Review of Literature:

A vitamin is an organic compound required as a nutrient in tiny amounts by an organism. A compound is called a vitamin when it cannot be synthesized in sufficient quantities by an organism, and must be obtained from the diet. Thus, the term is conditional both on the circumstances and the particular organism. For example, ascorbic acid functions as vitamin C for some animals but not others, and vitamins D and K are required in the human diet only in certain circumstances. Vitamins are classified by their biological and chemical activity, not their structure. Thus, each "vitamin" may refer to several vitamer compounds that all show the biological activity associated with a particular vitamin. Such a set of chemicals are grouped under an alphabetized vitamin "generic descriptor" title, such as "vitamin A," which includes the compounds retinal, retinol, and many carotenoids. Vitamer are often inter-converted in the body. The term vitamin does not include other essential nutrients such as dietary minerals, essential fatty acids, or essential amino acids, nor does it encompass the large number of other nutrients that promote health but are otherwise required less often. Vitamins have diverse biochemical functions, including function as hormones (e.g. vitamin D), antioxidants (e.g. vitamin E), and mediators of cell signaling and regulators of cell and tissue growth and differentiation (e.g. vitamin A). The largest number of vitamins (e.g. B complex vitamins) function as precursors for enzyme cofactor bio-molecules (coenzymes), that help act as catalysts and substrates in metabolism. When acting as part of a catalyst, vitamins are bound to enzymes and are called prosthetic groups. For example, biotin is part of enzymes involved in making fatty acids. Vitamins also act as coenzymes to carry chemical groups between enzymes. For example, folic acid carries various forms of carbon group – methyl, formyl and methylene - in the cell. Although these roles in assisting enzyme reactions are vitamins' best-known function, the other vitamin functions are equally important.

HISTORY

In 1749, the Scottish surgeon James Lind discovered that citrus foods helped prevent scurvy, a particularly deadly disease in which collagen is not properly formed, causing poor wound healing, bleeding of the gums, severe pain, and death. In 1753, Lind published his Treatise on the Scurvy, which recommended using lemons and limes to avoid scurvy, which was adopted by the British Royal Navy. This led to the nickname Limey for sailors of that organization. Lind's discovery, however, was not widely accepted by individuals in the Royal Navy's Arctic expeditions in the 19th century, where it was widely believed that scurvy could be prevented by practicing good hygiene, regular exercise, and by maintaining the morale of the crew while on board, rather than by a diet of fresh food. As a result, Arctic expeditions continued to be plagued by scurvy and other deficiency diseases. In the early 20th century, when Robert Falcon Scott made his two expeditions to the Antarctic, the prevailing medical theory was that scurvy was caused by "tainted" canned food.

In 1881, Russian surgeon Nikolai Lunin studied the effects of scurvy while at the University of Tartu in present-day Estonia. He fed mice an artificial mixture of all the separate constituents of milk known at that time, namely the proteins, fats, carbohydrates, and salts. The mice that received only the individual constituents died, while the mice fed by milk itself developed normally. He made a conclusion that "a natural food such as milk must therefore contain, besides these known principal ingredients, small quantities of unknown substances essential to life." However, his conclusions were rejected by other researchers when they were unable to reproduce his results. One difference was that he had used table sugar (sucrose), while other researchers had used milk sugar (lactose) that still contained small amounts of vitamin B.

The discovery of vitamins and their sources

Year of discovery Vitamin Source

1909 Vitamin A (Retinol) Cod liver oil

1912 Vitamin B1 (Thiamin) Rice bran

1912 Vitamin C (Ascorbic acid) Lemons

1918 Vitamin D (Calciferol) Cod liver oil

1920 Vitamin B2 (Riboflavin) Eggs

1922 Vitamin E (Tocopherol) Wheat germ oil, Cosmetic and Liver

1926 Vitamin B12 (Cyanocobalamin) Liver

1929 Vitamin K (Phylloquinone) Alfalfa

1931 Vitamin B5 (Pantothenic acid) Liver

1931 Vitamin B7 (Biotin) Liver

1934 Vitamin B6 (Pyridoxine) Rice bran

1936 Vitamin B3 (Niacin) Liver

1941 Vitamin B9 (Folic acid) Liver

In east Asia, where polished white rice was the common staple food of the middle class, beriberi resulting from lack of vitamin B was endemic. In 1884, Takaki Kanehiro, a British trained medical doctor of the Japanese Navy, observed that beriberi was endemic among low-ranking crew who often ate nothing but rice, but not among crews of Western navies and officers who consumed a Western-style diet. Kanehiro initially believed that lack of protein was the chief cause of beriberi. With the support of the Japanese navy, he experimented using crews of two battleships; one crew was fed only white rice, while the other was fed a diet of meat, fish, barley, rice, and beans. The group that ate only white rice documented 161 crew members with beriberi and 25 deaths, while the latter group had only 14 cases of beriberi and no deaths. This convinced Kanehiro and the Japanese Navy that diet was the cause of beriberi. This was confirmed in 1897, when Christiaan Eijkman discovered that feeding unpolished rice instead of the polished variety to chickens helped to prevent beriberi in the chickens. The following year, Frederick Hopkins postulated that some foods contained "accessory factors"—in addition to proteins, carbohydrates,

fats, et cetera—that were necessary for the functions of the human body. Hopkins was awarded the 1929 Nobel Prize for Physiology or Medicine with Christiaan Eijkman for their discovery of several vitamins.

In 1910, Japanese scientist Umetaro Suzuki succeeded in extracting a water-soluble complex of micronutrients from rice bran and named it aberic acid. He published this discovery in a Japanese scientific journal.

When the article was translated into German, the translation failed to state that it was a newly discovered nutrient, a claim made in the original Japanese article, and hence his discovery failed to gain publicity. Polish biochemist Kazimierz Funk isolated the same complex of micronutrients and proposed the complex be named "Vitamine" (a portmanteau of "vital amine") in 1912. The name soon became synonymous with Hopkins' "accessory factors", and by the time it was shown that not all vitamins were amines, the word was already ubiquitous. In 1920, Jack Cecil Drummond proposed that the final "e" be dropped to deemphasize the "amine" reference after the discovery that vitamin C had no amine component.

Throughout the early 1900s, the use of deprivation studies allowed scientists to isolate and identify a number of vitamins. Initially, lipid from fish oil was used to cure rickets in rats, and the fat-soluble nutrient was called "antirachitic A". Thus, the first "vitamin" bioactivity ever isolated, which cured rickets, was initially called "vitamin A", although confusingly the bioactivity of this compound is now called vitamin D.] What we now call "vitamin A" was identified in fish oil as a separate factor that was inactivated by ultraviolet light. In 1931, Albert Szent-Györgyi and a fellow researcher Joseph Svirbely determined that "hexuronic acid" was actually vitamin C and noted its anti-scorbutic activity. In 1937, Szent-Györgyi was awarded the Nobel Prize for his discovery. In 1943 Edward Adelbert Doisy and Henrik Dam were awarded the Nobel Prize for their discovery of vitamin K and its chemical structure.

There are several roles for vitamins and trace minerals in diseases: 1. Deficiencies of vitamins and minerals may be caused by disease states such as mal

absorption; 2. Deficiency and excess of vitamins and minerals can cause disease in and of themselves

(e.g., vitamin A intoxication and liver disease);

3. Vitamins and minerals in high doses may be used as drugs (e.g., niacin for hypercholesterolemia).

Vitamins are essential for the normal growth and development of a multi-cellular organism. The developing fetus requires certain vitamins and minerals to be present at certain times. If there is serious deficiency in one or more of these nutrients, a child may develop a deficiency disease. Deficiencies of vitamins are classified as either primary or secondary.

1. Primary Deficiency: A primary deficiency occurs when you do not get enough of the vitamin in the food you eat.

2. Secondary Deficiency: A secondary deficiency may be due to an underlying disorder that prevents or limits the absorption or use of the vitamin.

Types of Vitamins

Vitamins, one of the most essential nutrients required by the body and can be broadly classified into two main categories i.e., water-soluble vitamins and fat-soluble vitamins.

Water-soluble vitamins

Water-soluble vitamins cannot be stored in the body, so you need to get them from food every day. They can be destroyed by overcooking. These are easily absorbed by the body. Human body doesn't store large amounts of water-soluble vitamins. B-complex vitamins and vitamin C are water-soluble vitamins that are not stored in the body and must be replaced each day. These vitamins are easily destroyed or washed out during food storage and preparation. They are eliminated in urine so, body need a continuous supply of them in diets.

Proper storage and preparation of food can minimize vitamin loss. To reduce vitamin loss, refrigerate fresh produce, keep milk and grains away from strong light, and use the cooking water from vegetables to prepare soups. An excess of water soluble vitamins should not result in any side effects as they will disperse in the body fluids and voided in the urine.

Nine of the water-soluble vitamins are known as the B-complex group: Thiamin (vitamin B1), Riboflavin (vitamin B2), Niacin, Vitamin B6, Folate, Vitamin B12, Biotin, Pantothenic acid and Vitamin C. These vitamins are widely distributed in foods.

Major food sources of Water-Soluble Vitamins

Grains Fruits Vegetables Meats, EggsLegumes, Nuts, Seeds

Milk, Dairy

Thiamin X X X

Riboflavin X X

Niacin X X X

Biotin X X X

Pyridoxine X X X

Pantothenic acid X X X X X X

Vitamin B12 X X

Folate X X

Vitamin C X X

Fat-soluble vitamins

The fat-soluble vitamins include vitamins A, D, E and K - since they are soluble in fat and are absorbed by the body from the intestinal tract. The human body has to use bile acids to absorb fat-soluble vitamins. Once these vitamins are absorbed, the body stores them in body fat. When you need them, your body takes them out of storage to be used. Eating fats or oils that are not digested can cause shortages of fat-soluble vitamins.

Fat soluble vitamins should not be consumed in excess as they are stored in the body and an excess can result in side effects. An excess of vitamin A may result in irritability, weight loss, dry itchy skin in children and nausea, headache, diarrhea in adults.

Characteristics of the vitamins are:

1. Most of the vitamins have been artificially synthesized.2. Some of vitamins are soluble in water and others are fat-soluble.

3. Some vitamins are synthesized in the body. Some members of vitamin B complex are synthesized by microorganisms in the intestinal tract.

4. Vitamins are partly destroyed and are partly excreted.

5. Vitamins can be stored in the body to some extent, for example the fat-soluble vitamins are stored in the liver and subcutaneous tissue.

6. Vitamins can perform their work in very small quantities. Hence, the total daily requirement is usually very small.

First of all vitamin is that component of a balanced diet which the human body generally cannot manufacture on its own. So you must consume vitamin directly in the form of food or through

supplements as tonic or pills. The whole process of assimilation of vitamins depends on ingestion of food. Once you have it as a part of your meal, say for tomatoes, lemon, spinach and other stuffs, it is more helpful. Moreover you don't feel that you are a patient and need to have medicines for cure. But if the deficit of a particular vitamin is high, then supplementary dose of vitamins have to be given to the body for a particular period. The body's metabolism is also dependent on vitamins as on carbohydrates, fats, minerals and other basic components of a complete diet. But before adding the vitamin list to our routine diet, let's understand the importance of vitamins in life.

1. Vitamin A is referred to be a vitamin for growth and body repair. It is very vital in the formation of bone and tissues and also keeps your skin smooth. And if you are night blind, the cure is having more Vitamins A.

2. Vitamin B1 is an energy building vitamin which helps you to digest carbohydrates. It also keeps your heart and muscles stable.

3. Vitamin C is a very commonly pronounced vitamin worldwide. From kids to veterans, this vitamin is very essential as it protects your bones, teeth and gums. The ultimate medicine for curing scurvy and also resists any infection to grow in your body. Without its support collagen cannot be synthesized in the body.

4. Vitamin D is very important for children. The common disease seen in kids suffering from malnutrition is Rickets, which is actually caused by the deficit of Vitamin D. Bones cannot grow in a normal way if there is a lack of this vitamin. Direct sunlight is a natural source of vitamins apart from spinach and vegetables. In adults, Osteoporosis is caused due to lack of Vitamin D.

5. Vitamin E is a wound treating vitamin. It is very mush essential to prevent sterility and to break up blood clots. Damage of cells due to aging can be protected to supplement of this vitamin.

6. Vitamin B6 is necessary for production of antibodies

7. Vitamin B12 is required for carbohydrate and fat metabolism. This is a must for children's growth.

8. Vitamin B2 and Folic Acid help in the formation of red blood cells.

Vitamin Facts 1. A lot of the vitamins in fruits and vegetables are lost between the farm and your plate.

The longer the foods are stored before you eat them, the more nutrients are lost. Heat, light, and exposure to air all reduce the amount of vitamins, especially Vitamin C, thiamin, and folic acid.

2. About 25% of US households do not have balanced meals to meet the requirements that the body needs in digesting enough nutrients to sustain the body's health and fuel factors.

3. Research has shown that almost all varieties of disease can be produced by the deficiency of vitamins, minerals, amino acids, and other nutrients. Vitamins are vital for your skin. The most important factor of nutritional deficiencies is the intense processing and refining of foods like cereals and sugar.

4. The human body uses food to manufacture all its building blocks as well as to provide fuel. To do this, it performs several thousand different chemical reactions. Each reaction is controlled by "enzymes" and "coenzymes". Some of the coenzymes contain vitamins which the body cannot make by itself and which must be obtained from outside the body.

Sources of vitamins

Vitamins and minerals are essential for the maintenance of good health and the prevention of a number of diseases. In this article we look at the properties of vitamins A, B, C, D, E, K, and common food sources.

Types of vitamins.

There are two types of vitamins:

water-soluble vitamins B and C

Fat-soluble vitamins A, D, E and K.

Water-soluble vitamins cannot be stored in the body, so you need to get them from food every day. They can be destroyed by overcooking.

Vitamins and minerals are found in a wide variety of foods and a balanced diet should provide you with the quantities you need.

Vitamin A (retinol)

This vitamin is essential for growth and healthy skin and hair. It is a powerful antioxidant that plays a key role in the body's immune system. Vitamin A is found in the following animal products:

milk, butter, cheese and eggs

chicken, kidney, liver, liver pate

Fish oils, mackerel, trout, herring.

Another source of vitamin A is a substance called beta-carotene. This is converted by the body into vitamin A. It is found in orange, yellow and green vegetables and fruits.

Vitamin B Complex

The complex of B vitamins includes the following group of substances:

B1 - thiamine

B2 - riboflavin

B3 - nicotinic acid

B6 - pyridoxine

B12 - cobalamins

folate - folic acid.

The body requires relatively small amounts of vitamins B1, B2 and B3.

Vitamins B6 and B12 help the body to use folic acid and are vital nutrients in a range of activities such as cell repair, digestion, the production of energy and in the immune system. Vitamin B12 is also needed for the breakdown of fat and carbohydrate. Deficiency of either vitamin will result in anemia.

Vitamin B6 is found in most foods, so deficiency is rare.

The best dietary sources of the B vitamins, especially B12, are:

animal products (meat, poultry)

yeast extracts (brewers' yeast, Marmite).

Other good sources include:

asparagus, broccoli, spinach, bananas, potatoes

dried apricots, dates and figs

milk, eggs, cheese, yoghurt

nuts and pulses

fish

brown rice, wheat germ, wholegrain cereals

Dietary sources of vitamin B6 are similar to those for vitamin B12 and also include avocado, herring, salmon, sunflower seeds and walnuts.

Folic acid (folate)

Folic acid works closely in the body with vitamin B12. It is vital for the production of healthy blood cells.

Lack of folic acid is one of the main causes of anemia, particularly in people whose diet is generally poor. Vitamins B6 and B12 help the body use folate, so are often given alongside folic acid supplements.

In pregnancy, low folate levels increase the risk of the baby's spinal cord system not developing completely (spina bifida). All women are now advised to take folic acid supple8c7

Vitamin B-12 – Cyanocobalamin

Vitamin B-12 is a water soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood. It is one of the eight B vitamins. It is normally involved in the metabolism of every cell of the body, especially affecting DNA synthesis and regulation, but also fatty acid synthesis and energy production.

Vitamin B-12 is the name for a class of chemically-related compounds, all of which have vitamin activity. It is structurally the most complicated vitamin. Biosynthesis of the basic structure of the vitamin can only be accomplished by bacteria, but conversion between different forms of the vitamin can be accomplished in the human body. A common synthetic form of the vitamin, cyanocobalamin, does not occur in nature, but is used in many pharmaceuticals, supplements and as food additive, due to its stability and lower cost. In the body it is converted to the physiological forms, methylcobalamin and adenosylcobalamin, leaving behind the cyanide, albeit in minimal concentration. More recently, hydroxocobalamin, methylcobalamin and adenosylcobalamin can also be found in more expensive pharmacological products and food supplements. The utility of these is presently debated.

Historically, vitamin B-12 was discovered from its relationship to the disease pernicious anemia, which was eventually discovered to result from an effective lack of this vitamin due to problems

with the mechanisms in the body which normally absorb it. Many other subtler kinds of vitamin B12 deficiency, and their biochemical effects, have since been elucidated.

History of Vitamin B12

1824         The first case of pernicious anaemia and its possible relation to disorders of the digestive system is described by Combe.

 

1855 Combe and Addison identify clinical symptoms of pernicious anaemia.

 

1925 Whipple and Robscheit-Robbins discover the benefit of liver in the regeneration of blood in anaemic dogs.

 

1926 Minot and Murphy report that a diet of large quantities of raw liver to patients with pernicious anaemia restores the normal level of red blood cells. Liver concentrates are developed and studies on the presumed active principle(s) (“antipernicious anaemia factor”) are initiated.

 

1929 Castle postulates that two factors are involved in the control of pernicious anaemia: an “extrinisic factor” in food and an “intrinsic factor” in normal gastric secretion. Simultaneous administration of these factors causes red blood cell formation which alleviates pernicious anaemia.

 

1934 Whipple, Minot and Murphy are awarded the Nobel prize for medicine for their work in the treatment of pernicious anaemia.

 

1948 Rickes and associates (USA) and Smith and Parker (England), working separately, isolate a crystalline red pigment which they name vitamin B12.

 

1948 West shows that injections of vitamin B12 dramatically benefit patients with pernicious anaemia.

 

1949 Pierce and coworkers isolate two crystalline forms of vitamin B12 equally effective in combating pernicious anaemia. One form is found to contain cyanide (cyanocobalamin) while the other is not (hydroxocobalamin).

 

1955 Hodgkin and coworkers establish the molecular structure of cyanocobalamin and its coenzyme forms using X-ray crystallography.

 

1955 Eschenmoser and colleagues in Switzerland and Woodward and coworkers in the USA synthesise vitamin B12 from cultures of certain bacteria/fungi.

 

1973 Total chemical synthesis of vitamin B12 by Woodward and coworkers.

Terminology

The name vitamin B-12, known as vitamin B12 (commonly B12 or B-12 for short) generally refers to all forms of the vitamin. Some medical practitioners have suggested that its use be split into two different categories, however.

In a broad sense B-12 refers to a group of cobalt-containing vitamer compounds known as cobalamins: these include cyanocobalamin (an artifact formed as a result of the use of cyanide in the purification procedures), hydroxocobalamin (another medicinal form), and finally, the two naturally occurring cofactor forms of B-12: 5-deoxyadenosylcobalamin (adenosylcobalamin—AdoB-12), the cofactor of Methylmalonyl Coenzyme A mutase (MUT), and methylcobalamin (MeB-12), the cofactor of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR).

The term B-12 may be properly used to refer to cyanocobalamin, the principal B-12 form used for foods and in nutritional supplements. This ordinarily creates no problem, except perhaps in rare cases of eye nerve damage, where the body is only marginally able to use this form due to high cyanide levels in the blood due to cigarette smoking, and thus requires cessation of smoking, or else B-12 given in another form, for the optic symptoms to abate. However, tobacco amblyopia is a rare enough condition that debate continues about whether or not it represents a peculiar B-12 deficiency which is resistant to treatment with cyanocobalamin.

Finally, so-called Pseudo-B-12 refers to B-12-like substances which are found in certain organisms, including Spirulina (a cyanobacterium) and some algae. These substances are active in tests of B-12 activity by highly sensitive antibody-binding serum assay tests, which measure levels of B-12 and B-12-like compounds in blood. However, these substances do not have B-12 biological activity for humans, a fact which may pose a danger to vegans and others on limited diets who do not ingest B-12 producing bacteria, but who nevertheless may show normal "B-12" levels in the standard immunoassay which has become the normal medical method for testing for B-12 deficiency.

Structure

Structural Details for Vitamin B12

Vitamin B12 is the only known essential biomolecule with a stable metal-carbon bond, that is, it is an organometallic compound. The cobalt can link to:

1. a methyl group - as in methylcobalamin 2. a 5'-deoxyadenosine at the the 5' positon - as in

adenosylcobalamin (coenzyme B12

3. a cyanide group - as in Vitamin B12 - as supplied from drug companies

The particular link in the cobalamin has a profound effect upon the mechanism of the enyme reaction.

A methyl-nickel intermediate on acetyl-CoA synthase is also known, but only as an intermediate rather than a stable, isolable compound as the three cobalamins. Other organometals such as the methylmercury ion are highly toxic, it is interesting that there is an unfortunate connection between CH3Hg+ and methylcobalamin.

The core of the molecule is a corrin ring with various attached sidegroups. The ring consists of 4 pyrrole subunits, joined on opposite sides by a C-CH3 methylene link, on one side by a C-H methylene link, and with the two of the pyrroles joined directly. It is thus like a porphyrin, but with one of the bridging methylene groups removed. The nitrogen of each pyrolle is coordinated to the central cobalt atom. .

Links are to Chime pbd files to enable comparisons of the structures

The sixth ligand below the ring is a nitrogen of a 5,6-dimethylbenzimidazole. The other nitrogen of the 5,6-dimethylbenzimidazole is linked to a five-carbon sugar, which in turn connects to a phosphate group, and thence back onto the corrin ring via one of the seven amide groups attached to the periphery of the corrin ring. The base ligand thus forms a 'strap' back onto the corrin ring. An important aspect of the corrin ring, when compared to the porphyrin, is the relative flexibility of the corrin system, the corrin ring is also less flat when viewed from the side than is a porphyrin ring. This adds up to some considerable differences between the chemistry of a cobalt porphyrin and a cobalt corrin. In addition, the corrin only has a conjugated chain around part of the ring system, whereas a porphyrin is delocalised around the whole four pyrolle rings.

The center-piece in the structure is of course the cobalt(III), the octahedral coordination to five nitrogens and a carbon is common to all three cobalamins, and can be found in a number of simple coordination complexes. The simple complexes have attracted wide interest as models for cobalamins.

Cyanocobalamin crystals in polarized light

Synthesis

Vitamin B-12 cannot be made by plants or animals as only bacteria have the enzymes required for its synthesis. The total synthesis of B-12 was reported by Robert Burns Woodward and Albert Eschenmoser, and remains one of the classic feats of organic synthesis.

Species from the following genera are known to synthesize B-12: Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas. Industrial production of B-12 is through fermentation of selected microorganisms. The species most often used, Pseudomonas denitrificans and Propionibacterium shermanii, are frequently genetically engineered and grown under special conditions to enhance yield.

Functions

Vitamin B-12 is normally involved in the metabolism of every cell of the body, especially affecting the DNA synthesis and regulation but also fatty acid synthesis and energy production. However, many (though not all) of the effects of functions of B-12 can be replaced by sufficient quantities of folic acid (another B vitamin), since B-12 is used to regenerate folate in the body. Most "B-12 deficient symptoms" are actually folate deficient symptoms, since they include all the effects of pernicious anemia and megaloblastosis, which are due to poor synthesis of DNA when the body does not have a proper supply of folic acid for the production of thymine. When sufficient folic acid is available, all known B-12 related deficiency syndromes normalize, save those narrowly connected with the B-12 dependent enzymes Methylmalonyl Coenzyme A mutase (MUT), and 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), also known as methionine synthase; and the buildup of their respective substrates (methylmalonic acid, MMA) and homocysteine.

Coenzyme B-12's reactive C-Co bond participates in two types of enzyme-catalyzed reactions.

1. Rearrangements in which a hydrogen atom is directly transferred between two adjacent atoms with concomitant exchange of the second substituent, X, which may be a carbon atom with substituents, an oxygen atom of an alcohol, or an amine.

2. Methyl (-CH3) group transfers between two molecules.

In humans, only two corresponding coenzyme B-12-dependent enzymes are known:

1. Methylmalonyl Coenzyme A mutase (MUT) which uses the AdoB-12 form and reaction type 1 to catalyze a carbon skeleton rearrangement (the X group is -COSCoA). MUT's reaction converts MMl-CoA to Su-CoA, an important step in the extraction of energy from proteins and fats (for more see MUT's reaction mechanism). This functionality is lost in vitamin B-12 deficiency, and can be measured clinically as an increased methylmalonic acid (MMA) level. Unfortunately, an elevated MMA, though sensitive to B-12 deficiency, is probably overly sensitive, and not all who have it actually have B-12 deficiency. For example, MMA is elevated in 90-98% of patients with B-12 deficiency; however 25-20% of patients over the age of 70 have elevated levels of MMA, yet 25-33% of them do not have B-12 deficiency. For this reason, MMA is not routinely recommended in the elderly. The "gold standard" test for B-12 deficiency continues to be low blood levels of the vitamin. The MUT function cannot be affected by folate supplementation, which is necessary for myelin synthesis (see mechanism below) and certain other functions of the central nervous system. Other functions of B-12 related to DNA synthesis related to MTR dysfunction (see below) can often be corrected with supplementation with the vitamin folic acid, but not the elevated levels of homocysteine, which is normally converted to methionine by MTR.

2. 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), also known as methionine synthase. This is a methyl transfer enzyme, which uses the MeB-12 and reaction type 2 to catalyze the conversion of the amino acid Hcy back into Met (for more see MTR's reaction mechanism). This functionality is lost in vitamin B-12 deficiency, and can be measured clinically as an increased homocysteine level in vitro. Increased homocysteine can also be caused by a folic acid deficiency, since B-12 helps to regenerate the tetrahydrofolate (THF) active form of folic acid. Without B-12, folate is trapped as 5-methyl-folate, from which THF cannot be recovered unless a MTR process reacts the 5-methyl-folate with homocysteine to produce methionine and THF, thus decreasing the need for fresh sources of THF from the diet. THF may be produced in the conversion of homocysteine to methionine, or may be obtained in the diet. It is converted by a non-B-12-dependent process to 5,10-methylene-THF, which is involved in the synthesis of thymine. Reduced availability of 5,10-methylene-THF results in problems with DNA synthesis, and ultimately in ineffective production cells with rapid turnover, in particular blood cells, and also intestinal wall cells which are responsible for absorption. The failure of blood cell production results in the once-dreaded and fatal disease, pernicious anemia. All of the DNA synthetic effects, including the megaloblastic anemia of pernicious anemia, resolve if sufficient folate is present (since levels of 5,10-methylene-THF still remain adequate with enough dietary folate). Thus the best known function of B-12 (that which is indirectly involved with DNA synthesis and restoration of cell-division and anemia) is actually a facultative function which is mediated by B-12 conservation of active folate which can be used for DNA production.[13]

If folate is present in quantity, then of the two absolutely B-12 dependent reactions, the MUT reaction shows the most direct and characteristic secondary effects, focusing on the nervous system. Since the late 1990s folic acid has begun to be added to fortify flour in many countries, so that folate deficiency is now more rare. At the same time, since DNA synthetic-sensitive tests for anemia and erythrocyte size are routinely done in even simple medical test clinics (so that these folate mediated-biochemical effects are more often directly detected), the MTR dependent effects of B-12 deficiency are becoming apparent not as anemia (as they were classically), but now mainly as an elevation of homocysteine in the blood and urine (homocysteinuria). This condition may result in long term damage to arteries and in clotting (stroke and heart attack), but is difficult to separate from other processes associated with atherosclerosis and aging.

The B-12 dependent MTR reactions may have neurological effects through an indirect mechanism. Adequate methionine (which must otherwise be obtained in the diet) is needed to make S-adenosyl-methionine, which is in turn necessary for methylation of myelin sheath phospholipids. In addition, SAMe is involved in the manufacture of certain neurotransmitters, catecholamines and in brain metabolism. These neurotransmitters are important for maintaining mood, possibly explaining why depression is associated with B-12 deficiency. Methylation of the

myelin sheath phospholipids may also depend on adequate folate, which in turn is dependent on MTR recycling, unless ingested in relatively high amounts.

The specific myelin damage resulting from B-12 deficiency has also been connected to B-12 reactions related to MUT, which is needed to convert methylmalonyl coenzyme A into succinyl coenzyme A. Failure of this second reaction to occur results in elevated levels of methylmalonic acid (MMA), a myelin destabilizer. Excessive MMA will prevent normal fatty acid synthesis, or it will be incorporated into fatty acid itself rather than normal malonic acid. If this abnormal fatty acid subsequently is incorporated into myelin, the resulting myelin will be too fragile, and demyelination will occur. Although the precise mechanism(s) are not known with certainty, the result is subacute combined degeneration of central nervous system and spinal cord. Whatever the cause, it is known that B-12 deficiency causes neuropathies, even if folic acid is present in good supply, and therefore anemia is not present.

Human absorption and distribution

The human physiology of vitamin B-12 is complex, and therefore is prone to mishaps leading to vitamin B-12 deficiency. The vitamin as it occurs in foods enters the digestive tract bound to proteins, known as salivary R-binders. Stomach proteolysis of these proteins requires an acid pH, and also requires proper parietal cell release of proteolytic enzymes referred to as pepsin. (Even small amounts of B-12 taken in supplements bypasses these steps and thus any need for gastric acid, which may be blocked by antacid drugs).

The free B-12 then attaches to gastric intrinsic factor, which is generated by the gastric parietal cells in response to histamine, gastrin and pentagastrin, as well as the presence of food. The generation of this intrinsic factor-B12 complex will allow absorption of the vitamin as well as protect the vitamin from catabolism by intestinal bacteria. If this step fails due to gastric parietal cell atrophy (the problem in pernicious anemia), sufficient B-12 is not absorbed later on, unless administered orally in relatively massive doses (500 to 1000 mcg/day). Due to the complexity of B-12 absorption, geriatric patients, many of whom are hypoacidic due to reduced parietal cell function, have an increased risk of B-12 deficiency. The conjugated vitamin B-12-intrinsic factor complex (IF/B-12) is then normally absorbed by the terminal ileum of the small bowel. Absorption of food vitamin B-12 therefore requires an intact and functioning stomach, exocrine pancreas, intrinsic factor, and small bowel. Problems with any one of these organs makes a vitamin B-12 deficiency possible.

Once the IF/B-12 complex is recognized by specialized ileal receptors, it is transported into the portal circulation. The vitamin is then transferred to transcobalamin II (TC-II/B-12), which serves as the plasma transporter of the vitamin. Genetic deficiencies of this protein are known, also leading to functional B-12 deficiency.

For the vitamin to serve inside cells, the TC-II/B-12 complex must bind to a cell receptor, and be endocytosed. The transcobalamin-II is degraded within a lysozyme, and free B-12 is finally released into the cytoplasm, where it may be transformed into the proper coenzyme, by certain cellular enzymes (see above).

Hereditary defects in production of the transcobalamins and their receptors may produce functional deficiencies in B-12 and infantile megaloblastic anemia, and abnormal B-12 related biochemistry, even in some cases with normal blood B-12 levels. Individuals who lack intrinsic factor have a decreased ability to absorb B-12. This results in 80-100% excretion of oral doses in the feces versus 30-60% excretion in feces as seen in individuals with adequate intrinsic factor. The total amount of vitamin B-12 stored in body is about 2,000-5,000 mcg in adults. Around 80% of this is stored in the liver. Approximately 0.1% of this is lost per day by secretions into the gut as not all these secretions are reabsorbed. How fast B-12 levels change depends on the balance between how much B-12 is obtained from the diet, how much is secreted and how much is absorbed. B-12 deficiency may arise in a year if initial stores are low and genetic factors unfavourable or may not appear for decades. In infants, B-12 deficiency can appear much more quickly.

History of B-12 as a treatment for pernicious anemia

B-12 deficiency is the cause of pernicious anemia, a usually-fatal disease of unknown etiology when it was first described in medicine. The cure was discovered by accident. George Whipple had been inducing anemia in dogs by bleeding them, and then conducting experiments in which he fed them various foods to observe which diets allowed them fastest recovery from the anemia produced. In the process, he discovered that ingesting large amounts of liver seemed to most-rapidly cure the anemia of blood loss, and hypothesized that therefore liver ingestion be tried for pernicious anemia, an anemic disease of the time with no known cause or cure. He tried this and reported some signs of success in 1920. After a series of careful clinical studies George Minot and William Murphy set out to partly isolate the substance in liver which cured anemia in dogs, and found that it was iron. They found further that the partly isolated water-soluble liver-substance which cured pernicious anemia in humans was something else entirely different—and which had no effect at all on canines under the conditions used. The specific factor treatment for pernicious anemia, found in liver juice, had been found by this coincidence. These experiments were reported by Minot and Murphy in 1926, marking the date of the first real progress with this disease, though for several years, patients were still required to eat large amounts of raw liver or to drink considerable amounts of liver juice.

In 1928, the chemist Edwin Cohn prepared a liver extract that was 50 to 100 times more potent than the natural liver products. The extract was the first workable treatment for the disease. For their initial work in pointing the way to a working treatment, Whipple, Minot, and Murphy shared the 1934 Nobel Prize in Physiology or Medicine.

The active ingredient in liver was not isolated until 1948 by the chemists Karl A. Folkers of the United States and Alexander R. Todd of Great Britain. The substance was a cobalamin called vitamin B-12. It could also be injected directly into muscle, making it possible to treat pernicious anemia more easily. The chemical structure of the molecule was determined by Dorothy Crowfoot Hodgkin and her team in 1956, based on crystallographic data. Eventually, methods of producing the vitamin in large quantities from bacteria cultures were developed in the 1950s, and these led to the modern form of treatment for the disease.

Symptoms and damage from deficiency

Vitamin B-12 deficiency can potentially cause severe and irreversible damage, especially to the brain and nervous system. At levels only slightly lower than normal, a range of symptoms such as fatigue, depression, and poor memory may be experienced. However, these symptoms by themselves are too nonspecific to diagnose deficiency of the vitamin.

Vitamin B-12 deficiency can also cause symptoms of mania and psychosis.

Deficiencies

As the body stores vitamin B12, symptoms of deficiency can take up to four to five years of poor dietary intake or lack of intrinsic factor production to appear. Deficiency is more commonly linked to the inability to absorb the vitamin due to lack of intrinsic factor than to insufficient dietary intake.

Elderly people

Vitamin B12 deficiency is more common in the elderly than in younger people, with around 15 per cent of elderly men and women affected. This is usually because of decreased absorption due to reduced production of intrinsic factor or to a stomach disorder known as atrophic gastritis. Supplementation can prevent irreversible neurological damage if started early. Elderly people with vitamin B12 deficiency may show psychiatric or metabolic deficiency symptoms even before anemia is diagnosed. Screening for low vitamin B12 levels is necessary in elderly people with mental impairment, although it has also been found that deficiency states can still exist even when blood levels are higher than the traditional lower reference limit for vitamin B12. Patients who are most at risk of vitamin B12 deficiency include those with gastrointestinal disorders, autoimmune disorders, Type I diabetes mellitus and thyroid disorders, and those receiving long-term therapy with gastric acid inhibitors.

Blood

Vitamin B12 deficiency causes pernicious anemia with symptoms of tiredness, pallor, lightheadedness, breathlessness, headache and irritability. Red blood cells become abnormally enlarged and reduced blood platelet formation causes poor clotting and bruising. A high intake of

folic acid can prevent the red blood cell changes caused by vitamin B12 deficiency. It does not, however, prevent the nerve damage which may only become apparent in later stages and which may not be reversible. Strict vegetarians, whose folic acid intakes are high while their vitamin B12 intakes are low, may be at particular risk of nerve damage.

Immune system

Vitamin B12 deficiency leads to reduced numbers of white blood cells which causes increased susceptibility to infection. Recent research has shown that elderly patients with low vitamin B12 levels have impaired antibody response to bacterial vaccine, even when there are no clinical signs of deficiency.2

Brain and nervous system

Vitamin B12 deficiency eventually leads to a deterioration in mental functioning, to neurological damage and to a number of psychological disturbances including memory loss, disorientation, dementia, moodiness, confusion and delusions. Alzheimer's disease sufferers are often found to have low vitamin B12 levels, although it is unclear whether these are a contributing factor or a result of the disease.

Vitamin B12 deficiency leads to a loss of nerve-insulating myelin which begins at the peripheral nerves and eventually moves up to the spine causing decreased reflexes, abnormal gait, weakness, fatigue, poor vision and impaired touch or pain sensation. Other signs include tingling or loss of sensation and weakness in hands and feet, and diminished sensitivity to vibration and position sense.

Gastrointestinal system

Vitamin B12 deficiency causes poor cell formation in the digestive tract and leads to nausea, vomiting, loss of appetite, poor absorption of food, soreness of the mouth and tongue, and diarrhea.

Heart disease

Vitamin B12 deficiency may lead to increased levels of an amino acid called homocysteine, which has been linked to an increased risk of heart disease.

Other symptoms

Vitamin B12 is involved in production of the genetic material of the cell and deficiency may cause defective production which could lead to cancer. A 1997 Australian study found that low levels of vitamin B12 could contribute to chromosome damage in white blood cells.4 Low levels of Vitamin B12 may also contribute to diabetic neuropathy, poor vision, recurrent yeast infections and infertility. Vitamin B12 affects bone cells, and deficiency may be risk factor for osteoporosis.

Vitamin B-12 deficiency has the following pathomorphology and symptoms:

Pathomorphology includes: A spongiform state of neural tissue along with edema of fibers and deficiency of tissue. The myelin decays, along with axial fiber. In later phases, fibric sclerosis of nervous tissues occurs. Those changes apply to dorsal parts of the spinal cord, and to pyramidal tracts in lateral cords.

In the brain itself, changes are less severe: they occur as small sources of nervous fibers decay and accumulation of astrocytes, usually subcortically located, an also round hemorrhages with a torus of glial cells. Pathological changes can be noticed as well in the posterior roots of the cord and, to lesser extent, in peripheral nerves.

Clinical symptoms : The main syndrome of vitamin B-12 deficiency is Biermer's disease (pernicious anemia). It is characterized by a triad of symptoms:

1. Anemia with bone marrow promegaloblastosis (megaloblastic anemia)2. Gastrointestinal symptoms

3. Neurological symptoms

Each of those symptoms can occur either alone or along with others. The neurological complex, defined as myelosis funicularis, consists of the following symptoms:

1. Impaired perception of deep touch, pressure and vibration, abolishment of sense of touch, very annoying and persistent paresthesias.

2. Ataxia of dorsal cord type

3. Decrease or abolishment of deep muscle-tendon reflexes;

4. Pathological reflexes - Babinski, Rossolimo and others, also severe paresis.

During the course of disease, mental disorders can occur which include: irritability, focus/concentration problems, depressive state with suicidal tendencies, paraphrenia complex. These symptoms may not reverse after correction of hematological abnormalities, and the chance of complete reversal decreases with the length of time the neurological symptoms have been present.

Sources

Foods

Vitamin B-12 is naturally found in meat (especially liver and shellfish), milk and eggs. Animals, in turn, must obtain it directly or indirectly from bacteria, and these bacteria may inhabit a

section of the gut which is posterior to the section where B-12 is absorbed. Thus, herbivorous animals must either obtain B-12 from bacteria in their rumens, or (if fermenting plant material in the hindgut) by reingestion of cecotrope fæces. Eggs are often mentioned as a good B-12 source, but they also contain a factor that blocks absorption. Certain insects such as termites contain B-12 produced by their gut bacteria, in a manner analogous to ruminant animals. [25] An NIH Fact Sheet lists a variety of food sources of vitamin B-12.

Plants do not supply B-12 to humans. Vegan humans who eat only plant based foods must ordinarily take special care to supplement their diets accordingly. According to the U.K. Vegan Society, the only reliable vegan sources of B-12 are foods fortified with B-12 (including some soy products and some breakfast cereals), and B-12 supplements.[26]

While lacto-ovo vegetarians usually get enough B-12 through consuming dairy products, vitamin B-12 may be found to be lacking in those practicing vegan diets who do not use multivitamin supplements or eat B-12 fortified foods. Examples of fortified foods often consumed include fortified breakfast cereals, fortified soy-based products, and fortified energy bars. Claimed sources of B-12 that have been shown through direct studies of vegans to be inadequate or unreliable include, laver (a seaweed), barley grass, and human gut bacteria. People on a vegan raw food diet are also susceptible to B-12 deficiency if no supplementation is used.

Natural food sources of B-12

Vitamin B12 is found in foods that come from animals, including fish, meat, poultry, eggs, milk, and milk products. One half chicken breast, provides some .3 µg per serving or 6.0% of your daily value, (DV) 3 ounces of beef, 2.4 µg, or 40% of your DV, one slice of liver 47.9 µg or 780% of your DV, and 3 ounces of Molluscs 84.1 µg, or 1,400 % of your DV, while one egg

provides .6 µg or 10% of your DV. Vegans may have a challenge to ensure that they meet their daily allowance for B-12.

Supplements

Vitamin B-12 is provided as a supplement in many processed foods, and is also available in vitamin pill form, including multi-vitamins. Vitamin B-12 can be supplemented in healthy subjects also by liquid, strip, nasal spray, or injection and is available singly or in combination with other supplements.

Cyanocobalamin is converted to its active forms, first hydroxocobalamin and then methylcobalamin and adenosylcobalamin in the liver.

The sublingual route, in which B-12 is presumably or supposedly absorbed more directly under the tongue, has not proven to be necessary or helpful. A 2003 study found no significant difference in absorption for serum levels from oral vs. sublingual delivery of 500 µg (micrograms) of cobalamin.

Injection is sometimes used in cases where digestive absorption is impaired, but there is some evidence that this course of action may not be necessary with modern high potency oral supplements (such as 500 to 1000 µg or more). Even pernicious anemia can be treated entirely by the oral route. These supplements carry such large doses of the vitamin that the many different components of the B-12 absorption system are not required, and enough of the vitamin (only a few µg a day) is obtained simply by mass-action transport across the gut.

However, if the patient has inborn errors in the methyltransfer pathway (cobalamin C disease, combined methylmalonic aciduria and homocystinuria), treatment with intravenous or intramuscular hydroxocobalamin is needed.

Cyanocobalamin is also sometimes added to beverages including Diet Coke Plus and many energy drinks, (one example would be Chaser's Five Hour Energy Drink, which contains 8333% of the Recommended Daily Value of Vitamin B-12.) However, 500 µg would be needed to reverse biochemical signs of vitamin B-12 deficiency in older adults.

Recommendations

The Dietary Reference Intake for an adult ranges from 2 to 3 µg (micrograms) per day.

Vitamin B-12 is believed to be safe when used orally in amounts that do not exceed the recommended dietary allowance (RDA). The RDA for vitamin B-12 in pregnant women is 2.6 µg per day and 2.8 µg during lactation periods. There is insufficient reliable information available about the safety of consuming greater amounts of Vitamin B-12 during pregnancy.

The Vegan Society, the Vegetarian Resource Group, and the Physicians Committee for Responsible Medicine, among others, recommend that vegans either consistently eat foods fortified with B-12 or take a daily or weekly B-12 supplement. Fortified breakfast cereals are a particularly valuable source of vitamin B-12 for vegetarians and vegans.

Allergies

Vitamin B-12 supplements in theory should be avoided in people sensitive or allergic to cobalamin, cobalt, or any other product ingredients. However, direct allergy to a vitamin or nutrient is extremely rare, and if reported, other causes should be sought.

Side effects, contraindications, and warnings

Dermatologic: Itching, rash, transitory exanthema, and urticaria have been reported. Vitamin B-12 (20 micrograms/day) and pyridoxine (80mg/day) has been associated with cases of rosacea fulminans, characterized by intense erythema with nodules, papules, and pustules. Symptoms may persist for up to 4 months after the supplement is stopped, and may require treatment with systemic corticosteroids and topical therapy.

Gastrointestinal: Diarrhea has been reported.

Hematologic: Peripheral vascular thrombosis has been reported. Treatment of vitamin B-12 deficiency can unmask polycythemia vera, which is characterized by an increase in blood volume and the number of red blood cells. The correction of megaloblastic anemia with vitamin B-12 can result in fatal hypokalemia and gout in susceptible individuals, and it can obscure folate deficiency in megaloblastic anemia. Caution is warranted.

Leber's disease: Vitamin B-12 in the form of cyanocobalamin is contraindicated in early Leber's disease, which is hereditary optic nerve atrophy. Cyanocobalamin can cause severe and swift optic atrophy, but other forms of vitamin B-12 are available. [citation needed]

However, the sources of this statement are not clear, while an opposing view concludes: "The clinical picture of optic neuropathy associated with vitamin B-12 deficiency shows similarity to that of Leber's disease optic neuropathy. Both involve the nerve fibres of the papillomacular bundle. The present case reports suggest that optic neuropathy in patients carrying a primary LHON mtDNA mutation may be precipitated by vitamin B-12 deficiency. Therefore, known carriers should take care to have an adequate dietary intake of vitamin B-12 and malabsorption syndromes like those occurring in familial pernicious anaemia or after gastric surgery should be excluded."

Other medical uses

Hydroxycobalamin, or hydoxocobalamin, also known as Vitamin B-12a, is used in Europe both for vitamin B-12 deficiency and as a treatment for cyanide poisoning, sometimes with a large

amount (5-10 g) given intravenously, and sometimes in combination with sodium thiosulfate.[44]

The mechanism of action is straightforward: the hydroxycobalamin hydroxide ligand is displaced by the toxic cyanide ion, and the resulting harmless B-12 complex is excreted in urine. In the United States, the FDA approved (in 2006) the use of hydroxocobalamin for acute treatment of cyanide poisoning.

High vitamin B12 level in elderly individuals may protect against brain atrophy or shrinkage, associated with Alzheimer's disease and impaired cognitive function.

B12 synthesis is known to occur naturally in the human small intestine (in the ileum), which is the primary site of B12 absorption.  As long as gut bacteria have cobalt and certain other nutrients, they produce vitamin B12. Dr Michael Klaper argues that vitamin B12 is present in the mouth as well and intestines.  Furthermore, Dr Virginia Vetrano states that active Vitamin B12 coenzymes are found in bacteria in the mouth, around the teeth, in the nasopharynx, around the tonsils and in the tonsilar crypts, in  the folds at the base of the tongue, and in the upper bronchial tree. Absorption of the natural B12 coenzymes can take place in the mouth, throat, oesophagus, bronchial tubes and even in the upper small intestines, as well as all along the intestinal tract. This does not involve the complex enzyme mechanism for absorption (Intrinsic Factor) in the small intestine as required by cyanocobalamin. The coenzymes are absorbed by diffusion from mucous membranes.

About Fermentation

Introductory Information

Fermentation is connected in most people’s minds with wine making.  However, wine is just one of a multitude of products created by fermentation.  Cheeses, Yogurts, Miso, and sauerkraut are just a few of the foods created by fermentation.

Fermentation has recently been getting press again as a helpful way to make beans an even better source of nutrition. In the process of fermenting, large proteins and sugars are broken down by bacteria into smaller, more digestible forms. In addition, the bacteria produce other products such as vitamin B12 which are needed by the body.

Vitamin B12 is only produced by bacterial activity. A major source of B12 in the modern diet is from red meat, but it is again the activity of fermentation which produces it. A cow is a large fermentation vat which through thorough crushing and mixing of grasses with enzymes, produces an environment where bacteria break it down, providing nutrition to the cow. Fermentation does produce some B12 in the human digestive tract, but it is mostly produced later than when the body can absorb it.

Another benefit of fermented (and unpasteurized) foods is that they contain beneficial bacteria which are needed in our intestines. It is said that such 'living' foods can help to repopulate the intestine with proper bacterial strains. This helps with digestion and also provides a protective mechanism against other invasive bacteria and fungi which can cause serious problems when they get out of control.

There is a condition known as "antibiotic-associated colitis", which is most often seen in hospital situations where patients are undergoing long-term, wide spectrum antibiotic treatment. This treatment kills a large portion of the beneficial bacteria, leaving an open door for other bacteria (usually Clostridium difficale) to take over, causing pain, diarrhea, dehydration and possibly death. This bacteria is a normal occupant of the digestive tract, but is kept in check by the presence of other bacterial populations.

Vitamin B12 is an important vitamin for humans and animals. It is used to treat pernicious anaemia and peripheral neuritis, and is used as a dietary supplement. Vitamin B12 is also an important animal feed supplement as growth enhancer.

The term vitamin B12 is used to describe compounds of the cobalt corrinoid family, in particular those of the cobalamin group. The most used compound of this group is cyanocobalamin and as such the term vitamin B12 is sometimes used to refer to cyanocobalamin. In this specification the term vitamin B12 should be attributed its broad meaning so as to include all the cobalt corrinoids of the cobalamin group, which include in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and 5'desoxyadenosylcobalamin, characterised by a cyano, hydroxyl, methyl or 5'-desoxyadenosyl radical respectively. The methylcobalamin and 5'desoxyadenosylcobalamin compounds are known to be unstable to light in isolated form and are easily transformed to hydroxocobalamin in aqueous solution.

For this reason, almost all commercial vitamin B12 preparations consist of the stable cyanocobalamin, which as such is not the chemical form in which vitamin B12 can be found in nature. In this specification the term natural vitamin B12 is defined so as to comprise all chemical forms of vitamin B12 naturally occurring in nature, cyanocobalamin thus being excluded.

Vitamin B12 is produced industrially by microbial fermentation, using almost exclusively Pseudomonas denitrificans and Propionibacterium species. Contrary to Pseudomonas, Propionibacteria are food-grade. Processes using Propionibacterium species thus have the advantage that they allow to formulate natural vitamin B12 together with the biomass in which it is produced

Such processes avoid the conversion of natural vitamin B12 into the cyanocobalamin form by chemical processes including cyanidisation followed by extraction and purification steps using organic solvents. The chemical conversion step and any subsequent purification steps cause this production process to be expensive, unsafe to the operators and environmentally unfriendly.

Propionibacteria are Gram-positive bacteria capable of producing valuable compounds in a variety of industrial processes. Propionibacteria are, for instance, important in the production of

cheese, propionic acid, flavours and vitamin B12.

Propionibacteria are, as the name suggested, characteristic in the production of propionic acid. Glucose is commonly used as carbon source, but other substrates, i.e. fructose, mannose, galactose, glycerol and milk, can be used for growth. Besides propionic acid, acetic acid is produced under anaerobic conditions, with a ratio of 2:1 for propionic acid : acetic acid. The production of propionic acid is a clear advantage over other species as this compound is toxic in low levels for many other organisms, like lactic acid bacteria, acetic acid bacteria and yeasts. As a result there is little chance of contamination with other microorganisms during fermentation. The upper tolerance level for propionic acid for Propionibacterium is approximately 20-40 g/l (with fermentation around pH 7): this is the level where the propionic acid starts to inhibit growth. The undissociated propionic acid is the actual toxic component for Propionibacteria, as is shown by Nanba et al. for Propionibacterium shermanii. The specific growth rate decreases rapidly for undissociated propionic acid concentrations above 5 mM. This effect is also demonstrated by Blanc et al. for P. acidi-propionici, where the growth rate is drastically reduced above a pseudo critical value of 4 mM propionic acid. This implies that in fermentations with a pH around 7.0 propionic acid concentrations above 40 g/l are only reached with very low growth rates. This maximum amount of propionic acid produced in such fermentations results in a maximum of 25-35 g/l biomass that can be reached. Propionic acid concentration is thus one limiting factor for biomass growth and thereby for high vitamin B12 yield.

Several Propionibacterium species are capable to produce vitamin B12 in large scale fermentation processes. The process is described as a two-stage fermentation with a 72-88 hours anaerobic fermentation followed by a 72-88 hours aerobic phase. The vitamin B12 concentration in the cells rapidly increases in the aerobic phase, with typical values of 25-40 mg vitamin B12/l (see e.g. DE 1 239 694, U.S. Pat. No. 3,411,991, or in: Biochemical engineering and biotechnology handbook, 1991, B. Atkinson ed., Macmillan Publishers Ltd, pp: 1213-1220). Anaerobic growth followed by an aerobic phase with limited growth is important for economic production of vitamin B12 using Propionibacterium species. This requirement, however, limits the amount of biomass to 25-35 g/l as described above. Several attempts have been made to overcome the barrier of propionic acid toxicity in order to increase biomass and thereby the yield of vitamin B12.

Alternated anaerobic-aerobic phases are e.g. suggested to reduce the amount of acids (Ye et al., 1996, J. Ferment. Bioeng. 85: 484-491). In the aerobic phase the propionic acid is converted to less toxic acetic acid, with simultaneous formation of vitamin B12. The relative yield of vitamin B12 has been increased, but the final titre is rather low. This is probably due to inhibition early in the synthesis of vitamin B12 and/or other oxygen related products limiting the synthesis of vitamin B12. The final vitamin B12 produced with this method is 9 mg/l compared to 4.5 mg/l with the fully separated anaerobic and aerobic phases. Both values are rather low for vitamin B12 production with Propionibacteria.

The suggestion to use immobilized cells is mainly focused on the production of propionic acid (Rickert et al., 1998, Enzyme Microb. Technol. 22: 409-414). The propionic acid production is greatly enhanced. Use of this option for vitamin B12 production (which is not mentioned by Rickert et al.) will imply harvesting of the vitamin B12-containing cells with the immobilization material. This is only feasible when the additional cost for the immobilization equipment as well as the immobilization material itself are competitive with the current technology. Yongsmith et al. presented the production of vitamin B12 with immobilised cells of Propionibacterium sp. strain arl AKU1251. The maximum vitamin B12 concentrations is in the range of 14-16 mg/kg, which is no improvement of the production with freely suspended cells, as described before (Yongsmith et al. 1983, J. Ferment. Technol. 61: 593-598).

Although Propionibacteria can grow under aerobic conditions, the production of corrinoids (i.e. the general name for vitamin B12 and its precursors) is absent above a dissolved oxygen concentration of 0.19 mM=6 mg O2 /L. The lower the oxygen concentration the higher the corrinoid production is with a maximum corrinoid production under non-aerated conditions (Quesada-Chanto et al., 1998, Appl. Microbiol. Biotechnol. 49: 732-736). Oxygen concentration is a limiting factor for vitamin B12 synthesis.

Repeated fed-batch fermentation with an anaerobic phase followed by an aerobic phase and withdrawal of broth at the end of the aerobic phase is not possible. According to Quesada-Chanto et al. (1998), production of the corrinoids is optimal under anaerobic conditions, whereas small amounts of oxygen reduce the production of corrinoids. These findings are supported by the results obtained in example 1. A repeated fed-batch process with an aerobic and anaerobic phase in one fermenter therefore is, not economically feasible.

GB patent 846,149 describes a continuous process for the synthesis of vitamin B12. This process comprises fermenting Propionibacterium in a nutrient medium under anaerobic conditions in a first zone while adding nutrients to this zone, passing cell-containing medium from the first zone into a second zone which is under microaerobic conditions and withdrawing cell-containing medium containing vitamin B12 from this second zone. The concentration of cells and the volume of medium in both zones is maintained substantially constant by continuous fill and draw operations. This process leads to the synthesis of up to 12 mg/l of vitamin B12, which is no improvement compared with a more classical method of production.

There is thus still a need for Propionibacterium-based fermentation processes for the production of vitamin B12 with further improvements in efficiency and/or vitamin B12 yield.

A process for producing vitamin B12 (and precursors thereof having detectable vitamin B12 activity) which is not a continuous process and which comprises the steps of:

(a) culturing a strain of the genus Propionibacterium in a first fermenter under anaerobic conditions to obtain a culture of Propionibacterium,

(b) transferring at least part of the culture obtained in (a) to a second fermenter and subjecting this culture to oxygen,

(c) replacing in the first fermenter part of the volume transferred in (b) with fresh culture medium, and

(d) repeating steps (a), (b), and (c) at least once.

In this specification the term vitamin B12 should be attributed its broad meaning so as to include all the cobalt corrinoids of the cobalamin group, which include in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and 5'desoxyadenosylcobalamin, characterised by a cyano, hydroxyl, methyl or 5'-desoxyadenosyl radical respectively. The term vitamin B12 further comprises any vitamin B12 precursor having vitamin B12 activity as detectable in the turbidimetric bioassay based on the growth response of Lactobacillus leichmanii ATCC 7830 as described in detail in: the United States Pharmacopoeia, The National Formulary, 1995, pp. 1719-1721, United States Pharmacopoeial Convention, Inc., Rockville, Md.

One aspect of the process of the invention concerns the relation between the fraction of the anaerobic culture which is transferred in (b) and replenished with fresh medium in (c) and, on the one hand, the growth rate of the anaerobic culture (in (a)) and, on the other hand, the time interval between the subsequent withdrawals. Preferably, the withdrawal volume (which is transferred in (b) and replaced in (c)) relates to the total working volume of the first fermenter as a function of growth rate of the culture in the first fermenter and time interval between each draw according to the equation: ##EQU1##

In this equation the growth rate is expressed in h-1. Typically, the growth rate may vary over the applied time interval and an average value may be applied in the formula. The time interval between each draw is expressed in hours. Preferably, the growth rate during the anaerobic phase is maintained in the range between 0.03 and 1 h-1. The skilled person will appreciate that at constant growth rate, decreasing both the time interval and the withdrawal volume will approach a continuous process, which is not a preferred embodiment of the present invention.

One embodiment of the process of the present invention comprises the steps of:

(a) culturing a strain of the genus Propionibacterium in a first fermenter under anaerobic conditions,

(b) transferring at least 30 to 90%, preferably at least 40 to 90%, more preferably at least 50 to

90%,more preferably at least 60 to 90% and most preferably at least 70 to 90% of the culture volume obtained in (a) to a second fermenter and subjecting this culture to oxygen,

(c) replacing in the first fermenter the same volume as the one withdrawn in step (b) with fresh culture medium; and

(d) repeating steps (a), (b), and (c) at least once.

In another preferred embodiment of the process of the invention, the culture of a strain of the genus Propionibacterium under anaerobic conditions (step (a)) leads to at least 20 g/l dry biomass, preferably at least 30 g/l, preferably at least 40 g/l, preferably at least 50 g/l, preferably at least 60 g/l, preferably at least 70 g/l, preferably at least 80 g/l, more preferably at least 90 g/l and most preferably at least 100 g/l.

Knowing the withdrawal volume and the growth rate of the microorganism used, the skilled person can easily deduce from the formula the time interval between each draw and thus the duration of the whole process.

In another preferred embodiment of the process of the invention, the dissolved Oxygen concentration at inoculation of the anaerobic phase is less than 5% of air saturation, preferably less than 2.5%, and more preferably less than 1% of air saturation.

Preferably, the anaerobic conditions of the process of the invention are such that the oxygen uptake rate during the anaerobic phase (a) is no more than 2 mmol O2 l-1 h-1, preferably no more than 1 mmol O2 l-1 h-1, and most preferably approaches zero mmol O2 l-1 h-1, as measured by mass-spectometry and gas flow analysis (see e.g. in: Basic Bioreactor Design, 1991, K. van't Riet & J. Tramper, eds., Marcel Dekker Inc.).

In step (b) of the process of the invention, the culture is subjected to oxygen. Preferably, the oxygen uptake rate (OUR) during this aerobic phase of the process is at least 5 mmol O2 l-1 h-1, more preferably at least 20 mmol O2 l-1 h-1, still more preferably at least 40 mmol O2 l-1 h-1, and most preferably at least 80 mmol O2 l-1 h-1.

According to one embodiment of the invention there is provided a process wherein the aerobic second phase in (b) is performed in at least two serially connected aerobic fermenters. Most preferably the aerobic second phase in (b) is performed in plug flow mode, wherein e.g. the "second" aerobic fermenter comprises a series of aerobic fermenters.

Suitable culture media for the production of vitamin B12 with Propionibacteria are well-known in the art (cf. Biochemical engineering and biotechnology handbook, 1991, B. Atkinson ed., Macmillan Publishers Ltd, pp: 1213-1220). In a preferred embodiment of the invention the

culture medium is supplemented with 1-50 mM of one or more compounds selected from the group consisting of betaine, methionine and glutamine. Another preferred supplement for the culture medium is 5,6-dimethylbenzimidazole (DBI). DBI is preferably supplemented at 1-40 mg DBI per litre. Preferably, DBI is added to the culture medium at the start of the aerobic phase or during that phase.

In a preferred embodiment of the process of the invention the concentration of undissociated propionic acid is controlled such that it does not exceed 5 mM. This conveniently may be done by increasing the pH of the culture medium according to methods well known to the skilled person.

According to another embodiment of the invention, the temperature under anaerobic conditions is different from the temperature under aerobic conditions. Preferably, the temperature under anaerobic condition is higher than the temperature under aerobic conditions. More preferably, the temperature under anaerobic conditions is at least 2 degrees higher than the temperature under aerobic conditions, more preferably at least 4 degrees higher, more preferably at least 6 degrees higher, more preferably at least 8 degrees higher, more preferably at least 10 degrees higher and most preferably at least 12 degrees higher. For instance, the temperature under anaerobic conditions may be 36° C. and under aerobic conditions 24° C., or these temperatures may be 36° C. and 30° C., respectively.

In the process of the invention, preferably a strain of a Propionibacterium species is used which is selected from the group consisting of the classical or Dairy Propionibacteria as described in Bergey's manual of systematic bacteriology, 1986, J. B. Butler, Williams & Wilkins, p 1346-1353. This group comprises inter alia the species P. freundenreichii with subspecies freudenreichii and shermanii, P. thoenii, P. jensenii and P. acidipropionici. More preferably the strain P. freundenreichii CBS 929.97 is used. P. freundenreichii CBS 929.97 was deposited Jul. 10, 1997 at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands.

In one embodiment of the invention, the process of the invention is performed using Propionibacteria strains that are genetically modified by means of classical mutagenesis and/or recombinant DNA technology. Classically mutagenised strains can be propionic acid-resistant strains of the genus Propionibacterium, such as P. shermanii NOC 11012 and P. freudenreichii NOC 11013, as disclosed in U.S. Pat. No. 4,544,633. A propionic acid-resistant strain of Propionibacterium is herein defined as a strain which shows approximately equal (i.e. less than 10% difference) growth rates when compared in identical media with and without 20 g propionic acid/l.Transformed Propionibacteria strains that have been genetically modified by recombinant DNA technology are exemplified in J 08-056673.

One of the advantages of the process of the invention is the throughput increase of the

fermentation. The anaerobic phase is considerably reduced. For a growth rate of 0.06 h-1 the output of the anaerobic phase is three fermenter volumes in 72 hours, compared to one fermenter volume for the classical process. In addition, the invention reduces the turnaround time of the fermentation, with ten consecutive fills and draws, to 20% of the classical process.

Another advantage of the process of the invention is that the size of the fill and draw volumes may be relatively large. Consequently, the growth inhibiton caused by high concentration of propionic acid under anaerobic conditions does not occur, leading to an increase in the biomass and thereby an increase in vitamin B12 yield. The amount of vitamin B12 formed using the process of the invention may be 20 to as high as 200 mg/kg mesh.

 Twelve Points on Vitamin B12:

(1) Vitamin B12 is an essential nutrient. The chemical names for the form of B12 found most commonly in foods is hydroxocobalamin. In the body conversion occurs to the active forms adenosylcobalamin, and methylcobalamin. The usual synthetic form of the vitamin is cyanocobalamin which occurs only in small amounts naturally. The Recommended Daily Allowance (USA) is 2.4 micrograms per day. This increases by 0.2 micrograms during pregnancy and 0.4 micrograms during lactation. An article in the American Journal of Clinical Nutrition states that mild deficiency symptoms can occur in intakes of less than 6 micrograms per day. In foods vitamin B12 is at its highest in liver at around 100 and kidney at 55 micrograms per 100g. The oily fish contain 4-12; white fish 0-2; red meats 1-3 and white meats 0-2 micrograms per 100g. Eggs contain 2.5 and cheese 1-1.5 micrograms per 100g. Animal fats such as butter contain only trace levels.

(2) If taken orally as a constituent of foods, it is combined with first one factor called R-protein and later with intrinsic factor, both of which are produced in the stomach. This avoids its degradation by bacteria or its digestion so that it can it be absorbed in the small intestine. Passive (unbound) absorption accounts for only 1-3% of the total absorption.

(3) Vitamin B12 is a coenzyme , that is, it is required by at least 2 enzymes for their functionality. The first breaks down homocysteine to methionine. The other breaks down methylmalonyl-CoA to succinyl-CoA; if this does not occur then the methylmalonyl-CoA is converted to methylmalonic acid (MMA). Serum or urine levels of MMA are the most reliable test for B12 deficiency in the general tissues of the body.

(4) Vitamin B12 is an important nutrient in nervous tissue, helping to build myelin sheaths around nerves by carrying methyl groups to the nerve tissue. It is a potent brain detoxifier and the natural protective agent against the negative effects of the neurotransmitter glutamate which otherwise leaves nerves in a hyperactive state. The only form of the vitamin used in the nervous system is methylcobalamin. The Blood Brain Barrier (BBB) is a highly selective membrane protecting the delicate nerve tissues of the brain from mineral ions and other substances which would disturb its function. B12 does not easily cross the BBB. It is thus possible to have normal B12 levels in the body in general but a deficiency in nervous tissue. A test of homocysteine

levels in the cerebrospinal fluid is a reliable test for nervous system deficiency, whereas the MMA urine test is not.

(5) Around 5mg of vitamin B12 is stored in the liver and in the absence of intake, stores can last between 5 months and 30 years (due to recycling of the vitamin) before deficiency symptoms become apparent.

Early deficiency symptoms include unusual fatigue, faulty digestion, no appetite, nausea, or loss of menstruation.

Later symptoms include any of the following:

numbness and tingling of the hands and feet,nerve pains,nervousness,depression (from mild to psychotic),striking behavioural changes,paranoia,abnormal or hyperactive reflexes,abnormal coordination,impaired short term memory,confusion,impaired gait,spasticity,impaired vision,incontinence and frequent need to urinate,weakness and loss of muscle strength,inadequate melatonin metabolism leading to poorer sleep and daytime wakefulness,diarrhoea,fever,frequent upper respiratory infections,impotence,infertility,enlargement of the mucous membranes of the mouth, tongue, vagina, and stomach,macrocytic (pernicious) anemia,low platelet count and increased bleeding,neutropenia (low neutrophils in the blood - part of the immune system)impaired T-lymphocyte activity (part of the immune system) Not all symptoms occur in all cases of deficiency. Additionally, since deficiency of B12 leads to elevated homocysteine levels, it contributes to its resulting varied pathology including arterial disease.

(6) Vitamin B12 is protein-bound in foods and requires good protein digestion to be utilised. People with heavy yeast infection in the intestines have an impairment of intrinsic factor production as well as faulty digestion, and many of the mental symptoms within chronic fatigue syndrome may be related to B12. Also the symptoms of the terminal stage of AIDS are identical

to those of severe B12 deficiency. B12 absorption also requires normal levels of absorption in the small intestine, with relevance in for example, Crohn's Disease. Additionally hydroxocobalamin (or the synthetic cyanocobalamin) undergoes conversion to active forms in the liver and this conversion mechanism can be inadequate where liver function is impaired there are certain other nutrient deficiencies.

(7) Vitamin B12 is contained within a group of chemicals called corrinoids which occur in foods. The corrinoids close to B12 are termed B12 analogues and may be active or inactive in the body. Some inactive B12 analogues can be active in bone marrow but not in nervous tissue, thus making a diagnosis of deficiency by the presenting symptoms difficult. Current chemical analysis is not able to assess whether the analogue is active or inactive. This can only be verified by feeding the food to a human and testing the resulting MMA level. Inactive analogues interfere with active forms, quickening the onset of symptoms in marginal cases.

(8) As of 2004 no plant sources of vitamin B12 had been found, though many had been tested including various seaweeds, algae and fermented foods. Where claims have been made as to B12 being present in a plant source, it has not been based on the test for MMA levels, and any subsequent tests have found no reduction in MMA, proving the presence only of inactive analogues.

(9) Vitamin B12 is produced in the human intestine by some of the bacteria naturally present. A study in the 1950's verified that vegans with pernicious anaemia could be returned to normality by isolating the B12 analogues from their faeces and feeding them back orally, where it could be combined with intrinsic factor. This proved that the forms produced in the intestines contain active B12 analogues, but that insufficient is absorbed passively to prevent deficiency. A group of Finnish people living solely on fermented vegetable foods believed that vitamin B12 was produced by the fermentation processes. When their MMA levels were checked over a certain period they were found to be increasing, proving that normal methods of fermenting foods does not result in B12 production, but only its inactive analogues. It is assumed that passive absorption from B12 produced by gut bacteria in herbivorous animals is adequate due to the increased length of, and increased fermentation time in, their intestines. Additionally many “herbivores” eat insects and faeces.

(10) Vitamin B12 can be destroyed by cooking for a long enough time. There is thus an advantage to eating some raw animal protein. Raw food vegans, however, have not been shown to have any advantage over other vegetarian groups.

(11) Several foods are fortified with a synthetic form of the vitamin, including most yeast extracts and many breakfast cereals. This is how many vegans are maintaining their levels of B12 . This method of obtaining B12 is highly risky since levels cannot be guaranteed in each of these foods. The only sure way for a vegan to maintain levels of the vitamin is to take a synthetic form as a supplement.

(12) Inorganic mercury is known to accumulate on the BBB. It is thought that it oxidises the cobalt atom in methylcobalamin, making it far less able to cross the barrier. Inorganic mercury is thus a creator of deficiency of B12 in the nervous system and symptoms usually attributed to

mercury toxicity in the brain and nerves are due to its effect on B12. Inorganic mercury exposure is most commonly from mercury amalgam dental fillings and vaccines. Psychiatrist John Dommisse in the USA claims to have reversed 100% of his pre-Alzhemiers patients with the use of methylcobalamin as well as many cases of depression. Methylcobalamin is highly recommended by the Swedish Association of Dental Mercury Patients. It has been used successfully in the treatment of Chronic Fatigue Syndrome and Fibromyalgia - more than 60% of these patients have tested for low levels in the cerebrospinal fluid. It has been used successfully as part of treatments in Autism, Schizophrenia and Multiple Sclerosis and has great potential in Parkinson's Disease and Muscular Dystrophy as well as other neurological or psychological disorders. In such cases intramuscular injections or sublingual liquids or lozenges of methylcobalamin are maximally effective in combating deficiency symptoms since they by-pass the complex absorption process in the intestines. There is no evidence for toxicity of methylcobalamin. Up to 40mg per day has been used therapeutically in some cases. Large doses have been used since it must overwhelm the effect of any mercury on the BBB.

Fermentation:

Aerated condition with agitation bioreactor isused for growth of. Pseudomonas and biosynthesis of vitaminB12Flow chart for production of vitamin B12 from Pseudomonasdenitrificans:

Pseudomonas denitrificans

Inoculum cultivation

Preculture

Production culture

Types of Fermentation Process - 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 enzymatic reactions instead of chemical reactions aided by inanimate catalysts, sometimes operating at elevated temperature and pressure.

FERMENT OR

Batch Fermentation Process

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 inoculum of a pure culture is added to the fermenter, from a separate pure culture vessel.

Continuous Fermentation Process - 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 I in the rate of growth until the stationary phase is reached. This is because of limitation of one or more of the essential nutrients.

Aerobic Fermentation Process - A number of industrial processes, although called 'fermentations', are 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.

Anaerobic Fermentation Process - Basically a fermenter designed to operate under micro acrophilic 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

many anaerobic fermentations do, however, require mild aeration for the initial growth phase, and sufficient agitation for mixing and maintenance of temperature.

Surface Culture Method - In this method the organism is allowed to grow on the surface of a liquid medium without agitation. After an appropriate incubation period the culture filtrate is separated from the cell mass and is processed to recover the desirable product.

Sometimes the biomass may be reused. Examples of such fermentations are the alcohol production, the beer production and citric acid production. This method is generally time consuming and needs large, area or space.

Submerged Culture Method - In this process, the organism is grown in a liquid medium which is vigorously aerated and agitated in large tanks called fermentors. The fermentor could be either an open tank or a closed tank and may be a batch type or a continuous type and are generally made of non-corrosive type of metal or glass lined or of wood. In batch fermentation, the organism is grown in a known amount of culture medium for a defined period of time and then the cell mass is separated from the liquid before further processing while in the continuous culture, the culture medium is withdrawn depending on the rate of product formation and the inflow of fresh medium. Most fermentation industries today use the submerged process for the production of microbial products.

Semi Solid OR Solid State Methods - In this the culture medium is impregnated in a carrier such as bagasse, wheat bran, potato pulp, etc. and the organism is allowed to grow on this. This method allows greater surface area for growth. The production of the desirable substance and the recovery is generally easier and satisfactory.

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.

Continuous methane fermentation and the production of vitamin B12 in a fixed-bed reactor:

A fixed-bed reactor with acclimated methanogens immobilized on a loofah support was studied on a laboratory scale to evaluate the system producing methane from the mixture of CO(2) and H(2) gas, with the production of vitamin B(12) as a by-product. Fermentation using CO(2)/H(2) acclimated methanogens was conducted in a jar fermentor with hydraulic retention times (HRTs) of three and six days. The performance of the reactor was mainly dependent on the HRT. With an HRT of three days, the methane production rate and the vitamin B(12) concentration in the culture broth were 6.18 l/l-reactor/h and 2.88 mg/l-culture liquid; these values were 11.96 l/l-reactor/h and 37.54 mg/l-culture liquid for an HRT of six days. A higher total cell mass of methanogens retained 42.5 g dry cell/l-culture liquid was achieved in the HRT of six days. The loofah carrier immobilized almost 95% of the methanogens, which led to a more effective bio-reaction. It was also observed that the fermentation system had a better ability to buffer pH, especially for an HRT of six days.

Process for the production of fermentation broth with increased vitamin B12 content by synchronizing the bacterium population:A process for the production of a fermentation broth with increased vitamin B12 content by a fermentation process carried out with a methane-producing mixed bacterium population under anaerobic septic conditions in the presence of known nutrient components and precursors.

According to the invention, methanol, preferably in an amount of 0.5 v/v %, is added for some days, preferably for 3 days to the fermentation broth containing vitamin B12 used as the starting substance. Thereafter a minor part, preferably 10% of the fermentation broth, is removed and an equal volume of a nutrient broth containing the usual components in tenfold concentrations is added periodically in every 5th to 12th day, then the removal of fermentation broth is interrupted for 0 to 2 days, preferably for 1 day, and only 0.4 to 1.5 v/v% of methanol are added to the fermentation broth, and subsequently a minor part, preferably 10% of the fermentation broth, is removed daily and an equal volume of a nutrient broth containing the usual components in the usual concentrations is added together with 0.4 to 1.5 v/v % of methanol, depending on the biogas production of the fermentation broth. This series of operations is repeated periodically during the complete fermentation procedure taking into consideration the pH of the fermentation broth.

Influence of cobalt concentration on vitamin B12 production and fermentation of mixed ruminal microorganisms grown in continuous culture flow-through fermentors:

An experiment was conducted to determine the effects of dietary concentrations of Co on vitamin B12 production and fermentation of mixed ruminal microbes grown in continuous culture fermentors. Four fermentors were fed 14 g of DM/d. The DM consisted of a corn and cottonseed hull-based diet with Co supplemented as CoCO3. Dietary treatments were 1) control (containing 0.05 mg of Co/kg of DM), 2) 0.05 mg of supplemental Co/kg of DM, 3) 0.10 mg of supplemental Co/kg of DM, and 4) 1.0 mg of supplemental Co/kg of DM. After a 3-d adjustment period, fermentors were sampled over a 3-d sampling period. This process was repeated 2 additional times for a total of 3 runs. Ruminal fluid vitamin B12 concentrations were affected by Co supplementation (P < 0.01), and there was a treatment x day interaction (P < 0.01). By sampling d 3, cultures fed the basal diet supplemented with 0.10 mg of Co/kg had greater (P < 0.05) vitamin B12 concentrations than those supplemented with 0.05 mg of Co/kg of DM, and increasing supplemental Co from 0.10 to 1.0 mg/kg of DM increased (P < 0.01) ruminal fluid vitamin B12 concentration. Ruminal fluid succinate also was affected (P < 0.10) by a treatment x day interaction. Cobalt supplementation to the control diet greatly decreased (P < 0.05) succinate in ruminal cultures on sampling d 3 but not on d 1 or 2. Molar proportions of acetate, propionate,

and isobutyrate, and acetate:propionate were not affected by the addition of supplemental Co to the basal diet. However, molar proportions of butyrate, valerate, and isovalerate increased (P < 0.05) in response to supplemental Co. The majority of long-chain fatty acids observed in this study were not affected by Co supplementation. However, percentages of C18:0 fatty acids in ruminal cultures tended (P < 0.10) to be greater for Co-supplemented diets relative to the control. Methane, ammonia, and pH were not greatly affected by Co supplementation. The results indicate that a total (diet plus supplemental) Co concentration of 0.10 to 0.15 mg/kg of dietary DM resulted in adequate vitamin B12

production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermentors.

Production of vitamin B-12 in tempeh, a fermented soybean food:

Several varieties of soybeans contained generally less than 1 ng of vitamin B-12 per g. It was found that use of a lactic fermentation typical of tropical conditions during the initial soaking of the soybeans did not influence the vitamin B-12 content of the resulting tempeh. Pure tempeh

molds obtained from different sources did not produce vitamin B-12. It was found that the major source of vitamin B-12 in commercial tempeh purchased in Toronto, Canada, was a bacterium that accompanies the mold during fermentation. Reinoculation of the pure bacterium onto dehulled, hydrated, and sterilized soybeans resulted in the production of 148 ng of vitamin B-12 per g. The presence of the mold, along with the bacterium, did not inhibit or enhance production of vitamin B-12. Nutritionally significant amounts of vitamin B-12 were also found in the Indonesian fermented food, onto.

Microbial production of vitamin B12 :

One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates. The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition. In humans, the vitamin is required in trace amounts (approximately 1 microg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species. The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described. Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined.

Vitamin B12 production in fermented milk products:

Disclosed is a fermented milk product having an increased content of in situ produced vitamin B12, wherein at least bacteria selected from the group consisting of bacteria of the strain Propionibacterium freudenreichi spp shermani B369, bacteria of the subspecies Propionibacterium freudenreichi spp freudenreichi and bacteria of the species Lactobacillus reuteri are present, which result in a content of in situ produced vitamin B12 of preferably at least 120 % of the content of in situ produced vitamin B12 achieved in absence of at least bacteria selected from the group consisting of bacteria of the strain Propionibacterium freudenreichi spp shermani B369, bacteria of the subspecies Propionibacterium freudenreichi spp freudenreichi and bacteria of the species Lactobacillus reuteri. The invention also relates to a method of preparing such a fermented milk product having an increased content of in situ produced vitamin B12, wherein milk is fermented making use of a common starter culture, and to the use of the aforementioned bacteria for increasing the content of in situ produced vitamin B12 in a fermented milk product.

Vitamin B12 from Propionibacterium Shermanii’:

These and other mutantstrains are used in a two stage process with added cobalt.In a preliminary anaerobic phas (2-4days),5’-deoxyadenosylcobinamide is mainly produced ,in a second aerobic phase(3-4 days) the biosynthesis of 5,6-dimethylbenzimidiazole takes place, so that 5’-deoxyadenosylcobalamin can be produced. Only traces of other cobamides are

synthesized in this process. As an alternative to the two stage batch process in a fermenter, both stages can also be operated continuously in two tanks operated in cascade fashion. During their recovery process ,the cobalamins which are almost completely bound to the cell are brought into solution by heat treatment (10-30min at 80-120degree C,pH 6.5-8.5).They are then converted chemically into the more stable cyanocobalamin. The raw product 80% purity is used as a feed additive.Medium: Maintenance medium for P.shermanii includes (in.glL) “tryptone(10.0), yeast extract (10.0), filtered tomato juice (200.0), agar (10.0) and pH {to 7.2). The inoculated media isincubated for 4 days at 3O°C.Seed Culture Medium: This is different types and prepared according to stages.1. First stage medium: It is similar to maintenance medium but is devoid of agar. It is incubated for 2 days at 30°Cwithout agitation.2. Second stage medium: It includes corn steep 20g/L,glucose 90 g/l and pH maintained at 6.5. Stainless steel bioreactor is used. The medium is incubated at 30°C for 24hrs without aeration, pH is adjusted to 6.5.Main culture medium: The production media includes (in g/l)corn steep liquor (40.0), glucose (100.0), COCI2.6H2O (0.02),and pH (to 7.0). It is incubated at 30DC. The first 80 hrs isAllowed to proceed without aeration, with slight nitrogen with agitation and slight aeration (0.1 v/v/m). pH is adjusted to 7.0.Propionic organisms are grown on carbohydrate-based media in an ul1aerated conditions. These organisms produce cobaltocorrinoids. Cobalt supplement is necessary for cobalamins production. It also depends on the internal formation orexternal supply of 5,6-dimethylbenzimidazole (5,6-DBI). The mutant strains of p. shermal1ii.synthesis their own 5,6-DBI. It increases the yield up to 65 mg/L on pilot-scale. Aeration promotes formation of 5,6-DBI, but it decreases vitamin BI2 biosynthetic pathway at one of the stages. Therefore, at first stage (80 hrs) fermentation is conducted in absence of oxygen (anaerobic condition), but with little agitation until all the sugar in the media is utilized for the growth and the formation of cobinamide. This kind of operation will have no repressive effect. The subsequent stage (next 88 hrs) is followed with agitation and slight-aeration. Aeration at this stage induces biosynthesis of 5,6-DBI and cobinamide is converted in tocobalamin.

Isolation and Purification:The extraction process depends on the release of unstablecobalamin from the cells lysed and subsequent treatment of this with cyanide to convert into more stable cyanocobalamin. Cells are separated from the culture broth. Cells are lysed by heattreatment at 80 to l20°C for 1O-3O minutes at pH 6.5-8.5. Thecells on lysis release various cobalamin. These are then solubilizedwith potassium cyanide in the presence of sodium nitrite.The obtained cobalamin gets converted into cyanocobalamin. The purification of the product is done using adsorptionmethod for substances like amberlite IRC SO, Dowex Ix2,alumina, silanized silica gel, and Amberlite XAD2. It is then followed by elution with water-alcohol or water-phenol mixtures.

Production of vitamin B12 from Bacillus megaterium.

As long ago as the third decade of this century, vitamin B 12 was discovered indirectly through its effect on the human body by George Minot and William Murphy. Vitamin B 12 was purified and isolated for the first time in 1948, so that only eight years later, in 1956, its complex three-dimensional crystal structure was elucidated by Dorothy Hodgkin. The naturally occurring final products of the biosynthesis of vitamin B 12 are 5′-deoxyadenosylcobalamin (coenzyme B 12) and

methylcobalamin (MeCbl), while vitamin B 12 is defined as cyanocobalamin (CNCbl) which is the form which is principally prepared and dealt with by industry. In the present invention, unless specifically stated, vitamin B 12 always refers to all three analogous molecules.

The species B. megaterium was described for the first time by De Bary more than 100 years ago. Although generally classified as a soil bacterium, B. megaterium can also be detected in various other habitats such as seawater, sediments, rice, dried meat, milk or honey. It is often associated with pseudomonads and actinomyces. B. megaterium is, like its close relation Bacillus subtilis , a Gram-positive bacterium and is distinguished inter alia by its relatively distinct size, which gives it its name, of 2×5 μm, a G+C content of about 38% and a very pronounced sporulation ability. Even miniscule amounts of manganese in the growth medium are sufficient for this species to carry out complete sporulation, an ability which is comparable only with the sporulation efficiency of some thermophilic bacilli. Because of its size and its very efficient sporulation and germination, diverse investigations have been carried out in the molecular bases of these processes in B. megaterium , so that more than 150 B. megaterium genes involved in its sporulation and germination have now been described. Physiological investigations on B. megaterium classified this species as an obligately aerobic, spore-forming bacterium which is urease-positive and Voges-Proskauer negative and is unable to reduce nitrate. One of the most prominent properties of B. megaterium is its ability to utilize a large number of carbon sources. Thus it utilizes a very large number of sugars and has been found, for example, in corn syrup, waste from the meat industry and even in petrochemical waste. In relation to this ability to metabolize an extremely wide range of carbon sources, B. megaterium can be equated without restriction with the pseudomonads.

The advantages of the wide use of B. megaterium in the industrial production of a wide variety of enzymes, vitamins etc. are manifold. These include, firstly and certainly, the circumstance that plasmids transformed into B. megaterium prove to be very stable. This must be viewed in direct connection with the possibility which has now been established of transforming this species for example by polyethylene glycol treatment. Until a few years ago, this was still a major impediment to the use of B. megaterium as producer strain. The advantage of relatively well developed genetics must also be regarded in parallel with this, being exceeded within the Bacillusgenus only by B. subtilis . Secondly, B. megaterium has no alkaline proteases, so that scarcely any degradation has been observed on production of heterologous proteins. It is additionally known that B. megaterium efficiently secretes products of commercial interest, as is utilized for example in the production of α- and β-amylase. In addition, the size of B. megaterium makes it possible to accumulate a large biomass before excessive population density leads to death. A further favorable circumstance of very great importance in industrial production using B. megaterium is the fact that this species is able to prepare products of high value and very high quality from waste and low-quality materials. This possibility of metabolizing an enormously wide range of substrates is also reflected in the use of B. megaterium as soil detoxifier able to break down even cyanides, herbicides and persistent pesticides. Finally, the

fact that B. megaterium is completely apathogenic and produces no toxins is of very great importance, especially in the production of foodstuffs and cosmetics. Because of these many advantages, B. megaterium is already employed in a large number of industrial applications such as the production of α- and β-amylase, penicillin amidase, the processing of toxic waste or aerobic vitamin B12 productionThe use of Bacillus megaterium is of great economic interest because it has a number of advantages for use in the biotechnological production of various products of industrial interest. In the large-scale industrial fermentation of aerobic microorganisms, however, problems regularly arise, especially with an efficient oxygen supply to the bacterial cultures, which are associated with considerable losses of product yield.