in the cell. It is required for growth
Citric acid is formed via following pathways: (Flow Chart 1)
and production phase. Its optimum concentration in the
medium should be 0.02-0.025%. Fe2+ and Zn2+ have Citric acid accumulates in culture solutions of pH 1.8-
critical role to play in the growth and production of citric 2.0. In fungi different metabolic pathways are involved in the
acid. Both are essential for the production of citric acid in production of citric acid but 78% of the citric acid is produced
low concentration because their high concentration allows by the involvement of glycolysis and Tricarboxylic acid cycle.
vegetative growth. Iron also has deleterious effects on the The acetyl COA derived by EMP pathway condenses with
fungus but its action is counteracted by copper ions. Zn2+ Oxaloacetate of Kreb’s cycle to produce citric acid. Since it
at concentration 1-2 µM allows growth phase but its less has been observed that, during accumulation of citric acid,
concentration restricts growth. Its excess in the citric acid A.niger demonstrates decreased activity of condensing
producing culture reverses the production phase. It also has enzyme and almost no activity of Isocitrate dehydrogenase
its indirect role in the functioning of cAMP that enhances the and aconitase therefore another theory of its production
production phase. To remove the excess of trace elements came into light. In this pathway, glucose first splits up into
 Basic of Fermentation Technology

13
two 3-Carbon fragments followed by decarboxylation of heating the mixture to 80-90°C. Calcium citrate is filtered off
other fragment to yield a 4-Carbon compound. The 2-C on conventional filter and the filter cake is transferred to a
and 4-C compounds then combine to yield citric acid. In tank called acidulator where it is treated with Sulfuric acid to
methyl citrate pathway Propionyl COA formed through the precipitate calcium sulfate, which is then filtered. The dilute
β-oxidation of n-alkanes condenses with Oxaloacetate to filtrate containing citric acid is purified by treatment with
yield methyl citrate. activated charcoal and demineralized by passing it through
ion exchange resins. The purified solution is evaporated
Product recovery in a circulated granulator or circulating evaporator called
The crude fermented liquor is subjected to filtration to crystallizer. Crystals are removed by centrifugation. Citric
remove mycelia of fungi. Calcium citrate is precipitated acid so prepared is subjected to recrystallization. The
from the clear liquid by adding slurry of calcium hydroxide remaining mother liquor is added into the main stream prior
(add hydrated lime 1 part for every 2 parts of liquor added to limming and decolorization (Flow Chart 2).
over one hour period and temperature raised to 95°C) and

Flow Chart 1

Flow Chart 2
 Basic of Fermentation Technology

14
Single Cell Protein (SCP) the source of carbon for photosynthetic-autotrophically
growing cultures. Air contains only 0.03% carbon dioxide.
Definition -The dried cells of various groups of Accordingly, additional carbon dioxide must be supplied to
microorganisms like Bacteria, Yeasts, Molds, Higher fungi the cultures. Some algae can also be grown in lakes rich
and algae that have been considered for food or feed are in sodium carbonate. In sewage ponds, the algal growth
collectively referred to as Single Cell Protein. C.L. Wilson is limited by the extent of liberation of carbon dioxide and
in Massachusetts institute of technology coined the term ammonia by bacterial action. Slow and uniform liberation
SCP in 1966. People have eaten certain microorganisms of carbon dioxide is necessary to provide a uniform
as a portion of their diet e.g. Top fermenting yeasts supply of carbon source to the growing culture. Nitrogen
(Saccharomyces spp.) was recovered as a leavening sources suitable for algal growth include ammonium salts
agent for bread as early as 2500 B.C. Fermented milks or nitrates. The N-sources together with phosphorous and
and Cheeses produced by Lactic acid bacteria of genera mineral nutrients may be readily available in sewage ponds.
Lactobacilli and Streptococci were consumed by early However on other water supplies where synthetic media
Egyptians and Greeks and reached a higher state of are used in culturing algae, these nutrients may have to
development during the Roman era (100-50 B.C.). Pharaohs be supplied. Aseptic conditions are not maintained during
of Egypt prized wild mushrooms as a delicacy. Romans the large-scale growth of algae in ponds but the potential
regulated the grading and selling of mushrooms. People contamination by pathogenic bacteria may be given
of Chad regions of Africa and Aztec in Mexico have eaten serious consideration. The key factors in a large-scale algal
Spirulina spp. (Blue green Algae) for many generations. cultivation are agitation and flow rates. Agitation prevents
The purposeful cultivation of microorganisms for direct use sedimentation and keeps the cells in suspension while flow
in human food or animal feed is a fairly recent development. rate adjustments allows the detention period of algal culture
Considerable efforts have been expanded since World War- to exceed the generation time. This helps in maximum
II to develop mass cultivation of microbial cells. population development.
Nutritive value and use of SCP Cell recovery
Nutritive value of SCP varies with the microorganism Cells must be recovered by concentration, dewatering
used. Protein digestibility of SCP is expressed as and drying. The inorganic compounds like Aluminum
percentage that ranges from 65-96 for various cultures. sulfate, calcium hydroxide and cationic polymers have
Protein efficiency ratio ranges from 0.6-2.6. Food yeasts been investigated as flocculating agents but these do
are high in proteins and vitamins of B-12 category but not separate from algae. Algae may auto flocculate in
may be deficient in some amino acids such as cysteine shallow ponds at pH 9.5 or above without flocculants. Ion
and methionine. The UN protein advisory group has major exchange resins at pH 2.8-3.5 can also recover algae but
concern over the use of SCP for human beings because this method is expensive. Spirulina maxima float on the
of the following reasons: 1) The high nucleic content of the surface in clumps when maximum growth is attained. It can
SCP may elevate serum uric acid level, which may result be harvested by skimming at much lower cost than would
in kidney stone formation or gout. 2) Certain skin reactions be incurred by centrifugation.
may occur by consuming foreign proteins 3) Possibility
of carrying over of carcinogenic factors 4) possibility of SCP production by bacteria and actinomycetes
gastrointestinal reactions.
Bacteria are of interest for use in SCP production
Production of SCP because of following reasons: 1) High growth rate (20-
30 min. in bacteria, 2-3 hours in yeasts and 16 hours in
a. Production of SCP by algae: Algae can be grown algae, molds and higher fungi). 2) Bacteria can utilize a
photo synthetically or autotrophically in the Presence of variety of carbon and energy sources, renewable sources
sunlight, artificial light or heterotrophically in dark with as carbohydrates (starch, sugars and cellulose) and non-
organic carbon and energy sources. renewable sources as hydrocarbons and petrochemicals.
Microorganisms Actinomycetes are of interest because their growth patterns
and substrate utilization patterns are similar to bacteria.
The most important algal strains used for SCP production
include: Microorganisms
Chlorella sorokiniana, Scenedesmus acutus, S. Methylococcus capsulatus, Methylomonas methylovora,
quardicanda, S. obliqus, Spirulina maxima, S. platensis. Methylophilus methylotrophus, Pseudomonas spp.

Growth conditions Growth conditions

In an open circulating system particularly sewage Some important considerations for selecting bacterial
oxidation ponds, mixed culture of algae tend to predominate cultures suitable for use in SCP production are: High Specific
rather than a single strain. The limiting factor in the algae growth rate, yield on a given substrate, pH and temperature
growth on a large scale is illumination. Carbon dioxide is tolerance, less aeration requirement, genetic stability and
 Basic of Fermentation Technology

freedom from associated bacteriophage. Criterion for using having hydrocarbons and alcohols; lower concentrations
15
bacteria for SCP production is as follows: 1) Strains should are to be used. Ammonium salts or anhydrous Ammonia
not be pathogenic to plants, animals or human beings. 2) is suitable as nitrogen source. Phosphoric acid is used
Strains should not have potential for mating with known to adjust pH. Yeasts when grow on substrates like
pathogenic bacteria to yield pathogenic hybrids. Growth carbohydrates, hydrocarbons or alcohols, a lot of heat
condition requirements are: 1) Carbon and Nitrogen ration is liberated therefore heat tolerant varieties should be
in the medium should be 10:1 or less to favor high protein employed for production (e.g. Hansenula polymorpha can
content in cells and to prevent accumulation of lipids or grow at 37-42°C) and cooling water or refrigeration facilities
storage substances such as Poly-β-hydroxy butyrate. should be employed in the fermentation. The growth rate
Nitrogen is added as anhydrous ammonia or ammonium of yeasts under aerobic conditions depends upon the rate
salts and phosphates as phosphoric acid (feed grade) to of mass transfer of oxygen and substrate to and across the
avoid contamination with arsenic or fluorides found in crude cell surface. Yeast SCP production may or may not take
industrial phosphoric acid. Minerals are added as Magnesium place under sterile conditions. In either batch or long-term
and Manganese in water. Other minerals are added as continuous yeast production one must balance the need for
sulfates and hydroxides but not chlorides. pH is controlled contamination control by maintaining sterile conditions, with
in the range of 5-7. Temperature tolerance is important for the capital and operating costs of the equipment required.
bacteria grown on alcohol hydrocarbons. Maintenance of The temperature of fermentation is maintained at 36-37°C
sterile conditions is important to avoid contamination at and pH 4.5-6.0. Yields are 45% or more of the dry yeast on
pH 5-7. Different carbon sources may be used for bacteria the basis of the sugar fed.
these may be carbohydrates sugar, starch, cellulose, and
baggase, wheat bran, and wood, petroleum products diesel Cell recovery
oil, gas oil, and n-hexane, proteins (Collagen, meat packing Yeast cells range in size from 5-8µm and have a density
waste), methane, Alcohols (methanol) and n-alkanes. 1.04-1.09 gm/cm3. They can be recovered readily from the
medium by centrifugation. The cells are centrifuged in 2
Cell recovery
stages. In the first stage it is dewatered and yeast cream
A number of problems come in way in bacterial cell is separated. This is followed by two subsequent washing
recovery process because large volume of water is to be centrifugations. The final washed yeast cream usually
handled and bacteria have very small size in addition to contains 15-20% solids. In SCP by Kluyveromyces fragilis
that the bacterial cell densities are very close to water. grown on cheese whey, the entire growth medium is passed
Therefore they are separated by centrifugation, filtration through a three-stage evaporation to concentrate the solids
and electrochemical coagulation methods. After separation from 8-27% to give a feed grade product. The separated
they are spray dried. cells can either be drum or spray dried.

Production of SCP by yeasts Ethanol as Fuel

Yeasts are probably the most widely acceptable and Ethanol can be used as such or in blends with petrol as
used microorganism for SCP production. Yeasts have the a motorcar fuel. About 10% ethanol can be used without
capacity to grow on a variety of substrate including waste modifications in the engine but the only disadvantage it has
products that contain pentose sugars. Yeasts in general is that it increases the octane rating. Combustion engines
have several advantages over bacteria and algae these are: can be built to run straight on ethanol or on ethanol of
1) better public acceptance 2) lower nucleic acid content 3) lower concentration (80% ethanol and 20% water). Current
easier harvesting because of size and concentration 4) high interest in various parts of the world centers to use a blend of
protein content 5) production of vitamins of B-12 category ethanol called ‘Gasohol’, which is a blend of 90% unleaded
6) growth in substrates of low pH. petrol and 10% ethanol. Anhydrous ethanol is preferred for
this application but 96.5% ethanol can be used if mutually
Growth conditions miscible solvent such as isopropyl alcohol is added.
a. Raw materials used as substrates: Materials that
are employed to produce SCP by yeasts
Raw materials
Forty-five kilograms of fermentable sugars (glucose) is
include: 1) molasses from sugar industry 2) starch after
assumed to yield 18-23 kg or 23-28 litres of ethanol. The
hydrolysis 3) spent sulfite liquor from paper industry 4) acid
yield is same for starchy raw materials i.e. between 40-50%
hydrolysates from wood industry 5) whey from dairy industry
based on dry weight of carbohydrates. Complete hydrolysis
6) hydrolyzed starchy foods (grains and cull potatoes, fruit
of starch yields about 50 kg of glucose but conversion is
wastes etc.) 7) methane 8) methanol and ethanol 9) alkanes
never complete, therefore with 90% conversion the yield is
and paraffins 10) gas oil 11) combustion gas Substrates and
40-50%. For cellulosic raw materials yield is less because
nitrogen concentration for yeast growth should be adjusted
α-cellulose is quite resistant to enzymatic attack.
to provide C  N in the range of 7: 1 to 10: 1 to favor high
protein content. Concentration of carbohydrates in batch Sugar containing raw materials
culture should range from 1-5% while in continuous culture
 Basic of Fermentation Technology

16
Any sugar containing fruits, fruit juices or extracts, sugar- and succinate), which may consume up to 4-5% of the total
containing effluents from canneries, sugar beet, sugar cane, substrate.
sugar cane molasses, cheese whey or sweet sorghum may
C6H12O6 → 2 C2H5OH + 2 CO2 + 2 ATP (Gay
be used as raw materials.
Lusac’s Equation)
Starchy raw materials Glucose Ethanol carbon dioxide Energy
Cereal grains (wheat, rice, corn etc.), root crops
(cassava), tubers (potatoes) are gelatinized by heating and
Modes of fermentation
then hydrolyzed to fermentable sugars by enzymes. Batch fermentation: Most of the production of ethanol
is carried out on the same lines as Established hundreds
Cellulosic raw materials of years ago. These methods are based on the simple
Saw-mill residues, paper-mill residues, newsprints, batch fermentation of substrates inoculated with culture
potato peelings, rice straw, corn stover, peanut shells, cocoa microorganism at temperature 20-30 °C, initial pH adjusted
and coffee husk, tobacco stalks and wheat straw contain to 4.5 and the time varies between 36-72 hours. Final
α-cellulose, hemicellulose and lignin. These materials when ethanol concentration is usually 10-16%. Batch technology
used are delignified, hydrolyzed and then enzymatically has been preferred because of its ease of operation, low
treated with cellulases to convert them into fermentable requirement of sterilization, use of unskilled labor, low risk
sugars. of financial loss and easy management of feedstocks. It has
its disadvantages also such as low productivity due to long
Microorganisms turnaround times, expenditure of cooling as fermentation by
Most of the commercial production is carried out with this mode raises the temperature of the fermentation broth
Saccharomyces cerevisiae. For production of ethanol and initial lag in growth.
from pentose solutions from corn stover hydrolysis and Continuous fermentation: In continuous fermentation,
from spent sulfite liquor of wood industry Candida utilis is fresh medium along with inoculum (yeast)is fed to the
used and glucose is fermented with immobilized cells of S. fermenter and an equal volume of the fermented liquor is
cerevisiae. Many strains of S. cerevisiae readily produce withdrawn from the fermenter for the recovery of ethanol
10-13% ethanol by volume in batch fermentation of and yeast. Continuous ethanol production eliminates much
molasses solutions with initial fermentable sugars 20-25%. of the unproductive down time associated with the batch
Higher concentration of ethanol can be achieved in Fed- fermentation process. The microorganism inoculated in
batch culture. The fermentation of whey requires the use of the continuous mode should always be in its exponential
dairy yeast Kluyveromyces fragilis and K. lactis. growth phase because this increases the time dependent
productivity of ethanol. The rate at which medium in the
Nitrogen sources
fermenter is added or withdrawn is usually expressed as
Assailable Nitrogen sources such as ammonia, ‘Dilution rate’. It is denoted by a letter ‘D’. The time for which
ammonium salts, urea, or amino acids are added in addition the fermentation solution remains inside the fermentor for
to phosphorus in the form of ortho-phosphate. Minor fermentation is referred as ‘Residence Time’. The dilution
quantities of potassium, magnesium, calcium and trace rate is the ratio of withdrawn liquid to the volume of total
elements are also added. The addition of vitamins is rarely liquid in the fermentor. Hence, a dilution rate D = 0.1
required but thiamine has been often seen to accelerate the indicates that one-tenth of the fermentor liquid is withdrawn
rate of fermentation. S. cerevisiae requires the presence of per hour (or the residence time in the fermentor is 10 hours).
biotin. The best source of all required nutrients including the In continuous fermentation residence time and dilution rate
trace elements and vitamins is yeast extract. should be so adjusted to get higher ethanol production.

Fermentation Cell recycle: In Cell recycle, yeast cells are separated

from the withdrawn fermented solution by means of
Biochemical basis of fermentation: Yeasts, under centrifugation or by any comparable process or settling and
anaerobic conditions, metabolize glucose to ethanol returned to the fermentor. This is done to overcome low cell
primarily by way of the EMP pathway. The overall net density limitations and to maintain high cell concentration in
reaction involves the production of 2 moles each of ethanol, the fermentor.
carbon dioxide and ATP per mole of glucose fermented.
Therefore on a weight basis, each gram of glucose can Vacuum fermentation: This is a novel fermentation process
theoretically give rise to 0.51g of alcohol. The yield attained in which alcohol formed by fermentation is continuously
in practical fermentations however does not exceed 90- removed from the system so that it does not become toxic
95% of theoretical. This is due to the requirement for to the microorganism. This process was described by
some nutrient for to be utilized in the synthesis of new Cysewski and Wilke in 1977, 1978 and by Ramalingham
biomass and other cell maintenance related reactions. Side and Finn in 1977. The former took the advantage of high
reactions also occur in the fermentation (usually glycerol volatility of alcohol and conducted the fermentation at
 Basic of Fermentation Technology

17
temperature conducive for the growth of yeast and sufficient by the use of adsorbents, such as cellulose, or other dry
vacuum to boil off the alcohol while latter used vacuum plant materials (cracked grains). Inorganic desiccants have
lesser than what former had used and temperature 30 °C. also been suggested for this purpose. An endless loop of
yarn fibers is also used to remove water from ethanol water
Aeration azeotrope.
Aeration is done to keep the cells under suspension and
By-Products of ethanol fermentation
to maintain viability in cells. Therefore sufficient amount of
air should be sparged into the fermentor so that dissolved During the fermentation of ethanol many by-products
oxygen concentration (expressed as Oxygen Tension) may are also formed which must be removed effectively by
not exceed its toxic levels. the distillation system. The by-products more volatile
than the ethanol are mainly aldehydes with acetaldehyde
Distillation the principal compound. Methanol in small amounts is
Distillation process may be divided into two stages i.e. also formed by the hydrolysis of pectins present in the
Distillation proper and Rectification. In distillation proper, fermentation broth. The less volatile compounds formed
volatile components of the fermentor are separated from the as a by-product are Fusel oils which are a mixture of
insoluble solids and ethanol is concentrated to a distillate isomers of amyl alcohol 60-80%, isobutyl alcohol 15-30%
containing 30-96% ethanol by weight. In rectification, and n-propyl alcohol 0-10%. Separation of by-products:
ethanol is separated from other volatile components such The aldehydes are removed relatively easily due to the low
as aldehydes (acetaldehyde) and fuel oils (amyl alcohol 60- boiling point of acetaldehyde as a distillation head product;
80%, isobutyl alcohol 15-30%, and n-propyl alcohol 0-10%) however the separation is not complete. The residual
about 1 litre of acetaldehyde and 5 litre of fuel oil is produced aldehyde content in alcohol is typically 5-100 mg/L. Fuel
for every 1000 litre of ethanol produced. The amount of the oil, owing to its complex composition and limited solubility
volatile component depends upon the starting material used in water is more difficult to separate. Dry fuel oils boil in
for fermentation and fermentation process itself. the range from 128-137°C and normally, being even less
volatile than water, they remain in the bottom product. In the
Ethanol has a lower boiling point than water. presence of water however, the boiling point of the mixture
Concentration of ethanol is therefore always more in the falls below that of the water alone as a consequence of their
vapor phase of ethanol solution in equilibrium with the immiscibility. Fuel oil, therefore rises from the bottom of an
water. When distillation of the water and ethanol mixture alcohol-purifying column and concentrate in the middle of
is carried out, ethanol concentration of ethanol is always the column, from they are removed. In a continuous process
more in the distillate. Water and ethanol form an azeotrope. fuel oil is removed by bleeding from 6th to 15th plate of the
(Azetrope is a mixture of volatile substances that at a given rectification section and then fed to the fuel oil separator.
concentration has identical liquid and vapor compositions).
For ethanol and water this concentration is 96% by weight of Then specification of the industrial alcohol depends upon
ethanol and the boiling point of ethanol is 78.2°C. Therefore the intended use. Premium grade alcohol has low water
initially easy separation of ethanol from the broth becomes content, low odor ratings and high permanganate time test
increasingly difficult as concentration 96% by weight of values (presence of aldehydes).
ethanol is reached because volatilities of both water and a. Definitions
ethanol become identical. Boiling at this point gives a vapor
of same composition as that of boiling liquid and no further A. Denatured Spirit- Alcohol that is denatured by adding
concentration of ethanol can be achieved. 10% methanol is called denatured alcohol or denatured
spirit.
Anhydrous ethanol may be obtained from 96% ethanol
solution by forming a ternary azeotrope with benzene. B. Rectified Spirit- Alcohol from which fuel oil has been
This ternary azeotrope contains 74% benzene, 18.5% removed is called rectified spirit or rectified alcohol.
ethanol and 7.5% water and its boiling point is 68oC.
C. Anhydrous Alcohol- Alcohol that has very less water
Since this azeotrope contains more water than the
content is called anhydrous alcohol.
ethanol water azeotrope, it can be distilled overhead and
leaves anhydrous ethanol behind. Such a process usually D. Potable Alcohol- Alcohol that can be used for drinking
consists of two steps simple fractionation to produce near purposes is called potable alcohol.
azeotrope mixture followed by ternary distillation with an
added chemical agent yielding anhydrous ethanol. If the Different Types of Substrates for Industrial
extraneous material is less volatile than the feed, it is called fermentations
a solvent and the operation is called extractive distillation.
Substrates are used as components of media suitable
When the extraneous material is more volatile than the feed
for the growth of microorganisms or the production of
it is withdrawn with the overhead product and the operation
desirable microbial metabolites. Nutrient medium form
is called azeotrope distillation. In addition to these methods
the environment, in which microorganisms grow and from
water may also be removed from ethanol water azeotrope
 Basic of Fermentation Technology

18 which they draw substances necessary for the synthesis Water is an indispensable component of a medium. It is
of cellular components and to produce energy that runs much needed by microorganisms for fermentation process.
the biochemical machinery of the cell. Bio-chemically It is also needed in other processes like isolation of the cells
substrates may be classified as: 1) Simple-that contain the and products. In fermentation, we usually use tap water for
lowest amount of energy, e.g. water, carbon dioxide, oxygen, the preparation of culture media, separation and washing
nitrogen and inorganic ions, 2) High-energy-contain highest of microorganisms. The quality of water used depends
amount of energy and 3) Intermediary substrates-that are upon the process in which is used e.g. A water of low grade
formed during the transformation of simple substrates to the quality can be used for cooling purposes, whereas for the
high-energy substrates. The function of the nutrient medium preparation of media and isolation of cells and products etc.
is to ensure the growth of the microorganism and has to a water of high quality i.e. Potable water or treated water is
contain individual substrates in a form best accessible to the needed. The treated water can be produced by four ways:
microorganism, which usually means in solution. However, 1) By adjusting the chemical composition i.e. by removing
in some cases solid media are used and cells from gaseous salts or ions like iron, free chloride or chlorine, salts of
phase may sometimes utilize some substrates. Besides carbonic acid and adjusting the hardness. 2) By removing
major components medium also contains many other the extraneous materials by sedimentation, filtration etc.
components necessary for microbial growth. Physico- 3) by removing microorganisms from the water i.e. by the
chemical properties and medium composition adjustment processes like filtration, sterilization and disinfection. 4) By
help in the maintenance of the maximum rate and desirable removing the colloids i.e. by the process of osmosis. The
direction of the microbial process. quality of water always fluctuates and can be controlled by
various chemical and biological methods e.g. when levels
Complex media are prepared from natural raw materials
of ammonia in water increases, the water is said to be
of animal or plant origin; the exact chemical composition of
polluted and has to be treated by various chemical methods
these media is usually not known and individual components
or by growing certain organisms like ammonifying bacteria.
can be determined only with difficulty. A complex medium
Similarly when levels of salts of nitrous acid or nitric acid
is prepared by dissolving appropriate natural substances
rise above the normal limits, growing denitrifying bacteria
in water and is often further processed by hydrolysis,
also controls them. When iron levels in water increases,
clarification complemented by additional nutrients, growth
it causes discoloration of microorganisms such as yeasts
factors, and missing trace elements. Synthetic media are
and inhibit the biosynthesis of various substances e.g.
prepared by dissolving defined chemicals in distilled water to
antibiotics. It may also produce corrosion like effect in water.
eliminate completely all minerals and trace elements usually
The norms for high quality water are 20-100 germs per ml.
present in un-distilled water. Complex media are most often
The hardness of water is due to the presence of various
used for technological purposes while synthetic media are
salts of calcium and magnesium, which when rise above
employed only in special cases e.g. in the processing of
their normal limits, they lead to instability in product yields.
substrates from the chemical industry or utilization of some
In that case, water is deionized and appropriate portions of
waste materials. In laboratories usually synthetic media are
other salts are added. Such types of treatments are only
used despite their numerous shortcomings because they
possible in laboratory and at commercial scale; the cost of
are defined therefore it is possible to draw exact material
applicability becomes very high. The quality of ground water
and energy balances from changes in the levels of individual
now days is further deteriorating due to the increases use of
components. As a qualitative and quantitative combination
chemicals in agriculture and utilization of nuclear materials
of substrates, the medium must meet the microbiologist’s
in the warfare. So the demand of water for the commercial
demand. The consumption of substrates in the laboratory is
purpose has further increases and further prospects of
very small and it is quite within the range of microbiologist’s
research in this regard are needed.
to use more expansive substrates that cannot be utilized
on commercial scale due to certain reasons. According to Carbon sources
state of the medium, media can be classified as: 1) Solid
media. 2) Liquid media. 3) Gaseous media. The solid media They represent the main component of nutrient media
are present in the solid state and they are used immediately quantitatively. They are utilized for the biosynthesis of
on dissolving in distilled water. The liquid media are readily cell materials and are also used as main energy source.
utilized as they contain more water. These can be converted Carbon is also used to build carbohydrates, lipids, fats
onto solid ones or gels by the addition of certain thickening and proteins etc. in the cell and it is derived from various
agents like agar-agar, gums, and gelatin etc. The gases organic compounds added in the medium. Some new types
also play an important role in the growth and multiplication of substrates have also come into light such as synthetic
of the organisms and they also have a profound effect on alcohols, alkanes and many hydrocarbons. The nature of
the production of metabolites. The main components of microbial process, product purity and metabolism of the
a medium are as follows: 1) Water. 2) Carbon source. 3) inoculum microorganism may dictate the use of chemically
Nitrogen source. 4) Accessory substances. 5) Antifoam defined carbon sources such as glucose, sucrose, lactose
agents. or starch as well as complex raw materials like molasses,
sulfite liquors, wood hydrolysates, cellulose and raw
Water materials of petrochemical origin.
 Basic of Fermentation Technology

Carbohydrates temperatures while at high temperatures, they swell and

19
change into starch gel. The gelation is usually performed
They form a distinct group of organic compounds that at 100 °C in water. At 120-130 °C, the gel liquefies and
are divided into three categories as monosaccharides, may be easily degraded by enzymes. Starch is a mixture
disaccharides and polysaccharides. of two related polysaccharides, amylose (linearly arranged
Monosaccharides chain of α-glucosidically linked molecules of D-glucose with
1-4 bonds) and amylopectin (branched chain of D-glucose
Glucose-In fermentation process, glucose is the most molecules bonded also -glucosidically by 1-4 and less
frequently used monosaccharide (Grapes contain 25- often 1-6 bonds); one molecule contains more than one
30% of glucose) but it has its own disadvantages like thousand glucose residue. Acid hydrolysis of amylose
Catabolite repression and Pasteur effect. For industrial yields first maltose and then glucose. Enzymic degradation
purposes glucose is obtained by the hydrolysis of starch by amylases proceeds via amylodextrin and low molecular
both by chemical and enzymatic methods. Since it is weight dextrins to maltose.
rapidly assimilated therefore a continuous inflow of glucose
is required in growing cultures for the biosynthesis of Cellulose: It is a structural polysaccharide, forms a
complex compounds. It is sterilized separately from the substantial part of the plant cells and is found abundantly in
other components of the medium because at neutral pH nature. Isolation of pure cellulose is difficult because other
and at high temperature it undergoes Maillard’s reaction natural substances like lignin, hemicellulose and waxes
with amino acids to give dark brown colored solution. It accompany it. For industrial purposes, cellulose is obtained
is usually sterilized at pH 3.0 as a 30-50% solution (w/v); from wood and straw of various plants. Recently an intensive
under these conditions the solution remains colorless. In research has been initiated concerning cellulose-degrading
microbial processes, the so-called “Hydrol”, a waste product enzymes like cellulases. The cellulase is a multienzyme
in the manufacture of pure glucose, can also be used as a complex of three enzymes namely endoglucanase,
substrate. exoglucanase and α-glucosidase that act synergistically to
form glucose. A compound known as “Cadoxen” (25-30%
Disaccharides and oligosaccharides ethylene diammine in water and 4.5-5.2% cadmium) has
the capacity to disrupt the crystalline structure of cellulose,
1. Sucrose- It is obtained from sugar cane or sugar beet
which renders it susceptible to rapid and total hydrolysis
and is available in different levels of purity. It is much
to form soluble products. Other agricultural wastes, which
needed for the fermentation by fungi, yeast and in the
can be used as carbon sources are: Molasses from sugar
fermentation of microorganisms those biosynthesize
industry, Barley water and its products from brewing
antibiotics, amino acids and organic acids.
industry, sulfite waste liquor from paper and pulp industry,
2. Lactose- Lactose is present in the milk of mammals at acid-wood hydrolysates from wood and paper industry,
the levels of 3-8% and is prepared by the evaporation of vegetable oils and animal fat from oil industry and alcohol
whey that remains after the processing of milk to butter. and hydrocarbons from petrochemical industry.
Only a few species of microorganisms can assimilate
Molasses: It is a thick, syrup-like, viscous and dark colored
lactose; yeasts with a few exceptions do not assimilate
liquid obtained as a byproduct in the production of raw and
lactose. Penicillium chrysogenum can assimilate lactose
refined sugar from sugar cane or sugar beet (remains after
in low but in efficient levels. The main disadvantage of
the crystallization of main fraction). It is named after its
the lactose is its varying quality, which can be attributed
source as Cane or Beet molasses. Cane molasses contains
to different milk processing operations.
a high level of sugars
Polysaccharides Barley and its products: Barley and its products such
These substances represent a suitable source for as malt, wort and malt extract. Malt is prepared by the
bacteria, yeast and fungi and are used in the biosynthesis of germination of barley. During malting amylases and
many products. They are macromolecular, forming colloidal pectinases are only partially activated since the process
solutions or exhibiting negligible solubility in water, for this takes place at low temperatures and in a medium containing
reason they must often be converted into a soluble form only vegetable liquor. In the brewery malt is crushed,
before being used in nutrient media. mixed with water and kept at a higher temperature; under
these conditions the enzyme reactions proceed rapidly
Starch: It is one of the most important polysaccharide and completely as far as the degradation of malt starch is
both biologically and industrially. It is accumulated as a concerned. Wort is obtained from malt residue by filtration. It
reserve substance in fruits, seeds, and bulbs of higher is used as a raw material for Beer brewing. After the addition
plants in the form of granule of different shapes and sizes. of hops it is boiled and filtered. The resulting solution is
It is usually denoted according to its origin i.e. according to hopped wort; after cooling it is inoculated by a culture of
the plant from which it is manufactured i.e. Potato starch brewer’s yeast and fermented. Malt extract is prepared from
from potatoes, Maize starch from maize, Wheat starch from wort by filtration and evaporation; it is thick syrup containing
wheat, Rye starch from rye and Barley starch from barley. 80% dry weight that may be vacuum dried to a powder.
Starch granules are hygroscopic and do not changes at low
 Basic of Fermentation Technology

20
Sulfite waste liquor-It is a waste product in the production and therefore are used for the production of fodder yeasts.
of pulp from wood by the sulfite method. It is a brown-yellow Vegetable oils and Animal Fats-Majority of the vegetable
liquid with a density of 5 to 90 B at 20°C. Its specific weight is oils are easily metabolized by microorganisms therefore
1.045 to 1.060. It contains 8 to 14% dry weight that consists they are used as a carbon sources in many microbial
of 18 to 20% minerals and 80 to 90% organic substances. processes especially in the production of antibiotics. They
The inorganic part is composed chiefly of sulfur dioxide, are also often used as an antifoam agent. Fats on the other
sulfite and calcium sulfate. A part of the calcium is bound to hand are solid at room temperature and their liquefaction
lignin as a calcium lignosulfate salt, which is water soluble requires high temperature. For this reason they are not so
in acidic media. Other inorganic compounds present in the often used as substrates.
liquor are salts of heavy metals such as copper, arsenic
Alcohols-Synthetic alcohols, especially ethanol and
and lead. These heavy metals ions in their minute amount
methanol have importance as potential raw materials
also affect the growth of microorganisms. The organic
for microbial processes. Ethanol is available in sufficient
compounds found in the liquor i.e. lignin and carbohydrates,
quantities, has a reasonable price, standard quality and
are released from wood during treatment with the sulfite
homogeneity. Its physico-chemical properties place it
cooking acid. A short cooking at low temperature yields solid
among surface-active substances; hence it increases the
pulp containing higher levels of hemicelluloses and lignin;
foaming of nutrient media. However, the foam is thin and
therefore the resulting liquor contains lower amount of these
facilitates a better distribution of air into liquid. Synthetic
substances. On the other hand long and intensive cooking
alcohol is easy to produce in pure form and is highly water-
at higher temperature produces soft pulp having very
soluble. Its disadvantage is the presence microorganism
less lignin and hemicelluloses resulting in liquor of higher
inhibiting substances like crotonaldehyde. Methanol is
contents of these substances. The liquor further contains
also water-soluble and has similar properties like that
small amounts of volatile acids like acetic and formic acid.
of ethanol. In comparison with ethanol it has a higher
Further fermentation of sulfite liquor depends upon how
volatility and toxicity. Hydrocarbons-Microorganisms can
much carbohydrates are released during cooking and
utilize almost all hydrocarbons to larger or some smaller
pulping. The amount of carbohydrates varies according to
extent. Bacteria can assimilate all hydrocarbons while
the species of the pressed wood and technology of pulping.
yeasts can assimilate only some of them. The best carbon
The composition differs in liquors from conifers and from
sources among hydrocarbons are n-alkanes. Alkanes can
deciduous trees. The total carbohydrates in the coniferous
be divided in to several groups according to the number of
wood liquor usually include 30-40% mannose, 30-35%
carbon atoms they contain. These are: C1- C9 n-alkanes,
glucose, 10% galactose, 15% xylose, and 5-10% arabinose.
C10-C20 n-alkanes and C20 and higher n-alkanes. C1-C9
In deciduous trees, the liquor contains 50% xylose, 15-
n-alkanes-Methane is the most widely used alkane in this
20% arabinose, 10% methylpentoses, 10% mannose, and
group because it is readily available. It is assimilated by
about 10% uronic acids. In addition they contain higher
most of the bacteria but not readily by yeasts. An amount of
levels of furfural, which originates from pentoses during
1 Kg of methane yields 0.8 kg of dry cell matter. Due to their
cooking at high temperature and pressure. The furfural
yield and narrow range of possible sources the alkanes of
affects negatively on the growth of microorganisms. The
this group are not suitable for microbial industry. C10-C20
sulfite waste liquor cannot be used directly for fermentation
n-alkanes -The alkanes of this group are readily assimilated
in the form obtained from the cooker since 1) it contains
by almost all microorganisms including bacteria, yeasts,
a considerable amount of sulfur dioxide, which has to
actinomycetes and molds. The yield of cell mass per
be removed. 2) Has fluctuating pH values, which can be
consumed alkane of this group is 90-100%. C20 and higher
adjusted with calcium carbonate or calcium hydroxide. 3) it
alkanes-Assimilation of this group of alkanes is slower
may contain insignificant levels of assimilable nitrogen and
because alkanes of this group do not exist in liquid state at
phosphorus and these nutrients must therefore be added.
normal cultural temperatures.
Sulfite liquors are used especially for the production of
ethanol and production of fodder yeasts. Nitrogen sources
Acid wood hydrolysates: The resulting pulp after the Substances containing nitrogen in various forms may
sulfite waste liquor has been removed still contains a serve as sources of nitrogen for microbial growth. The
considerable amount of carbohydrates other than cellulose. range of nitrogen assimilation is much wider than the
These are removed by low concentrations of acids, which carbon assimilation. Many microorganisms, especially
hydrolyse hemicelluloses to soluble sugars; this process lower fungi, utilize nitrogen from inorganic nitrogen sources
is called as prehydrolysis. Prehydrolysis removes only like inorganic ammonium salts. Ammonium sulphate, the
hemicelluloses and not cellulose in contrast to the complete cheapest of these compounds is often used in combination
hydrolysis that cleaves all the polysaccharides including with ammonium hydroxide and gaseous ammonia for
cellulose. The prehydrolysis is carried out on deciduous continuous regulation of pH. Another very good source of
wood, cereals, rape or rice straw, and reed grass. The nitrogen is urea; since it decomposes at high temperature
straw prehydrolysates contain higher amount of pentoses is should be sterilized by filtration. Quality wise nitrogen
 Basic of Fermentation Technology

21
sources cannot match with the pure laboratory chemicals either dry or in the form of paste. The extract is prepared
because of the economic reasons. Although many by autolysis or plasmolysis of Baker’s or Brewer’s yeast. To
microorganisms assimilate inorganic nitrogen, they often remove bitter hop substances, Brewer’s yeast is washed at
grow faster in the presence of organic nitrogen sources. At pH 6.5 to 7.0 prior to treatment. Yeast cells must be kept
present many suitable nitrogen sources of plant and animal in a viable state during washing and filtration in order to
origin are available as waste products. prevent losses of cell components. Baker’s yeast does
not need washings. The autolysis is performed in a large
Inorganic and synthetic organic nitrogen sources
vessel under continuous stirring at a temperature above
Ammonium sulphate is readily soluble in water; the purest 75°C. The yeast is then transferred to a vessel containing
form used in microbial industry is technical ammonium sodium chloride solution, which induces cell plasmolysis.
sulphate that contains a minimum of 20.7% nitrogen. It is then filtered or centrifuged to separate cell walls and
The so-called coke and gas sulphates are considerably membranes. The liquid is rapidly concentrated at 37oC
contaminated with tar substances and are used only to prevent degradation of thermolabile substances and
exceptionally as substrates. Diammonium hydrogen also to prevent contamination. Drying the liquid form in an
phosphate, which is used especially in yeast production, is atomizer or spontaneously in vacuum makes the powered
a source of both nitrogen and phosphorus. Ammonia is used form of the yeast extract. Yeast extract is a mixture of amino
either in the form of 25% aqueous solution or in gaseous acids, peptides, water, soluble vitamins and carbohydrates.
form. Urea is used only exceptionally in microbial industry Soybean and Peanut flour-Soybean flour is an important
i.e. in the production of fine and high-grade products, due to source of nitrogen. Its most important constituents are
its relatively high price. proteins 44-47%, fat 5.5-6.0% and phosphorus 0.62-0.65%.
It is obtained by the extraction of oil from soybeans. Peanut
Natural sources of nitrogen flour is also obtained after the extraction of oil from peanuts.
Corn Steep Liquor-It is obtained as byproduct in the It’s important constituents are nitrogen, fat and phosphorus.
production of maize starch. It contains biologically active The composition of proteins is variable and depends on the
substances that differ qualitatively and quantitatively source of raw material. Both peanut and soybean flours
according to the quality of the corn used and its are used as substrates in the biosynthesis of antibiotics.
technological processing. These substances are released Peanut flour is suitable for the biosynthesis of Penicillin and
into the corn steep liquor during corn steeping and some Lysine while Soybean flour is suitable for the biosynthesis
microbial processes, especially lactic acid fermentation of Chlortetracycline. Peanut flour is not suitable for the
participating in the release, occur during steeping process. biosynthesis of Tetracyclines.
Moyer and Coghil first used corn steep liquor in the Antifoam agents
Penicillin fermentation in 1946 and that enhanced the yield
of antibiotic many folds. Corn steep liquor contains nitrogen Media when are rich in natural substances; foaming
substances, lactic acid, reducing substances and ashes occurs in the microbial processes. This foam poses
with many trace elements. An important criterion of the difficulties when it is uncontrolled. The outflow of a large
quality of corn steep liquor is the ratio of amino nitrogen to amount of foam from the fermentor may reduce the volume
total nitrogen, which should be about 0.4 i.e. approximately of the culture and may lead to contamination. Various
2% of amino nitrogen dry matter. This ratio indicates the antifoam devices and certain natural or synthetic antifoam
intensity of lactic fermentation during steeping. Another agents are employed to control foam in a fermentation
criterion is the content of amino acids, which should be system. The effect of these substances is based on their
higher than 20%. ability to reduce the surface tension of the liquids. The term
‘Antifoam’ is used for the substances that are added before
Potato liquor and fermented extracts from bran and oil the foaming actually occurs in fermentation while the term
seeds-Sometimes liquor from potato starch industry and ‘Defoam’ applies to a substance added afterwards to knock
fermented extracts from bran and oil seeds are used as down the foam. The natural antifoam agents used are soya,
substrates in microbial industry. Potato liquor is obtained rape, coconut, sunflower and mustard oils. Antifoam agents
as follows: The liquid formed by pressing potato pulp in of animal origin are tallow and deodorized fish fat. Mineral
starch production is subjected to sulphuriation and lactic oils may also be used. Another group of substances used
acid fermentation by Lactobacillus delbrueckii and carefully are alcohols. The most common compound of this group
dried. Though it contains many reducing substances yet is Octadecanol either pure or in combination with lard oil
it lacks amino acids like arginine, glutamic acid, histidine, or mineral oil. Specially designed antifoam agents are also
leucine, isoleucine, lysine, methionine, phenylalanine, available; among them are silicon oils. The silicon oils are
proline, threonine and valine. The fermented extract of usually used in water based emulsions containing 10%
bran and oil seeds is prepared as follows: Wheat bran and silicon oil. They are most effective in bacterial or yeast
ground tobacco seeds are mixed with water and a small cultures but are less effective in cultures of filamentous
amount of lactic acid and superphosphate is added. After six microorganisms. Since the microorganism does not
days of lactic acid fermentation at 50oC the liquid is filtered assimilate silicon oils, that is why silicon oils are very efficient
and evaporated in vacuum. Yeast extract-It is available
 Basic of Fermentation Technology

22
and a single dose at the start of the fermentation is usually or on top of the yogurt to be mixed by the consumer). 4) Soft
sufficient to prevent the formation of the foam. A similar frozen yogurt (Flavored and Unflavored). 5) Hard frozen
effect is exhibited by polyalcohol’s with molecular weight of yogurt (Flavored and Unflavored). 5) Frozen yogurt sticks.
2000 and alkylated glycols. Phosphorus and magnesium 6) Fluid yogurt drinks. 7) Fruit topped with yogurt 8) Spray
are important compounds of nutrient agar medium since dried yogurt products for confectionary, bakery and soups.
they participate in energy transduction, especially in
reactions mediated by ATP. Microorganisms need calcium, Yogurt ingredients
potassium, sulfur and sodium; therefore are added to the a. Dairy ingredients: The dairy ingredients for the
medium for microorganisms. Though trace elements such preparation of yogurt include whole, partially defatted milk,
as iron, cobalt, copper and zinc are indispensable, they are condensed skim milk, cream, and non-fat dry milk. All the
usually present as impurities in other components and in dairy ingredients should be of very high bacteriological
water. Enrichment by these trace elements is necessary only quality. Mastitis milk, rancid milk, partially fermented milk;
when analysis of synthetic media or raw materials indicate milk containing antibiotics and sanitizing chemical residues
their shortage. A major element in media is phosphorus, cannot be used for yogurt production. Since yogurt is a
which can be added both in organic and inorganic form. The manufactured product, it may have variations from the quality
compound most frequently used in industrial microbiology standards established by the marketing considerations.
is Diammonium hydrogen phosphate that also serves as Therefore it becomes necessary to standardize the milk
nitrogen source. Superphosphate is used in the production used in the yogurt production. The milk fat levels in yogurt
yeast; it is a mixture of calcium hydrogen phosphate and range from 1.0 -3.25%. Yogurt can be classified on the basis
calcium sulphate with variable content. Growth Factors and of fat present as: 1) Yogurt -contains minimum of 3.25% fat,
Vitamins Growth factors and vitamins are usually present 2) Low-fat yogurt -containing not less than 0.5% and not
in natural sources of carbon or nitrogen compounds. When more than 2.0% milk fat, 3) Non-fat yogurt -contains less
these natural substrates are used as the only source of than 0.5% milk fat. In all the categories of yogurt, a minimum
vitamins, it becomes sometimes necessary to add additional milk solid non-fat and minimum titrable acidity stipulated is
growth factors and vitamins in a pure form. Vitamins of the 8.25% and 0.5% respectively. In order to standardize the
B-complex category are necessary as growth factor in the milk solids non-fat, cream, partially skimmed milk, and skim
production of Baker’s yeast. The production of amino acids milk alone or in combination, concentrated skim milk, non-
by auxotrophic mutants requires the presence of certain fat dry milk, or other milk derived ingredients may be used.
vitamins; shortage or excess of these substances in the The milk-derived ingredients include casein, sodium and
medium affects the yield. Production of certain secondary calcium caseinates, whey; whey protein concentrates alone
substances is greatly increased when precursors of or in combination.
these substances are added into the medium e.g. when
phenylacetic acid or phenylacetamide is added into the Sweeteners
medium; biosynthesis of Penicillin G increases but when
Sucrose is the major sweetener used in yogurt
phenoxyacetic acid is added into the medium; biosynthesis
production. Sometimes corn sweeteners and honey may
of Penicillin V is enhanced.
also be used. The level of sucrose in yogurt mix appears to
Yogurt affect the production of lactic acid and flavor in the yogurt.
Sucrose concentrations above 4% inhibit the growth of S.
Yogurt is defined as the end product of a controlled thermophilus. Sucrose may be added in a dry, granulated,
fermentation of high solids whole milk with a symbiotic free-flowing, crystalline form or as a liquid of 670Brix.
mixture of Streptococcus thermophilus and Lactobacillus Commercial yogurt has an average of 4.06% lactose, 1.85%
bulgaricus in the ratio of 1:1 for the manufacture of high- galactose, 0.05% glucose and pH of 4.15. In frozen yogurt
grade yogurt. Among the various cultured dairy products it is 6% corn syrup solids are added. Non-nutritive sweeteners
unique because it is the only product in which acetaldehyde like calcium-saccharine, maltol, and sorbitol alone or in
in relatively high concentration is desired as an essential combination may be used for diabetics. Sometimes lactase
flavor component. Similar sour milk products are called as is added to break the lactose of milk.
‘Matzoon’ in Armenia, Matsoni in Georgia, Leben in Syria,
Egypt and Algeria and Matsurad or Jodda in Sycilly. Stabilizers

Forms of yogurt They produce smoothness, body texture, gel structure,

reduce wheying off or syneresis, and increase shelf life.
At present yogurt is sold and used in various forms in Stabilizers function through their ability to form gel structure
different parts of the world. These forms are as follows: 1) in water, thereby leaving less free water for syneresis. In
Plain Yogurt (Set type and Stirred type). 2) Flavored yogurt addition, some stabilizers perform their action by forming
without fruits (lemon or Vanilla). 3) Yogurt with fruit puree a complex with casein. A good stabilizer should not impart
(French style or Swiss style or Continental style -Whole or any flavor, should be effective at low pH values, and should
sliced fruits are mixed uniformly throughout the yogurt and be easily dispersed in the normal working temperatures in
Sundae style -Fruits may be filled in the bottom of the cup a dairy plant. The stabilizers generally used are gelatin,
 Basic of Fermentation Technology

23
vegetable gums like carboxy methylcellulose, locust of the system is depressed. Depletion of oxygen and the
bean, carob, guar and seaweed gums like alginates and availability of the formate ion stimulate the growth of rods.
carrageen an. Sometimes agar-agar or pectin is also used. Until the pH value reaches 4.2 Streptococci grow very
Calcium chloride is added to control whey separation. slowly. Below pH 4.2 Lactobacilli grow rapidly and dominate
the fermentation. The major portion of the lactic acid and
Fruit preparations for flavoring yogurt acetaldehyde necessary for the characteristic flavor of
Fruit preparations are present at the level of 15-20% yogurt is contributed by the lactobacilli but the second part
in the final product. Generally fruits are added to meet of the fermentation is greatly aided by the initial metabolic
the market demand. In every type of flavored yogurt, the activity of the coccus component. Yogurt should be cooled
composition of basic fruit preserve remains the same while at pH value of 4.2-4.3 to obtain a high-grade product.
its pouring styles are different. Fruit preserve consists of
Flavors of yogurt
55% sugar and a minimum of 45% fruit which is cooked
until the final soluble solid content is 68% or higher (65% Typical flavor of yogurt can only be detected in plain
in the case of certain fruits). Frozen fruits and juices are yogurt because it is a product of bacterial symbiosis. The
the usual raw materials. Commercial pectin, 150grade, is production of flavor depends upon the proportion of rods
normally utilized at a level of 0.5% in preserves and the pH and cocci and their combined metabolic activity. The
is adjusted to 3.0-3.5 with a food grade acid, viz. citric acid distinctive flavor is contributed by lactic acid (odorless) and
during manufacturing of the preserve. Calcium chloride and by acetic acid, which are volatile and have strong odors.
certain food grade phosphates are also used in several fruit Milk components and other ingredients in the mix also play
preparations. In Fruit on Bottom or Eastern Sundae style a role in providing a background flavor. To obtain a good
yogurt, 59ml fruit preserve on bottom is followed by 177ml flavor one should keep in mind that 1) starter strain should
of inoculated yogurt mix on the top. No flavor or sweetener be selected with a view to obtain a good symbiosis 2)
is added. It is incubated till pH becomes 4.2 and then proportion of rods to cocci should be equal 3) rapid cooling
refrigerated. In western Sundae style yogurt, the top yogurt should be done at pH 4.2. If the pH value falls below 4.2,
layer contains flavors and colors while in Swiss style yogurt, in large tanks that require a long time to cool down to 10oC
yogurt and mixed fruit layers are mixed together. then the desired cocci to rods ratio and flavor balance is
lost. The product so obtained will be too sour and coarse.
Flavors and colors
Characteristics of a good yogurt
Only certified flavors and colors should be used for
the preparation of yogurt. It should meet the following 1) The body of yogurt should possess a relatively high
requirements. It should 1) exhibit true color and flavor of viscosity and should be firm and cohesive enough to be
the fruit when blended with yogurt, 2) be easily dispersible removed and eaten with a spoon. 2) There should be very
in yogurt without causing texture defects, phase separation little wheying off during normal handling for mixing, cooling,
or syneresis. pumping and packaging. 3) It should have a smooth, rich
texture free from lumps, granules or graininess. 4) There
Microorganisms should be no gas packets, fissures or gassy effervescence.
The culture is specified as a mixture of Lactobacillus Factors affecting body characteristics of yogurt
bulgaricus and Streptococcus thermophilus in 1:1 ratio.
The inoculum culture can be regenerated from a lyophilized The factors are: 1) Fat and SNF concentration in the
culture tubes on media containing milk as a component. mix. 2) Stabilizers used. 3) Control over weighing and
blending of ingredients in the mix. 4) Heat treatment of the
Microbiology of yogurt mix 5) Concentration of protein by new process such as
ultrafiltration. 6) Concentration of calcium and magnesium
Streptococcus thermophilus is commonly referred as
ions. 7) Type of starter culture used 8) Incubation conditions
coccus and Lactobacillus bulgaricus as rod. Equal numbers
used. 9) Initial pH value of the mix. 10) Handling during pre
of both of these bacteria are desirable for the production
and post incubation operation. 11) Sucrose concentration
of tartness and green flavor of yogurt. The fermentation is
in the mix.
carried out within a temperature range of 35 -45°C, optimum
being 40°C. During fermentation, both rods and cocci work a. Defects and their causes in yogurt
together to produce yogurt. Rod shaped Lactobacillus
bulgaricus grows first slowly but their weak proteolytic activity 1) Graininess -If coagulum becomes exceedingly
liberates sufficient amount of the amino acids (glycine and firm before stirring, the finished yogurt tends to grainy. In
histidine) and peptides to stimulate cocci. Streptococcus addition, the use of rennet to obtain a good body invariably
thermophilus now grows vigorously to produce moderate leads to graininess. 2) Coarse Texture -Disturbance of the
amounts of lactic acid, acetic acid, acetaldehyde, diacetyl yogurt mix just before the gelling stage gives rise to coarse
and formic acid. When the pH of the yogurt mix (initial 6.3- texture. 3) Granular feeling in Mouth -Inadequate mixing
6.5) drops below 5.5, the rapid growth of cocci is arrested of the powdered products causes a granular feeling in the
and that of rods is favored. Oxidation-Reduction potential mouth. 4) Gas packets and Fissures -These are caused by
 Basic of Fermentation Technology

24
the trapped carbon dioxide or hydrogen gas produced by heating at such high temperature denatures the proteins of
contaminant flora like E. coli, Bacillus spp. and yeasts. 5) the milk that releases some peptides required for the growth
Slimy feeling in Mouth -Slime producing strains when used of bacteria. It is then cooled to 37-38°C and inoculated with
in starter culture would result in a mouth feeling similar to 5% milk starter culture. The incubation at 37-38°C for 18-
the white of eggs. 6) Off Taste and Off Odor -These develop 24 hours yields Acidophilus milk, which is quickly cooled to
due to faulty fermentation. 5-7°C and bottled when final acidity of the finished product
reaches 1.0%.
Kefir
Bulgaricus Butter Milk Or Bulgarian Milk
It is a historic and old product from Caucasus region of
Russia. Kefir grains are used as starter culture that can be It is prepared from cow or mare’s milk, which is fermented
reused several times. The kefir grains are gelatinous white by a pure culture starter of Lactobacillus bulgaricus. This
or cream-colored irregular granules from the size of wheat is particularly consumed in various parts of the world
grain to walnut. These are made up of a polysaccharide particularly Balkan countries. The starter produces the
called ‘Kefiran’, which is associated with denatured milk required acidity and flavor. It can also be prepared from cow
protein. These are insoluble in water and swell up when or buffalo’s skimmed milk.
soaked in water to form a jelly like product. Bacteria and
yeasts are present within the folds of the grains and there Method of preparation
may be some symbiotic relationship between the grains The milk is heated at 95°C for 30 minutes and cooled
and microorganisms. Various kinds of microorganisms to 37-38°C. It is then inoculated with 2% milk starter
are involved in the kefir fermentation these include: culture prepared with Lactobacillus bulgaricus. The milk
Saccharomyces kefir, Torula kefir, Lactobacilli, Streptococci, is incubated until the acidity reaches 1.4%. The product is
and Leuconostoc spp. Coliforms, micrococci and spore cooled to 7°C. After incubation a curd like smooth mass is
forming rods contaminate the fermentation. formed that is diluted and churned. The butter is removed.
Buttermilk left has its acidity in terms of lactic acid (0.25%).
Method of preparation
The final product lacks aroma but has a pleasant flavor. It
Fresh cow’s milk is pasteurized at 85 °C for 30 minutes. can be used as a substituent for milk but it has very less
It is inoculated with kefir grains taken from the previous sugar content. It has diuretic effect when taken in large
batch after cooling to 25 °C. The incubation at 23 °C yields a quantities (Flow Chart 3).
soft curd, which after being agitated forms a beer like foam
and fuzziness from which kefir grains come upward with Role of heat treatment at high temperature in
the evolution of carbon dioxide. Kefir grains are separated culture dairy products
and washed in cold water. They are stored in cold water Heat treatment at 85-95 °C for 30 minutes or equivalent
at 4 °C or are dried in a warm oven. The kefir grains have is an important step in the manufacture of cultured dairy
alcoholic, yeasty sour with tangy effervescent flavor. Fresh products. The heat treatment 1) produces a relatively
kefir grains have activity up to 8-10 days while dried grains sterile medium for the exclusive growth of starter culture. 2)
may remain active for 8-12 months. They contain lactic acid Removes air from the medium to produce more conducive
0.8%, alcohol 1%, carbon dioxide and flavor compounds medium for microaerophilic lactic cultures to grow. 3)
(acetaldehydes, diacetyl and acetone). Effects thermal breakdown of milk constituents, especially
proteins, releasing peptones, sulfhydryl groups which
Acidophilus Milk or Reform Yogurt
provide nutrition and anaerobic conditions for the starter. 4)
It is a product formed by fermenting milk with an authentic denatures and coagulates milk albumins and globulins that
culture of Lactobacillus acidophilus. The organism has its enhance the viscosity and produce custard like consistency
unique features that it can survive in the severe conditions of in the product.
intestinal tract of human beings, animals and birds. Its ability
to initiate growth in, or form colonies on media containing Flavor production in cultured dairy products
bile salts should be used as a distinguishing characteristic. The characteristic flavor of cultured dairy products
The strains of man and animals have certain differences is produced by the activity of lactic culture by metabolic
like DNA of human isolates generally had lower G+C ratio transformation of milk constituents. An important
than that of pig and chicken biotypes. The acidophilus milk transformation of milk components into an essential flavor
has its therapeutic value in controlling intestinal disorders compound in cultured dairy products involves citrate
but its mode of action has not yet been elucidated. metabolism. Milk contains an average of 0.2% citrate
and it exhibits the greatest seasonal fluctuation (0.07-
Method of preparation
0.4%). Citrate is converted into ‘Diacetyl’ by flavor bacteria
The acidophilus milk is an extremely sour product. Leuconostoc cremoris and Streptococcus lactis sub sp.
The finished product contains very little, if any metabolic Diacetylactis. Leuconostocs ferment citrate only when there
byproduct other than lactic acid. It is prepared from partially is sufficient acid development during the fermentation.
skimmed milk, which is heated at 120°C for 15 minutes. The Because leuconostocs do not produce much acid from the
 Basic of Fermentation Technology

25
lactose of the milk, therefore require an associative growth acetic acid and propionic acid. In koumiss and Kefir alcohol
with lactic acid producing strain. Streptococcus lactis sub is an important component characteristic of the product.
sp. diacetylactis on the other hand produces sufficient Torula kefir yeast and Saccharomyces kefir are responsible
lactic acid, which required for the fermentation of citrate to for the production of alcohol. One very important compound
produce diacetyls. The literature contains conflicting reports causing flavor problems in cultured milks is 3-methylbutanal.
on the biosynthetic pathways involved for the production of This imparts a malty flavor to cultured milks and ripened
diacetyls in aroma bacteria. Diacetyl synthesized by flavor cream butter. Carbon dioxide plays an essential role in the
bacteria in dairy products does not accumulate indefinitely. flavor impact of cultured buttermilk, kefir and koumiss. The
Once the concentration of diacetyl precursor, citrate falls gas entrapped in the thickened milk provides lift, fizz, or
below a critical value, the diketone is rapidly converted into effervescence to these cultured products. Carbon dioxide is
a flavorless compound, Acetone. An enzyme called diacetyl derived from lactose by heterolactic bacteria. Fermentation
reductase is widely distributed among flavor bacteria and of citrate by aroma bacteria also produces considerable
other contaminating psychotropic bacteria commonly found amount of carbon dioxide. The minor metabolic products,
in dairy environment, catalyzes conversion of diacetyl although found in small or even trace amounts, may be
into acetone. Acetaldehyde is another important flavor important in maintaining a desirable flavor balance in
compound but in cultured creamy butter, cultured buttermilk cultured dairy products. Any shift in the flavor balance in
and cultured sour cream it is undesirable because it imparts cultured dairy products may result in the organoleptic
a flavor defect referred to as green or yogurt flavor. It is a very perception of off-flavor. Rancid flavor present in milk is
important flavor component in yogurt and related products. It derived from straight chain free fatty acids up to 18-carbons
is primarily derived from lactose although other mechanisms by the action of lipase enzyme. Cream flavor is derived
for production of carbonyl residue are found among lactic from isomeric 18-carbon unsaturated fatty acids by auto
acid bacteria. For example, metabolism of threonine and oxidation, 4-cis-Heptenol is produced. Butter flavor is due to
deoxyribonucleic acid results in acetaldehyde formation by β -lactones derived from hydroxy fatty acids by ring closure.
bacteria. Many bacteria produce acetaldehyde; major ones Sharp sour flavor is produced by the fermentation of lactose
are streptococcus lactis subsp. diacetylactis, Streptococcus to lactic acid by starter culture. Aldehydes, ketones, alcohols
thermophilus, Lactobacillus bulgaricus. Lactic Streptococci and esters combined to give milk a cowy flavor. Acetic acid
also produce a variety of carbonyl compounds that impart contributes significant culture flavors. (Flow chart 4, 5)
flavor important ones are: volatile fatty acids, formic acid,

Flow Chart 3
 Basic of Fermentation Technology

26

Flow Chart 4
 Basic of Fermentation Technology

27

Flow Chart 5

Starter cultures acid from lactose, citric acid from various compounds
or are involved in the fermentation of sugars to produce
Cultures used to start the fermentation in the preparation alcohols, diacetyls, or other flavor and aroma compounds.
of butter, ripening of cream, souring of milk in cheese The microorganisms of the starters may grow in association
manufacture; preparation of various fermented milks and or in succession to produce the desired product. Various
preparation of fermented cereal products are called “Starter purposes for which they may be used are:
Cultures”. They contain microorganisms that produce lactic
 Basic of Fermentation Technology

28
Aroma production milk product. The samples of dahi from south India
showed predominance of Lactobacilli. But samples of dahi
Aroma producing microorganism is Streptococcus lactis from North India showed predominance of Streptococci. The
that usually grows in fermented milk products. It produces species of lactic acid bacteria occurring most commonly in
lactic acid from lactose and also ferments citric acid to dahi include L. bulgaricus, Streptococcus thermophilus, S.
produce acetic acid, carbon dioxide, acetyl methyl carbinol, faecalis, S. lactis, L. casei and L. plantarum, in order of their
diacetyls and 2,3-butylene glycol. frequency.
Butter production Soy sauce: Soy sauce is a dark brown liquid with a salty
During butter ripening Streptococcus lactis produces taste and distinct aroma, which is made by
lactic acid, volatile acids as acetic acid, propionic acid and fermenting soybeans, wheat, and salt with a mixture
carbon dioxide. Some neutral 4-carbon compounds like of mold, yeast and bacteria. It is a seasoning agent used
diacetyls, acetyl methyl carbinols and 2,3-butylene glycol as a substitute for salt in the preparation of food as well
add to the flavor and aroma. as a table condiment. It enhances the flavor of the food
Batter fermentation and adds color into meats, sea foods, vegetables etc.
The fermentation of soy sauce is essentially a process of
In case of idli, dosa, jalebi, vada and bhatura, culture of enzymatic hydrolysis of proteins, carbohydrates and other
the previous batch is added as starter culture that starts the constituents of soybeans and wheat to peptides, amino
fermentation. The starter causes leavening and acidification acids, sugars, alcohols acids and other low-molecular
of the batter that produces a pleasant flavor in the batter. compounds by the enzymes of microorganisms. The
brewing of soy sauce originated in China many centuries
Preparation of fermented milk products ago and later was introduced in Japan and other oriental
In fermented milk products preparation, the flavor and countries. Soy sauce is consumed in almost every oriental
body is so related with the starter that without good starter country and has different names in different countries. It
culture, the product is sure to become unsatisfactory. is known as Chiang-yu in China, Shoyu in Japan, Kecap
in Indonesia, Kangjang in Korea, Toyo in Philippines and
Dahi or curd See-iew in Thailand. Japan leads the soy sauce industry
Dahi is the most important sour milk product used in the world. It has not only the largest fermentation plants
throughout India. Dahi is the common term used in India for but also has the most advanced technology. Japanese
any kind of sour milk. But sour milk from different parts of shoyu is produced primarily in the areas near Tokyo and in
India varies very much in quality, consistency and microflora. Chiba prefecture. Japanese shoyu is mainly of three types:
Dahi of Northern India is much thicker than in Southern India Koikuchi, Usukuchi and Tamari. Nearly 90% of the Japanese
where dahi is generally used in diluted form. In some parts shoyu is of Koikuchi type, with dark reddish brown color
of India Cane sugar is added to milk to prepare sweet dahi. and strong flavor and made from a nearly equal mixture
Sweet dahi is liked very much in Northern parts of India. of soybeans and wheat using Aspergillus oryzae hydrolytic
Dahi sold in the market is prepared from fresh raw milk, enzymes followed by vigorous lactic acid and alcoholic
but it is advisable to prepare dahi from boiled milk. Dahi yeast fermentations. It is pasteurized at a relatively high
prepared from fresh raw milk is fermented by natural flora temperature. Ten percent of shoyu is Usukuchi type, light in
i.e. no starter is added. When it is prepared from boiled milk, color with maximum total nitrogen content 1.2%. Third type
a little portion of dahi from the previous day is added and of shoyu is Tamari in which major portion is soybean and
milk is allowed to curdle at room temperature. As dahi is smaller portion is wheat. The highest quality shoyu is made
prepared without special care regarding its mother starter, by fermentation but second and third grades contain some
its quality varies from place to place and is never uniform. portion of chemical hydrolysates.

Method of preparation of dahi: Dahi of good quality Preparation of soy sauce: Soy sauce is prepared by
should be prepared in the following way: Good fermentation of a mixture of soybeans and

quality whole milk or skim milk may be used for the Cereal usually wheat and salt. However, in recent years,
purpose. Milk should be heated to 85 to 95 °C for half an defatted soybean meals and flakes have taken place. Today,
hour or boiled for 10 minutes. It is then cooled to 45 °C. A more than 90% of soy sauce production in Japan is from
small portion of the starter from good quality dahi is then defatted soybean products. In addition to the fermentation
inoculated into the milk. The amount of starter added will process, a chemical process in which acid hydrolyzes
vary with the type of the starter, condition of the milk and the proteins and carbohydrates is also being used in
incubation period. The milk is then incubated at temperature some western countries. In this method acid hydrolysis
near about 40 0C. After 24 hours of incubation dahi of good usually results in a complete breakdown of the substrate
quality may be obtained. than enzyme hydrolysis. However, acid hydrolysis cannot
perform many other specific reactions or interactions of
Microflora of dahi: Very little information is available hydrolyzed products as carried out by the multiple enzyme
regarding the microflora of this important system produced by molds, yeasts and bacteria. That is
 Basic of Fermentation Technology

29
why chemically hydrolyzed product does not possess the adding a koji starter culture called “Tane Koji” in Japan in
flavor and odor of the soy sauce prepared by fermentation cooked cereals and /or soybean substrate. Different types
process. of tane koji (soy sauce koji, miso koji) are available for
commercial use in making soy sauce, miso and others.
Treatments of the raw materials
Preparation of tane koji: Tane koji is prepared by using
Soaking soybeans: In preparation for fermentation, naturally selected or mutant strains of Aspergillus oryzae
selected soybeans are cleaned by ashing and soaked for or A. soyae to give desirable starter for a particular
10-15 hours at room temperature. The water is changed fermentation. Although strains used for preparation of koji
every few hours to prevent acidification by bacteria. Weight starter are different, the method for preparation is similar.
of beans should increase 2.1-2.5 times during soaking. Polished rice is soaked in water overnight, drained, steamed
Cooking soybeans: Hydrated soybeans are cooked for 1 for 1 hour and mixed thoroughly with 2% wood ash as a
hour in steam at 10-14 lb/sq in. ina rotary cooker (capacity 1 source of trace elements. The mixture is then inoculated
ton). They are then cooled rapidly. When defatted soybean with spores of selected strains of A. oryzae and spread out
meals or flakes are used, they are first moistened by on trays in layers approximately 1.5 cm deep. And covered
spraying with water amounting to about 130% of soybean with a moistened cloth to favor the growth of mold mycelium.
weight and then are steamed at 13lb/sq in. for 45 minutes. After incubation at 30 °C for 5 days, the rice is well covered
with mycelium and with green to yellowish green spores of
Roasting wheat: Whole wheat or wheat flour is essential A. oryzae. The spores are harvested, dried at 50 °C and
for production of typical Japanese stored at 15 °C. A koji starter is usually composed of a blend
of spores of different strains in a definite proportion, so that
shoyu flavor. Usually low protein flour is used. Wheat is
various enzymes are produced in proper amounts during
roasted in sand for several minutes at 170-180 °C and then
the preparation of koji.
the grains are crushed into 4-5 pieces in a roller mill. The
roasting process adds flavor and color to the resulting soy Preparation of soy sauce koji: Soy sauce koji is prepared
sauce and in addition destroys surface microorganisms and from a mixture of roasted wheat and steamed soybeans
facilitates enzymatic hydrolysis. inoculated with a koji starter (soy sauce Tane Koji) consisting
of selected strains of Aspergillus oryzae grown on polished
Effect of addition of wheat to the fermentation mixture:
rice. Inoculated soybean and wheat mixture is placed in
According to Yokotsuka (1964),
wooden or stainless steel porous trays of depth 30-40 cm
the addition of wheat to the fermentation mixture and several meters in length and width. The trays are then
serves several functions. Firstly, the mold grows better incubated at 25-35 °C. Aeration and moisture is carefully
and produces more enzymes on a mixture of wheat and controlled. This step is completed in 45 hours because this
soybeans than on wheat or soybeans alone. Secondly, the prevents the development of Mucor spp. and bacteria and
addition of roasted, crushed wheat to the cooked soybeans enhances the development of proteolytic enzymes. The end
would minimize the growth of undesirable bacteria i.e. product is called Soy sauce koji, which is a mixture of fungal
moisture of cooked soybeans is 60%, which is ideal for hydrolytic enzymes on soybean wheat mixture substrate.
bacterial growth whereas moisture of soybeans and Soy sauce koji of superior quality has a dark green color,
wheat mixture (1:1) is about 45%, which is adequate for pleasant aroma, sweet but bitter taste and high protease
mold growth but not for bacteria. Thirdly, wheat serves as and amylase activity.
a precursor of sugars, alcohols, organic acids, and flavor
Fermentation
compounds. Lastly, wheat is rich in glutamic acid.
The soy sauce koji is mixed with 1.2-1.5 volumes of
Addition of salt: Commercial salt is generally preferred
salt brine (23% w/v salt) to make mash called “Moromi”
for making soy sauce because it may carry an inoculum
in Japan. Moromi is fermented in large concrete tanks or
of halophilic and halotolerant bacteria and yeasts. Salt is
wooden vats for 8-12 months. Since koji is not prepared
added in such quantities that it prevents spoilage and/or
under aseptic conditions, one would expect the presence
food poisoning bacteria and permits the development of
of yeasts and bacteria in moromi. However pure cultures of
flavor and aroma forming bacteria and yeasts.
Pediococcus soyae, Saccharomyces rouxii and Torulopsis
Role of salt: In addition to giving a salty taste, sodium spp. are added to the mash at the start of fermentation and
chloride acts as a preservative and also at one month after the start of fermentation to accelerate
has a selective action on microorganisms that grow in the the fermentation and to improve the flavor of the final
fermentation substrate. product. High salt content ensures the development of
flavor enhancing yeasts while lower brine to koji ration
Koji and tane koji result in decreased utilization of total nitrogen. Traditional
Koji is a Japanese name given to a preparation consisting fermentation starts in April and takes a year to complete.
of mold growth on cooked cereals and/or soybeans. Koji In general low temperature fermentation gives better
serves as an enzyme source for converting complex plant results, because the rate of enzyme inactivation is slow and
constituents to simpler compounds. Koji is prepared by enzymes remain active for a longer time.
 Basic of Fermentation Technology

30
Pressing: The matured moromi is pressed in a hydraulic used for the fermentation of oriental foods, have shown
press at 100kg/sq cm. (1379lb) for 2-3days into a liquid that they do not produce Toxins. But, on the other hand
part, known as raw soy sauce, and a solid cake. When they resist accumulation of certain toxins which otherwise
whole soybeans are used as raw materials, soy sauce will be produced by other microorganisms in the food. This
oil, consisting chiefly of ethyl esters of higher fatty acids, could be considered as a protection of the product against
is produced during fermentation and appears at the upper other harmful microorganisms. A good example of such a
layer of raw soy sauce. This oily layer must be removed protective role is demonstrated by Rhizopus oligosporus,
and has no potential use. This is one of the reasons that the mold species used for the production of Tempeh. This
defatted soybean meal is used instead of whole soybeans mold species does not produce aflatoxins. On the contrary,
as raw materials in soy sauce fermentation. if aflatoxins are already present in the growth substrate,
R.oligosporus could lower its contents to about 40% of its
Pasteurization and bottling: The raw soy sauce liquid
original content. In addition it was found that R. oligosporus
is pasteurized at 70-80 °C in a kettle or heat exchanger,
inhibits growth, sporulation, and aflatoxin production by
cooled, filtered to remove precipitates and stored. The
Aspergillus flavus.
final product is bottled for market. The bottles are usually
made of plastic or glass and sometimes benzoic acid or Mold starters
propyl or butyl ester of p-hydroxy benzoic acid is added as
preservative. The mold species that are traditionally used for
fermentation of foods in different parts of the world
belong to different genera. Species of Rhizopus, Mucor,
Benefits of pasteurization
and Aspergillus are used for the fermentation of foods
1) Flavor, color and clarity is produced by the removal throughout the orient, with the exception of Japan. In
of oil particles mixed with heat coaguable substances 2) Japan, it is restricted to species of Aspergillus including A.
Inactivation of enzymes occur, which gives a stable product oryzae and A. soyae. These species are used in Tane koji,
3) Concentration of compounds like aldehydes, acetals, which is used as starter culture for the preparation of soy
phenolic compounds, mercaptans, organic acids, pyrazines, sauce, miso etc. Tane koji is prepared by growing the mold
furfurals, and β-diketones is increased 4) Because of on steamed rice. In other Asian countries, Ragi type starter
the increase in phenolic compounds and organic acids cultures are in common use. Cultivating molds on cakes
resistance to spoilage by film forming yeasts occur. made of rice or wheat flour, which has not been steamed or
cooked, makes these starters. The difference in preparing
Role of Mold in foods fermented by molds growth substrates for manufacture of two types of inocula
Synthesis of enzymes: Molds, in these food fermentations is thought to be the cause of a natural selection of mold
synthesize enzymes that decompose complex compounds, species which are developing in each of the type starters
including proteins, carbohydrates and fats into smaller over many centuries, when they were produced with non-
molecules. At the same time, other compounds may be aseptic, traditional methods.
synthesized from the food substrates. These complex
Yeast fermentations
changes are accompanied by changes in the original
properties of the raw materials. Taste, flavor, texture, When yeasts are involved in the fermentation process,
color, palatability and other properties of the raw materials production of alcohols improves the aroma of the product.
are usually modified in such a way that product becomes In addition, alcohol at a certain concentration makes the
more attractive to the consumer. In addition to this general substrate unsuitable for microorganisms, which may create
function of producing enzyme, in certain products the mold undesirable properties in the product. Combined with the
has a special role to play. organic acids that are produced by lactic acid bacteria, the
inhibitory effect of alcohol on undesirable microorganisms is
Mold growth: Mold growth on certain products contributes
increased. (Flow Chart 6)
to the appearance of the food, which is desired by the
consumer. Neurospora spp. provides oncom (fermented Miso
food) cake with a coating of its pink orange colored and
powdery conidia. Rhizopus oligosporus covers tempeh cake Miso is a paste like product made by fermenting cereals,
with a clean white mycelium surface layer and additionally soybeans and salt with molds, yeasts and bacteria in
has the function of binding together the soybean into a Japan. This product is generally known as Bean Paste. It
solid, compact cake. has the consistency of peanut butter, some smooth and
some chunky and its color varies from light yellow to reddish
Synthesis of coloring compounds: The function of brown. It has distinctive pleasant aroma resembling that of
Monascus purpureus during fermentation of Angkak soy sauce, and it is typically salty (degree of saltiness may
(fermented food) is the production of red colored compound vary) and may sometimes has sweet taste. It is like soy
monascorubin and a yellow pigment monascoflavin in sauce is used as a flavoring agent in cooking as well as
soaked rice. table condiment and can be used in place of soy sauce. The
Protection of the product: Molds, which are traditionally product is known as Chiang in China, Doenjang in Korea,
 Basic of Fermentation Technology

31
Tao-Chico in Thailand, and Tauco in Indonesia. All mean also be made according to the length of fermentation and
bean paste. In Japan, there are many types of miso and fermentation temperature. This can be understood from the
they can be prepared by varying the ratios of soybeans to following table: (Table 1)
cereals, salt content, length of fermentation and addition of
other ingredients such as hot pepper, which is very popular General method of preparation of Japanese rice
in China and Korea. miso
Cooking Soybeans: Whole soybeans are generally
used for the preparation of miso, but sometimes dehulled
soybeans or full fat soybean grits are also used for making
white or yellow rice miso. Soybeans used should be of
large size because the ratio of hull to cotyledons is lower in
large sized beans. They should have high water absorbing
capacity and when cooked under described conditions,
the beans should be homogeneously soft with fine texture
and bright color. To prepare the whole soybeans for
fermentations, they are washed, soaked in water for about
20 hours at 16 °C and drained. The soaked beans are then
cooked in water (white miso) or steamed at a temperature
11 °C for about 20 minutes in a closed cooker and slightly
mashed Table 2.

Preparation of rice miso koji

For Rice miso koji preparation, polished rice is used.
Since it is essential that the mold mycelium quickly
penetrates the rice kernels, the rice is soaked in water
(15°C) overnight or until moisture content is about 35%.
Excess water is drained off, and the soaked rice is steamed
at atmospheric pressure for 40 minutes. After cooling to
35°C, tane koji (koji starter culture), which is a blended
mixture of several different strains of A. oryzae, prepared as
described in the soy sauce preparation, is sprayed over the
rice and mixed well. The inoculated rice is then incubated
in a temperature and humidity controlled room. In about
40 -48 hours at 30 -35°C. The rice is completely covered
with white mycelium of the inoculate culture. Harvesting
is done while koji is white and before any sporulation has
occurred. At this time, koji has a pleasant odor, lacks any
musty or moldy odors, and is quite sweet in taste. The koji
is removed from the fermentor and mixed well with salt to
stop any further development of the mold.

Yeast and bacterial fermentation

Next fermentation is carried out under anaerobic
conditions by yeast and bacteria. Cooked and slightly
mashed soybeans are mixed with the salted koji and
inoculated with a starter culture containing pure culture of
Yeasts (Saccharomyces rouxii, Torulopsis spp.) and bacteria
(Pediococcus halophilus, Sterptococcus faecalis). Miso
Flow Chart 6 from the previous batch can also be used as an inoculum
Japanese miso can be categorized into three major of yeast and bacteria. Sufficient water is added to bring the
groups as follows: moisture content equal to 48%. The mixture, now known
as Green Miso, is thoroughly blended and tightly packed
1) Rice miso -it is prepared from rice, soybeans, and salt into a vat or tank for fermentation at 25 – 30 °C. During
2) Barley miso -it is prepared from barley, soybeans and the fermentation period, the green miso is transferred from
salt 3) Soybean miso -it is prepared from soybeans and one vat to another at least twice to improve fermentation
salt. Each group can further be subdivided into white, light conditions. Fermentation time varies widely depending
yellow, red according to color, and sweet, medium salty, upon the type of miso. It is one week for white miso, 1 -3
and salty according to taste. Similar classifications can months for salty miso and one year for soybean miso.
 Basic of Fermentation Technology

32
Table 1

Soybean : Rice : Salt Type Color Taste Fermentation Time Fermentation Temperature

100 : 200 : 35 White miso Bright light yellow Sweet 2-4 days 50 °C

100 : 60 : 45 Salty miso Light yellow Salty 30 days 30 –35 °C

100 : 50 : 48 Red salty miso Yellow red Salty 60days 30-35 °C

Table 2

Protein Isoelectric point Protein Isoelectric point

Egg albumin ~ 4.6 γ1-Globulin 6.6

Haemoglobin 6.8 Lysozyme 11.0

Pepsin ~ 1.0 Myoglobin 7.0

Serum Albumin 4.9 Chymotrypsinogen 9.5

β- Lactoglobulin 5.2 -- --

Aging and packaging soybeans that most people find unpleasant. Because of its
high protein content and universally acceptable taste and
At the end of fermentation, the fermented mass is kept texture, it can be a potential source of low -cost proteins.
at room temperature for about 2 weeks to ripen. The aged
product is then blended mashed, pasteurized (60 – 70°C Preparation of tempeh
for 30 minutes), and packaged. Traditionally, miso is sold in
wooden kegs of various sizes. Presently, it is sold in sealed Microorganism: The mold used for tempeh fermentation
polythene bags or tubes. For packaging into the plastic was reported earlier to be Rhizopus oryzaebut in Indonesia,
bags, miso must first be pasteurized at 60 –70°C for 30 a strain identified as Rhizopus oligosporus saito NRRL 2710
minutes to prevent swelling. Sorbic acid or its potassium is considered better producer of a good product. This strain
salt is also added at a level of less than 1 gm/kg of miso is characterized by sporangiospores showing no striations
(Flow Chart 7). and being very irregular in shape under any condition of
growth. The sporangiospores are short unbranched and
Dehydrated miso powder arise opposite to rhizoids that are much reduced in length
and branching. It’s all isolates also show Chlamydospores.
This product has become increasingly popular. The Rhizopus oligosporus is highly proteolytic, which is important
dehydration is carried out by freeze-drying process. in tempeh fermentation because of the high protein content
Dehydration process does not affect the flavor of the of the substrate. Two proteolytic enzyme systems were
product. It has its potential use as an ingredient in the observed in the fungus; one has an optimum pH at 3.0 while
instant mix products. (Flow chart 7) the other at 5.5. Both the enzyme systems have maximum
activities at 50 – 55 °C and are fairly stable at pH 3 -6.
Tempeh They rapidly denature at pH below 2 or above 7. In addition
Tempeh or Tempe kedelee is a cake -like product to high protease activity, the mold possesses strong lipase
that originated in Indonesia and is widely consumed in activity, low amylase and no detectable pectinase activity.
the regions of Malaysia and Indonesia. It is prepared by Preparation of soybeans for fermentation: The Full
fermenting dehulled and briefly cooked soybeans with a -fat soybeans are soaked in water overnight at room
mold, Rhizopus; the mycelia bind the soybean cotyledons temperature. The soybean cotyledons can be mechanically
together in a firm cake. The raw tempeh has a clean, fresh, cracked into 4-5 pieces, so that they absorb water easily
and yeasty odor. When sliced and deep -fat fried, it has a and this also reduces the soaking time from 20 hours to 30
nutty flavor, pleasant aroma, and texture that are familiar minutes. They are dehulled by hand or by a simple roller
and highly acceptable to almost all people around the mill. The grits of beans are boiled in water for 30 minutes.
world. Unlike most of other fermented soybean products They are then drained and spread to cool and surface
that are used as flavoring agents or relishes, tempeh is drying.
used as a main dish and meat substitute in Indonesia. It
is easy to cook and does not possess the beany flavor of
Basic of Fermentation Technology

33
Fermentation
A pure culture of R. oligosporus spore suspension
(prepared by adding a few milliliters of sterilized distilled
water to the slant culture) already grown on potato dextrose
agar and incubated at 28 °C for 5-7 days is mixed with
the cooled and surface dried soybeans at the rate 106
spores/100gm of cooked soybeans. The inoculated
soybeans are tightly packed into an appropriate container
(petri dish) for incubation to obtain a final product in which
a white mycelium developed abundantly but black spores
are minimal. Rhizopus oligosporus like many molds require
air to grow, but it does not require as much aeration as
many other molds. In fact, too much aeration causes
spore formation and may dry the beans resulting in poor
growth. Therefore, it is important to properly pack the
beans for fermentation. Many workers have successfully
tried different types of packing materials. These are petri
plates, aluminum foils or metal trays, with perforated or
woven mesh bottoms and covers or perforated plastic
film covers or perforated plastic bags and tubings. When
the fermentation is complete, the beans are covered with
and bound together by white mycelia. Thus raw tempeh
looks like a firm white cake and has an attractive and
slightly yeasty odor. Prolonged fermentation often causes
the product to become obnoxious due to the enzymatic
breakdown of proteins (Flow Chart 8).

Flow Chart 8

Flow Chart 7
 Basic of Fermentation Technology

34
Idli Soaking
Idli is a breakfast food in most parts of India especially Generally, the ingredients are soaked separately in water
popular in southern India. It is acidified and leavened at room temperature for 5 to 10 hours before grinding to
through fermentation by hetero fermentative acid bacteria prepare the batter. Parboiled rice semolina can frequently
rather than by the activity of the yeast. It is closely related be used while dry black gram dhal flour has been found
to sourdough bread of the western world, but it does not unsuitable for the preparation of idli. If the idli batter is to
depend upon wheat or rye as a source of protein to retain be made without inoculation, it is essential that cereal and
the carbon dioxide gas during leavening. The importance legume be soaked, ground with water and incubated at
of idli lies in 1) its high degree of acceptability as a food room temperature. Hot soak or hot grind will destroy the
2) its protection against food poisoning and transmission organisms essential for fermentation and, unless they are
of pathogenic microorganisms, because of acidity and 3) replaced by an inoculum, the fermentation will not proceed
the fact that idli fermentation can be used in many parts properly.
of the world using various combinations of cereal grains
and legumes to produce acid, leavened bread, or pancake
Proportion of water and salt to other ingredients
like products 4) No wheat or rye flour is needed. Idli is a The amount of water added to the rice and dhal batter
small, white, acid leavened, and steamed cake made by the has varied from 1.5 to 2.2 times the dry weight of the
bacterial fermentation of a thick batter made from carefully ingredients. Batter should be rather thick for idli. Generally
washed rice (Oryza sativa) and dehulled black gram dhal salt 0.8 to 1% is added to the batter as a seasoning before
Phaseolus mungo. Idli cakes are soft, moist and spongy fermentation.
and have a desirable sour flavor. It is served like a pancake
with butter, honey, and jam or with other sauces. It can also Fermentation time
be consumed directly “out-of-hand” following steaming or Fermentation time varies from 14-24 hours, with overnight
the cake may be deliciously flavored with fried mustard being the traditional time interval for the preparation of idli.
seeds and chopped coriander leaves. The unflavored cakes The fermentation time must be sufficient to allow a definite
are eaten with chutney and /or samber, a thin-spiced soup leavening of the batter and allow for the development of
of dhal and vegetables. pleasant acid flavor.
Details of manufacture of Idli Inoculum
Ingredients: Idli preparation contains a number of different Ordinarily, the microorganisms developing during initial
ingredients these are 1) Rice 2) Black gram dhal 3) Salt 4) soak and then during the overnight fermentation are
water. Dehulled soybeans or Bengal gram can be used as a sufficient to leaven idli. The Central food technological
substitute for black gram dhal and a number of cereal grains Research Institute recommends adding one tablespoonful
can replace rice. However, there may be marked change of buttermilk to each pound of its dry idli mix. The
in the texture and flavor when using substituted materials. microorganisms that develop during overnight soaking
It has been reported that rice variety and its physical are: Leuconostoc mesenteroides, Streptococcus faecalis,
characteristics are very important to produce a good quality Pediococcus cerevisiae, Lactobacillus fermentum,
idli. White Kar and IR20 varieties of rice have given much Torulopsis candida, Trichosporon pullutans, Torulopsis
better performance in the production of idli, especially the holmii etc.
White Kar variety because of its high amylose content, low
amylopectin content better gelatinization, and better water Incubation temperature
uptake ability.
Ordinarily, the idli fermentation is carried out at room
Proportion of cereal to legume temperature. It generally means that temperature of 25 °C
to 30 °C is probably an optimum temperature.
Ordinary idli consists of three parts rice and one part
black gram dhal plus salt to taste. Kancheepuram idli is Steaming
prepared from one part rice and one part black gram dhal
The fermented batter is steamed as soon as the product
plus cashew nuts, ghee, salt, pepper, ginger and cumin
has become leavened and acidified for 15-30 minutes at
added to taste. Normally proportions of rice to black gram
atmospheric pressure to get a soft, spongy and sour tasted
dhal varies from 4:1 to 1:4, the 2:1 being the best. It has
and flavored idli. The fermented batter is poured into the
been seen that when black gram dhal proportion is less
cups of an idli steamer, which is placed in a covered pan
than 25%, the steamed idli was hard and organoleptically
and steamed until the starch is gelatinized and idli cakes
unacceptable whereas when it is more than 50%, the
are soft and spongy. They are generally consumed on the
product obtained is too sticky to be acceptable. Thus, not
same day and there is no effort to preserve the product.
only can the ingredients be varied, but the proportions can
The acid content of the product retards the growth of food
also be varied within a wide range and still an acceptable
poisoning and food spoilage causing microorganisms.
product is obtained.
 Basic of Fermentation Technology

35
Microbiology of fermentation Method of dosa preparation
In a study of sequence of microorganisms that Ingredients: The ingredients used for dosa preparation are
developed during soaking of ingredients and subsequently similar to as that of idli preparation i.e.rice, black gram dhal,
during fermentation of the batter at 30 °C, it has been salt and water. Rice may be substituted by wheat, bajra
found that Leuconostoc mesenteroides and Streptococcus (Pennisetum typhoideum), maize, or kodri and black gram
faecalis developed concomitantly and then continued dhal may be substituted by sprouted peas, cowpeas (Vigna
to multiply following grinding. The number of these two catjang), field beans (Dolichos lablab) or soybeans. Fresh
species remained very high until 23 hours while in the later groundnut oilcakes may also be substituted for black gram
stages Pediococcus cerevisiae was also found. During this dhal.
stage idli is being steamed and consumed. Thus, these
Soaking and batter formation: Generally, equal quantities
two species are responsible for the acid production and
of rice and dehulled black gram dhal are soaked in water at
leavening of the batter. The usual aerobic contaminants
room temperature separately for 5-10 hours. It is common
present on the ingredients are eliminated partly by careful
practice that finely ground powders are used to prepare
washing and partly by acidic conditions produced by the
batter. The finely ground powders are mixed with water at
fermentation. The fermenting microorganisms appear to be
temperatures ranging from 480 to 980 °C, best at 80 °C for
present on the ingredients and if the product has to be made
the preparation of batter. Water is added in the range of 2.0
daily, there might be some advantage in adding a bit of the
to 2.2 times the initial dry weight of ingredients to prepare a
freshly fermented batter to the newly ground batter. Yeasts
batter of viscosity desired for dosa. Salt is added from 0.8
like Torulopsis and Trichosporon can possibly leaven the
to 1.0% as a seasoning before fermentation.
batter if present in sufficient number, but it is highly unlikely
that they produce the acid characteristics of idli. Fermentation Time: Traditionally, dosa batter is kept
overnight for fermentation. The fermentation time should be
Jalebi sufficient to allow a definite leavening and acidification of
To make jalebi, refined wheat flour (Maida), dahi and the batter and to allow for the development of a pleasant
water are mixed into a thick batter and fermented for 14- acid flavor.
16 hours. The fermented batter is deep fat fried in spiral
Inoculum: The natural microflora developed during the
shapes and immediately immersed in sugar syrup (600B to
soaking operation and then at the overnight fermentation
750B) for a minute or two and eaten.
is sufficient to leave the dosa batter. However, fermented
Microbiology of jalebi fermentation batter of the previous batch may also be used as a
starter culture for fermentation. The microorganisms that
Inoculum in jalebi ads from the natural microflora develop during the overnight soaking are: Leuconostoc
includes Lactobacillus fermentum, Streptococcus lactis, mesenteroides, Streptococcus faecalis, lactobacillus
Streptococcus faecalis, Lactobacillus buchneri and fermentum, Pediococcus cerevisiae, Trichosporon
Saccharomyces spp. pollutants, Trichosporon beigelii, and Candida kefyr etc.
Changes during jalebi fermentation Incubation temperature: Ordinarily, the dosa fermentation
is carried out at room temperature. In the tropics, this
During fermentation, pH decreases from 4.4 to 3.3,
generally means a temperature of 25-30 °C.
volume of the batter increases about 9%; both amino
nitrogen and free sugar decrease. Fermentation containers: In Indian homes, fermentation
is customarily carried out in utensils that have sufficient
Dosa capacity to hold the batter and clean to avoid excessive
It is a thin crisp, fried, pancakes like staple food of contamination. The containers are covered with a clean
southern India and is also gaining much popularity in other damp cloth to prevent the entry of insects.
parts of the country. Like most of the fermented foods Grinding of the Ingredients: Stone mortars are used for
consumed in India and other Asiatic countries, dosa is grinding the ingredients in Indian homes. These provide
prepared by natural fermentation. Dosa batter is very similar excellent control of the particle size in batter. The ingredients
to idli batter, except that both the rice and black gram dhal are very finely ground to a thin paste and then salt is added
are finely ground. The batter of dosa is thinner than the idli in it.
batter. Following fermentation, the dosa is quickly fried as a
thin, fairly crisp pancake and eaten directly. The unflavored Harvesting and preservation: As soon as the batter
crisp pancake may be rolled onto a cooked mixed vegetable becomes leavened and acidified, it is spread onto a hot
mixture and eaten with samber, which is a thin richly spiced and greasy griddle where it assumes the shape of a crisp
soup of dhal and vegetables. It is an important source of pancake. The spreading of dosa on the griddle is an art
protein and calories in the diet and nutrition of south Indians. that matures with practice. Sometimes a mixture of cooked
Since is easily digested therefore it is often used as food for different vegetable is poured onto the crisp dosa and the
infants and invalids. sides are rolled, which now becomes ready to be eaten with
a spiced soup of dhal and vegetables popularly known as
Samber.
 Basic of Fermentation Technology

36
Microbiology of dosa fermentation: Traditional dosa Fermented sausages
batter fermentation has revealed the Occurrence and
role of several bacteria alone or in combination with Sausages are cylindrically shaped, fermented, solidified,
yeasts in bringing about various biochemical changes but and heat stabilized emulsions formed by mixing together
Leuconostoc mesenteroides appears in the fermentation proteins, fats, water, salt and flavorings. Color, flavor and
early and brings about the leavening in batter. It is also texture of sausages depend on the type of meat, which
along with Streptococcus faecalis involved in the acid is mixed and comminuted together with ice, salt, spice
production. These organisms appear to be present on the flavorings, curing agents and selected meat trimmings, to
ingredients and develop during fermentation. Therefore it form a sausage emulsion. The word “sausage” is derived
becomes an advantage that previous batch inoculum is from the Latin, salus; meaning salted or literally, preserved
used as a starter for fermentation. Yeasts, if are present in meat. The term “salus” undoubtedly was used by the
sufficient number can leave the batter but they have no role Romans to denote meat preserved through the use of salt.
to play in acid production, which is characteristic of dosa Basically all sausages are comminuted meats. Products
batter fermentation. Pediococcus cerevisiae appear very differ primarily because they are spiced in varied fashion
late in the fermentation when the batter becomes ready to and because of differences in methods of processing.
be fried on hot and greasy griddle. (Flow Chart 9) Classification of sausages
Sausages may be loosely classified into three general
categories: 1) Fresh sausages. 2) Cooked or Smoked
sausages and. 3) Dry Sausages. Some sausages,
especially the dry and semi-dry types depend on bacterial
fermentation of the production of their characteristic
flavors, while in manufacture of some of the more common
sausages such as frankfurters bacteria play no role. No
distinct classification can be made based on spice formulae
because basic spices are used in virtually all products.
Fresh sausages: Fresh sausages are always kept under
refrigeration and are fried, boiled, or Cooke thoroughly
before serving. They are prepared from selected cuts of
fresh meats, principally pork, or beef that has not been
previously cured. Examples of fresh sausages are fresh
Pork sausages that are prepared selectively from pork meat
and fresh sausages that may also contain a percentage
of beef and other meats in addition to pork meat. Pork
trimmings are put through a grinder, seasoning is added
and mixed, and the product is stuffed into natural casings
or sold in bulk.
Cooked or smoked sausages: These can be divided
into two categories -1) smoked and un cooked and 2)
smoked and cooked. Both these categories of sausages
are prepared from cured meats and must be kept under
refrigeration prior to preparation for serving. Smoked and
uncooked sausages -include Smoked country-style Pork
sausages that are subjected to mild curing and then placed
in casings and smoked and cooked before serving. Other
varieties like mettwurst, smoked country-style sausage and
polish sausage, all contain beef and pork mixed in various
proportions. Smoked and cooked sausages -these are
prepared from both uncured and cured meats. They are
processed by cooking and smoked after cooking. Examples
of these are Frankfurters (60% beef and 40% pork), Bolonga
(cured beef and pork), Berliner sausages (cured, coarsely
ground pork and finely chopped beef), and German type
Mortadella (Cubes of fat pork and pistachio nuts), Liver
sausages (pork liver and gelatin), Blood sausages or
Bluwurst diced, cooked fat pork plus finely ground cooked
Flow Chart 9 meat, blood and gelatin.
 Basic of Fermentation Technology

37
Dry sausages: Dry sausages are prepared by curing proportion of fillers in sausages should not exceed 3.5%.
freshly comminuted meats added with Curing agents and When corn syrup and milk solids are used their proportion
spices for 2 to 3 days. They are then placed in casings and should not exceed 2%.
are processed by carefully controlled air-drying. When pork
Curing agents: The common curing agents for sausage
is used in such sausages, it is subjected to a light smoke in
preparation are salt, sodium nitrite and/or nitrate and sugar.
order to destroy live trichinae. The principal dry sausages
Three percent of salt on the basis of the meat ingredients
are either salamis or cervelats. Salamis are generally more
is frequently used in sausages. Sodium nitrite and/or nitrate
highly seasoned than cervelats.
are usually added along with the salt to the beef portion of
Meat Ingredients for sausages: The meat ingredients the sausage emulsion. The proportion of sodium nitrite may
used chiefly in preparing sausages are those parts of the not exceed ¼ oz per 100 lb of meat; if sodium nitrate is used
animal that do not have a ready sale as such. These are with nitrite, not more than 2 oz should be employed per 100
classified according to their “water binding” properties i.e. lb of meat. Sugar is added in many sausage products at a
their ability to retain moisture during thermal processing. level of 0.5-1.0%. Other curing agents such as potassium
Meats considered to have best binding properties are nitrate (saltpeter), potassium nitrite, vinegar and flavorings
skeletal tissue from the beef animal, and include bull meat, are also added.
shank meat, chucks and boneless cow meat. Medium
Seasonings: Variation in seasoning is one factor that
binders are head meat, cheek meat, and lean pork
is responsible for the large number of sausage varieties.
trimmings. The meat with low binding properties usually
These seasonings may be added as ground natural spices,
contains large proportions of fat or are non-skeletal or
extracted oils, and oleoresins, or a mixture of the two.
smooth muscles. Examples are regular pork trimmings,
Spices that are usually employed include allspice, black
beef brisklets, heart, giblets and tongue trimmings. Meat
pepper, cardamom, cinnamon, coriander, garlic, mace,
tissues classified as “Filler Meats” and considered to have
nutmeg, paprika and sage. Most seasonings for pork
little or no binding properties include ox lips and tripe, pork
sausage contain 1.75-2.0 lb of salt, 6-8 oz dextrose, 4-5
stomachs, skin, snout, lips, and particularly defatted pork
oz pepper, 2-3 oz sage and 0.25 oz of ginger, Per 100 lb
tissue; their use in sausages must be severely limited if the
of meat.
quality of the product is to be maintained. The muscles of
beef in sausages enhances flavor as well as contributes to Other additives: The other condiments used in meat
color and texture. The beef muscles contain water-soluble products are pistachio nuts, mono sodium glutamate,
nitrogenous extractives that act favorably on flavor. Fat in ascorbates and isoascorbates. Pistachio nuts are used
the sausages enhances flavor and changes the texture of in headcheese, meat loaves and Braunschweiger.
the sausages. They become tender and juicy, but total fat in Monosodium glutamate is occasionally employed in pork
sausage must not exceed 50%. sausage at a level of 0.1% to enhance flavor. Ascorbic acid
or d-isoascorbic acid (erythrobic acid) is used at a level of ¾
Ice and salt: The addition of ice (moisture) assists in
oz or ⅞ oz of their sodium salts, in each 100 lb of sausage
controlling the temperature of the emulsion while it is
product. These are commonly used in smoked sausage to
comminuted. Otherwise the chopping temperature will
assure minimum color development and retention. Coloring
exceed 60oF, which will lead to instability of emulsion and
matter and dyes, which are approved by the regulatory
promote bacterial growth. The moisture in the final cooked
bodies may be mixed with the rendered fat, applied to
sausage should not exceed four times the meat proteins
animal and artificial casings, and applied to such casing
plus 10%. For fresh sausages that have not been heat-
enclosing products. The following coloring matter and
processed the limit is four times plus 3%. Water and salt
dyes are acceptable 1) Natural coloring matters -alkanet,
make meat proteins soluble, which then stabilizes the fat
annatto, carotene, cochineal, green chlorophyll, saffron and
globules in the sausage emulsion. Salt also adds to flavor.
turmeric 2) Coal tar dyes -all food certified dyes.
Binders or fillers: Cereal flour, potato flour, soy flour,
Sausage casings: Sausage casings may be natural
bread or cracker crumbs, milk powder, casein are some
casings prepared from some part of alimentary tract of
of the binders that are used both to hold together and to
cattle, sheep or hogs, or they may be cellulosic casings.
extend the meat ingredients in the sausages. The flours
These latter are frequently used on frankfurters and may be
that are made from cereals as corn, durum wheat, and rye
clear or colored.
and from potato starch absorb water highly; however, they
must not readily ferment when mixed with it. The soy flour a. Method of preparation of frankfurter sausage
with its low fat content enables making sausage flour high
in protein content. Rice and cracker flours are also high in Frankfurters are the most popular of all the sausage
protein content. Binders or fillers improve color, provide products. More frankfurters are consumed than any other
better binding properties, improve slicing characteristics, type of smoked and cooked category of sausage; they
change or improve flavor and reduce cost of the product. represent 25% of all sausage sold. The meat formulation of
Their value depends on their ability to absorb moisture in ordinary frankfurters is 40-60% beef and 60-40% pork. Beef
the emulsion and retain it throughout heat processing. The content includes Bull meat, boneless chuck, plates, hearts
and trimmings while pork ingredients are filler meats such
 Basic of Fermentation Technology

38
as tongue, snout, lips and other byproducts in proportion sausage makers developed products that would keep
not exceeding 20%; more will result unsatisfactory product. under the climatic conditions of their particular area. Since
Fillers employed are dry skim milk, corn syrup solids and artificial refrigeration and canning processes were unknown
sometimes cereals. Total filler content should not exceed therefore sausage makers of Italy and southern France,
3.5%. Meat used for preparation was pre-cured, but at developed dry sausage products. German and Hungarians
the present time the curing agents are normally added produced definite types of dry sausages.
at the time of chopping. Salt sugar and curing salts are
c. Action of microorganisms in sausages
added at the level of 3 lb of salt, ½ lb of dextrose, ¼ lb
oz of sodium nitrite and 2 oz of sodium nitrate, for each Paulo and Smith (1977) have shown that Micrococci
100 lb of meat. The more common spices and seasonings dominate the surface of stuffed sausage during the early
used are pepper, nutmeg, mace, cinnamon, mustard and stages of fermentation. These are killed at pH 5.5 and
garlic. After chopping and mixing, frankfurters are stuffed are not found in the sausage after heat treatment of
into casings and linked. They then held at refrigeration or drying. The results corroborated the research carried out
ambient temperatures for varying periods of time prior to by Sison (1967) in Philippines on the native chorizo-type
heat processing to permit completion of curing process. sausages. The major functions of micrococci during the
Ascorbic acid may be used to obviate the need for this fermentation are the reduction of nitrate to nitrite and the
holding period. The sausages are then heated and smoked. production of catalase. Lactic acid bacteria rarely reduce
Immediately after smoking, they are cooked with a spray the nitrates to nitrites. Lactic acid bacteria were also found
of hot water at a temperature of 77-82 °C. Simply raising in the fermentation. The activity of the lactic acid bacteria
the smoke house temperature finishes some frankfurters; is the conversion of sugars to lactic acid by EMP pathway.
such dry-processed products are usually given a brief hot Streptococcus diacetylactis produces diacetyls and acetoin
water shower to plump the sausages and provide better that imparts nutty flavor and aroma to some sausage.
peeling characteristics. The frankfurters are then cooled Staphylococci also actively reduce nitrates to nitrites.
by cold water showering to an internal temperature slightly Excessive nitrite level in sausage has been noted when
above ambient; the remaining heat is usually sufficient to using high nitrate concentrations with micrococci, producing
dry the product prior to its placement in the holding cooler a defect in sausage called “nitrite burn”. The micrococci are
at 2-7 °C. The total processing time may be as short as 65 also lipolytic, and they produce lipase during early stages of
minutes (when the cure proceeds rapidly) or as long as 2.5 fermentation. This results in an increase in free fatty acids,
hours. Colored cellulosic casings may be used to add color, volatile fatty acids and carbonyl compounds after 28 days
or dye may be mixed with the hot shower water, which must of drying. Lactic acid bacteria produce varying amounts
then be recirculated; occasionally frankfurters are colored of hydrogen peroxide, which is destroyed by the catalase
by dipping. Certified coal tar colors can also be used. of micrococci. Another important function of lactic acid
Frankfurters processed in cellulosic casings may have their bacteria is the inhibition of Staphylococci. This inhibition
casings peeled from them after processing to produce the or suppression also suppresses the enterotoxin production
product sold as skinless variety. by Staphylococci. This inhibition is more pronounced as
the ratio of lactic acid bacteria to Staphylococci increases
b. History of sausage
and as temperature of the fermentation decreases. The
Sausage, one of the oldest forms of processed food, was beneficial effects of lactic acid bacterial starter cultures
developed some thousand years before the birth of Christ. in inhibiting Staphylococci and enterotoxin production in
It started by slow stages from simple process of salting fermented sausages have also been demonstrated.
and drying meats by the aborigines and was originated in
part as a means of preserving meat that could not once be Sauerkraut
consumed. The American Indians combined chopped, dried These are German terms for sour cabbage, which is
meat with dried berries and pressed these ingredients into a generally prepared from shredded cabbage. The yellow
cake for use when food was scarce. Similar drying of meat -white shreds are approximately 2-5 mm in width and as
was a common place along the shores of the Mediterranean long as 20 cm.
centuries before the rise of Roman Empire. The ancient
Romans were extremely fond of a sausage made of fresh Standards for sauer kraut
pork and white pine nuts, chopped fine and seasoned
It must contain at least 0.75% lactic acid and less than
with cumin seed, bay leaves and black pepper. Salami is
10% of the total acid can be volatile. The pH must not
mentioned in Grecian literature of about 5th century B.C.
exceed 4.1. The strainable brine should amount to about
Sausage was made and eaten by Babylonians (1500 B.C.).
10% of the total weight of sauerkraut and should contain
The fact that a city of Salamis existed on the east coast of
from 0.7 to 3.0% NaCl.
Cyprus in Aegean Sea about 449 B.C. provides foundation
for a supposition that salami sausage may have originated Preparation for fermentation
in this ancient Grecian city. The various types of sausages
as we know today were developed in certain European Properly matured sound heads of cabbage are trimmed
localities because of local climate conditions. European to remove the damaged parts and outer green or dirty
 Basic of Fermentation Technology

39
leaves. The cabbage is then sliced to shreds of size 0.16 to Influence of Temperature
0.08 cm in thickness. The shredded cabbage is conveyed
to vats or tanks for salting and fermentation. At low temperature (7.5 °C), fermentation is very slow.
Leuconostoc mesenteroides grows slowly attaining an
Role of salt acidity of 0.8-0.9% in terms of lactic acid in a month. Acidity
is important for its preservative effect. Other lactobacilli
Salt plays a primary role in the preparation of sauerkraut; and pediococci cannot grow at this low temperature. The
therefore its concentration is carefully controlled. According sauerkraut may not be completely fermented for 6 months
to legal standards salt must not be less than 2% and must or more or until the temperature rises to a temperature
not be more than 3%. Most of the producers of sauerkraut suitable for the growth of higher lactic acid producing
add salt in the concentration of 2.25 to 2.5%. Salt extracts lactics. At a temperature of 18 °C with a salt concentration
water from shredded cabbage by the process of osmosis, of 2.25%, a total acidity of 1.7-2.3% as lactic acid will be
thus forming fermentation brine. It suppresses the growth of attained, with an acetic to lactic acid ratio of about 1:4 in
some undesirable bacteria that might cause deterioration of about 20 days. At higher temperature i.e. 23 °C, the rate
the product and at the same time makes conditions favorable of fermentation will be greater so that a brine acidity of
for the growth of lactic acid bacteria. Salt also contributes 1.0-1.5% (lactic acid) may be attained in 8 to 10 days.
to the flavor of finished product by yielding a proper salt Active growth of Lactobacillus plantarum and Lactobacillus
acid ratio. The use of too little salt causes softening of brevis may be initiated in 3-5 days and the kraut may be
tissues and produces a product lacking in flavor. Too much completely fermented in approximately one month. At still
fermentation and over salting may produce a product with higher temperature of 32 °C, the rate of fermentation may
a sharp, bitter taste. It may also cause darkening of the be very rapid and an acidity of 1.8-2.0 may be attained in
color of product and may favor the growth of pink yeasts. 8 to 10 days. The major share of the acid produced will
Brine begins to form once the shreds are salted and tank is result from the growth of homofermentative bacteria L.
closed when it is filled to the proper level. Then a weight is plantarum and P. cerevisiae. The flavor of the sauerkraut
placed over these shreds so that it squeezes the water out will be inferior, similar to an acidified cabbage. At the higher
of the shreds. The weight may be of wood (old method) or temperature sauerkraut will darken rapidly unless canned
some plastic bag filled with water may be placed (modern immediately. It will have a poorer shelf life than sauerkraut
method). fermented at lower temperature. This sauerkraut also has
Microbiology of sauerkraut fermentation low percentage of acetic acid and will not attain as high a
total acidity, even though the pH is lower. It will also subject
Pederson first described the lactic acid bacteria that to yeast spoilage, partly because of its low content of carbon
he observed in fermenting sauerkraut. He found that the dioxide. It is also low in ascorbic acid content.
fermentation was initiated by the species of Leuconostoc
mesenteroides. This species was followed by gas forming Influence of salt
rods and finally by non-gas forming rods and cocci. Salt plays a primary role in the preparation of sauerkraut;
Leuconostoc mesenteroides is a heterofermentative therefore its concentration is carefully controlled. According
bacteria and it grows more rapidly than other lactic acid to legal standards salt must not be less than 2% and must
bacteria. It is active over a wide range of temperature and not be more than 3%. Most of the producers of sauerkraut
salt concentrations. It produces acid and carbon dioxide add salt in the concentration of 2.25 to 2.5%. Salt extracts
that rapidly lowers the pH, thus inhibiting the activity of water from shredded cabbage by the process of osmosis,
undesirable microorganisms and enzymes that may soften thus forming fermentation brine. It suppresses the growth of
the shredded cabbage. The carbon dioxide replaces some undesirable bacteria that might cause deterioration of
the air and creates an anaerobic condition favorable to the product and at the same time makes conditions favorable
prevent oxidation of ascorbic acid and natural color of for the growth of lactic acid bacteria. Salt also contributes
the cabbage. It also stimulates the growth of many lactic to the flavor of finished product by yielding a proper salt
acid bacteria. While this initial fermentation is developing, acid ratio. The use of too little salt causes softening of
the heterofermentative species of lactobacillus brevis and tissues and produces a product lacking in flavor. Too much
homofermentative species of lactobacillus plantarum and fermentation and over salting may produce a product with
sometimes Pediococcus cerevisiae begin to grow rapidly a sharp, bitter taste. It may also cause darkening of the
and contribute to the major end products like lactic acid, color of product and may favor the growth of pink yeasts.
carbon dioxide, ethanol, acetic acid. Minor products also Brine begins to form once the shreds are salted and tank is
appear in the fermentation. The minor products are a closed when it is filled to the proper level. Then a weight is
variety of volatile compounds e.g. diacetyls, acetaldehyde placed over these shreds so that it squeezes the water out
and primary carbonyls. of the shreds. The weight may be of wood (old method) or
Control of fermentation some plastic bag filled with water may be placed (modern
method).
Temperature, salt concentration and sanitary conditions
are the primary environmental factors controlling the
sauerkraut fermentation.
 Basic of Fermentation Technology

40
Acetylcholine content in sauerkraut at household level and consumed directly while limited
amounts of cabbage-based kimchi are canned in factories
Sauerkraut is known to provide certain laxative and sold in the market. Kimchi is available throughout the
properties; both sauerkraut and its juice have been used as year and, served three times a day, is a staple in the diet
purgative. The strain of l. plantarum produces acetylcholine along with cooked rice and accessory side dishes. It is a
in the presence of choline, while simultaneously fermenting favorite food unique in its complex of sour, sweet and hot
carbohydrates. This acetylcholine is of significance in nerve pepper flavors accompanied by carbonation derived from
activity. fermentation with natural microflora. Kimchi differs from
Defects and spoilage of sauerkraut sauerkraut in two respects: 1) It has, optimally much less
acid and 2) It is carbonated.
Pink-Kraut: Pink-kraut was observed first by Butjagin
(1904) Wchmer (1905) and Henneberg
1916. Brunkow et. al (1925) and Fred and Peterson 1922
noted that this cause was the growth of pigmented yeast
i.e. asporogenous yeasts presumably members of genus
Rodotorula. Pederson and Kelly (1938) observed that Pink-
kraut usually contained a salt content greater than 2.5%.
They associated the growth of yeasts with any factor that
would inhibit a normal fermentation or that would suppress or
adversely affect the heterofermenting bacteria. Sometimes
pink-kraut was observed in vats of sauerkraut only a few
feet away from an area of soft-kraut. The latter condition
arises due to insufficient salt concentration. Stamer (1975)
reported that L. brevis produces a red pigment under certain Flow Chart 10
condition that can be related to discoloration or darkening
of sauerkraut. The red color occurs between pH 4.4 and Methods of preparation for typical korean kimchi
5.2 and is most readily generated under aerobic conditions. (Tongbaechu-Kimchi or Kakduggi-kimchi)
Chemical reducing agents like ascorbic acid, cystein or
glutathione inhibit this color formation. Materials for kimchi preparation include 1) fresh
vegetables (major vegetables are Korean cabbage, and
Slimy or Ropy Kraut: This is generally caused by dextran radish; minor vegetables are garlic, green onion, ginger, leaf
formation induced by the L.mesenteroide and is transitory mustard, hot pepper, parsley pear, chest nut and carrot),
in nature. This species prefers to ferment fructose rather 2) Jeotkal (Korean pickled fish) 3) fresh fish 4) seasoning
than glucose, therefore in the fermentation of sucrose; the agents (table salt, sesame seeds, sugar, monosodium
fructose is fermented leaving the glucose which interacts to glutamate, chenggak (type of seaweed), pear etc. additional
form slimy, ropy water insoluble dextrans. These vary from minor ingredients may also be added depending on the
an almost solid, gelatinous mass to ropy slime surrounding household maker these are: saeujeot (pickled shrimp),
the bacterial cell. meolchijeot (pickled anchovy), whangsegijeot, frozen
Other Defects: Discoloration caused by autochemical Pollack, oyster, shrimp and small octopus. The ratio of major
oxidation. Loss of acidity caused bgrowth of molds and to minor ingredients varies depending upon the household
yeasts. Spoilage caused by molds and yeasts cause off- maker; the range generally is 70-90 to 30-10. Although the
flavors and off-odors (yeasty and rancid). Slimy, softened proper combination of minor ingredients is reported to be
kraut caused by aerobic growth of asporogenous yeasts. the key to good-tasting kimchi, the most important factor
seems to be the salt concentration. Salting of cabbage can
Advantages of the acid food fermentations: 1) They be done at 5 to 7% for 12 hours or in 15% saline solution
render foods resistant to microbial spoilage and development for 3-7 hours followed by rinsing and draining. Optimum salt
of food toxins 2) They generally preserve the food between concentration during kimchi fermentation is approximately
the time of harvest and consumption 3) they make the 3% and is adjusted by experience at the household level.
food less likely to transfer pathogenic microorganisms 4) Fermentation of kimchi in the homes is usually carried out
they modify the flavor of the original ingredients and often at ambient temperature. Using 3% salt concentration, the
improve the nutritional value. (Flow Chart 10) optimum fermentation period is one day at 30 °C, 2 to 3
days at 20 °C, 12-15 days at 10oC and 30-60 days at 5 °C.
Kimchi Optimum acidity of kimchi is 0.4 to 0.8% (as lactic acid).
Kimchi is the general name given to a group of Higher acidity makes the product unacceptable.
fermented acid vegetable foods with a long tradition in
Microorganisms
Korea. More specific names are used for these pickled
vegetables depending on the raw materials, processing Kimchi is fermented by the microflora of the region.
methods, seasons and localities. Most kimchi is prepared Organisms isolated include lactic acid bacteria, such as
 Basic of Fermentation Technology

41
Leuconostoc mesenteroides, Streptococcus faecalis, harvested while still immature. Fully grown (ripe) ones
Lactobacillus brevis, Pediococcus cerevisiae, Lactobacillus are undesirable for pickling because they are too large,
plantarum, and aerobic bacteria, such as Achromobacter, change color and shape, are full of mature seeds, and
flavobacterium and pseudomonas spp. In the later stages are too soft for most commercial uses. After harvesting
of fermentation yeasts and molds appear that are reportedly the cucumbers are immediately transferred to the salting
causes of softening. station to avoid sweating i.e. growth of undesirable
softening microorganisms. Unsound, decomposed, broken
Preservation of kimchi or crushed, distorted (wilt, rots, crooks, nubbins, etc.) are
Kimchi is preserved at low temperature (below 5 °C) sorted out. They are then graded in a mechanical grader
for a very short period of time. During storage at elevated into 4 or more sizes. Three types of cucumber pickles
temperature, rancidity and soft rot are accelerated by the are made: 1) Fresh pack -These are held in salt brine
microbial action. Thus shelf life is very short in summer for as long as 2 days, then packed into jars or cans and
months. pasteurized. They undergo marginal fermentation. 2) Salt
Stock Pickles -From these pickles a variety of processed
Biochemical changes in kimchi products are prepared. They undergo complete lactic acid
fermentation. 3) Fermented Dill Pickles -They also undergo
The initial pH 5.5-5.8 falls to an optimum of 4.5-
complete lactic acid fermentation. Dill herb is also added in
4.0. Optimum acidity (as lactic acid) is 0.4-0.8%. Salt
these types of pickles.
concentration remains constant during fermentation. Kimchi
fermented at low temperature (6-7 °C) contains more lactic, Brining techniques for salt stock
succinic, oxalic, tartaric, malonic, maleic, and glycolic acid
than that fermented at 22-23 °C. Vitamins B1, B2, B12 and There are two general methods for preparing salt stock
niacin reach the highest levels (twice the initial level) when pickles for fermentation these are Dry Salting and Brining
kimchi possesses the most palatable taste and decrease Dry Salting
when kimchi becomes sour. Vitamin C and carotene content
decreases upon ripening. (Flow Chart 11) For cucumbers, dry salting is done after first adding
salt brine to cover the bottom of the tank (at least 12 in.)
to form a cushion. This prevents bruising, breaking, or
crushing the fresh cucumbers. Dry salt is then added at the
rate of about 22.5 kg for every 450 kg of small cucumbers
and 29.25 kg for every 450 kg large cucumbers. When full,
the tank is covered with a wooden lid very tightly. Brine
forms by osmosis. If the brine formed by osmosis does not
cover cucumbers then 400 salometer brine is added to the
desired level. The brine should be recirculated a day or two
after tank is filled in order to equalize the concentration of
salt throughout the brine. For long storage 60o salometer
brine is used. In industry 100 salometer is equal to 2.64%
NaCl by weight or 100o salometer is equal to 26.359 g NaCl
at 15.5°C (saturated solution of salt).
Brine salting: Brine salting process is preferred over
dry salting because dry salting yields soft,
Flow Chart 11
flabby, shriveled pickles that do not fill out properly when
Cucumber Pickles processed. Therefore most picklers mostly use brine-salting
The cucumber (Cucumis sativus) is popular both as technique for fermenting cucumbers. For brine salting ‘low’
a fresh and as a pickled vegetable. It is grown widely in or ‘high’ brine process may be used. In low brine salting, a
temperate climates although originally of semitropical salt brine of 25-30°C salometer is added into cucumbers
origin. Cucumbers for pickling must be grown from whereas in high brine technique, 40°C salometer salt is
varieties known to have regular form, firm texture, and added into cucumbers and tank is closed to air tight first
good pickling characteristics. Earlier varieties used by covering it with polythene sheet and then by lid. The
for pickling were monoecious plants but new varieties cucumbers are handled by the same procedure as described
developed by hybridization method have preponderance in dry salting except brine is used to cover the cucumbers.
of female flowers and are called Gynoecious. These new Now a days, molded plastic and fiberglass tanks are being
cultivars often have greater vigor and uniformity than the used in place of wood or concrete tanks. These plastic
open pollinated ones formerly grown. In addition, several and fiberglass tanks have several advantages. These are
of the hybrids are early maturing so they can be used to 1) They are not subject to biological degradation or metal
advantage in harvest scheduling. Pickling cucumbers are corrosion 2) They do not have to be maintained during the
 Basic of Fermentation Technology

42
off season, as do wooden tanks 3) As all the valves and Post fermentation
piping are made of plastic, the problem of metal corrosion
and contamination is eliminated 4) They are properly When fermentable carbohydrates are exhausted,
designed, closures are nearly airtight so problems of loss microbial growth is restricted to the surface of brines
of acidity is reduced. exposed to air; the spoilage bacteria may become
established on the surface of improperly managed tanks.
Microbiology of the cucumber fermentation At the end of fermentation, total acidity is 0.9% and pH is
equal to 3.3.
A rapid development of the microorganism causes a
spontaneous fermentation as soon as the lid of the tank Fermented dill pickles
is closed after adding brine. The rapidity of fermentation is
directly related to the temperature of the brine, concentration Cucumbers when are subjected to bacterial fermentation
of the salt in brine, availability of fermenting materials and in dill flavored, spiced, salt brine; the product obtained is
relative number of microorganisms available on cucumbers. called Dill pickles. They have their distinctive flavor and
Fresh cucumbers contain numerous and varied microflora aroma due to the products of fermentation of lactic acid
including many potential spoilage microorganisms and a bacteria and to the blending of flavor and aroma of dill herb
small number of lactic acid bacteria (5-103 acid forming and spices that were added to the brine.
bacteria per gram of cucumber). When cucumbers are Method of preparation: The larger size cucumbers are
brined at 5-8% NaCl range and allowed to undergo natural washed and placed in suitable containers, together with the
fermentation, the salt solution supports the fermentation requisite amount of dill weed (which was earlier cured in
by a sequence of various types of microorganisms. This vinegar, salt and brine) and dill spice and brine solution.
sequence is categorized into four stages 1) Initiation 2) Dill pickles are generally fermented in low salt (5% NaCl)
Primary fermentation 3) Secondary fermentation and 4) brine solution. Vinegar is added to retard the growth of
Post-fermentation undesirable microorganisms (by decreasing the pH value).
Initiation The fermentation is carried out at a temperature between
21 °C and 26.7 °C for 3-4 weeks. A curing period of 3-4
This stage may include growth of many facultative and weeks further is also necessary. During this period, the flesh
strictly anaerobic microorganisms originally present on the of pickles becomes entirely translucent and acidity about
fresh material, the growth of undesirable microorganisms 0.5-1.2% (lactic acid). In addition, there is small amount of
such as Gram-negative and spore-forming bacteria is volatile acid (acetic acid); lactic acid bacteria and yeasts
inhibited as the pH gets lowered and lactic acid bacteria produce ethanol and other minor products.
become established. The quality of the final product
depends largely of the rapidity with which lactic acid bacteria Microbiology
are established and undesirable bacteria are excluded. In the beginning, a wide variety of unrelated
Primary fermentation microorganisms start growing in the fermentation but soon
lactic acid bacteria predominate them. At low temperatures
In this stage lactic acid bacteria (Leuconostoc, Leuconostoc mesenteroides play an important role in the
Lactobacilli and Pediococci) and both fermentative and fermentation. Once Leuconostoc starts predominating other
oxidizing yeasts are the predominant active microflora. species like Lactobacillus brevis, Lactobacillus plantarum
They grow in brine until the fermentable carbohydrates begin to grow in the fermentation and ultimately complete
are exhausted or until there is production of lactic and the fermentation.
acetic acids. In normal fermentation the undesirable
microorganisms are excluded within 10-14 days. Buffering Packaging
capacity and the fermentable carbohydrate content present Fermented dill pickles are marketed in bulk plastic
in the medium are the important factors that govern the containers and glass containers covered with acidified
extent of fermentation by lactic acid bacteria and the extent brine, closed and pasteurized at 74 °C for 15 minutes.
of subsequent fermentation by yeasts.
Spoilage of cucumber pickles
Secondary fermentation
In Cucumber pickles, most of the deterioration is caused
Pediococci, Lactobacillus brevis, and Lactobacillus by the microorganisms; chemical defects are generally
plantarum and fermentative yeasts are responsible for the caused by metallic contamination, auto-chemical and
completion of lactic acid build up in this final stage of the physico-chemical reactions. Microorganisms damage the
fermentation. The acid tolerant yeasts still remain in the tissues by their cellulolytic or pectinolytic enzymes that
medium after the lactic acid bacteria are inhibited by low pH result in loss of texture of firmness. Gaseous deterioration
values and continue to grow till fermentable carbohydrates caused by microorganisms, resulting in the production of
are exhausted. internal cavities or distorted stock caused by excessive gas
pressure is another common spoilage. This defect is known
as bloater or floater spoilage.
 Basic of Fermentation Technology

43
Softening of cucumber pickles Production of Industrial Enzymes
Softening occurs most frequently after brining of the Introduction
cucumbers for dill or salt stock pickles. The entire skin of the
cucumbers become slippery and can be removed easily. Enzymes are biocatalysts produced within the living
Such condition of pickles is sometimes referred to as ‘Slip cells to bring about specific chemical changes. They
or Slippery pickles’ in industry. When softening progresses are present in all living cells and the metabolic reactions
into the deeper layers of cells and more and more pectic common to all cells are catalyzed by these enzymes. The
materials, present in the middle lamella separating the use of enzymes in industry dates back to some centuries
individual cells of the cucumber are attacked, the condition before the discovery of enzymes. For example use of barley
is known as ‘Mushy Pickles’. A variety of bacteria of Gram- malt for starch conversion in brewing is a notable example.
positive type (Bacillus subtilis, B.pumilis, B.polymyxa, The field of enzymology was opened by Buchner brothers
B.stearothermophilus) and yeasts such as Saccharomyces who showed that cell free extracts from yeasts fermented
fragilis, and Rhodotorula that produce pectinolytic and sugar to produce alcohol and carbon dioxide. In oriental
cellulolytic enzymes cause mushy deterioration. countries microorganisms are directly employed as enzyme
source for the preparation of products such as shoyu,
Gaseous Spoilage of Cucumber Pickles miso, natto, and sake. Other important processes in which
enzymes are primarily used are cheese making, leavening
A number of genera of bacteria and yeasts cause
of bread, manufacture of vinegar, tanning of leather etc.
gaseous spoilage in cucumber pickles. Undesirable yeasts
It was Takamine who laid the foundation for the industrial
produce gas because they utilize lactic acid and cause a
production of microbial enzymes by developing process for
rise in the pH. The fermenting yeasts have been identified
producing diastase from fungi. Boidin and Effront of France
as belonging to genera Brettanomyces, Hansenula,
were the first to produce industrial enzymes from bacteria.
Saccharomyces and Torulopsis. Among bacteria,
So far more than 1300 enzymes have been identified, out
Lactobacillus brevis, Lactobacillus plantarum cause
of which nearly 100 have been obtained in crystalline form.
serious bloater spoilage. The major gases formed during
spoilage are generally hydrogen and/or carbon dioxide. Commercial production of industrial enzymes
Nitrogen purging of the fermenting brine is used to reduce
undesirable levels of carbon dioxide that otherwise might Selection of microorganisms: The potential
result in bloater formation. (Flow Chart 12) microorganisms are isolated from soil, decaying Organic
matter or air and are tested individually for their capability to
produce the desired product; this process is called primary
screening. A few members of this potential natural isolates
will possess the desired characteristics. It is also customary
to grow the selected organisms on their substrates.
In certain cases such as in the case of pectinases the
organisms is induced to secrete the desired enzyme.
The selected strains are maintained in pure form by
lyophilization, on agar slant, or soil culture. The isolates are
periodically checked for purity and for the retention of their
original activity. Secondary screening is conducted in flasks
or small fermentors. This evaluates the true potential of the
organism to produce the desired product both qualitatively
as well as quantitatively. Once potentiality of the organism
is established, investigations are undertaken to work out a
suitable medium and optimization of other conditions like
pH, temperature, aeration etc. for the maximal enzyme
production are carried out. Continuous maintenance of
strains may cause degeneration and ability to produce
the desired enzyme. Therefore, periodic re-isolation and
reevaluation is necessary. The strains are also continually
improved to enhance their capabilities by various physical
agents such as UV treatment or by chemical methods.
Recombinant DNA technology has also playean important
role in the strain improvement programme.
Flow Chart 12
 Basic of Fermentation Technology

44
Methods of cultivating microorganisms: Several the bran can be stirred. The chamber is also provided with
methods of culturing the microorganisms are being cooling coils and an inlet for aeration. Bran is loaded into
employed industry that can be classified as follows: 1) the drum and moistened with dilute acid and sterilized with
Solid Culture -i) Conventional koji culture ii) Bulk koji culture steam. After cooling, the inoculum prepared in bran culture
iii) Rotary drum culture 2) Liquid culture -i) Stationary ii) is added and mixed. After charging, the air is passed slowly
Submerged These methods are explained in the coming into the drum which is maintained at a temperature of 28-
section. 30 °C. The spores germinate within 5-6 hours during which
period drum is rotated slowly for 15-20 minutes after every
Preparation of starter culture: Working cultures are first
2 hours. The drum is rotated continuously for 5-6 hours
prepared from stock culture (i.elyophilized or soil culture).
at a speed of 1 rpm or less. The growth of the fungus is
These cultures are tested for their potency from time to
completed within 60 hours. The moldy bran is then spread
time. In certain countries like Japan, there are firms that
in the form of layers on paper for drying. After drying, the
specialize in supplying pure fungal spores called Tane koji
moldy bran is ground and utilized as such, as an enzyme
of desired strains of fungi to large manufacturing units. In
source or to extract the enzyme.
such cases spores are directly inoculated into the growth
medium. Starter inoculums for large fermentations (both Extraction process: Powdered moldy bran prepared
for solid and liquid fermentations) have to be progressively from the foregoing procedures is utilized for extraction and
built up. The quantity of inoculum required depends upon purification of the enzyme. The moldy mass is extracted
the batch size and it varies between 0.01 to 0.001 of the with cold water (1-2 °C) or with solvents like ethanol. The
volume of the medium and is expressed as number of cells, common procedure is to employ counter current extraction
weight of cell mass or just on the volume basis. Generally system which gives a better and clear extract of enzyme.
the amount of inoculum is kept as low as possible. Large The quantity of water utilized generally is 5-10 times the
Scale Mold Fermentation using Fungi weight of the moldy bran. The clear extract thus obtained
is utilized for concentration and purification of the enzyme.
Bran process: Wheat bran is moistened with 0.2 to 0.3N
HCl and autoclaved at 15lb pressure for1 hour. Addition of Submerged fermentation: After selecting a suitable
acid improves sterilization and inhibits growth of undesirable microorganism capable of producing the desired enzyme
microorganisms. Sterilization can also be carried out by and standardizing the conditions for its maximum output,
direct injection technique and continuously stirring the mass large scale fermentation is taken up. This involves various
SO that bran particles come in direct contact with steam. operations that are discussed below: The substrate
When dilute acid is used for moistening bran, it is sufficient for growing microorganism is designed, based on the
to hold the medium for 15-30 minutes in live steam to obtain availability of cheap raw materials and types of enzyme
practical sterility. The cooked bran is then cooled to room to be produced. Composition of media recommended for
temperature and inoculated with inoculum grown earlier, at different enzymes consists of components selected from
1% level. It has been reported that 0.4% dry spore inoculum starch hydrolysates, wheat bran extract, milled cereal
is sufficient for good growth of fungus. The inoculated bran products, soybean meal, peanut meal, corn steep liquor,
is mixed well and transferred to trays having false bottoms. distiller’s solubles, yeast extracts and other organic and
Layers of 5 centimeter are considered good for uniformly inorganic nitrogenous compounds and mineral salts. The
good growth. The trays are placed one above the other liquid nutrient medium is charged into cleaned fermentors
8 to 10 cm apart. Spores germinate within 3-4 hours and and sterilized by means of steam. The medium is constantly
temperature begins to rise after 5-6 hour. Aeration is started kept stirred during sterilization. The sterilization is carried
at this stage and continued till growth is completed. It takes out for 2-3 hours depending on the size of the batch.
48-120 hours for the fermentation to complete depending Generally the holding temperature is 121 °C for 20 minutes.
upon the microorganism. After the completion of growth, the For large fermentors a continuous high temperature and
trays are shifted to drying tunnels with a central exhaust. short time regime is adopted. Direct heating with steam
The hot air is blown into the tunnel and air temperature is is preferred when medium is thick and viscous. After
not allowed to rise above 40 °C. Different modification of the sterilization medium is cooled to the desired temperature
above mentioned process are adopted by different enzyme and pH is adjusted to optimum, the inoculum is introduced
producing companies. under aseptic conditions. In commercial production of
microbial enzymes generally aerobic microorganisms
Bulk-koji process: In this method the fungus is cultivated
are used for fermentation. In such cases aeration and
on thick layers of bran up to 25cm t50cm high. The chamber
agitation is started soon after inoculation. The fermentor is
has a false bottom and air is circulated under pressure from
kept under constant pressure to avoid contamination. The
the bottom of the chamber. The chambers have a floor
agitator speed and quantity of air depends on the size of the
area of 2 to 30 meters by 6 to 10 meters. Temperature
batch, medium composition, and the requirements of the
and humidity of the air in the chamber are automatically
selected microorganism. After inoculation microorganisms
controlled.
establish themselves and passing through a lag phase,
Rotary drum method: In this method, the fermentation begin to grow exponentially. The standard fermentors are
vessel consists of a rotating drum fitted with baffles so that provided with aseptic sampling devices that allow samples
 Basic of Fermentation Technology

45
to be withdrawn for routine checking of contaminants, cell sulphite, magnesium sulphite, sodium sulphite, or by other
population, morphological observations and enzyme yield. substances added to bring the pH of the solution to its
Fermentation is carried out for 1-7 days. Most of the enzymes isoelectric point to separate the protein. The former process
are secreted into the medium by the microorganism and is called as ‘Salting Out’ and latter referred to as ‘Isoelectric
extracellular enzymes appear in the medium during early Precipitation’. Water soluble solvents such as ethanol,
part of the logarithmic phase. In case of intracellular methanol, isopropyl alcohol, acetone, dioxane etc. are also
enzymes cell disruption techniques are employed to recover used for precipitating the enzyme. The precipitated enzyme
the enzymes. The enzyme rich material obtained in any of is separated by process of centrifugation or filtration. The
the three procedures i.e. moldy bran, cultural extract, or cell organic solvents are removed by low temperature drying
autolysates is concentration and purified after solution is and the inorganic salts by dialysis. Enzyme now gets
stabilized by the addition of chemical s such as ammonium concentrated manifolds. To obtain an enzyme in pure form,
phosphate, ascorbic acid, calcium salts, hydrochloric acid, the enzyme solution is absorbed on ion exchange resins,
phosphoric acid, sodium citrate, sodium phosphate, sodium inert earth, calcium phosphate, aluminum hydroxide,
sulphite or organic substances such as gelatin, gums, colloidal iron etc. after adjusting the pH to the optimum
Arabic gum etc. level. Highly purified enzyme is freeze-dried or crystallized.
Purification of enzymes: The fermentation solution is Enzyme activity
subjected to centrifugation or filtration to obtain a clear
liquid free of suspended particles. Concentration of the Commercial enzymes are evaluated according to their
enzyme is brought by employing techniques such as specific activity per unit volume or weight. The activity
vacuum evaporation and fractionation. In fractionation, all is usually measured in ‘Enzyme Units’. An enzyme unit
substances in the crude enzyme solution except the desired indicates an amount of chemical change catalyzed by a
enzyme are separated out. Enzyme is then purified by definite quantity of enzyme. The activity can be standardized
precipitation, absorption, and crystallization. Precipitation by blending the enzymes with inert materials such as
of enzyme is carried out with salts such as ammonium diatomous earth, glucose, sucrose etc (Flow Chart 13).

Flow Chart 13
 Basic of Fermentation Technology

46
Production of Vitamins 26-28 °C for 4-5 days. The fermentation is submerged,
aerated but high levels of aeration may inhibit mycelial
Riboflavin production and reduces the product yield. When Candida
spp. are used for riboflavin production, the vessels made
Microbiologically produced Riboflavin has long been
of steel cannot be used because the organism is very
available in yeast and related preparations in association
sensitive to traces of iron, therefore the vessels may be
with many other vitamins of the B-complex category.
lined with plastic. Cobalt at proper concentration (stimulates
Riboflavin is essential for the growth and reproduction of
the ascomycete fermentation) can be added into the
both humans and animals. Thus it is often incorporated
fermentation broth as it partially counteracts the iron toxicity.
into the feed of the animals. By fermentation process the
Candida fermentation can be carried out at low pH, which
riboflavin content of the medium can be raised up to 7
eliminates the bacterial contamination and less sterilization
gm/L. Various microorganisms involved in the fermentation
is required.
of Riboflavin are as follows: a) Ascomycetes- Ermothecium
ashbyii and Ashbya gossypii are the commercial strains. b)
Bacteria- recovered from the acetone butanol fermentation
e.g. Clostridium butylicum, C. acetobutylicum and
Mycobacterium smegmatis also produce riboflavin. c)
Yeasts- Candida guilliermondia, C. flareris and Mycocandida
riboflava are the non-commercial strains. (Figure 3)

Fermentation process for ascomycetes

Ascomycetes for the production of Riboflavin require
semi purified sugars (glucose) and crude organic nutrients
like corn steep liquor, animal stick liquor and meat scraps.
Glucose may be totally replaced by corn oil, however low
levels of corn oil may be added to the glucose to stimulate
riboflavin yields. pH is adjusted to 6.5-7.5 and temperature Figure 3

Mechanism of riboflavin accumulation

Kaprálek (1962) and Stárka (1957) demonstrated that fermentation of riboflavin progresses through three phases.
First phase is a rapid growing phase with no riboflavin production. The substrate is utilized and oxidized; pH decreases as
pyruvic acid accumulates. In the second phase glucose gets exhausted, sporulation begins, pyruvate decreases, ammonia
accumulates because of deaminase activity, pH becomes alkaline, Rapid synthesis of cell bound riboflavin occurs in the
form of FAD and rapid catalase activity causes disappearance of cytochromes. In the third phase autolysis of the cells
occurs with the release of riboflavin into the medium (Flow Chart 14).

Flow Chart 14
 Basic of Fermentation Technology

47
Recovery of riboflavin
On completion of fermentation, the solids were dried to a
crude product for feed supplement. For a crystalline product,
broth is heated for 1 hour at 15lb pressure to solubilize the
riboflavin. Insoluble matter was removed by centrifugation
and riboflavin is recovered by conversion to the less
soluble form by chemical and microbiological methods. The
precipitated riboflavin was then dissolved in water or polar
solvents or in an alkaline solution, oxidized by aeration and
recovered by recrystallization from aqueous or polar solvent
solution or by acidification of the alkaline solution.

Carotenoid
Carotenes
Carotenes are precursors of vitamin A. Some carotenes
are normally present in foods and have an essential
biological function to perform. They are used as food
supplement to prevent or cure vitamin deficiency diseases.
In addition other pigmented carotenoids are used both
as food additives for intensifying or modifying the color in
fats, oils, cheese, and beverages and also as animal feed
supplement to enhance the color of such foods as egg yolks
and chicken flesh. Though carotenoids are widely found in
plants and animals, only microorganisms and plants have
the necessary systems to synthesize a wide range of these
products.

Microorganisms
Many species of algae and fungi (e.g. Neurospora
crassa, Penicillium sclerotium, Phycomyces blakesleeanus)
and also yeasts (Rhodotorula) were considered for use in
β-Carotene production but were found unsuitable. Some Flow Chart 15
particular fungi in mucorales group and choanophoraceae
family of Phycomyces concentrated the interest on the Recovery and Purification of β-Carotene
development of industrial fermentation. Particularly
Crystallization
Blakeslea trispora have received extensive study for their
ability to produce β-Carotene. This microorganism is It is the formation of solid particles within a homogenous
heterothallic in nature. High concentrations of β-Carotene phase. It may occur as the formation of solid particles in a
are produced only when both the mating types are present vapor, as solidification from a liquid melt or as crystallization
in the medium. from liquid solution. Crystallization from solution is important
industrially because of the variety of materials that are
Growth conditions marketed are in crystalline form. In industrial crystallization
The medium should be viscous and rich in vegetable from solution, the two-phase mixture of mother liquor
oils, kerosene and surface-active agents, β or β- ionones and crystals of cell sizes that occupy the crystallizer and
are added during the incubation. withdrawn as product is called Magma. Crystals have been
classified into seven classes these are: cubic, hexagonal,
Activators of β-carotene production trigonal, tetragonal, orthorhombic, monoclinic, and triclinic.
β-Ionone, a precursor of β-Carotene is not directly A given material may crystallize in two or more different
incorporated into the β-Carotene but it activates the classes depending upon the conditions or crystallization e.g.
enzymes required for carotene production. β-Carotene calcium carbonate occur commonly in nature in hexagonal
production can also be enhanced by the presence of form (as calcite) but also it occurs in the orthorhombic form
dimethyl formamide, α-pyrrolidone and succinimide in (as aragonite). Under ideal conditions, a growing crystal
addition to β-ionones. (Flow Chart 15) maintains geometric similarity during growth; such a crystal
is called invariant. Unless the crystal is a regular polyhedron,
 Basic of Fermentation Technology

48
the rates of growth of various faces of an invariant crystal Organic solvent + Erythromycin + water β Erythromycin
are not equal. hydrate

Principles of crystallization Biopolymer recovery is also obtainable by salt addition

e.g. Xanthan gum is a polyanion and calcium ion can
Crystallization may be analyzed from the standpoint of be used to form gel precipitate. Alginate biopolymer is
purity, yield, energy requirements, rates of nucleation and recoverable from algal biomass by cell removal (filtration),
growth. Purity- crystals are purified from the mother liquor followed by calcium chloride precipitation of the biopolymer.
by filtration, centrifugation and then washing the crystals
with fresh solvent. The effectiveness of these purification 2) Solvent driven precipitations are useful in the
steps depends upon the size and uniformity of the crystals. production of microbial biopolysaccharides including
Equilibrium- Equilibrium in crystallization process is reached dextrans and xanthan gums. The biogum fermentations are
when the solution is saturated and equilibrium relationship typically aerobic and produce a highly viscous final broth
for bulk crystals is the solubility curve. Yields- In many with xanthan production, final broth pasteurization kills
industrial crystallization processes, the crystals and mother Xanthomonas cells. After adding KCl and then methanol
liquor are in contact long enough to reach equilibrium, and or isopropyl alcohol the gum polysaccharide directly
the mother liquor is saturated at the final temperature of the precipitates out. Dextran recovery is achieved by alcohol
process. The yield of the process can then be calculated or acetone precipitation. In solvent driven precipitation for
from the concentration of the original solution and the the production of bulk polysaccharide, the modest product
solubility at the final temperature. Supersaturation- In the value requires efficient recovery and reuse of solvent as
formation of a crystal two steps are required 1) the birth of a well as good solvent removal for food or pharmaceutical
new particle 2) its growth to macroscopic size. The first step grade product. 3) Protein precipitation techniques- The
is called Nucleation. techniques result in a phase change to form a precipitate,
require some alteration of protein solution conditions to
Types of crystallizers render the original, thermodynamically stable one phase
Commercial Crystallizers are different in several ways. system unstable with respect to precipitation. The various
The difference lies in how the crystals are brought into methods for causing the needed reduction in solubility of
contact with supersaturated liquid. The first technique protein include: 1) Added high salt concentration to give
called the ‘Circulated liquid method’, a stream of precipitates by salting out 2) pH adjustments to protein’s
supersaturated solution is passed through a fluidized bed of pH of neutral charge, the isoelectric point, at which point
growing crystals, within which supersaturation is released the protein has minimum solubility 3) Reduction of medium
by nucleation and growth. The saturated liquid then is dielectric constant to enhance electrostatic interaction by
pumped through a cooling or evaporating zone, in which e.g. addition of miscible organic solvent 4) Addition of non-
supersaturation is generated and finally the supersaturated ionic polymers that reduce the amount of water available
solution is recycled through crystallizing zone. In the for protein solvation 5) Addition of polyvalent metal ion to
second technique called ‘Circulating Magma method’, the form reversibly a protein precipitate. The method of choice
entire magma is circulated through both crystallization and includes considerations not only of the protein concentration
supersaturation steps without separating the liquid from needed and cost of separation technique, but also the purity
solid. Supersaturation as well as crystallization occurs in of the final product compared to precipitating agent (Table
the presence of crystals. In both the methods feed solution 2).
is added to the circulating stream between crystallizing and Recovery and purification of microbial products
supersaturating zone.
When biosynthesis of products in a fermentor takes
Precipitation place this becomes necessary to isolate the product and
Precipitation phenomenon is used to obtain products convert it into a form suitable for the required purpose. The
from the broth or some times, to remove impurities from isolation procedures differ considerably depending upon
the ongoing fermentation process. There are a number of the location of the product i.e. intracellular or extracellular
processes where insoluble precipitates are isolated. Since and also depend upon the concentration and stability of the
organic solutes have solubilities dependent on the solution product. Sometimes microorganisms also produce many
temperature, pH, composition, ionic strength and dielectric other organic products apart from the main product that may
constant therefore precipitation can be brought about in complicate the process of isolation. The simplest situation
many ways as follows: is the isolation of microbial cells when they represent the
desired end product. The basic process to isolate the
1) By adding precipitant to react with solute and microbial products is shown in the following figure: (Flow
producing an insoluble product, often a salt e.g. procaine Chart 15)
hydrochloride + penicillin β Procaine-penicillin.
After cultivation, the culture fluid is usually processed
Organic solvent + streptomycin + sulfuric acid β in order to facilitate the separation of microorganisms. The
Dihydrostreptomycin sulfate treatment depends upon the composition of the fluid, the
 Basic of Fermentation Technology

49
type of cultured microorganisms and the product; it may the diminishing distance between particles inside the pores.
include an adjustment of pH, heating and /or addition of For rigid or non-compressible particles the filter cake may
substances that coagulate the microbial cells. After such be considered as a system comprising a large number of
treatments the microorganisms are separated by filtration or capillary channels through which the fluid flows according
centrifugation. Further treatments depend on whether the to Poiseuille’s law. Then
product is intracellular or extracellular in the supernatant.
The microorganisms themselves, or the filtrate, may, after
suitable processing by pressing, evaporation, and etc.
constitute the end product. If the product is contained
in the cells, it is necessary first to disintegrate them; the
method of disintegration again depends upon the type of
microorganism, physiological state and composition of the Where u = linear flow rate; V= filtrate volume; T= time;
cell wall. Following disintegration, the cell walls are separated P= pressure drop through filter cake; L= thickness of filter
and product is isolated by the methods shown in the above cake; A= filter area; and = viscosity of fluid
figure. Products contained in the supernatant are isolated-
depending on their chemical properties-by precipitation,
extraction, adsorption, dialysis, ultrafiltration, evaporation
etc.; these methods are often used in combination. After
isolation, the products are further processed to a form
they are to be used for the said purpose (in medicine, food
industry, agriculture, etc.) (Table 3)
Table 3

Grape Fermenting Wine

Parameter Juice
(mls) 7 days 14 days 21 days Food Fermentation Practical (1) -To study the wine
% Tartaric acid production by the fermentative activity of Yeast cells.
% Acetic acid I. Principle
Alcohol Wine is a product of the natural fermentation of the
juices of grapes and other fruits such as peaches, pears,
Taste
plums, and apples by the action of yeast cells. This
Aroma biochemical conversion of juice to wine occurs when the
Clarity yeast cells enzymatically degrade the fruit sugars, fructose
and glucose firstly into acetaldehyde and then into alcohol.
Mechanical separation of microorganisms Grapes containing 20-30% sugar contents will yield
wines with an alcohol content of 10-15%. Also present in
The choice of the method of mechanical separation grapes are acids and minerals whose concentration are
and the appropriate equipment depend on the type increased in the finished product and are responsible for
of microorganism (Bacteria, yeast, actinomycetes or the characteristic taste and bouquet of different wines. For
filamentous fungi), composition of the medium (synthetic red wines crushed grapes must be fermented with their
or complex) and the absence or presence of suspended skins to allow extraction of their color into juices while white
particles. Basically, the separation of microorganisms is wines are produced from the juice of white grapes without
carried out using one or two main methods, filtration and skins. The commercial production of wine is a long and
centrifugation. exacting process. First the grapes are crushed to express
Filtration juice called “must”. Potassium metabisulfite is added to the
must to retard the growth of acetic acid bacteria, molds, and
It may be defined as the separation of suspended wild yeasts that are endogenous to grapes in the vineyard.
particles from a liquid by means of a pressure difference A wine producing yeast Saccharomyces cerevisiae var
through a permeable partition. The diameters of the ellipsoideus is used to inoculate the must that is used to
particles may be smaller than the openings in the filter inoculate the must, which is then incubated for 3-5 days
so that initially they pass through these openings. When, under aerobic conditions at 21-32oC. This is followed by
however, the filter pores become clogged with the particles, an anaerobic incubation period. The wine is then aged for a
the filter begins to retain further particles almost completely. period of one to 5 years in aging tanks or wooden barrels.
As filtration proceeds the thickness of the particle cake on During this time the wine is clarified of any turbidity and
the filter increases and the flow rate of the filtrate decreases. formation of esters responsible for characteristic flavor are
The gradual decrease in flow rate is caused partly by produced. The clarified product is then filtered, pasteurized
clogging of the pores on the filter surface and partly also by at 60oC for 30 minutes and bottled.
 Basic of Fermentation Technology

50 II. Materials required 40-60 minutes; and incubated at 21-28oC for 12-18 hours
till the pH reaches 4.9-5.0. The starter culture now is cooled
Fifty mls of white grape juice broth culture of
to 5-10 °C and is kept at the same temperature until used.
Saccharomyces cerevisiae incubated for 48 hours at 25oC.
It is not a good idea to hold ripe starter for more than 24
Five hundred mls of pasteurized Welch commercial white
hours.
grape juice. Phenolphthalein solution 1%. Sodium hydroxide
0.1N and sucrose. One litre Erlenmeyer flask, One holed VI. Fermentation
rubber stopper containing a 2” glass tube plugged with
Weigh desired quantity of milk and adjust its SNF to
cotton plug, Pan balance, Spatula, Glassine paper, 10 ml
10-12% by adding whole dry milk. Add sugar at the rate of
graduated cylinder, Ebulliometer, Burette and Pipettes for
10%. Heat it to 80-90 °C for 20 minutes. Cool the milk to
titration.
45-48 °C (for S. thermophilus) and inoculate with 5% yogurt
III. Procedure starter culture. Mix well. Keep the milk in clean and sterile
container for setting. Incubate the milk containers at 45 °C
Pour 500 ml of white grape juice into one litre Erlenmeyer
for 3-4 hours till a firm coagulum is obtained. Remove the
flask. Add 20gm sucrose and 50 ml of Saccharomyces
product from incubator and keep it at 5 °C till it is consumed.
cerevisiae containing grape juice broth culture (10% starting
culture). Close the flask with the stopper containing cotton Calculation of Milk Solid Non Fat (MSNF)
plugged air vent. Incubate the wine at 25 °C. After 2nd
Milk at a temperature of 60oF is added up to the brim
and 4th day of incubation, add 20gm more sucrose to the
of the cylinder and lactometer is gently dropped into it.
fermenting wine. Now again incubate at 25oC for 21 days.
Reading on the lactometer is noted down. This reading is
IV. Total acidity (expressed as % Tartaric acid) corrected as follows:
To 10 ml of aliquot of fermenting wine, add 10ml distilled  Add one for every 10oF rise in temperature
water and 5 drops of phenolphthalein indicator. Mix and
 Add 0.5 for upper meniscus of the milk
titrate it with 0.1N NaOH solution. (Table 2)
This now is called as Corrected lactometer reading
(CLR). The MSNF is calculated by the formula as: MSNF =
CLR/4 + 0.2 × Fat + 0.14
E.g. If lactometer reading observed at 70oF comes out
to be 27 then CLR is equal to 27+1+0.5 (for meniscus) =
28.5 and MSNF = 28.5/4 + 0.2× Fat + 0.14
Calculation of Acidity in Yogurt
Take one gram of yogurt in titration flask and dilute
it with 5 ml of distilled water. Add to it a few drops of
phenolphthalein indicator. The solution is colorless. Now
add from the burette 0.1N NaOH solution drop wise till
Theory the color of solution changes to light pink. This is the end
Milk of high SNF (10-12 or 15%) is used for the point. Repeat the experiment till a concordant set of three
preparation of yogurt. It is different from curd in the sense readings is obtained.
that, in yogurt milk of high SNF is inoculated with pure culture General Calculations
of Lactobacillus bulgaricus and Streptococcus cremoris or
Streptococcus thermophilus in the ratio of 1:1 while in curds Take one gram of yogurt in titration flask and dilute
natural flora acts as inoculum. After fermentation, the acidity it with 5 ml of distilled water. Add to it a few drops of
of the final product is measured in terms of lactic acid (gm phenolphthalein indicator. The solution is colorless. Now
per 100ml of yogurt). add from the burette 0.1N NaOH solution drop wise till
the color of solution changes to light pink. This is the end
V. Procedure point. Repeat the experiment till a concordant set of three
Preparation of starter culture: The inoculum of readings is obtained.
Lactobacillus bulgaricus is maintained in Micro-inoculum VII. Theory
broth of composition: Yeast extract 20g, Peptone 5g,
Dextrose 10g, Potassium dihydrogen phosphate 2g, Natural microflora present on cabbage produces lactic
Sorbitan monooleate complex 0.1g or Tween-80 few drops, acid from carbohydrates. In due course of time, after the
and distilled water one litre. The inoculum of Streptococci is accumulation of lactic acid to certain extent, all proteolytic
maintained in Nutrient broth of composition: Beef extract 3g, and other microorganisms are eliminated from the product
Peptone 5g, NaCl 5g, and distilled water one litre. Cultures except lactic acid tolerant Lactobacilli. These bacteria
of both the strains are to be mixed in 1:1 ratio in Peptonized further produce more lactic acid resulting in lowering of the
milk of composition: Skim milk powder 10g, Peptone 5g, pH of the product significantly after 3-4 weeks. Because of
and distilled water 100 ml; pH 6.5 sterilized at 85-95oC for this lowering in pH other organisms do not find any access
to grow in the same product.
 Basic of Fermentation Technology

51

VIII. Procedure Weigh the salt (2.25%) i.e. 11.25 gm for ½ kg of cabbage
shredding. Put the outer leaves that were kept aside, at the
Wash cabbage with clean water. Remove the outer
bottom of a glass jar. Take one lot of shredded cabbage
leaves. These leaves are kept aside for their further use.
and layer onto the leaves inside the glass jar. Sprinkle ¼th
Remove the case and other undesirable area. Prepare
of the salt on the cabbage and wait for its absorption in the
lots of cabbage weighing ½ kg each and slice them into
shredded cabbage. Similarly layer the rest of the shredded
shredding or small pieces of 0.16-0.08 cm in thickness.
 Basic of Fermentation Technology

52
cabbage and sprinkle the salt onto it till all the cabbage and therapeutic value and has been successfully tried in cases of
salt finishes for the preparation of sauerkraut. The addition chronic colitis and gastro-intestinal disorders in general. It is
of salt serves two main functions. Firstly, it draws moisture prepared by the inoculation of pure culture of Lactobacillus
out of cabbage that dissolves the salt forming a brine acidophilus. It is a very acidic product. The acidophilus
solution, which acts as a fermenting medium for Lactobacilli milk has not gained popularity as that of other fermented
and also equally distributes them in the medium. Secondly, milk products because of its taste and off-flavor. When it is
it inhibits the growth of proteolytic bacteria. Now place sweetened with sugar, it is called as sweet acidophilus milk.
the plastic bags filled with water as a weight to press the Sweet acidophilus milk is gaining popularity now a day.
cabbage and close the lid of the glass jar. Fermentation was
carried out at 25oC for 3-4 weeks. Note down the change in Medium for maintenance for lactobacillus
pH and color after every week. acidophilus
Microbiology of Sauerkraut Fermentation Pederson The organism is maintained in Tomato Juice Agar
first described the lactic acid bacteria that he observed in of Composition: Tomato juice 400ml equivalent to 20g
fermenting sauerkraut. He found that the fermentation was tomatoes, Peptone 15g, Skimmed milk powder 10g, Agar-
initiated by the species of Leuconostoc mesenteroides. This Agar 1.5-2.0%; pH 5.0; sterilize at 15lb pressure for 15
species was followed by gas forming rods and finally by non- minutes.
gas forming rods and cocci. Leuconostoc mesenteroides is
Procedure for ordinary acidophilus milk
a hetero fermentative bacteria and it grows more rapidly
than other lactic acid bacteria. It is active over a wide Take adequate quantity of low fat milk. Boil or steam it
range of temperature and salt concentrations. It produces for 20 minutes. Cool it to a temperature of 28-300C. Pass
acid and carbon dioxide that rapidly lowers the pH, thus carbon dioxide gas through it for 1-2 minutes. Inoculate it
inhibiting the activity of undesirable microorganisms and with 1-2% inoculum of Lactobacillus acidophilus. Incubate
enzymes that may soften the shredded cabbage. The at 37-40 °C for 30-40 hours.
carbon dioxide replaces the air and creates an anaerobic
condition favorable to prevent oxidation of ascorbic acid and Procedure for sweetened acidophilus milk
natural color of the cabbage. It also stimulates the growth Inoculate cold pasteurized sweetened low fat milk with
of many lactic acid bacteria. While this initial fermentation is 1-2% of pure culture of Lactobacillus acidophilus. No
developing, the hetero fermentative species of lactobacillus incubation is done. Inoculated milk is held under refrigeration
brevis and homofermentative species of lactobacillus at 7°C or below for 30-40 hours or till it is consumed. It
plantarum and sometimes Pediococcus cerevisiae begin tastes exactly like low fat milk.
to grow rapidly and contribute to the major end products
like lactic acid, carbon dioxide, ethanol, acetic acid. Minor Food Fermentation Practical (5) -Production of Lactic
products also appear in the fermentation. The minor acid
products are a variety of volatile compounds e.g. diacetyls,
Materials required
acetaldehyde and primary carbonyls.
Pure culture of Lactobacillus delbrueckii B-70 and
Role of temperature in sauerkraut fermentation Production medium of composition: Sucrose 100g/L, Yeast
Lower the initial temperature better is the product extract 20g/L, Potassium dihydrogen orthophosphate
formation. It is considered that the initial temperature of 2.5g/L, Calcium carbonate 10%, Agar-Agar 2% (for solid
18.3oC produces superior quality sauerkraut because at medium); pH 7.0-7.2; Sterilize at 15 lb pressure for 15
lower temperature hetero fermentative lactic acid bacteria minutes. Vitamin B-complex+ Aspartic acid +Folic acid
exert a greater effect. combination may be used in place of yeast extract.

Spoilage of Sauerkraut Medium for inoculum preparation

Common spoilage signs found in sauerkraut are Inoculum is prepared in Glucose Yeast Extract medium
discoloration, off-flavor, off-odor caused by yeast and mold of composition: Glucose 10%, Yeast extract 2% and
growth, loss of acidity and slimy product due to the dextran Potassium dihydrogen orthophosphate 0.25%.
formation by Leuconostoc mesenteroides. The proteolytic I. Procedure
activity of molds and yeasts and also by asporogenous
yeasts produces product pink in color. Such type of spoilage Take two litres of production medium in laboratory
is known as “Pink kraut”. fermenter and inoculate it with 10% of inoculum of L.
delbrueckii B-70 prepared in Glucose Yeast extract medium.
Food Fermentation practical (4) -Preparation of Sweet Incubate it at 37 °C for 5-7 days. The medium is gently
Acidophilus milk stirred during the incubation period to keep the calcium
I. Theory carbonate in suspension.

The fermented acidophilus milk is known for its

 Basic of Fermentation Technology

Recovery of lactic acid II); add solution-I into solution-II and adjust pH to 5.2, make
53
volume 100ml and heat it to 60oC for 10-15 minutes.
Filtration -The suspension is filtered with conventional
laboratory filters. Acidification -To the filtrate add I. Procedure
concentrated sulfuric acid to form precipitates of calcium
Dispense 100ml of production medium in two 250ml
sulfate. Filter and wash the precipitates with water. The
conical flasks and sterilize it at 15lb pressure for 15 minutes.
washings are added into the filtrate. Removal of Impurities
Inoculate the flasks with Streptomyces spp. culture that
-The filtrate is treated with activated charcoal to remove
was preserved in nutrient broth medium at the rate of 5%.
the impurities. Filter again. Concentration –The filtrate
Incubate the flaks on shaker at 37 °C for 96 hours.
is evaporated on a steam bath to concentrate it to 25%
solids and then again subjected to evaporation till 50% Standard curve for starch
solids are obtained. Removal of Heavy metals -The heavy
metals like lead is removed by adding sodium or potassium Prepare dilutions of starch in acetate buffer from 0.1% to
ferrocyanide into the concentrated mass and filtering it. The 0.01%. To each of these dilutions, add 0.2ml Iodine solution
filtrate contains lactic acid, which can be purified by passing and add water to dilute it to 10ml. Measure the optical
it through ion exchange resin column. The lactic acid so density at 520nm. For control take 1ml of distilled water
obtained may have 93-95% purity. in place of starch dilution and to this add iodine solution;
observe optical density in a similar manner.
Food Fermentation Practical (6) –Production of
Amylase enzyme and its estimation Estimation of amylase
Materials required Prepare Control, Blank and Digest. Control -To 6ml of
0.1% starch solution, add 1ml of 1% calcium chloride, 2ml
Inoculum -An Amylase producing strain of Streptomyces of hydrochloric acid, and 1ml of distilled water and 0.2ml of
spp. preserved in Nutrient medium of composition: Beef iodine solution. Measure optical density at 520nm. Blank
extract 3g, Peptone 5g, NaCl 5g, and distilled water one litre. -To 6ml of 0.1% starch solution, add 1ml of 1% calcium
Calcium chloride solution 1% in water, Starch solution 0.1% chloride solution, 1ml of enzyme extract, 2ml of hydrochloric
in 0.05M acetate buffer pH 5.2 (dissolve 1mg starch in 1ml acid and 2ml of iodine solution. Measure optical density at
of acetate buffer), Production medium of composition: Beef 520nm. Digest -To 6ml of 0.1% starch solution, add 1ml of
extract 3g, Peptone 5g, NaCl 5g, and distilled water one 1% calcium chloride solution and 1ml of enzyme extract.
litre. Add starch 10g per litre into the nutrient broth medium. This is incubated at 30 °C for 10 minutes. Now add 2ml of
Sodium acetate-Acetic acid buffer solution (0.05M) -Sodium HCl and 0.2ml of iodine solution. Measure optical density
acetate 2.72g dissolve in 100ml distilled water (solution-I), at 52 nm.
Acetic acid 1.15g dissolve in 100ml distilled water (solution-

Calculation of Enzyme activity

I. Theory Materials required

The yeast Saccharomyces cerevisiae converts Molasses; fermentation jar (10 L); micro distillation
fermentable sugars (glucose, fructose and sucrose) into unit; Test tubes; conical flasks; Standard volumetric flasks
ethanol and carbon dioxide. In large-scale production of (25ml); Urea; DNS reagent (3,5-Dinitosalicylic acid 1%,
alcohol, molasses is used as substrate. Blackstrap molasses Phenol 0.2%, Sodium carbonate 0.05%, Sodium hydroxide
contains 45-55% w/v fermentable sugar as sucrose, which 1% and Sodium potassium tartarate 20%); Dichromate
is metabolized by yeast through Embden-Meyerhoff-Parnas reagent (Dissolve 34 gm of Potassium dichromate in 500
Pathway to produce ethanol and carbon dioxide as the end ml of distilled water, add 325 ml of concentrated Sulfuric
products. acid slowly keeping the flask in ice bucket); YPD medium
of composition: Yeast extract 10gm, Peptone 20 gm,
 Basic of Fermentation Technology

54 Dextrose 20gm and Agar-Agar 20gm; Slant culture of yeast Black gram dhal 3) Salt 4) water. Dehulled soybeans or
S. cerevisiae. Bengal gram can be used as a substitute for black gram dhal
and a number of cereal grains can replace rice. However,
I. Procedure
there may be marked change in the texture and flavor
Preparation of Inoculum: Prepare 250ml GYE / YPD when using substituted materials. It has been reported
broth medium. Add 50ml in separate flasks and autoclave that rice variety and its physical characteristics are very
at 15lb pressure for 15 min. Inoculate slant culture of yeast important to produce a good quality idli. White Kar and IR20
aseptically in each 50ml flask. Incubate the flasks at 28 °C varieties of rice have given much better performance in the
for 16-18h. production of idli, especially the White Kar variety because
of its high amylose content, low amylopectin content better
II. Inoculum for fermentation gelatinization, and better water uptake ability.
Dilute molasses to 12 oBrix (1.1Kg molasses in 8 litre of Proportion of cereal to legume: Ordinary idli consists of
water). Adjust pH to 5.5 with 10N Sulphuric acid. Sterilize at three parts rice and one part Black gram dhal plus salt to
10lb pressure for 30min and then cool. Inoculate this 250 ml taste. Kancheepuram idli is prepared from one part rice
molasses medium (2 flasks) with 10% of the culture grown and one part black gram dhal plus cashew nuts, ghee,
in YPD. Incubate the culture in shaker for 12h at 28 °C. salt, pepper, ginger and cumin added to taste. Normally a
III. Fermentation medium proportion of rice to black gram dhal varies from 4:1 to 1:4,
the 2:1 being the best. It has been seen that when black
Dilute the Black strap molasses with tap water to 22 0Brix gram dhal proportion is less than 25%, the steamed idli was
(2.1Kg molasses in 8 litre water). Adjust pH to 5.5 with 10N hard and organoleptically unacceptable whereas when it is
Sulphuric acid. Add 200mg Sodium dihydrogen phosphate more than 50%, the product obtained is too sticky to be
and 200mg Urea per litre respectively. Maintain pH 5.5. acceptable. Thus, not only can the ingredients be varied,
Sterilize medium at 10lb pressure for 30min. Inoculate with but the proportions can also be varied within a wide range
10% v/v inoculum and incubate at 28 °C for 48-72h for and still an acceptable product is obtained.
fermentation. After the fermentation has ceased close the
mouth of the flask with an airtight bung to provide anaerobic I. Procedure
conditions so that alcohol production may take place. White polished rice is carefully washed and soaked for
Withdraw 10ml sample at every 12h interval and estimate 5-10 hours. Black gram dhal is carefully washed and soaked
alcohol and sugar concentration. Plot a graph with time on for 5-10 hours. The rice is then drained and coarsely ground
X axis and alcohol and sugar concentration on Y- axis. in a stone mortar or other grinder. The black gram dhal is
Alcohol estimation drained and finely ground in a stone mortar. The rice and
black gram dhal slurries are combined to form a rather thick
Preparation of standard curve for alcohol concentration: batter, which is stirred with hands. Salt is added to taste.
Prepare 1-10% v/v alcohol in Other seasonings, such as chilies are occasionally added.
The batter is placed in a warm place to ferment overnight.
Various test tubes. Take 1ml of the various concentration
In the morning, the batter is poured into the cups of an idli
of alcohol into 25ml of distilled water in 100ml distillation
steamer, which is placed in a covered pan or cooker and
flask fitted with Liebig condenser. Distill and collect 15ml
steamed until the starch is gelatinized and the idli cakes are
of the distillate in a 50ml volumetric flask containing 25ml
soft and spongy.
Pot. Dichromate solution. Make up the volume to 50ml.
Keep the alcohol-Pot. dichromate complex at 60 °C for 30 Food Fermentation Practical (9) -Preparation of
min. Measure OD at 600nm. Plot a standard curve with Dosa
concentration of alcohol on X-axis and OD at 600nm on
Y-axis. II. Ingredients

Determination of Alcohol Concentration from The ingredients used for dosa preparation are similar to
fermentation Medium as that of idli preparation i.e. rice, black gram dhal, salt and
water. Rice may be substituted by wheat, bajra Pennisetum
Take 1ml sample and mix it with 25ml of distilled water. typhoideum, maize, or kodri and black gram dhal may be
Distill as above and measure OD at 600nm. Find the alcohol substituted by sprouted peas, cowpeas (Vigna catjang),
concentration from the standard graph. field beans (Dolichos lablab) or soybeans. Fresh groundnut
oilcakes may also be substituted for black gram dhal.
A. If molasses could not be obtained for laboratory
exercise, use YPD medium with 50g/L sucrose for inoculum III. Soaking and batter formation
development and with 150g/L sucrose for preparation of
fermentation broth. Generally, equal quantities of rice and dehulled black
gram dhal are soaked in water at room temperature
Food Fermentation Practical (8) -Preparation of Idli separately for 5-10 hours. It is common practice that finely
ground powders are used to prepare batter. The finely
Ingredients: Idli preparation contains a number of different
ground powders are mixed with water at temperatures
ingredients these are 1) Rice 2)
 Basic of Fermentation Technology

55
ranging from 48o to 98 °C, best at 80 °C for the preparation the crisp dosa and the sides are rolled over it, which now
of batter. Water is added in the range of 2.0 to 2.2 times becomes ready to be eaten with a spiced soup of dhal and
the initial dry weight of ingredients to prepare a batter of vegetables popularly known as Samber. Food Fermentation
viscosity desired for dosa. Salt is added from 0.8 to 1.0% as Practical (10) -Production of Citric acid by Aspergillus niger
a seasoning before fermentation. in media containing molasses and sucrose.
IV. Fermentation time VII. Theory
Traditionally, dosa batter is kept overnight for Many microorganisms like molds (Aspergillus niger,
fermentation. The fermentation time should be sufficient to A. awamori, Penicillium janthinallum, Trichoderma
allow a definite leavening and acidification of the batter and viridae, Mucor piriformis, etc.), yeasts (Candida lipolytica,
to allow for the development of a pleasant acid flavor. C.tropicalis, C.citrica, Hansenula, Rodotorula, Pichia,
Torulopsis etc.) and bacteria (Bacillus licheniformis, Bacillus
V. Incubation temperature
subtilis, Brevibacterium flavus, Corynebacterium species)
Ordinarily, the dosa fermentation is carried out at have the capability to produce citric acid. Commercially
room temperature. In the tropics, this generally means a spores of Aspergillus niger are employed to produce citric
temperature of 25-30 °C. acid. It accumulates in culture solutions of pH 1.8-2.0. In
fungi different metabolic pathways are involved in the
VI. Harvesting and preservation production of citric acid but 78% of the citric acid is produced
As soon as the batter becomes leavened and acidified, by the involvement of glycolysis and Tricarboxylic acid cycle.
it is spread onto a hot and greasy griddle where it assumes The acetyl COA derived by EMP pathway condenses with
the shape of a crisp pancake. The spreading of dosa on Oxaloacetate of Kreb’s cycle to produce citric acid. Citric
the griddle is an art that matures with practice. Sometimes acid is an important chemical used in medicines, flavoring
a mixture of cooked different vegetable is poured onto extracts, food and candies and in dyeing industry.

Flow Chart 16
 Basic of Fermentation Technology

56
VIII. Materials required sucrose) of 250ml flasks. Each set having three flasks
containing 100ml production medium. One set having
Molasses; Sucrose; Flasks; Test tubes; Sodium
molasses and sucrose in the second set. Label the flasks
hydroxide; Ammonium dihydrogen orthophosphate; Trace
of each set as control flask, Methanol containing flask, and
element solution of composition: Zinc sulfate 3mg/100ml,
flask without methanol. Add 3ml of methanol in the flask
Copper sulfate 680mg/100ml, Magnesium sulfate
labeled as methanol flask. Sterilize both the sets at 15 lb
2.0g/100ml and EDTA 2.0g/100ml; Dissolve in 100ml
pressure for 15 minutes. Cool the flasks and inoculate all
distilled water and incubate at 25oC for 3 days.
the flasks of both the sets with spores of Aspergillus niger
Composition of production medium of citric acid except the control flask. Incubate the flasks at 25 °C for
4-5 days on shaker. After the fermentation is over, filter the
Molasses or Sucrose 45gm/300ml, Ammonium contents. Compare the acidity of the filtrate with the control
dihydrogen orthophosphate 0.75gm/300ml, Potassium flask.
dihydrogen orthophosphate 0.3gm/300ml, Tween-80
0.6gm/300ml; pH for molasses 5.0-6.0 and for sucrose 2.0- II. Acidity
3.0 Add trace element solution before sterilization. Sterilize Titrate the filtrate against 0.1N NaOH using
at 15lb pressure for 15 minutes. phenolphthalein as an indicator. Appearance of pink color
I. Procedure is the end point. Note down the volume of filtrate used in
titration and calculate the strength of citric acid in filtrate.
Prepare two sets (one for molasses and second for

General calculations