Sanjeet
Fermentation is a metabolic
process that converts sugar to acids, gases and/or alcohol.
It occurs in yeast
and bacteria,
but also in oxygen-starved muscle cells, as in the case of lactic acid fermentation. Fermentation is
also used more broadly to refer to the bulk growth of microorganisms
on a growth medium. French
microbiologist Louis Pasteur is often
remembered for his insights into fermentation and its microbial causes. The
science of fermentation is known as zymology.
Fermentation takes place in the absence of oxygen (when the electron transport chain is unusable) and
becomes the cell’s primary means of ATP (energy) production. It turns NADH
and pyruvate
produced in the glycolysis step into NAD+
and various small molecules depending on the type of fermentation (see examples
below). In the presence of O2, NADH and pyruvate are used to
generate ATP in respiration. This is called oxidative phosphorylation,
and it generates much more ATP than glycolysis alone. For that reason, cells
generally benefit from avoiding fermentation when oxygen is available.
Exceptions include obligate anaerobes,
which cannot tolerate oxygen. The first step, glycolysis, is common to all
fermentation pathways:
C6H12O6
+ 2 NAD+ + 2 ADP + 2 Pi → 2 CH3COCOO−
+ 2 NADH + 2 ATP + 2 H2O + 2H+
HISTORY
The use of fermentation,
particularly for beverages, has existed since the Neolithic and has been documented dating
from 7000–6600 BCE in Jiahu, China, 6000 BC in Georgia, 3150 BC in ancient Egypt, 3000 BCE in Babylon, 2000 BC in pre-Hispanic Mexico,
and 1500 BC in Sudan. Fermented foods have a religious
significance in Judaism and Christianity. The Baltic god Rugutis was worshiped as the
agent of fermentation. The first solid
evidence of the living nature of yeast appeared between 1837 and 1838 when
three publications appeared by C. Cagniard de la Tour, T. Swann, and F. Kuetzing,
each of whom independently concluded as a result of microscopic investigations
that yeast is a living organism that reproduces by budding. It is perhaps because wine,
beer, and bread were each basic foods in Europe that most of the early studies
on fermentation were done on yeasts, with which they were made. Soon, bacteria
were also discovered; the term was first used in English in the late 1840s, but
it did not come into general use until the 1870s, and then largely in
connection with the new germ
theory of disease.
Louis Pasteur (1822–1895), during the 1850s and
1860s, showed that fermentation is initiated by living organisms in a series of
investigations. In 1857, Pasteur showed that lactic acid fermentation is caused
by living organisms. In 1860, he demonstrated that bacteria cause souring in milk, a process formerly
thought to be merely a chemical change, and his work in identifying the role of
microorganisms in food spoilage led to the process of pasteurization. In 1877, working to improve the
French brewing
industry, Pasteur
published his famous paper on fermentation, "Etudes sur la Bière",
which was translated into English in 1879 as "Studies on
fermentation". He defined fermentation (incorrectly) as "Life without
air", but correctly showed that specific types of microorganisms cause
specific types of fermentations and specific end-products. Although showing
fermentation to be the result of the action of living microorganisms was a
breakthrough, it did not explain the basic nature of the fermentation process,
or prove that it is caused by the microorganisms that appear to be always
present. Many scientists, including Pasteur, had unsuccessfully attempted to
extract the fermentation enzyme from yeast. Success came in 1897 when the German chemist Eduard Buechner ground up yeast, extracted a
juice from them, then found to his amazement that this "dead" liquid
would ferment a sugar solution, forming carbon dioxide and alcohol much like
living yeasts. The "unorganized ferments" behaved just like the
organized ones. From that time on, the term enzyme came to be applied to all
ferments. It was then understood that fermentation is caused by enzymes that
are produced by microorganisms. In 1907, Buechner won the Nobel
Prize in chemistry
for his work. Advances in microbiology
and fermentation technology have continued steadily up until the present. For
example, in the late 1970s, it was discovered that microorganisms could be mutated with physical and chemical
treatments to be higher-yielding, faster-growing, tolerant of less oxygen, and
able to use a more concentrated medium. Strain selection and hybridization developed as well, affecting most
modern food
fermentations.
CHEMISTRY
Fermentation
products contain chemical energy (they are not fully oxidized), but are
considered waste products, since they cannot be metabolized further without the
use of oxygen. The chemical equation
below shows the alcoholic fermentation of glucose,
whose chemical formula is C6H12O6.
One glucose molecule is converted into two ethanol
molecules and two carbon dioxide molecules:
C6H12O6 → 2 C2H5OH
+ 2 CO2
The fermentation
unit in industrial microbiology is analogous to a chemical plant in the
chemical industry. A fermentation process is a biological process and,
therefore, has requirements of sterility and use of cellular enzymic reactions
instead of chemical reactions aided by inanimate catalysts, sometimes operating
at elevated temperature and pressure. Industrial fermentation processes may be
divided into two main types, with various combinations and modifications. These
are batch fermentations and continuous fermentations.
Batch
fermentations
A tank of fermenter is filled with the
prepared mash of raw materials to be fermented. The temperature and pH for
microbial fermentation is properly adjusted, and occasionally nutritive
supplements are added to the prepared mash. The mash is steam-sterilized in a
pure culture process. The inoculums of a pure culture are added to the
fermenter, from a separate pure culture vessel. Fermentation proceeds, and
after the proper time the contents of the fermenter, are taken out for further
processing. The fermenter is cleaned and the process is repeated. Thus each
fermentation is a discontinuous process divided into batches.
Continuous fermentation
Growth of microorganisms during batch
fermentation confirms to the characteristic growth curve, with a lag phase
followed by a logarithmic phase. This, in turn, is terminated by progressive
decrements in the rate of growth until the stationary phase is reached. This is
because of limitation of one or more of the essential nutrients. In continuous
fermentation, the substrate is added to the fermneter continuously at a fixed
rate. This maintains the organisms in the logarithmic growth phase. The
fermentation products are taken out continuously. The design and arrangements
for continuous fermentation are somewhat complex.
Aerobic fermentations
It is carried on by microorganisms under
aerobic conditions. In older aerobic processes it was necessary to furnish a
large surface area by exposing fermentation media to air. In modern
fermentation processes aerobic conditions are maintained in a closed fermenter
with submerged cultures. The contents of the fermenter are agitated with
au impeller and aerated by forcing sterilized air.
Anaerobic fermentations
Basically a fermenter designed to operate
under micro-aerophilic or anaerobic conditions will be the same as that
designed to operate under aerobic conditions, except that arrangements for
intense agitation and aeration are unnecessary. Much anaerobic fermentation does,
however, require mild aeration for the initial growth phase, and sufficient N
agitation for mixing and maintenance of temperature.
ALCHOLIC FERMENTATION
Ethyl alcohol can be produced by fermentation
of any carbohydrate containing a fermentable sugar, or a polysaccharide that
can be hydrolysed to a fermentable sugar. It
indicates that a sugar is the substrate and that the process is anaerobic.
Selected strains of Saccharomyces cerevisiae are commonly employed for
fermentation. It is imperative that the strain must have a high tolerance for
alcohol, must grow vigorously and produce a large quantity of alcohol. In recent years the production of industrial
alcohol by the fermentation process has declined because of the-increased cost
of raw materials and the rapid developments of synthetic ethanol production.
Industrial alcohol will probably continue to be obtained on a diminished scale
from certain processes. For example alcohol is obtained as the end product in
the processes designed to reduce biological oxygen demand (BOD) of some
industrial wastes, including whey and sulphite waste of paper mills. The large
amount of carbon dioxide evolved from decarboxylation of pyruvate during the
fermentation period is recovered and converted to solid carbon dioxide.
Alcoholic beverages
These products of alcoholic fermentations originated
in spontaneous fermentation processes are of great antiquity. However, it is
only in recent years that modern methods of industrial microbiology have been
applied to their manufacture. In principle, the production of alcoholic
beverages is similar to the production of industrial ethyl alcohol. These
fermentation processes do not suffer competition from synthetics products. This
is because the character of the beverage is dependent upon interactions between
varieties of biological factors that have not yet been denned in chemical or
physical terms. In beverage production refinements are introduced with respect
to flavour, aroma, colour, and sanitation that are not necessary in the making
of industrial alcohol. The type of beverage
produced is determined by the nature of the plant material employed for
fermentation. In all these processes the method of preparing the fermentation
medium is a factor of prime importance.
Beer
It is made by the yeast fermentation of grains to
ethanol and carbon dioxide. There are five major steps in the manufacture of beer
or ale from grain. These are malting, mashing, 'fermenting, maturing,
and finishing. Malting and mashing are concerned with the
conversion of starch into fermentable form such as maltose or glucose. The
chief raw material is malt, which is germinated barley that has been
dried and ground. It contains stanch, proteins, and high concentration of
amylases and proteinases. Amylases convert the starch into fermentable sugar.
Mould amylase derived from Aspergillus oryzae is sometimes used for the
same purpose. Ground malt is mashed in warm water to bring about the digestion
of starch and proteins. The aqueous extract contains dextrins, maltose, and
other sugars, protein breakdown products, minerals and various growth factors.
This is a rich nutrient medium and is called beer wort. The beer wort is
filtered and hops are added, Hops are the flowers of Humulus
lupulus. They are added for flavour, colour, and for aroma and for mild
antibacterial activity to prevent the growth of spoilage bacteria.A large
inoculum of selected strain of Saccharomyces cerevisiae is added to the
wort to bring about a vigorous fermentation. Yeasts are classified as 'top
yeasts' or 'bottom yeast*.Top yeasts float on the surface of a
fermenting mixture and are employed in making ale. Bottom yeasts settle
in the fermentation tank and are used in making beer. Beer fermentation
takes place at 6 to 12°C., whereas ale fermentation is complete in five to
seven days at 14 to 23°C. The alcoholic content of beer is between 3 to 6
percent, that of ale is somewhat higher.The fermented wort is refrigerated at
0°C for two weeks to several months to remove the harsh flavour and other
undesirable characteristics. Some of the harshness attributed to higher
alcohols disappears as they are oxidized or esterified during aging. Finishing
process consists of carbonation, cooling, filtering and dispensing into
barrels, bottles, and cans. Bottled or canned beer is usually pasteurized at
60°C for 20 minutes to kill yeasts and other microorganisms. As an alternative,
the beer may be passed through a filter to remove microorganisms, and then
aseptically dispensed into sterile cans. The composition of American lager beer
is as follows:
Alcohol
|
3.8 percent
|
Dextrins
|
4:3 percent
|
Proteins
|
0.3 percent
|
Ash
|
0.3 percent
|
C02
|
0.4 percent
|
It also contains appreciable amounts of
vitamins, particularly riboflavin. In addition, there area number of minor
constituents, some of which are important for flavour and aroma.
Wines
Wine is the product made by the normal alcoholic
fermentation of the juice of sound, ripe grapes and the usual cellar treatment.
Beverages produced by the alcoholic fermentation of other fruits and certain
vegetable products-are also called wines for example, peach wine, orange wine,
cherry wine. Wine making is a much simpler process. It can be made by a direct
fermentation of sugars, i.e. glucose and fructose, instead of starch which
requires hydrolysis to yield sugars. Many fruits have the wine yeast Soccharomyces
cerevisiae var. ellipsoideus on them. All that is necessary is to crush the
fruits. An alcoholic fermentation starts spontaneously. The characteristic
qualities of famous wines are attributed in part to strains of yeast found in
certain localities. However, undesirable moulds, wild yeasts, and bacteria are
also likely to be present and the fermentation may not give a predictably good
product. Many wine makers now destroy natural yeasts by adding sulphur dioxide
to the raw juice. The grapes are crushed carefully and the juice is collected.
To the raw juice or must; sulphur dioxide is added as sodium
metabisulphite. The must is then inoculated with a starter culture-of a
selected strain of S. cerevisiae var. elliposideus. At the start the
must is aerated slightly to promote vigorous yeast growth. Once the fermentation
sets in, the rapid production of carbon dioxide maintains anaerobic condition.
The temperature of fermentation is usually 25 to 30°C and the process may
extend from few days to 2 weeks. The yield of ethanol varies from 7 to 15
percent (by volume). The wine is placed in large casks to settle, clarify and
age for two to five years to develop a good flavor and aroma. Wines are endless
in their varieties and differ in so many attributes that it is difficult to
classify them. According to colour, the two most basic types are red and
while wine. In making red wines the grapes are crushed and
stemmed but the skins and seeds are left in the must. While wines are
made from white grapes or from the juice of grapes from which the skins have
been removed. Dry wines are those which contain too little sugar to be
detected by taste. In sweet wines the sugar content is high enough to be
detected by taste. Sparkling wines contain carbon dioxide. They are made
effervescent by secondary fermentation in closed containers, generally in the
bottle itself. Still wines are those which do not contain carbon
dioxide. Fortified wines contain added alcohol in the form of brandy.
Distilled liquors
Yeast action is limited by the amount of
alcohol present, and at about the level of 18 percent by volume its action
ceases. To produce the so called hard liquor, for higher levels of
alcohol, distillation is required. Distilled alcoholic beverages may be divided
into three major classes depending on the nature of the solution distilled:
Ø
The
products starting from a starchy substance and needing enzymes.
Ø
The
products starting directly from a sugar substrate.3. The type of liquor produced by adding flavor
substances to quite pure ethanol,
which has been obtained by distillation and rectification.
Malt whisky is prepared by fermentation and subsequent
distillation of malted barley. Grain whisky is prepared in a similar
manner from a mixture of malted and unmalted barley with unmalted maize. Malt
and grain whisky are matured and finally blended to form Scotch whisky.
Bourbon is whisky prepared from a mash in which maize is the predominant
grain. Irish Whisky is manufactured from a mash in which rye grain
predominates. Arrak (Far East) and sake (Japan) are fermented
beverages piepared from rice. Rice starch is hydrolysed by amylases derived
from moulds, principally Aspergills oryzae. Brandy is obtained from distillation of fermented
fruit juice, that is wine. Rum is produced by distillation of fermented
molasses or other sugarcane byproducts. Gin is prepared by extracting
juniper berries with alcohol and distillation of alcohol. Cordials and liqueurs
are sweetened alcoholic distillates from fruits flowers, leaves, etc. Vinegar may
be defined as the condiment made from sugary or starchy material by alcoholic
and subsequent acetic acid fermentations. The word vinegar is derived from
French term vinaigre, meaning, 'sour wine' (vin=wine, aigre=sour).
Vinegar is the product resulting from the conversion of ethyl alcohol to acetic
acid by a group of widely distributed bacteria of the genus Acetobacter. Thus it can be produced
from any alcoholic material, ranging from alcohol-water mixtures to various
fruit wines. The composition of a vinegar will depend somewhat upon on she
nature of the raw material that has undergone alcoholic and acetous
fermentations. Vinegar is a solution containing at least 4 percent acetic acid
and small amounts of alcohol glycerol, esters, reducing sugars, pentosans,
salt, and other substances. Depending on the raw materials, vinegars are
differentiated as wine vinegar, apple
cider vinegar, malt vinegar, and others. The
microorganisms that produce acetic acid from ethyl alcohol are species of Acctobac.ct,—A. orkannt, A. orleansis, A.schutzenbachi, A.Aceti and others.
The biochemical reaction by which they form acetic acid from ethanol is as
follows :-:-
2CH3 CH2OH + 02 à 2CH3CHO
+ 2H2O
2CH3 CHO + O2 à 2CH3COOH
Some of the Acelobacter
species do not stop with acid production but continue the oxidation to
carbon dioxide.
CH3COOH + O2 à 2CO2
+ 2H2O
Thus selection of proper organisms is important for
vinegar fermentation. The organisms should carry the reaction as near to
completion without destroying acetic acid by oxidation. In addition they must
be tolerant to ethanol. The basic methods of vinegar production are
known as the slow process: or
Orleans method, the rapid generator process and submerged fermentation in an acetatof. Vinegar is commonly made at
home from cider, grape juice, etc. in a barrel. Yeast fermentation is used for
the production of alcohol. The alcoholic solution is transferred to a vinegar
barrel and alcohol concentration is adjusted between 10 to 13 per cent. The
alcoholic solution is inoculated and acidified by adding 10 to 25 per cent of
pure vinegar. When vinegar fermentation is complete the vinegar is bottled and
stoppered tightly. This is to prevent further oxidation of acetic acid by Acetobacter when the alcohol
concentration drops to 1 to 2 per cent. During acetic fermentation the bacteria
develop as a gelatinous pellicle on the surface of the liquid. The organisms
thus have access to both ethyl alcohol and oxygen If the pellicle is disturbed
and sinks to the bottom ('Mother of
Vinegar’), acetification stops, until another pellicle forms. The Orleans
or the French process
employs wooden vats or asks of about 200 litres capacity. These are filled
one-third with a good grade of vinegar. This constitutes the starter or the
culture. At weekly intervals, 10 to 15 liters of wine are added. After five
weeks 10 to 15 liters of vinegar are drawn off each week and the same amount of
wine is added. Air is admitted to the barrels through holes above the level of
the vinegar medium. This is a slow continuous process and requires constant
attention and maintenance. However it produces a high quality of vinegar.Vinegar
is manufactured by more rapid methods, using the generator (German process). Generators are of various sizes and
shapes. They may be as large as 15 feet in diameter and 20 feet high. The
generator is equipped with a false perforated bottom, through which air enters
and supports beechwood shavings. Near the top of the generator, there is a
false top or perforated plate over which is arranged a rotating sprinkler, or
sparger
Lactic
acid production
The use of lactic acid fermentation as a good
preservation method is another ancient art of unknown origin. Lactic acid
fermentation was investigated by Pasteur as one of his first microbiological
problems. Lactic acid is commonly produced from the usual cheap sources of
fermentable carbohydrates such as acid or enzyme hydrolysed corn and potato
starches, molasses, and whey. Whey, the
watery part of milk separated from curd during cheese making, is widely used in
the manufacture of lactic acid. Whey represents a satisfactory medium for the
growth of certain bacteria. It contains a relatively large amount of lactose
and proteiuaceous substances, minerals, and some essential vitamins. The
homofermeutative lactobaeilli such
as Lactobacillus bufgariau, L.
delbrueckii, etc., grow raipdly and convert the lactose to the single
end product, lactic acid. The typical fermentation process involved in
making commercial calcium lactate and the principal grades of lactic acid is
described in brief. Pasteurized whey is inoculated with a starter containing L. bulgaricus. To prepare a
sufficient amount of inoculum the culture is built up by successive transfers
in sterile skim milk, pasteurized skim milk, and finally, when fermentation is
carried out at a temperature of 430C to discourage the growth of
undesirable organisms. Fermenters and accessory equipment are fabricated with
type 316 stainless steel to resist the corrosiveness of lactic acid.
Combinations of glass and fluorocarbon resins (teflon) are also employed in the design of piping system,
valve, filters, etc. During the fermentation, lime (Ca(OH)2) is
added intermittently to neutralize the acid and to promote a good yield of
calcium lactate, At the end of fermentation, the lactalbumin is coagulated by
heat, When lactalbumin settles, the solution of calcium lactate is decanted off
and filtered. It is then treated with decolourizing carbon and filter aids,
filtered, evaporated, and crystallized. The crystals are further purified and
sold as calcium lactate or converted to lactic acid. Various procedures are
followed in producing the different grades of lactates and lactic acid. Lactic acid
has many uses. It is used as ail
acidulant in confectionery, fruit juices, and essences. It may be used in the
curing of meat and in canned vegetable and fish products. Lactic acid is used in various chemical
industries. The lactates also have important
uses. Calcium lactate is used in baking powders and bread,
and in the treatment of calcium deficiency. Iron lactate is used in the
treatment of anemia. Sodium lactate is used to help in the retention of
moisture by such products as tobacco and as a plasticizer. Lactic acid fermentation from whey also helps in
the removal of pollution of our environment.
Conclusion:
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