Microbial Biotech Final PPT This is introduction to microbial technology course material..pdf

Microbial Biotechnology (Biot. 3105)
Addis Ababa Science and Technology University
College of Applied
Department of Biotechnology
By:
Alena B. and Woinshet M.

CHAPTER-ONE
Introduction and overview
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Cont.….d

1.1. Brief on Microbial Physiology and Habitat
Molecular phylogenies divide all living organisms into three domains:
1. Bacteria
2. Archaea and
3. Eukarya (eukaryotes: protists, fungi, plants, animals).
Ø Microbes are organisms that are too small to be seen by the unaided eye.
Ø They include bacteria, fungi, protozoa, micro algae, and viruses.

1.1.1. Prokaryotes and Eukaryotes
ØCellular organisms fall into two classes depending on internal organization of their cells.
ØThe cells of eukaryotes contain a true membrane-bounded nucleus, which in turn contains a set of
chromosomes that serve as the major repositories of genetic information in the cell.
ØEukaryotic cells also contain other membrane-bounded organelles that possess genetic information,
namely mitochondria and chloroplasts.
Ø In the prokaryotes, the chromosome (nucleoid) is a closed circular DNA molecule, which lies in the
cytoplasm, is not surrounded by a nuclear membrane, and contains all of the information necessary
for the reproduction of the cell.
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Ø Prokaryotes also have no other membrane-bounded organelles whatsoever.
Ø Bacteria and archaea are prokaryotes, whereas fungi are eukaryotes.
Ø The choice of a fungus (such as the yeast Saccharomyces cerevisiae) or a
bacterium (such as Escherichia coli) for a particular application is often dictated by
the basic genetic, biochemical, and physiological differences between prokaryotes
and eukaryotes.

Fig. 1 Prokaryotes and eukaryotes cells

v Microbes can be classified in terms of their oxygen intake into three main
classifications:
1. Aerobes where the growth depends on a plentiful supply of oxygen to make cellular
energy.
2. Strictly anaerobes, by contrast, which are sensitive to even low concentration of oxygen
and are found in deep oil reservoirs.
3. Facultative microbes, which can grow either in the presence or reduced concentration
of oxygen.

Ø Microbes live in familiar settings such as soil, water, food, and animal intestines,
atmospheric, as well as in more extreme settings such as rocks, glaciers, hot springs,
and deep-sea vents.
Ø The wide variety of microbial habitats reflects an enormous diversity of
biochemical and metabolic traits that have arisen by genetic variation and natural
selection in microbial populations.

Ø The microbial world encompasses most of the phylogenetic diversity on Earth, as all Bacteria, all
Archaea, and most lineages of the Eukarya are microorganisms.
Ø Microbes live in every kind of habitat and their presence invariably affects the environment in
which they grow.
Ø Their diversity enables them to thrive in extremely cold or extremely hot environments.
Ø Their diversity also makes them tolerant of many other conditions, such as limited water
availability, high salt content, and low oxygen levels.

Fig.2 Microorganisms in a cold environment: Ice algae in Antarctica.

Fig.3 Microorganisms in a hot environment: Algae growing in a hot pool in New Zealand

Ø Halophilic microorganisms grow in brine ponds encrusted with salt,
Ø Thermophilic microorganisms grow on smoldering coal piles or in volcanic hot
springs, and
Ø Barophilic microorganisms live under enormous pressure in the depths of the seas.
Ø Some bacteria are symbionts of plants; other bacteria live as intracellular parasites
inside mammalian cells or form stable consortia with other microorganisms.
Ø Not every microbe can survive in all habitats, though. Each type of microbe has
evolved to live within a narrow range of conditions.

1.2. The use and application of microbes in Biotechnology
1.2.1. Beneficial and harmful aspects of microorganisms
vThe beneficial effects of microbes derive from their metabolic activities in the environment,
their associations with plants and animals, and from their use in food production and
biotechnological processes.
vBacillus thuringiensis (Bt) is a soil bacterium that produces a protein with insecticidal
qualities.
vTraditionally, a fermentation process has been used to produce an insecticidal spray from
these bacteria.
vIn this form, the Bt toxin occurs as an inactive protoxin, which requires digestion by an
insect to be effective.

vThere are several Bt toxins and each one is specific to certain target insects.
vCrop plants have now been engineered to contain and express the genes for Bt
toxin, which they produce in its active form.
vWhen a susceptible insect ingests the transgenic crop cultivar expressing the Bt
protein, it stops feeding and soon thereafter dies as a result of the Bt toxin binding
to its gut wall.

v Fungi are particularly effective in colonizing dry wood and are responsible for
most of the decomposition of plant materials by secreting powerful extracellular
enzymes to degrade biopolymers (proteins, polysaccharides, and lignin).
v They produce a huge number of small organic molecules of unusual structure,
including many important antibiotics.
v Historically, humans have exploited some of this microbial diversity in the
production of fermented foods such as bread, yogurt, and cheese.

Foods Made Using Microorganisms

v Microorganisms used to clean up contaminated environments bioremediation and
biodegradation.
v The elimination of a wide range of pollutants and wastes from the environment is an absolute
requirement to promote a sustainable development of our society with low environmental impact.
v Biological processes play a major role in the removal of contaminants and biotechnology is
taking advantage of the astonishing catabolic versatility of microorganisms to degrade/convert
such compounds.
v Scientists can inject microbes into the ground to clean up or deactivate groundwater pollution.
v This process, called bioremediation, modifies bacteria that naturally break down toxins so we
can clean up chemical spills, waste dumps, and even radioactive waste sites faster and more
efficiently than without their help.

vSome soil microbes release nitrogen that plants need for growth and emit gases that maintain
the critical composition of the Earth's atmosphere.
vOther microbes challenge the food supply by causing yield-reducing diseases in food-
producing plants and animals.
vIn our bodies, different microbes help to digest food, ward off invasive organisms, and
engage in skirmishes and pitched battles with the human immune system in the give-and-take
of the natural disease process.
v Some microbes are pathogenic and cause diseases to human beings, plants and animals. E.g.
Mycobacterium tuberculosis, Mycobacterium leprae, Staphylococcus epidermidis,
Staphylococcus aureus, Streptococcus pyogenes, Variola, etc.

.

1.3. Scopes of Microbial Biotechnology
vMicrobial biotechnology is defined as any technological application that uses
microbiological systems, microbial organisms, or derivatives thereof, to make or
modify products or processes for specific use.

ü Microbial biotechnology, enabled by genome studies, will lead to breakthroughs
such as:
I. Improved vaccines and better disease-diagnostic tools,
II. Improved microbial agents for biological control of plant and animal pests,
III. Modifications of plant and animal pathogens for reduced virulence,
IV. Development of new industrial catalysts and fermentation organisms, and
V. Development of new microbial agents for bioremediation of soil and water.

v Microbial genomics and microbial biotechnology research is critical for advances in:
§ food safety,
§ food security,
§ biotechnology,
§ value-added products,
§ human nutrition and functional foods
§ plant and animal protection
§ Fundamental research in the agricultural sciences.

§ Cultures such as those in Mesopotamia, Egypt, and India developed the
process of brewing beer.
§ It is still done by the same basic method of using malted grains (containing
enzymes) to convert starch from grains into sugar and then adding specific
yeasts to produce beer.
§ In this process the carbohydrates in the grains were broken down into
alcohols such as ethanol.
§ Ancient Indians also used the juices of the plant Ephedra vulgaris and used to call it
Soma.

§ Later other cultures produced the process of Lactic acid fermentation which
allowed the fermentation and preservation of other forms of food.
§ Although the process of fermentation was not fully understood until Louis
Pasteur's work in 1857, it is still the first use of biotechnology to convert a
food source into another form.
§ Combinations of plants and other organisms were used as medications in many
early civilizations.

§ Since as early as 200 BC, people began to use disabled or minute amounts of infectious
agents to immunize themselves against infections.
§ These and similar processes have been refined in modem medicine and have led to many
developments such as antibiotics, vaccines, and other methods of fighting sickness.
§ In the early twentieth century scientists gained a greater understanding of microbiology and
explored ways of manufacturing specific products.
§ In 1917, Chaim Weizmann first used a pure microbiological culture in an industrial process,
that of manufacturing com starch using Clostridium acetobutylicum, to produce acetone,
which the United Kingdom desperately needed to manufacture explosives during World War
I.

§ Indian-born Ananda Chakrabarty, working for General Electric, had developed a
bacterium (derived from the Pseudomonas genus) capable of breaking down crude oil,
which he proposed to use in treating oil spills. Revenue in the industry is expected to
grow by 12.9% in 2008.
§ Microbial biotechnology has applications in four major industrial areas, including
health care (medical), crop production and agriculture, nonfood (industrial) uses of
crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and
environmental uses.

§ For example, one application of biotechnology is the directed use of organisms for the
manufacture of organic products (examples include beer and milk products).
§ Another example is using naturally present bacteria by the mining industry in bioleaching.
§ Biotechnology is also used to recycle, treat waste cleanup sites contaminated by industrial
activities, and also to produce biological weapons.

§ Many microbes are responsible for many beneficial processes such as industrial
fermentation (e.g. the production of alcohol and dairy products), antibiotic
production and as vehicles for cloning in higher organisms such as plants.
§ Scientists have also exploited their knowledge of microbes to produce
biotechnologically important enzymes such as Taq polymerase, reporter genes
for use in other genetic systems and novel molecular biology techniques such
as the yeast two-hybrid system.

• Bacteria can be used for the industrial production of amino acids.
Corynebacterium glutamicum is one of the most important bacterial species with
an annual production of more than two million tons of amino acids, mainly L-
glutamate and L-Iysine.
• A variety of biopolymers, such as polysaccharides, polyesters, and polyamides,
are produced by microorganisms.
• Microorganisms are used for the biotechnological production of biopolymers with
tailored properties suitable for high-value medical application such as tissue
engineering and drug delivery.

§ Microorganisms are used for the biosynthesis of xanthan, alginate, cellulose, cyanophycin,
poly (gamma-glutamic acid), levan, hyaluronic acid, organic acids, oligosaccharides and
polysaccharide, and polyhydroxyalkanoates.
• Microorganisms are beneficial for microbial biodegradation or bioremediation of -domestic,
agricultural and industrial wastes and subsurface pollution in soils, sediments and marine
environments.
• The ability of each microorganism to degrade toxic waste depends on the nature of each
contaminant

o Since most sites are typically comprised of multiple pollutant types, the most effective
approach to microbial biodegradation is to use a mixture of bacterial species and strains,
each specific to the biodegradation of one or more types of contaminants.
o There are also various claims concerning the contributions to human and animal health by
consuming probiotics (bacteria potentially beneficial to the digestive system) and/or
prebiotics (substances consumed to promote the growth of probiotic microorganisms).

CHAPTER-TWO
Important microorganisms for Biotechnologically Applications

2.1. Criteria for Selecting Microbes for Applications
Ø Although the well-known ubiquity of microorganism implies that almost any natural
ecological entity may provide microorganisms, the soil is the preferred source for
isolating organisms.
Ø Because it is a vast reservoir of diverse organisms.
ØIndeed, microorganisms capable of utilizing virtually any carbon source will be found
in soil.
Ø In recent times, other ‘new’ habitats, especially the marine environment, have been
included in habitats to be studied in searches for bioactive microbial metabolites.

2.2. Microbial Isolation (Classical and Emerging Methods)
1. Enrichment with the substrate utilized by the organism being sought
Ø If the organism being sought is one which utilizes a particular substrate, then soil is
incubated with that substrate for a period of time.
ØThe conditions of the incubation can also be used to select a specific organism.
ØIn the search for α-amylase producers, the soil may be enriched with starch and
subsequently suitable soil dilutions are plated on agar containing starch as the sole
carbon source.
Ø Clear halos form around starch-splitting colonies against a blue background when
iodine is introduced in the plate.

2. Enrichment with toxic analogues of the substrate utilized by the organism being
sought
Ø Toxic analogues of the material where utilization is being sought may be used for
enrichment, and incubated with soil.
Ø The toxic analogue will kill many organisms which utilize it.
Ø The surviving organisms are then grown on the medium with the non-toxic
substrate.
Ø Under the new conditions of growth many organisms surviving from exposure to
toxic analogues over-produce the desired end-products.

Types of culture collections
Ø There are various kinds of culture collections. The best known are:
I. American Type Culture Collection (ATCC).
II. National Collection of Type Cultures (NCTC)- specialized and may
handle only pathogenic microorganisms, in Colindale, London, UK or
III. National Collection of Industrial Bacteria (NCIB)- for industrial microorganisms
in Aberdeen, Scotland.

Handling culture collections
Ø Cultures are expensive to purchase.
Ø They are usually, however, supplied at a discount when used for reaching.
ØAn industrial process may be initiated with organisms obtained through the Patent
Office in connection with a patent.
Ø Often only one vial of such an organism is usually available.
ØOnce growth has been obtained from that vial the organism should be multiplied and
stored in one or more of the several manners.

Ø No matter what the source of a valuable organism, it is important that several replicates
are stored immediately for fear of contamination.
Ø If the tests show that the expected antibiotic or other desired metabolite is being
produced in the expected quantity then stored organisms are retained.
Ø The stocks of those organisms which proved negative at first sampling should not be
discarded in a hurry because further examination may show that poor productivity was
due to factors extrinsic to the organism such as an inadequate medium.

Ø In order to identify the organisms they must be properly labeled and accurate records
kept of the handling of the organism.
ØDate of transfer, the medium and the temperature of growth, etc., must all be carefully
recorded to afford a means of assessing the effect of the preservation method.

Methods of preserving microorganisms
Ø Several methods have been devised for preserving microbial cultures.
Ø None of them can be said to apply exclusively to industrial microorganisms.
Ø Furthermore, no one method is suitable for preserving all organisms.
Ø The method most suited to any particular organism must therefore be determined by
experimentation unless the information is already available.

The principles involved in preserving microorganisms are:
(a) reduction in the temperature of growth of the organism
(b) dehydration or desiccation of the medium of growth and
(c) limitation of nutrients available to the organism.
All three principles lead to a reduction in the organisms’ metabolism

2.3. Strain Improvement and Maintenance
Gram stain
a. Gram stain divides the bacteria into Gram positive & Gram negative. The basic procedure
goes like this:
i. Take a heat fixed bacterial smear.
ii. Flood the smear with crystal violet for 1 minute, then wash with water. [primary stain]
iii.Flood the smear with iodine for 1 minute, then wash with water.
iv.Flood the smear with ethanol quickly, then wash with water. [decolor]
v. Flood the smear with safranin for 1 minute, then wash with water. [counterstain]

§ Blot the smear, air dry and observe.
Examine under microscope
i. Gram positive bacteria‐violet
ii. Gram negative bacteria‐pink

Cultural characteristics
These provide additional information for the identification of a bacterium
A. On solid medium the following characters are observed
I. Shape: circular, irregular, radiate or rhizoid.
II. Size: The size of the colony can be a useful characteristic for identification.
III. The diameter of a representative colony may be measured.
IV. Elevation:
V. Margin: Entire, wavy, lobate, filiform
VI. Surface: smooth, wavy, rough, granular, papillate, glistening etc.
VII. Size in mm
VIII.Texture: dry, moist, mucoid, brittle, viscous, butyrous (buttery).
IX. Color: colorless, pink, black, red, bluish‐green.

B. In a fluid medium following characters are observed
i. Degree of growth ‐ Absence, scanty, moderate, abundant etc.
ii. Present of turbidity and its nature.
iii. Presence of deposit and its character.
iv. Nature of surface growth.
v. Ease and disintegration and odor.

Biochemical tests
1. Indole Test
Principle: to determine the ability of the organism to split tryptophan molecule into Indole.
Indole is one of the metabolic degradation
product of the amino acid tryptophan.
Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminatin
g tryptophan with the production of Indole, Pyruvic acid and ammonia.
This test is performed to help
differentiate species of the family Enterobacteriaceae.
Media and Reagents Used: Trypton broth contains tryptophan. Kovac’s reagent
contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—
yellow in color.

Procedure:
Inoculate Trypton broth with for 18 to 24 hr
s. at 37°c
Add 15 drops of Kovac’s reagent down the i
nner wall of the tube.
Interpretation: -
Development of bright red color at the inter
face of the reagent.

2. Methyl Red (MR)
Both tests are used to differentiate species of the family Enterobacteriaceae.
Media and Reagents Used: Glucose Broth Methyl Red indicator for MR test
Principle of MR test:
To test the ability of the organism to produce and maintain stable acid end pro
ducts from glucose fermentation and to overcome the buffering capacity of th
e system. This is a qualitative test for acid production.

Procedure: ‐
Inoculate the MR/VP broth with a pure culture of the test organism and incubate at 35°f
or 48 to 72 hrs.
• Add 5 drops of MR reagent to the broth
Result interpretation:
• Positive result is red (indicating pH below 6)
• Negative result is yellow (no acid production)

3. Citrate Utilization test
This test is identification of enterobacteria.
The ability of an organism to use citrate as its only sole source of carbon and ammonia as its on
ly source of nitrogen test.
Principle:
The test organism is cultured in a medium which contains sodium citrate, an ammonium salt an
d the indicator bromothymol blue.
Growth in the medium is shown by turbidity and a change in colour of the indicator from light
green to blue, due to alkaline reaction following citrate utilization.

Procedure: Inoculum is streaked over the slant of Simmon’s citrate agar in a
tube and incubated for 24‐48 hrs.
Result interpretation: Growth on the slant and change in colour
to blue of the medium indicates positive result.

4. Oxidation‐Fermentation (OF) test (Hugh & Leifson)
Principle:
§ Oxidation fermentation test is used to determine the oxidative
or fermentative metabolism of a carbohydrate or its non utilization.
§ Fermentation is a anaerobic process and bacterial fermenter
of carbohydrates are usually facultative anaerobes.
§ Oxidation is a aerobic process and bacterial oxidisers are usually strict aerobes

• Procedures:
Test employs a semi‐solid medium in tubes containing the carbohydrate under test (usuall
y glucose) and a pH indicator.
‐Two tubes are inoculated and one is immediately sealed with paraffin oil to produce ana
erobic conditions
Result interpretation: Oxidizing organisms produce an acid
reaction in the open tube only. Fermenting organisms
produce an acid reaction throughout the medium in both tubes.

5. Motility Test
ü To differentiate species of bacteria that are motile
from non‐motile. Media and Reagents Used:
üMotility media contains tryptose, sodium chloride, agar, and a color i
ndicator.
How to Perform Test: Stab motility media with inoculating needle.
Reading Results: If bacteria is motile,
there will be growth going out away from the stab line, and test is posit
ive. If bacteria is not motile, there will only be growth along the stab li
ne. A colored indicator can be used to make the results easier to see.
change or is reddish.

6. Glucose Fermentation & Gas Production
To differentiate species of the family Enterobacteriaceae. This tests
for the bacteria’s ability to ferment glucose and produce gas and/or
an acid end product. Media and Reagents Used:
Glucose broth contains beef extract, gelatin peptone, and glucose
A phenol red indicator is added to indicate an acid end‐product.
A Durham tube is added to indicate gas production. How to Perform Test:
Inoculate broth with inoculating loop.
Results
A positive result for acid is yellow after indicator is added (indicating glucose fermentatio
n) A positive result for gas is a bubble in the Durham tube.
A completely negative result has no color change or reddish color and no bubble.

7. Immunological Testing
v Uses serology
v Study and diagnostic use of antigen-antibody interactions in blood serum
Two categories of immune testing: –
1. Direct testing (looks for antigens)
2. Indirect testing (looks for antibodies)
How to choose? Consider suspected diagnosis, cost, and speed of result

Genetic Testing
v Genetic testing involves the direct examination of the DNA molecule itself.
vA scientist scans a patient's DNA sample for mutated sequences.
vThere are two major types of gene tests.
vIn the first type, a researcher may design short pieces of DNA ("probes") whose sequences are
complementary to the mutated sequences.
vThese probes will seek their complement among the base pairs of an individual's genome.
vIf the mutated sequence is present in the patient's genome, the probe will bind to it
and flag the mutation.
vIn the second type, a researcher may conduct the gene test by comparing the sequence
of DNA bases in a patient's gene to disease in healthy individuals or their progeny.

Strain improvement
v Ability of any organism to make any particular product is predicated on its capability
for the secretion of a particular set of enzymes.
v The production of the enzymes, themselves depends ultimately on the genetic make-
up of the organisms.
v Improvement of strains can therefore be put down in simple term as follows:

(i) regulating the activity of the enzymes secreted by the organisms
(ii) in the case of metabolites secreted extracellularly, increasing the permeability of the
organism so that the microbial products can find these ways more easily outside the
cell
(iii) selecting suitable producing strains from a natural population
(iv) manipulation of the existing genetic apparatus in a producing organism
(v) introducing new genetic properties into the organism by recombinant DNA
technology or genetic engineering.

Manipulation of the Genome of Industrial Organisms in Strain Improvement
ü The manipulation of the genome for increased productivity may be done in one of
two general procedures
(a) manipulations not involving foreign DNA
(b) manipulations involving foreign DNA

a. Genome manipulations not involving Foreign DNA or Bases: Conventional
Mutation
üThe properties of any microorganism depend on the sequence of the four nucleic
acid bases on its genome: adenine (A), thymine (T), cytosine (C), and guanine (G).
üThe arrangement of these DNA bases dictates the distribution of genes and hence
the nature of proteins synthesized.
üA mutation can therefore be described as a change in the sequence of the bases in
DNA (or RNA, in RNA viruses).

§ It is clear that since it is the sequence of these bases which is responsible for the type of
proteins (and hence enzymes) synthesized, any change in the sequence will lead
ultimately to a change in the properties of the organism.
§ Mutations occur spontaneously at a low rate in a population of microorganisms. It is this
low rate of mutations which is partly responsible for the variation found in natural
populations.
§ An increased rate can however be induced by mutagens, (or mutagenic agents) which
can either be physical or chemical.

A. Physical agents
I. Ionizing radiations- X-rays, gamma rays, alpha-particles and fast neutrons
II. Ultraviolet light- The mutagenic range of ultraviolet light lies between wave length 200
and 300 nm.
B. Chemical mutagens
These may be divided into three groups:
I. Those that act on DNA of resting or non-dividing organisms: Nitrous acid , Alkylating
agents, NTG (nitrosoguanidine) and Nitrogen mustards:
II. DNA analogues which may be incorporated into DNA during replication- e.g 2-amino
purine-resemble adenine, 5-bromouracil resemble- thymine
III. Those that cause frame-shift mutations. E.g Acridines (C13H9N)

b. Methods involving DNA foreign to the organism (recombination)
Genetic engineering
o Genetic engineering, also known as recombinant DNA technology, molecular cloning
or gene cloning.
oHas been defined as the formation of new combinations of heritable material by the
insertion of nucleic acid molecules produced by whatever means outside the cell, into
any virus, bacterial plasmid or other vector system so as to allow their incorporation
into host organisms in which they do not naturally occur but in which they are capable
of continued propagation.

§ The DNA to be inserted into the host bacterium may come from a eukaryotic cell, a
prokaryotic cell or may even be synthesized chemically.
§ The vector-foreign DNA complex which is introduced into the host DNA is sometimes known
as a DNA chimera after the Chimera of classical Greek mythology which had the head of lion,
the body of a goat and the tail of a snake.
§ Genetic engineering has enabled the crossing of the species barrier, in that DNA from one
organism can now be introduced into another where such exchange would not be possible
under natural conditions.
§ With this technology engineered cells are now capable of producing metabolic products vastly
different from those of the unaltered natural recipient.

Procedures for the Transfer of the Gene in Recombinant DNA Technology (Genetic
Engineering)
The steps involved in in vitro recombination or genetic engineering:
1. Dissecting a specific portion from the DNA of the donor organism.
2. Attachment of the spliced DNA piece to a replicating piece of DNA (or vector), which can
be from either a bacteriophage or a plasmid.
3. Transfer of the vector along with the attached DNA (i.e., the DNA chimera) into the host
cell.
4. Isolation (or recognition) of cells successfully receiving and maintaining the vector and its
attached DNA.

CHAPTER-THREE
Microbial Fermentation Technology

3.1. Fermentation
• Is a process in which microorganisms, such as fungi and bacteria, break
down organic substances, such as sugars, anaerobically and it produces
substances such as alcohol and organic acids.
• Fermentation also produces chemical energy, such as ATP, that is important for
biological processes. It is used to make products such as wine, beer and bread.
• There are many types of fermentation that produce different end products from
pyruvate or its derivatives.
• The two most commonly used fermentations by humans are ethanol and lactic
acid fermentation
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Alena B. and Woinshet M. 69

3.1.1. Types of Fermentation
v Fermentation has been classified into SSF and SmF mainly based on the type of
substrate used during fermentation.
1. Solid-State Fermentation (SSF)
v SSF utilizes solid substrates, like bran, bagasse, and paper pulp.
vThe main advantage of using these substrates is that nutrient-rich waste materials can be
easily recycled as substrates.

v In SSF the substrates are utilized very slowly and steadily, so the same substrate
can be used for long fermentation periods.
v Hence, this technique supports controlled release of nutrients.
v SSF is best suited for fermentation techniques involving fungi and microorganisms
that require less moisture content.

2. Submerged Fermentation (SmF)
v SmF utilizes free flowing liquid substrates, such as molasses and broths.
v The bioactive compounds are secreted into the fermentation broth.
v The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented
with nutrients.
v This fermentation technique is best suited for microorganisms such as bacteria that require
high moisture content.
v In SmF technique purification of products is easier.
v SmF is primarily used in the extraction of secondary metabolites that need to be used in
liquid form.

v Some common substrates used in submerged fermentation are soluble sugars,
molasses, liquid media, fruit and vegetable juices, and sewage/waste water.
v Various bioactive compounds such as antibiotics, pigments, enzymes,
hypercholestrolemic agents, antioxidants, antihypertensive agents, antitumor agents,
bio surfactants and bioactive peptides have been extracted using fermentation.

üFermentation is the primary technique for the production of various enzymes.
ü Both fungi and bacteria yield an invaluable array of enzymes when fermented on
appropriate substrates.
üBoth solid-state and submerged fermentation are used for enzyme production.
üSmF is usually implemented in case of bacterial enzyme production, due to the
requirement of higher water potential.
üSSF is preferred when enzymes have to be extracted from fungi, which require
lesser water potential.

3.2. Bioreactors
§ Bioreactors are cylindrical vessels with hemispherical top and bottom,
made of stainless steel and glass ranging in size from some liter to cube
meters.
§ It is an apparatus for growing organisms such as bacteria, viruses, or yeast
that are used in the production of d/t products.
§ Under optimum conditions of gas flow rates, temperature, pH, dissolved
oxygen level, and agitation speed, the microorganisms or cells will
reproduce at a rapid rate.

§ The sizes of the bioreactor can vary over several orders of magnitudes.
§ The microbial cell (few mm³), shake flask (100-1000 ml), laboratory fermenter (1 –
50 L), pilot scale (0.3 – 10m³) to plant scale (2 – 500 m³) are all examples of
bioreactors.
§ The design and mode of operation of a fermenter mainly depends on the production
organism, the optimal operating condition, product value and scale of production.
§ The design also takes into consideration the capital investment and running cost.

• Large volume and low value products like alcoholic beverages need simple fermenter and
do not need aseptic condition.
• High value and low volume products require more elaborate system of operation and
aseptic condition.
• Bioreactors differ from conventional chemical reactors in that they support and control
biological entities.
• As such, bioreactor systems must be designed to provide a higher degree of control over
process upsets and contaminations, since the organisms are more sensitive and less stable
than Chemicals.

The general requirements of the bioreactor are as follows:
1. The vessel should be robust and strong enough to withstand the various
treatments.
2. The vessel should be able to be sterilized and to maintain stringent
aseptic conditions over long periods of the actual fermentation process.
3. The vessel should be equipped with stirrers or mixers to ensure mass
transfer processes occur efficiently.
4. It should have sensors to monitor and control the fermentation process.

5. It should be provided with inoculation point for aseptic transfer in inoculum.
6. Sampling valve for withdrawing a sample for different tests.
7. Baffles should be provided in case of stirred fermenter to prevent vertex formation.
8. It should be provided with facility for intermittent addition of an antifoam agent.
9. In case of aerobic submerged fermentation, the tank should be equipped with the
aerating device.
10. Provision for controlling temperature and PH.

3.3. Fermentor
§ Fermentors may be grouped in several ways: shape or configuration, whether aerated or
anaerobic and whether they are batch or continuous.
1. Submerged fermentation system stirred tank reactors
üStirred tank reactor is the choice for many fermentation processes.
üStirred tank reactors have the following functions: homogenization, suspension of solids,
dispersion of gas-liquid mixtures, aeration of liquid and heat exchange.
üThe Stirred tank reactor is provided with a baffle and a rotating stirrer is attached either at
the top or at the bottom of the bioreactor.

üThe industry prefers stirred tanks because in case of contamination or any other
substandard product formation the loss is minimal.
ü The Stirred tank reactors offer excellent mixing and reasonably good mass transfer rates.
üThe cost of operation is lower and the reactors can be used with a variety of microbial
species.

SmF methods:
- Batch fermentation.
- Fed-batch fermentation.
- Continuous fermentation.
- Semi-continuous fermentation.
Batch fermentation.
1. Considered to be a closed system.
 The sterilized media in the fermenter is inoculated with the microorganism.
 Incubation is allowed under the optimum conditions (aeration, agitation, temperature).
 During entire fermentation nothing is added except air, antifoam and acid/base.

Fed-batch fermentation
vIt is enhancement of batch fermentation.
vContinue adding the nutrients (feeding) in a small doses during the fermentation.
vThe method in controlling nutrients feeding process is by measuring methods.
vThe main advantage of fed-batch fermentation is the elimination of catabolite
repression (feed-back inhibition).

Continuous fermentation
§ It is an open system.
§ Continuously sterile nutrient is added and the converted nutrient is taken out
from the fermentor.
§ In continuous process cell loss as a result of outflow must be balanced by growth
of the microorganism.

Important factors for continuous fermentation:
vThe system must be stable for at least 500 hours.
vMaintaining sterile conditions for all period of fermentation time.
vThe composition of nutrients must be constant all the time.
vMaintaining the strain stability for constant high production yield.
Semi-continuous fermentation
Semi-continuous fermentations, in which a fraction of a fermentation is replaced with fresh
media at regular intervals.

2. Airlift bioreactor
§ Airlift fermenter (ALF) is generally classified as pneumatic reactors without any
mechanical stirring arrangements for mixing.
§ The turbulence caused by the fluid flow ensures adequate mixing of the liquid. It is ideally
suited for aerobic cultures.
§ The draft tube is provided in the central section of the reactor.
§ The introduction of the fluid (air/liquid) causes upward motion and results in circulatory
flow in the entire reactor.
§ The air/liquid velocities will be low and hence the energy consumption is also low.
§ ALFs can be used for both free and immobilized cells.

3. Fluidized bed reactor
üFluidized bed bioreactors (FBB) have received increased attention in the recent years.
üMost of the FBBs developed for biological systems involving cells as biocatalysts are
three phase systems (solid, liquid & gas).
üUsually fluidization is obtained either by external liquid re-circulation or by gas fed to the
reactor.
üIn the case of immobilized enzymes the usual situation is of two-phase systems involving
solid and liquid but the use of aerobic biocatalyst necessitate introduction of gas (air) as
the third phase.

Fig.5 Fluidized bed bioreactors

4. Bubble column reactor
v Bubble column fermenter is a simplest type of tower fermenter consisting of a tube
which is air sparged at the base.
v It is an elongated non-mechanically stirred fermenter with an aspect ratio of 6:1.
v This type of fermenter was used for citric acid production.

Solid State Fermentation
vThere are many biotechnological processes that involve the growth of
microorganisms on solid substrates in the absence or near absence of free water.
vThe most regularly used solid substrates are cereal grains, legume seeds, wheat
bran, lignocellulose materials such as straws, sawdust or wood shavings, and a
wide range of plant and animal materials.
vMost of these compounds are polymeric molecules, insoluble or sparingly soluble
in water, but are mostly cheap, easily obtainable and represent a concentrated
source of nutrients for microbial growth.

§ In SSF technique, microorganisms are grown on and inside the humidified solid
substrate.
§ Many of the filamentous fungi basically live and grow on solid substrate.
§ The efficiency of the SSF basically depends on: Energy, Economy and
Environment.
§ In SSF, substrate itself is the source of energy and requires no medium for growth
of micro-organism.

§ It is more cost effective (smaller vessels lower water consumption, reduced waste
water treatment costs, lower energy consumption, and less contamination problems).

Applications:
§ Many high value products such as extra-cellular enzymes, primary metabolites, and
antibiotics could be produced in SSF.
§ It is estimated that nearly a third of industrial enzyme produced in Japan is made by
SSF process.
§ Production of organic and ethanol from starchy substrates.
§ Digestibility of fibers and lignocelluloses materials for both human and animal
consumption.

Laboratory scale SSF bioreactor
• Laboratory-scale bioreactors typically have a working volume that varies from about
0.2 L to 20 L.

Industrial scale SSF bioreactor
• For large scale production at industrial level, SSFr employs either tray type or drum
type fermenter.
• In large-scale industrial bioreactor cultivation volumes ranging from 10,000 to
500,000 L.
• A leading enzyme manufacturer in India, ‘BIOCON’ uses tray type fermenter for large
capacity production of immunosuppressants.

Fig. 7 drum bioreactor

Fig. 8 Tray type bioreactors

Upstream Processing and Downstream Processing
• What is Bioprocessing?
 Is a process w/c uses living cells or their components (e.g., bacteria, enzymes, chloroplasts) to obtain desired
products such as ethanol and biodiesel, therapeutic stem cells, gene therapy vectors, and new vaccines and etc
 Bioprocessing includes two important processes - Upstream and downstream processes.
 Upstream and downstream bioprocessing are the two main stages of a
bioprocess or fermentation that involves the production of biologics
using host cell proteins.
Upstream Bioprocessing
 The process of converting raw materials into a form that can be used in a biologic manufacturing process.
 deals with the identification, screening, culture, and growth of the
organism in a bioreactor.

Downstream bioprocessing
vThe term "downstream bioprocessing" refers to the steps that take place after
the initial bioprocessing steps, which involve the production of a biological
agent
vDownstream bioprocessing deals with the harvesting, testing, purification,
and packaging of the product.
qThe various steps of Downstream Processing involve: Separation; Cell
disruption; Extraction; Isolation; Purification; Drying; Separation of
particles.
ØBoth stages require controlled conditions and quality assurance.

Similarities Between Upstream and Downstream Bioprocessing
 The two main parts of a bioprocess are upstream and downstream bioprocessing.
 Both processes involve living organisms, particularly microorganisms.
 These processes are carried out on bioproducts that are both industrially and
medicinally important.
 When it comes to making bioproducts, both processes are crucial.
 During both processes, contamination should be avoided.
Difference Between Upstream and Downstream Bioprocessing
 Product development happens in the upstream bioprocessing stage, while product
harvesting happens in the downstream bioprocessing stage. As a result, the key
distinction between upstream and downstream bioprocessing is this.

CHAPTER-FOUR
Application of Microbes in Industrial Biotechnology

4.1. Alcohol Beverages
ØEthyl alcohol, CH3 CH2 OH (synonyms: ethanol, methyl carbinol, grain alcohol,
molasses alcohol, grain neutral spirits, cologne spirit, wine spirit), is a colorless, neutral,
mobile flammable liquid with a molecular weight of 46.47, a boiling point of 78.3 and
a sharp burning taste.
ØIt is rarely found in nature, being found only in the unripe seeds of Heracleum
giganteun and H. spondylium.

Uses of Ethanol
(i) Use as a chemical feed stock: In the chemical industry, ethanol is an intermediate in
many chemical processes because of its great reactivity. It is thus a very important
chemical feed stock.
(ii) Solvent use: Ethanol is widely used in industry as a solvent for dyes, oils, waxes,
explosives, cosmetics etc.
(iii) General utility: Alcohol is used as a disinfectant in hospitals, for cleaning and lighting
in the home, and in the laboratory second only to water as a solvent.
(iv) Fuel: Ethanol is mixed with petrol or gasoline up to 10% and known as gasohol and
used in automobiles.

Substrates
ØSubstrate used will vary among countries. Thus, in Brazil sugar cane is the major source of
fermentation alcohol.
Ø In the United States enormous quantities of corn and other cereals are grown and these are
the obvious substrates.
Fermentation
ØWhen the nitrogen content of the medium is insufficient nitrogen is added usually in the
form of an ammonium salt.
ØThe heat released must be reduced by cooling and temp. should not exceed 35-37°C. The
pH is usually in the range 4.5-4.7.

ØAlcohol-resistant yeasts, strains of Saccharomyces cerevisiae are used, and nutrients
such as nitrogen and phosphate lacking in the broth are added.
Ø During the fermentation contaminations can have serious effects on the process: sugars
are used up leading to reduced yields; metabolic products from the contaminants may not
only alter the flavor of the finished product, but metabolites such as acids affect the
function of the yeast.

Distillation- After fermentation the fermented liquor or ‘beer’ contains alcohol as well as
low boiling point volatile compounds such as acetaldehydes, esters and the higher boiling,
fusel oils.
ØThe alcohol is obtained by several operations. First, steam is passed through the beer
which is said to be steam-stripped.
ØThe result is a dilute alcohol solution which still contains part of the undesirable volatile
compounds.
ØSecondly, the dilute alcohol solution is passed into the center of a multi-plate aldehyde
column in which the following fractions are separated: esters and aldehydes, fusel oil,
water, and an ethanol solution containing about 25% ethanol.

ØThirdly, the dilute alcohol solution is passed into a rectifying column where a constant
boiling mixture, an azeotrope, distils off at 95.6% alcohol concentration.
ØTo obtain 200° proof alcohol, such as is used in gasohol blending, the 96.58% alcohol is
obtained by azeotropic distillation.
ØThe principle of this method is to add an organic solvent which will form a ternary
(three-membered) azeotrope with most of the water, but with only a small proportion of
the alcohol.
ØBenzene, carbon tetrachloride, chloroform, and cyclohezane may be used, but in
practice, benzene is used.

ØAzeotropes usually have lower boiling point than their individual components and that of
benzene-ethanol-water is 64.6°C.
ØOn condensation, it separates into two layers. The upper layer, which has about 84% of the
condensate and the heavier, lower portion, constituting 16% of the condensate
Ø In practice, the condensate is not allowed to separate out, but the arrangement of plates
within the columns enable separation of the alcohol. Four columns are usually used. The
first and second columns remove aldehydes and fusel oils, respectively, while the last two
towers are for the concentration of the alcohol.

Fig. 1 Fermentation Production of ethanol

Barley beers
ØThe word beer derives from the Latin word bibere meaning to drink.
ØThe process of producing beer is known as brewing.
Ø Beer brewing from barley was practiced by the ancient Egyptians as far back as 4,000
years ago, but investigations suggest Egyptians learnt the art from the peoples of the
Tigris and Euphrates where man’s civilization is said to have originated.
ØThe use of hops is however much more recent and can be traced back to a few hundred
years ago.

Production of Beer
Types of Barley Beers
• Barley beers can be divided into two broad groups: top-fermented beers and bottom
fermented beers.
• This distinction is based on whether the yeast remains at the top of brew (top-fermented
beers) or sediments to the bottom (bottom-fermented beers) at the end of the fermentation.
Bottom-fermented beers
• Bottom-fermented beers are also known as lager beers because they were stored or
lagered (from German lagern = to store) in cold cellars after fermentation for
clarification and maturation.

ØYeasts used in bottom-fermented beers are strains of Saccharomyces uvarum.
ØSeveral types of lager beers are known.
ØThey are Pilsener, Dortumund and Munich, and named after Pilsen (former
Czechoslovakia) Dortmund and Munich (Germany), the cities where they originated.
Most of the lager (70%-80%) beers drunk in the world is of the Pilsener type.

i) Pilsener beer: This is a pale beer with a medium hop taste with alcohol content 3.0-3.8%.
It is lagered for about two weeks.
ii) Dortmund beer: This is a pale beer, but it contains less hops and less bitter than Pilsener.
However it has more body (i.e., it is thicker) and aroma.
The alcohol content is also 3.0-3.8%, and is classically lagered for slightly longer: 3-4
months.
ØThe brewing water is hard, containing large amounts of carbonates, sulphates and
chlorides.

iii) Munich: This is a dark, aromatic and full-bodied beer with a slightly sweet taste,
alcohol content varying from 2 to 5%.
Top-fermented beers- brewed with strains of Saccharomyces cerevisiae.
i) Ale: ale (Pale ale) is England’s own beer. English ale is a pale, highly hopped beer
with an alcohol content of 4.0 to 5.0%
ØIt is very bitter and has a sharp acid taste and an aroma of wine because of its high ester
content.

ii) Porter: This is a dark-brown, heavy bodied, strongly foaming beer produced from dark
malts. It contains less hops than ale and consequently is sweeter. It has an alcohol content
of about 5.0%.
iii) Stout: Stout is a very dark heavily bodied and highly hopped beer with a strong malt
aroma.
ØIt is produced from dark or caramelized malt; sometimes caramel may be added.
ØIt has high alcohol content, 5.0-6.5% (w/v).

Raw materials for brewing: barley, malt, adjuncts, yeasts, hops, and water.
Barley has the following advantages:
üIts husks are thick, difficult to crush and adhere to the kernel. This makes malting as well as
filtration after mashing, much easier.
ü The thick husk is a protection against fungal attack during storage.
üThirdly, the gelatinization temperature (i.e., the temperature at which the starch is converted
into a water soluble gel) is 52-59°C much lower than the optimum temperature of alpha-
amylase (70°C) as well as of beta-amylase (65°C) of barley malt.

• Adjuncts- are starchy materials hydrolyzed to fermentable sugars.
used when malt lacks in proteins, starch and sugars
• Water -the mineral and ionic content and the pH of the water have profound effects on
the type of beer produced.
Hops- are the female flower clusters
or seed cones of the hop vine
Humulus lupulus, which are used
as a flavoring and preservative
agent in nearly all beer made today.

ØBrewer’s yeasts- Yeasts in general will produce alcohol from sugars under anaerobic
conditions.
ØBrewing yeasts are able, besides producing alcohol, to produce from wort sugars and
proteins a balanced proportion of esters, acids, higher alcohols, and ketones which
contribute to the peculiar flavor of beer.
ØMainly Saccharomyces strains use in the production of Barley beers. (S. cerevisiae
and S. uvarum)

• Sacch. cerevisiae strains have a stronger respiratory system than Sacch uvarum.
Brewery Processes
1. Malting
2. Cleaning and milling of the malt
3. Mashing
4. Mash operation
5. Wort boiling treatment
6. Fermentation
7. Storage or lagering
8. Packaging

Malting - The purpose of malting is to develop
amylases and proteases in the grain.
• These enzymes are produced by the germinated barley to enable it to break down the
carbohydrates and proteins in the grain to nourish the germinated seedling before its
photosynthetic systems are developed enough to support the plant.
• As soon as the enzymes are formed and before the young seedling has made any
appreciable in-road into the nutrient reserve of the grain, the development of the seedling is
halted by drying. These enzymes are reactivated during mashing and used to hydrolyze
starch and proteins and release nutrients for the nourishment of the yeasts.

Cleaning and milling of malt- The barley is transported to the top of the brewing tower.
ØSubsequent processes in the brewery process occur at progressively lower floors.
Lagering and bottling are usually done on the ground level floor.
Mashing- Mashing is the central part of brewing.
ØThe purpose of mashing is to extract as much as possible the soluble portion of the malt
and to enzymatically hydrolyze insoluble portions of the malt and adjuncts.

Mashing determines:
a. The nature of the Wort
b. The nature of the nutrients available to the yeasts
c. The type of beer produced
Ø The aqueous solution resulting from mashing is known as Wort.
Starch breakdown during mashing
Ø Starch forms about 55% of the dry weight of barley malt. Of the malt starch 20-25% is
made up of amylose. The key enzymes in the breakdown of malt starch are the alpha and
beta-amylases.

ØThe breakdown of the malt proteins, albumins, globulins, hordeins, and gluteins starts
during malting and continues during mashing by proteases.
Three mashing methods:
a) Decoction methods- part of the mash is transferred from the mash tun to the mash kettle
where it is boiled.
b) Infusion methods- the mash is never boiled, but the temperature is gradually raised.
c) The double mash method- the starchy adjuncts are boiled and added to the malt.

Mash separation- At the end of mashing, husks and other insoluble materials are removed
from the wort in two steps.
ØFirst, the wort is separated from the solids.
ØSecond, the solids themselves are freed of any further extractable material by washing or
sparging with hot water.
Wort boiling- wort is boiled for 1-1½ hours in a brew kettle.
ØWhen corn syrup or sucrose is used as an adjunct it is added at the beginning of the
boiling. Hops are also added, some before and some at the end of the boiling.

Fig. 3 Brew kettles

The purpose of boiling:
a) To concentrate the wort, which loses 5-8% of its volume by evaporation
b) To sterilize the wort before its introduction into the fermentor.
c) To inactivate any enzymes so that no change occurs in the composition of the wort.
d) To extract soluble materials from the hops, which not only aid in protein removal, but also
in introducing the bitterness of hops.
e) To precipitate protein

f) To develop color in the beer.
Ø Color is formed by several chemical reactions including caramelization of sugars,
oxidation of phenolic compounds, and reactions between amino acids and reducing
sugars.
g) Removal of volatile compounds: volatile compounds such as fatty acids which could
lead to rancidity in the beer are removed.
ØDuring the boiling, agitation and circulation of the wort help increase the amount of
precipitation and flock formation.

ØPre-fermentation treatment of wort: The hot wort is not sent directly to the fermentation
tanks. If dried hops are used then they are usually removed in a hop strainer.
ØDuring boiling proteins and tannins are precipitated while the liquid is still warm.
ØSome more precipitation takes place when it has cooled to about 50°C.
Ø The warm precipitate is known as trub and consists of 50-60% protein, 16-20% hop
resins, 20-30% polyphenols and about 3% ash. Trub is removed either with a centrifuge,
or a whirlpool separator which is now more common.

ØWort which is fed into a flat centrifuge, is thrown at the side of the equipment and finds
its way out through an outlet on the periphery.
Ø The heavier particles (trub) are thrown to the center and withdrawn through a centrally
located outlet. The separated wort is cooled in a heat exchanger.
ØWhen the temperature has fallen to about 50°C further sludge known as ‘cold break’
begins to settle.
ØThe cooled wort is now ready for fermentation. It contains no enzymes but it is a rich
medium for fermentation. It has therefore to be protected from contamination

Fermentation- The cooled wort is pumped or allowed to flow by gravity into
fermentation tanks and yeast is inoculated or pitched in at a rate of 7-15 x 106 yeast
cells/ml, usually collected from a previous brew.
Top fermentation - This is used in the UK for the production of stout and ale, using
strains of S. cerevisiae.
ØYeast is pitched in at the rate of 0.15 to 0.30 kg/hl (1.5–3g/l) at a temp. of 15-16°C. The
temp. is allowed to rise gradually to 20°C over a period of about three days. At this
point it is cooled to a constant temp.

Ø The entire primary fermentation takes about six days. Yeasts float to the top during this
period, they are scooped off and used for future pitching.
ØIn the last three days the yeasts turn to a hard leathery layer, which is also skimmed off.
ØSometimes the wort is transferred to another vessel in the so-called dropping system
after the first 24-36 hours.
ØThe transfer helps aerate the system and also enables the discarding of the cold-break
sediments.

ØBottom fermentation- Wort is inoculated to the tun of 7-15 x 106 yeast cells per ml of
wort.
ØThe yeasts then increase four to five times in number over three to four days.
ØYeast is pitched in at 6-10°C and is allowed to rise to 10-12°C, within 3 - 4 days; it is
cooled to about 5°C at the end of the fermentation.
ØCO2 is released and this creates a head called Krausen, which begins to collapse after 4-5
days as the yeasts begin to settle.

ØFormation of some beer components- During wort fermentation in both top and bottom
fermentation anaerobic conditions predominate; the initial oxygen is only required for cell
growth.
ØFermentable sugars are converted to alcohol, CO2 and heat which must be removed by
cooling.

Monitoring following fermentation progress
ØThe progress of fermentation is followed by wort specific gravity.
Ø During fermentation the gravity of the wort gradually decreases because yeasts are
using up the extract. However alcohol is also being formed.
ØAs alcohol has a lower gravity than wort the reading of the special hydrometer (known
as a saccharometer) is even lower.

Lagering (bottom-fermented beers) and treatment (top-fermented beers)
a) Lagering: At the end of the primary fermentation, the beer, known as green beer is
harsh and bitter. It has a yeasty taste arising probably from higher alcohols and
aldehydes.
ØThe green beer is stored in closed vats at a low temperature (around O°C), for max.
periods 6 months to mature and make it ready for drinking.
ØDuring lagering secondary fermentation occurs. Yeasts are sometimes added to induce
this secondary fermentation, utilizing some sugars in the green beer.

ØThe secondary fermentation saturates the beer with CO2, indeed the progress of secondary
fermentation is followed by the rate of CO2 escape from a safety valve.
ØSometimes actively fermenting wort or Kraeusen may be added. At other times CO2 may
be added artificially into the lagering beer.
ØMaterials which undesirably affect flavor and which are present in green beer e.g. diacetyl,
hydrogen sulfide, mercaptans and acetaldehyde are decreased by evaporation during
secondary fermentation.

ØAn increase occurs in the desirable components of the beer such as esters. Any tannins,
proteins, and hop resins still left are precipitated during the lagering period.
ØIn some countries the turnover time from brewing, lagering, and consumption could be as
short as three weeks.
ØThis reduction has been achieved by artificial carbonation and by the manipulation of the
beer due to greater understanding of the lagering processes. Thus, in one method used to
reduce lagering time, beer is stored at high temperature (14°C) to drive off volatile
compounds e.g. H2S, and acetaldehyde.

ØThe beer is then chilled at –2°C to remove chill haze materials, and thereafter it is
carbonated. In this way lagering could be reduced from 2 months to 10 days.
Ø Lagering gives the beer its final desirable organoleptic qualities. The beer is filtered
through kieselghur or through membrane filters to remove protein-tannin complexes and
yeast cells.
b) Beer treatment (for top-fermented beers): They are treated in casks or bottles in
various ways. In some processes the beer is transferred to casks at the end of fermentation
with a load of 0.2-4.00 million yeast cells/ml.

ØIt is primed to improve its taste and appearance by the addition of a small amount of
sugar mixed with caramel. The yeasts grow in the sugar and carbonate the beer. Hops are
also sometimes added at this stage.
Ø It is stored for seven days or less at about 15°C. After ‘priming’, the beer is ‘fined’ by the
addition of isinglass.
Ø Isinglass - a gelatinous material from the swim bladder of fish, precipitates yeast cells,
tannins and protein-tannin complexes.
ØThe beer is thereafter pasteurized and distributed.

Packaging
vThe beer is transferred to pressure tanks from where it is distributed to cans, bottles and
other containers.
vThe beer is not allowed to come in contact with oxygen during this operation; it is also
not allowed to lose CO2, or to become contaminated with microorganisms.
vThe beer is added to the tanks under a CO2, atmosphere, bottled under a counter
pressure of CO2, and all the equipment is cleaned and disinfected regularly.

Ø Bottles are thoroughly washed with hot water and sodium hydroxide before being
filled.
ØThe filled and crowned bottles are passed through a pasteurizer, set to heat the bottles at
60°C for half hour.
ØThe bottles take another half hour to cool down.
ØSome of the larger breweries now carry out bulk pasteurization and fill containers
aseptically.

Grape wines
ØWine is a product of the normal alcoholic fermentation of the juice of ripe grapes.
ØAny fruit with a good proportion of sugar may be used for wine production.
ØThus, citrus, bananas, apples, pineapples, strawberries etc., may all be used to
produce wine. Such wines are always qualified as fruit wines.
Ø The production of wine is simpler than that of beer in that no need exists for
malting since sugars are already present in the fruit juice being used.

Production of Wine
vCrushing of Grapes- Selected ripe grapes of 21° to 23° Balling are crushed to release
the juice which is known as must after the stalks have been removed.
vThese stalks contain tannins which would give the wine a harsh taste if left in the must.
The skin contains most of the materials which give wine its aroma and color.
v For the production of red wines the skins of black grapes are included, to impart the
color.

vGrapes for sweet wines must have a sugar content of 24 to 28 Balling so that a residual
sugar content is maintained after fermentation.
v The chief sugars in grapes are glucose and fructose; in ripe fruits they occur in about the
same proportion.
vGrape juice has an acidity of 0.60-0.65% and a pH of 3.0-4.0 due mainly to malic and
tartaric acids with a little citric acid.

v During ripening both the levulose content and the tartaric acid contents rise.
vNitrogen is present in the form of amino acids, peptides, purines, small amounts
of ammonium compounds and nitrates

Fermentation
i) Yeast used: The grapes themselves harbor a natural flora of microorganisms (the
bloom)
ØNowadays the must is partially sterilized by the use of sulphur dioxide, a bisulphate
or a metabisulphite which eliminates most microorganisms in the must leaving wine
yeasts. Yeasts are then inoculated into the must.

vThe yeasts which are used: Saccaromyces cerevisiae and Sacch. ellipsoideus
Other yeasts which have been used for special wines are:
üSacch. fermentati,
üSacch. oyiformis
ü Sacch. bayanus.

vWine yeasts have the following characteristics:
(a) growth at the relatively high acidity (low pH) of grape juice
(b) resistance to high alcohol content (higher than 10%)
(c) resistance to sulfite.
(ii) Control of fermentation
(a) Temperature: Heat is released during the fermentations. The fermentation is cooled and
the temperature is maintained at around 24°C with cooling coils mounted in the fermentor.

b) Yeast Nutrition: Yeasts normally ferment the glucose preferentially although some
yeasts e.g. Sacch. elegans prefer fructose.
vTo produce sweet wine glucose-fermenting wine yeasts are used leaving the fructose
which is much sweeter than glucose.
vMost nutrients including macro- and micro-nutrients are usually abundant in must;
occasionally, however, nitrogenous compounds are limiting.

vThey are then made adequate with small amounts of (NH4)2 SO4 or (NH4)2
HPO4.
v Oxygen: oxygen is required in the earlier stage of fermentation when yeast
multiplication is occurring.
v In the second stage when alcohol is produced the growth is anaerobic and this
forces the yeasts to utilize such intermediate products as acetaldehydes as
hydrogen acceptors and hence encourage alcohol production.

iii) Flavor development: Although some flavor materials come from the grape most of it
come from yeast action.
vThe flavor of wine has been elucidated with gas chromatography and has been shown
to be due to alcohols, esters, fatty acids, and carbonyl compounds, the esters being the
most important.
vDiacetyl, acetonin, fusel oils, volatile esters, and hydrogen sulfide have received
special attention. Autolysates from yeasts also have a special influence on flavor.

Fig.4 Flow of wine making

vAgeing and Storage - The fermentation is usually over in 3-5 days.
vAt this time ‘pomace’ formed from grape skins (in red wines) will have risen to the
top of the brew.
vFor white wine, the skin is not allowed in the fermentation.
vAt the end of this fermentation the wine is allowed to flow through a perforated
bottom if pomace had been allowed.

v When the pomace has been separated from wine and the fermentation is complete or
stopped, the next stage is ‘racking’.
vThe wine is allowed to stand until a major portion of the yeast cells and other fine suspended
materials have collected at the bottom of the container as sediment or ‘lees’.
v It is then ‘racked’, during which process the clear wine is carefully pumped or
siphoned off without disturbing the lees.
vThe wine is then transferred to wooden casks (100-1,000 gallons), barrels (about 50
gallons) or tanks (several thousand gallons).

vThe wood allows the wine only slow access to oxygen. Water and ethanol evaporate
slowly leading to air pockets which permit the growth of aerobic wine spoilers e.g. acetic
acid bacteria and some yeasts.
vThe casks are therefore regularly topped up to prevent the pockets. In modern tanks
made of stainless steel the problem of air pockets is tackled by filling the airspace with
an inert gas such as carbon dioxide or nitrogen.

vDuring ageing desirable changes occur in the wine.
v The reaction is responsible for the rich flavor developed during the ageing of some
wines e.g. Bordeaux.
vCultures which have been implicated in this fermentation are Lactobacillus sp and
Leuconostoc sp. A temperature of 11-16°C is best for ageing wines, High temperature
probably functions by accelerating oxidation.

v Clarification- the wine is allowed to age in a period 2 - 5 years, depending on the
type of wine.
vAt the end of the period some will have cleared naturally. For others artificial
clarification may be necessary.
vThe addition of a fining agent is often practiced to help clarification.
v Fining agents react with the tannin, acid, protein or with some added substance to
give heavy quick-settling coagulums. In the process of setting various suspended
materials are adsorbed.

vThe usual fining agents for wine are gelatin, casein, tannin, isinglass, egg albumin,
and bentonite.
vIn some countries the removal of metal ions is accomplished with potassium
ferrocyanide known as blue fining.
vIt removes excess ions of copper, iron, manganese, and zinc from wines.

vPackaging- before packing in bottles the wine from various sources is sometimes blended
and then pasteurized.
vIn some wineries, the wine is not pasteurized, rather it is sterilized by filtration.
vIn many countries the wine is packaged and distributed in casks.

4.2. Enzymes
Fermentation for Enzyme Production
ØMost enzyme production is carried out in deep submerged
fermentation; a few are best produced in semi-solid media.
Semi solid medium- This system, also known as the Koji or moldy
bran method of solid state fermentation is still widely used in Japan.
ØThe medium consists of moist sterile wheat or rice bran acidified with
HCl; mineral salts including trace minerals are added.

ØAn inducer is also usually added; 10% starch is used for amylase, and gelatin and
pectin for protein and pectinase production respectively.
ØThe organisms used are fungi, which appear amenable to high enzyme production
because of the low moisture condition and high degree of aeration of the semi-
soluble medium.

ØThe moist bran, inoculated with spores of the appropriate fungi, is distributed
either in flat trays or placed in a revolving drum.
ØMoisture (about 8%) is maintained by occasionally spraying water on the trays
and by circulating moist air over the preparation.
ØThe temperature of the bran is kept at about 30°C by the circulating cool air.

ØThe production period is usually 30-40 hours, but could be as long as seven days.
ØThe optimum production is determined by withdrawing the growth from time to
time and assaying for enzyme.

ØGrowth in a semi-solid medium seems sometimes to encourage an enzyme range
different from that produced in submerged growth.
ØThus, Aspergillus oryzae on semi-solid medium will produce a large number of
enzymes, primarily amylase, glucoamylose, and protease.
Ø In submerged culture amylase production rises at the expense of the other
enzymes.

Submerged production
ØMost enzyme production is in fact by submerged cultivation in a deep fermentor.
ØSubmerged production has replaced semi-solid production wherever possible
because the latter is labor intensive.
ØControlling temperature, pH and other environmental factors in a fermentor also
easier with submerged.

ØThe medium must contain all the requirements for growth, including adequate
sources of carbon, nitrogen, various metals, trace elements, growth substances, etc.
ØHowever, a medium adequate for growth may not be satisfactory for enzyme
production.
ØFor the production of inducible enzymes, the inducers must be present.
ØThus, pectic substances need to be in the medium when pectinolytic enzymes are
being sought.

ØSimilarly, in the production of microbial rennets soy bean proteins are added into
the medium to induce protease production by most fungi.
ØThe inducer may not always be the substrate but sometimes a breakdown or end-
product may serve.
ØFor example, cellobios may stimulate cellulose production.
Ø Sometimes some easily metabolizable components of the medium may repress
enzyme production by catabolite repression. Strong repression is often seen in
media containing glucose.

ØThus, -amylase synthesis is repressed by glucose in Bacillus licheniformis and B.
subtilis.
ØFructose on the other hand represses the synthesis of the enzyme in B.
stearothermophilus.
ØIn many organisms protease synthesis is repressed by amino acids as well as by
glucose.
ØIt is therefore usual to replace glucose by more slowly metabolized carbohydrates
such as partly hydrolyzed starch.
ØHigh enzyme yield may also be obtained by adding constantly, low amounts of the
inducer.

Ø End-product inhibition has also been widely observed. Some specific amino acids
inhibit protease production in some organisms.
ØThus, isoleucine and proline are involved in the case of B. megaterium while
sulphur amino acids inhibit protease formation in Aspergillus niger.
ØTemperature and pH requirements have to be worked out for each organism and
each desired product.

ØThe temperature and pH requirements for optimum growth, enzyme production,
and stability of the enzyme once it is produced are not necessarily the same for all
enzymes.
Ø The temperature adopted for the fermentation is usually a compromise taking all
three requirements into account.
The oxygen requirement is usually high as most of the organisms employed in
enzyme production are aerobic.

ØVigorous aeration and agitation are therefore done in the submerged fermentations
for enzyme production.
ØBatch fermentation is usually employed in commercial enzyme fermentation and
lasts from 1 - 7 days.
ØContinuous fermentation, while successful experimentally, does not appear to
have been used in industry.

Enzyme Extraction
ØDuring enzyme extraction care is taken to avoid contamination.
ØIn order to limit contamination and degradation of the enzyme the broth is cooled
to about 20°C as soon as the fermentation is over.
ØStabilizers such as calcium salts, proteins, sugar, and starch hydrolysates may be
added and destabilizing metals may be removed with EDTA.

ØAntimicrobials if used at all are those that are normally allowed in food such as
benzoates and sorbate.
ØMost industrial enzymes are extra-cellular in nature. In the case of cell bound
enzymes, the cells are disrupted before centrifugation and/or vacuum filtration.
ØThe extent of the purification after the clarification depends on the purpose for
which the enzyme is to be used.
ØSometimes enzymes may be precipitated using a variety of chemicals such as
methanol, acetone, ethyl alcohol or ammonium sulfate.

Ø The precipitate may be further purified by dialysis, chromatography, etc., before
being dried in a drum drier or a low temperature vacuum drier depending on the
stability of the enzymes to high temperature.
ØUltra-filtration separation technique based on molecular size may be used.

Packaging and Finishing
ØNowadays, enzymes are packaged preferably in liquid form but where solids are
used, the enzyme is mixed with a filler and it is now common practice to coat the
particles with wax.

4.3. Other Industrial Microbial Products
Production of antibiotics
ØAntibiotics are chemical substances that can inhibit the growth and even destroy
harmful microorganisms.
ØThey are derived from special microorganisms or other living systems and are
produced on an industrial scale using a fermentation process.
ØAfter the discovery of penicillin, other antibiotics were sought. In 1939, work began
on the isolation of potential antibiotic products from the soil bacteria streptomyces. It
was around this time that the term antibiotic was introduced.

ØSelman Waxman and associates discovered streptomycin in 1944.
ØSubsequent studies resulted in the discovery of a host of new, different antibiotics
including actinomycin, streptothricin, and neomycin all produced by Streptomyces.
ØOther antibiotics that have been discovered since include bacitracin, polymyxin,
viomycin, chloramphenicol and tetracyclines.
ØSince the 1970s, most new antibiotics have been synthetic modifications of
naturally occurring antibiotics.

Raw Materials
vThe compounds that make the fermentation broth are the primary raw materials
required for antibiotic production.
vThis broth is an aqueous solution made up of all of the ingredients necessary for the
proliferation of the microorganisms.
vTypically, it contains a carbon source like molasses, or soy meal, both of which are
made up of lactose and glucose sugars.

ØThese materials are needed as a food source for the organisms. Nitrogen is another
necessary compound in the metabolic cycles of the organisms.
ØFor this reason, an ammonia salt is typically used. Additionally, trace elements needed
for the proper growth of the antibiotic-producing organisms are included.
ØThese are components such as phosphorus, sulfur, magnesium, zinc, iron, and copper
introduced through water soluble salts.
ØTo prevent foaming during fermentation, anti-foaming agents such as lard oil,
octadecanol, and silicones are used.

The Manufacturing Process
ØAlthough most antibiotics occur in nature, they are not normally available in the
quantities necessary for large-scale production.
ØFor this reason, a fermentation process was developed. It involves isolating a desired
microorganism, fueling growth of the culture and refining and isolating the final
antibiotic product.
Ø It is important that sterile conditions be maintained throughout the manufacturing
process, because contamination by foreign microbes will ruin the fermentation.

Starting the culture
ØBefore fermentation can begin, the desired antibiotic-producing organism must be
isolated and its numbers must be increased by many times.
ØTo do this, a starter culture from a sample of previously isolated, cold-stored
organisms is created in the lab.
ØIn order to grow the initial culture, a sample of the organism is transferred to an
agar-containing plate.
ØThe initial culture is then put into shake flasks along with food and other nutrients
necessary for growth.

Ø This creates a suspension, which can be transferred to seed tanks for further growth.
Ø The seed tanks are steel tanks designed to provide an ideal environment for growing
microorganisms.
ØThey are filled with the all the things the specific microorganism would need to
survive and thrive, including warm water and carbohydrate foods like lactose or
glucose sugars.
ØAdditionally, they contain other necessary carbon sources, such as acetic acid,
alcohols, or hydrocarbons, and nitrogen sources like ammonia salts.

ØGrowth factors like vitamins, amino acids, and minor nutrients round out the
composition of the seed tank contents.
ØThe seed tanks are equipped with mixers, which keep the growth medium moving,
and a pump to deliver sterilized, filtered air.
ØAfter about 24-28 hours, the material in the seed tanks is transferred to the primary
fermentation tanks.

Fermentation
ØThe fermentation tank is essentially a larger version of the steel, seed tank, which is
able to hold about 30,000 gallons.
ØIt is filled with the same growth media found in the seed tank and also provides an
environment inducive to growth.
ØHere the microorganisms are allowed to grow and multiply. During this process,
they excrete large quantities of the desired antibiotic.

ØThe tanks are cooled to keep the temperature between 23-27.2 ° C.
ØIt is constantly agitated, and a continuous stream of sterilized air is pumped into
it.
ØFor this reason, anti-foaming agents are periodically added.
ØSince pH control is vital for optimal growth, acids or bases are added to the tank
as necessary.

Isolation and purification
ØAfter three to five days, the maximum amount of antibiotic will have been produced
and the isolation process can begin.
ØDepending on the specific antibiotic produced, the fermentation broth is processed by
various purification methods.
ØFor example, for antibiotic compounds that are water soluble, an ion-exchange method
may be used for purification.
ØIn this method, the compound is first separated from the waste organic materials in the
broth and then sent through equipment, which separates the other water-soluble
compounds from the desired one.

ØTo isolate an oil-soluble antibiotic such as penicillin, a solvent extraction method is
used.
ØIn this method, the broth is treated with organic solvents such as butyl acetate or
methyl isobutyl ketone, which can specifically dissolve the antibiotic.
ØThe dissolved antibiotic is then recovered using various organic chemical means.
ØAt the end of this step, the manufacturer is typically left with a purified powdered
form of the antibiotic, which can be further refined into different product types.

Refining
ØAntibiotic products can take on many different forms. They can be sold in solutions
for intravenous bags or syringes, in pill or gel capsule form, or they may be sold as
powders, which are incorporated into topical ointments.
ØDepending on the final form of the antibiotic, various refining steps may be taken
after the initial isolation.

ØFor intravenous bags, the crystalline antibiotic can be dissolved in a solution, put
in the bag, which is then hermetically sealed.
ØFor gel capsules, the powdered antibiotic is physically filled into the bottom half
of a capsule then the top half is mechanically put in place.
ØWhen used in topical ointments, the antibiotic is mixed into the ointment.

Ø From this point, the antibiotic product is transported to the final packaging
stations.
ØHere, the products are stacked and put in boxes. They are loaded up on trucks and
transported to various distributors, hospitals, and pharmacies.
ØThe entire process of fermentation, recovery, and processing can take anywhere
from five to eight days.

Production of Vitamins
ØVitamins are defined as essential micronutrients that are not synthesized by mammals.
ØMost vitamins are essential for the metabolism of all living organisms, and they are
synthesized by microorganisms and plants.
ØVitamins are usually used as dietary supplements e.g. ergo-sterol (pro-vitamin D),
riboﬂavin, B12, etc., while vitamin C (ascorbic acid) is mostly used as a food ingredient.

ØWhile some vitamins are chemically synthesized many are now produced by means of
selected microorganisms.
ØMicroorganisms can be successfully used for the commercial production of many
of the vitamins e.g. thiamine, riboflavin, pyridoxine, folic acid, pantothenic acid,
biotin, vitamin B12, ascorbic acid, P-carotene (pro-vitamin A), ergosterol (pro-
vitamin D).

Riboflavin (Vitamin B2) production
ØRiboflavin is used for human nutrition and therapy and as an animal feed additive.
ØPure riboflavin has needle-shaped, practically odorless, orange-yellow crystals, which
begin to darken at about 240°C. and completely decompose at about 280°C.
ØWater solutions show a characteristic yellowish-green fluorescence.
Ø Riboflavin is slightly soluble in water (12 mg in 100 ml at 27.5° C; 19 mg at 40° C.)
and in several organic solvents. It is very soluble in alkali.

ØThe crude concentrated form is also used for feed. It is produced by both synthetic
and fermentation processes.
ØTwo closely related ascomycete fungi, Eremothecium ashbyii and Ashbya gossypii,
are mainly used for the industrial production.
Ø In the fermentation production of riboflavin by Ashbya gossypii, the culture
medium, comprising glucose (corn sugar), corn steep liquor (byproduct of corn wet
milling), and animal stick liquor (a packing-house byproduct of wet rendering), is
prepared in a mixing tank.

ØThe medium is pumped at a controlled rate through a steam jet heater, where by injection of
high-pressure steam the solution is almost instantaneously heated to 135° C.
ØThe hot solution circulates through insulated pipes to retain the high temperature for 5
minutes, then through additional pipes or coils surrounded by cold water to reduce the
temperature to 28° to 30°C.
Ø Through steam-sterilized pipe lines, the cooled solution is pumped to a sterile fermentation
vessel.
ØThis is a closed tank equipped with a jacket or coils by which the tank contents may be
maintained at a uniform temperature of 28°c.

ØIn the bottom of the tank are fine-porosity stone or perforated coils through which
sterile air is supplied.
ØA mechanical agitator assists in providing adequate air distribution.
ØAfter the sterile culture medium is transferred to the tank a small volume of a day-
old culture of Ashbya gossypii is added, and sterile air is introduced through the air
distribution system.

ØBy the fourth day, the maximum yield of riboflavin has been obtained, and the culture
medium has acquired a beautiful, intense yellow color.
ØTwo types of products can be produced. A potent riboflavin concentrate, ideally suited
to enriching poultry and livestock feeds, can be had by evaporating the water from the
fermented medium to prepare a syrup of about 30% solids.
ØThe syrup is converted to a dry powder by a drum or spray drier. The drum drier has a
pair of cylindrical rolls, mounted horizontally, which are steam heated.

Ø In the spray-drier method, the syrup is sprayed into a chamber through which heated
air is passed; the air absorbs the water; and the dry riboflavin concentrate is
mechanically removed to packaging equipment.
ØConcentrates containing 25,000 micrograms of riboflavin per gram (2.5% riboflavin)
are thereby produced.
Ø Pure crystalline riboflavin may be recovered from the fermented solution.

Modified Compounds (Biotransformation)
vBiotransformation means alteration of chemicals such as nutrients, amino
acids, toxins, and drugs in the body.
v It is also needed to render nonpolar compounds polar.
vThe series of chemical changes occurring in a compound, especially adrug, as a resu
lt of enzymatic or other activity by a living organism.

ØBiotransformation encompasses the use of biological systems to catalyze the
conversion of one compound to another.
ØThe catalyst part of the biological system can thereby consist of: whole cells
cellular extracts, or isolated enzymes.
Øone can principally follow three different approaches with various degrees of
complexity:
1. Use a purely chemical strategy.
2. Use a chemo enzymatic route- combining chemical and bio catalytic steps.

Ø In this case, the biocatalyst is preferentially used to perform the key reaction
requiring high selectivity or speciﬁcity or to replace environmentally intolerable
reaction steps.
3. Use a biological total synthesis by fermentation or multistep biotransformation.
ØEnzymes and whole-cell biocatalysts have several attractive properties.

ØBiocatalysis is normally performed in an aqueous environment but can, in many
cases, also be conducted in solvent mixtures, liquid–liquid two-phase systems, and
even in pure organic solvents.
Ø A relevant practical example is the use of esterases and lipases to catalyze
esteriﬁcations in organic solvents such as vinyl acetate.

Reactor for Bio catalytic Reactions
vNo special equipment is needed for biocatalysis in many cases and ordinary stirred
tanks, used in large-scale chemical synthesis with temperature and pH control, are
sufﬁcient.
vFermentors are used for the production of the enzymes, if they are not commercially
available. They are stirred vessels that allow a sterile (mono septic) operation.
vThis means that only the organism with the desired enzyme used for the biocatalysis
is allowed in the bioreactor.

vThe product recovery processes are key steps after the bio catalytic reactions and
make use of conventional unit operations and employ currently established and
available techniques
vIn addition to liquid-liquid extraction and crystallization a variety of other unit
operations like chromatographic separations, membrane separations and drying
operations are standard processes for product recovery and puriﬁcation.

• Table 1. Some Examples of Biotransformation Products Used in the Ton Scale
Acrylamide ∼ 250 000 tons
Aspartame 10 000 tons
Nicotinamide 15 000 tons
L-Carnitine Several hundred tons
(S) Naproxen >1000 tons
Lysine >1000 000 tons
Glucose–Fructose syrup 12 000 000 tons
Vitamin C >100 000 tons
Citric acid 1000 000 tons

210
1/29/2024 Alena B. and Woinshet M.

5.1 Dairy products
1. Cheese
ØCheese is a highly proteinaceous food made from the milk of some herbivores.
ØCheese is originated in the warm climates of the Middle East some thousands of years
ago.
ØThe scientific study and manipulation of milk for cheese manufacture is however just
over a hundred years old.
Ø Most cheese in the temperate countries of the world such as Western Europe and the
USA is made from cow’s milk.
211

ØThe composition of cow’s milk varies according to the breed of the cattle, the
stage of lactation, the adequacy of its nutrition, the age of the cow, and the
presence or absence of disease in the breasts (udders), known as mastitis.
Ø In some subtropical countries milk from sheep, goats, the lama, yak, or ass is also
used.
ØSheep milk is used specifically for the production of certain special cheese types
in some parts of Europe.
212

ØMilk from the water buffalo may be used in India and other countries, while milk
from the reindeer and the mare may be used in northern parts of Scandinavia and in
Russia, respectively.
Ø Cheese made from the milk of goat and sheep has a much stronger flavor than that
made from cow’s milk. Because the fat in goat and sheep milk contain much lower
amounts of the lower fatty acids, caproic, capryllic, and capric acids.
213

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