FERMENTATION

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 P_i → 2 CH₃COCOO⁻ + 2 NADH + 2 ATP + 2 H₂O + 2H⁺

Pyruvate is CH₃COCOO⁻. P_i is phosphate. Two ADP molecules and two P_i are converted to two ATP and two water molecules via substrate-level phosphorylation. Two molecules of NAD⁺ are also reduced to NADH. In oxidative phosphorylation the energy for ATP formation is derived from an electrochemical proton gradient generated across the inner mitochondrial membrane (or, in the case of bacteria, the plasma membrane) via the electron transport chain. Glycolysis has substrate-level phosphorylation (ATP generated directly at the point of reaction). Fermentation has been used by humans for the production of food and beverages since the Neolithic age.

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

C₂H₅OH is the chemical formula for ethanol. Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules. This is known as glycolysis.

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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