Production of Vitamin B12 (Cyanocobalamine)
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Vitamins Production by Fermentations - Microbial production is the only source of vitamin Bl2 whilst of an the other water-soluble vitamins now available commercially only riboflavin (vitamin B2) is manufactured to any significant extent, microbiologically.
There are reports that ascorbic acid is also produced by microbial fermentation but the details are not available. None of the fat soluble vitamins is produced industrially by microbiological methods but one compound, β-carotene which is converted by animals to vitamin A, can be prepared by microbial synthesis.
Processes for Microbial Synthesis of Vitamin B12 - It seems probable that the only primary source of vitamin Bu in nature is the metabolic activity of the microorganisms. It is synthesized by a wide range of bacteria and Streptomycetes, though not to any extent by yeasts and fungi. While over 100 fermentation processes have been described for the production of vitamin Bl only half a dozen have apparently been used on a commercial scale.
Production of Vitamin B12 (Cyanocobalamine)
The steps involved in production of Vitamin B12 are:
a) Selection of microorganisms
b) Inoculum preparation
c) Fermenting media
d) pH
e) Temperature and fermentation process
f) Aeration and agitation
g) Addition of Antifoaming agents
h) Recovery
a) Selection of microorganisms:- The organisms involved in vitamin B production are Sterptomyces griseus, Bacillus megaterium, B. coagulance, Pseudornonas dentrificans, Propionibacterium sherman/i and a Pseudomonas spp. Normally, the vitamin B is produced on large scale by using submerged fermentation method. Fermentation is usually completed within 3 to 5 days. B
b) Inoculum preparation:- For every commercial fermentation the preparation of inoculum is essential prior to the addition of the culture into the fermenting medium. Here also one can prepare the stock culture from the selected strains. The pure culture of Streptomyces olivaceus is inoculated and grown in 100-250 ml of inoculum medium (usually Bennet’s agar medium is used) contained in flasks. Thus seeded flasks are to be kept on mechanical shakers during incubation for proper aeration. After the growth these flasks are utilized for inoculating into fermenting medium. Usually 50% of the inoculum is sufficient for the initiation of fermentation.
C) Fermenting medium:- The fermenting medium for vitamin B12 usually consists of carbohydrates, proteins, a source of cobalt and other salts. In the composition, cobalt plays very important role in giving high yields of vitamin B12 . The composition of vitamins B12 fermentation medium is as follows:
Composition of Vitamin B12 medium:
Distillers solubles :4.0%
(Soya meal, bean meal)
Dextrose: 5.0-10.0%
CaCO3: 0.5%
C0Cl3.6H20: 1.5tolOp.p.m.
pH7.0
After sterilization of the medium in the fermentor, the fermentation medium is ready for inoculation with the seed lot cultures to initiate the production of vitamin 812.
d) pH:- Once the fermentation is started there is rapid consumption of sugar in the first 24 hours. After 2 to 4 days, lysis of mycelium begins resulting in the rise of pH. The stabilization of the mash is practiced by reducing the pH to about 5 with sulphuric acid and adding small amounts of reducing agent.
e) Temperature and fermentation process:- A temperature of 27°C is satisfactory during fermentation in the production flasks. The duration of the fermentation is about 3 to 4 days, or until mycelium lysis begins to occur. Thus, most of the vitamin 812 remains within microbial cells until autolysis sets in, and therefore the recovery of the vitamin from the fermentation broth is simplified by harvesting before autolysis has become serious.
f) Aeration and Agitation:- The growth of the Streptomyces strain mainly depends upon the rate of aeration and the speed of agitation. Aeration rates higher than the optimum results in foaming. The optimum rate of aeration is about 0.5 volume air/volume medium/minute.
g) Addition of antifoaming agents:- In this fermentation the formation of foaming is a very serious problem. There are several antifoaming agents available which suppress foam formation. They include: soyabean oil, corn oil, lard oil, and silicones.
h) Recovery:- As we know already, during the fermentation period, most of the vitamin B is present in the mycelium. It is released into the medium at the end of the fermentation period. But, a large amount of vitamin B is still present in the mycelia. Heating the mixture to boiling at pH5 or below releases the vitamin B from the mycelium.
The mycelium with other solids is filtered or centrifuged. Then the filtered broth is treated with cyanide to bring about the conversion of cobalamin to cyanocobalamin. The adsorption of cyanocobalamin from the solution is practiced by passing it through an adsorbing agent packed in columns. Adsorbing agents such as charcoal, bentonite, fuller’s earth and ion exchange resins are used. After removal of cyanocobalamin from the adsorbent, it is treated with water-acetone to form an organic solvent of vitamin B then it is concentrated and crystallized.
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Production of Riboflavin (Vitamin B2)
(a) Media preparation and biosynthesis of Riboflavin.
For Riboflavin production, basic medium consists of corn steep liquor 2.25%, commercial peptone 3.5%, soybean oil 4.5% but it can be supplemented further by addition of different peptones, glycine, distiller’s solubles, or yeast extract. The glucose and inositol increase the production of riboflavin.
The medium should be kept at 26-28°C at pH 6.8 for 4-5 days incubation. After inoculation the submerged growth of Ashbya gossypi is supported by insufficient air supply. The excess air inhibits mycelial production and reduces the riboflavin yield. The fermentation progresses through three phases.
(b) First phase: In this phase, rapid growth occurs with small quantity of riboflavin production. The utilization of glucose occurs resulting into decrease in pH due to accumulation of pyruvate. By the end of this phase, the glucose is exhausted and growth ceases.
(c) Second phase: Sporulation occurs in this phase. The pyruvate decreases in concentration. Ammonia accumulates because of an increase in deaminase activity. The pH reaches towards alkalinity.
(d) Third phase: There is a rapid synthesis of cell-bound riboflavin (FMN and FAD). This phase is accompanied by rapid increase in catalase activity subsequently cytochromes disappear.
As the fermentation completes, the autolysis takes place which releases free riboflavin into the medium as well as retained in the nucleotide form. It is also observed that certain purines also stimulate riboflavin production without simultaneous growth stimulation.
The riboflavin is present both in solution and bound to the mycelium in the fermentation broth. The bound vitamin is released from the cells by heat treatment (lh, 120°C) and the mycelium is separated and discarded. The riboflavin is then further purified. The crystalline riboflavin preparation of high purity have been produced using Saccharomyces fermentation with acetate as sole C source.
Uses: It is essential for the growth and reproduction of both humans and animals and, thus, it often is recommended as a feed additive for the animal nutrition. The riboflavin deficiency in rats causes stunted growth, dermatitis, and eye damage. Ariboflavinosis is a disease in humans caused by riboflavin deficiency.
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Production of Penicillin
Penicillium species required the production medium which contains lactose (1%), Calcium carbonate (1%), corn steep liquor 8.5%, glucose (1%), sodium hydrogen phosphate (0.4%) phenyl acetic acid (O.5g). The pH is kept between 5 and 6 and temperature for incubation is 23-25°C. Aeration and agitation are necessary.
Fermentation: Penicillin is produced by Penicillum chrysogenum Q-176, a fungus that can be grown in stirred fermenters. The inoculum under aerobic condition (seed) can be produced when there is glucose in sufficient amount in the medium. If particular penicillin is produced specific precursor (substance added prior or simultaneously with the fermentation which incorporated without any major change in the molecules) is added in the medium for e.g. phenyl acetic acid or its derivatives such as ethanol amide to get penicillin G. The antifoam agents such as vegetable oil (corn or soybean oil) is added to the medium before sterilization.
The spore suspension is inoculated in flasks, each containing 15 g barley seeds. These flask are vacuum dried, to which sterilized quartz is added.
The preparation of inoculum takes place on barley seeds. The flask containing 15 g barks seeds are to be mixed with mother culture, and incubated at 25°C for 7 days. The spores develops on barley seeds are suspended in distilled water to make spore suspension. After testing the antibioc activity, the seeds containing flasks are ready for seeding in fermenter. Three phases of growth can be differentiated during cultivation of Penicillium chrysogenum.
(a) First phase. In this phase, growth of mycelium occurs; yield of antibiotic is quite lo Lactic acid present in corn steep liquor is utilized at a maximum rate by the microorganism. Lactose is used slowly. Ammonia is liberated into the medium resulting into rise in pH.
(b) Second phase. There was intense synthesis of penicillin in this phase, due to rapid consumption of lactose and the ammonium nitrogen (NH3 N). The mycelial mass increases; the pH remains unchanged.
(c) Third phase. The concentration of antibiotic decreases in the medium. The autolysis of mycelium starts liberation of ammonia and slight rise in pH.
Recovery: When the fermentation cycle (7 days) is completed, the whole batch is harvest for recovery. Its activity disappears on evaporation to dryness, hydrolyzed to penicilloic acid. Penicillin has tendency that it remains in aqueous phase at normal pH and in solvent phase at acidic pH. This property of penicillin is used in recovery of potassium penicillin from natural solution. Once the fermentation is completed the broth is separated from fungal mycelium and processed by absorption, precipitation and crystallization to yield the final product. This basic product can then be modified by chemical procedures to yield a variety of semi synthetic penicillins such as ampicillin, amoxycillin, etc.
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ALCOHOL FERMENTATION
BEER
Beer is made of malt, as well as other sources of starch such as rice and corn, water, hops and yeast as the fermentation agent. The purpose of the brewing process is the conversion of the starch contributed by the malt and the adjuncts corn or rice to maltose and dextrins. This conversion is accomplished by the enzymes present in the malt. The solution of carbohydrates obtained (called wort) is then boiled with hops cooled and fermented to beer containing alcohol, carbon dioxide, and residual dextrins. The finished beer has as major constituents water, dextrins, alcohol and carbon dioxide and as minor though important constituents, unfermented sugars, proteins and aromatics such as hops-resins etc.
Beer production:- The ingredients used to produce beer are malt, adjuncts, water, hops, yeast and a few other additions.
Malt and Malting:- Malt is prepared from barley, wheat, oat and it is cleaned of dust and other foreign materials. The actual process of malting barley is carried out in malt house. Malt contains the enzymes which degrade the starch of the malt itself and additional starchy adjuncts, such as corn starch and rice starch, to dextrins and maltose. The dextrins remain to give body to the beer, and the maltose is fermented to the alcohol and carbon dioxide. The malt is also the source of the beer proteins which are important for foam and of the flavor typical of the beer.
For processing into malt, the barley is cleaned and soaked in water so that water is then drained off and sprouting continued for 5 to 7 days, until the desired growth of the embryo has been obtained. During this time, the malt has to be turned and is thus aerated frequently to prevent local overheating and achieve uniformly controlled growth. At the end of the period necessary for optimum growth (measured by the length of the growth of the germ or acrospore and the hardness of the endosperms), the sprouts are killed by killing the malt, duration and killing steps are very important for colour, aroma, and level of enzymes. After killing, the sprouts and rootlets are removed and the malt is aged for several weeks. It is then ready for use in the brewery.
During malting, certain enzymes are increased and others are newly formed: -amylase is increased, and ct-amylase is newly formed. a- amylase is important for attack on the starch (Iiquefaction), and - amylase for final sugar formation. This conversion starts in the malting process, increasing the proportion of lower carbohydrates, and it is even more important for production of the work during mashing. The overall composition of the malt is as follows:
Starch: 59%
Sugars: 10%
Gums: 10%
Cellulose: 5%
Protein: 10%
Fat: 2.5%
Ash: 2%
Brewing process:- The malt is transferred to the brewery for grinding and other raw materials used for brewing include adjuncts (rice or corn) as additional sources of starch and hops.
The hops are plant belonging to the family Cannabidaceae. This is a climbing herbaceous dioecious plant. Only pistillate flowers which are cone like are used in brewing. Staminate flowers are destroyed to make the pistillate flowers seedless. Chemically hops contain 50% of hydrocarbons and the remainder are esters, alcohols and carbonyl compounds. Other than this, it contains hop-resin and hop-oil. The hop resins are contained in small yellow aggregates below the petals. They are the major flavor contributors, being responsible for the characteristic bitterness in beer. These resins also have preservative properties. Hops also contains hop-oil responsible for the strength of the beer. Brewers usually use hop flowers (many people mistake them as leaves because of their green colour) directly and some may use hop extracts.
The other raw material of importance, often not thought of as such, is water. Water should have the right hardness and calcium content for enzyme activity and must not have off flavours. The mineral content of the water influences the final beer flavour.
After getting the malt from the malt house, the ground malt is mixed with water to let the amylases convert the molecules. The brewers use mash tub for holding the mash for certain period of time at a selected temperature to assure maximum conversion. Meanwhile, the adjuncts are boiled in the cooker to achieve gelatinization. The pregelatinized adjuncts are added to the main bulk of the brew in the mash tub where they go through final stages of conversion until no free starch remains and the desired ratio of dextrin and maltose is obtained. The total mash consisting of adjuncts and malt mash, including the barley husks is heated at about 72°C, when enzyme activity ceases. It is then passed through the lauder tub or mash filter. During filtration, the spent grain solids and the insoluble barley husks are removed. This filtered solution, now is called wort. The wort is then boiled with hops in the brew kettle for 1.5 hour. This solubilizes the valuable hop constituents, while undesirable proteins and tanning are coagulated and at the same time the wort is sterilized.
From the brew kettle the wort passes via the hop strainer, where the insoluble part of the hops (about 70 percent of the originat hop dry weight) is removed, to the hot wort settling tank. After air cooling, the brew is now moved to the fermentors. The yeast is added (Saccharomyces cerevisiae var ellipsoides) and the wort is fermented to beer. Fermentation takes place at a low temperature (6-12°C) for a period of 8 to 10 days. At the end of the fermentation the yeast settles and the beer is removed and stored in tanks for further clarification, aging and carbonation. Keg beer is sold unpasteurized in the package of sterile filtered prior to filling. The final beer (lager beer contains:
Alcohol: 3.8%
Dextrins: 4.3%
Proteins: 0.3%
Ash: 0.3%
CO2 : 0.4%
It also contains an appreciable amount of vitamins such as riboflavin.
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WINE
Wine is produced by the normal alcoholic fermentation of juices of ripe grapes. It can also be produced by direct fermentation of sugars(Glucose or Fructose). Wine is also produced from peaches, oranges, cherries, etc. Accordingly they are called peach wine, orange wine, cherry wine etc.
Fermentation practices differ from winery to winery and also with the type of the wine to be produced. But general procedure is more or less the same. Wines are endless in their varieties. Based on the colour two basic types are recognized mainly red wine and white wine. In making red wine the grapes are crushed and steamed and the skins and seeds are left in the must. White wine is made from white grapes or the grapes in which the skins and seeds are removed. Dry wines are those which contain more sugars. Sparkling wines are those which contain CO2. It is made by secondary fermentation in closed containers. Still wines are those which do not contain CO2. Fortified wines contain added alcohol in the form of Brandy.
WHITE WINE
The quality of wine depends on the type of the grapes, its maturity and health. The grapes are first separated from the stems and crushed. The crushed grapes provide the must. Rapid processing is to be carried out to inhibit the growth of undesirable microorganisms especially the acetic acid bacteria. Besides this oxidative, enzyme catalyzed processors occur on prolonged standing of the must which may affect the colour and the quality of wine. But certain rest period is required to facilitate the flow of free run juice. The must should be treated immediately with SO in order to inhibit oxidative enzyme action and also to prevent growth of undesirable microorganisms. Sometimes must is directly sulfited. Sulfating is necessary to prevent bacterial fermentation of the malic acid of the must. Next step is to make must free from particulate matter by centrifugation, filtration or sedimentation which occurs on standing. Removal of sediment reduces the susceptibility of the must to oxidation and improve the colour and aroma of the wine. Alternatively oxidation of enzymes can also be stopped by treating the must at high temperatures for short time (85°C). This also kills the microorganisms. In this case inoculation with fermenting yeast is absolutely essential. But even if the must is not heated it is desirable to inoculate the must with pure culture of yeast. If the fermentation is brought about in large fermentors increase of temperature becomes a problem. If the temperature goes above 38°C the fermentation stops because of inactivation and loss of viable yeasts. Besides these fermentation brought about at higher temperatures leads to loss of aroma and quality of wine. Therefore temperature should not exceed 20°C.