Liver extracts cure pernicious anaemia in human beings. This vitamin is present in a very small amount in every animal tissue including human blood but it is synthesized only by microorganisms such as Butyribacterium rettgeri, Bacillus megaterium, Streptomyces olivaceus, Micromonospora sp., Klebsiella pneumoniae etc. High yields have been obtained from Propionibacterium freudenreichii, P. shermanii and Pseudomonas denitrificans.
Vitamin B12 is not a single compound, but a group of closely chemically related cobamides. These cobamides are also called pseudo B12 group. They consist of cobalt porphyrin nucleus to which is attached ribose and phosphate. Various cobamide differ in the purine, benzimidazole, or other base, found in the nucleotide like portion of the molecule. Vitamin B12 analogue having other heterocyclic bases are either produced by microorganisms or are produced after the addition of these substances in the culture medium.
Fermentation- The nutritionally rich crude medium with glucose as a major carbon source is used in a two stage process with added cobalt chloride. In a preliminary anaerobic phase (2-4 days), deoxyadenosylcobinamide is mainly produced; in a second phase, which is aerobic (3-4 days) the biosynthesis of 5,6 dimethyl benzimidazole takes place, so that 5- deoxyadenosyl cobalamine (B12) can be produced. This compound is completely intracellular and bound to the cell which after heat treatment released in solution form after 10 min at 80-120°C at pH 6.5-8.5. This process is applicable in case of Propionibacterium freudenreichii ATCC6207 and P. shermanii, ATCC3673.
While using Peudomonas denitrificans, there is one-stage process that occurs during the entire fermentation. Similar to the previous process, cobalt is added but here we also add 5,6- dimethylbenzimdazole a supplement. It has also been observed that addition of betaine induce the yield. In place of glucose, other cheap C source such as hydrocarbon and higher alcohol are also found promising. Methanol proved better carbon source.
Riboflavin (Vitamin B2)
Kuhn, Gyorgy and Wagner Jauregg in 1933 isolated riboflavin (=lactoflavin) from whey of milk where it is present in free riboflavin form. It is also present in other foods (liver, heart, kidney, eggs) as flavoproteins which contains the prosthetic group FMN (flavin mononucleotide) or FAD (Flavin adenine dinucleotide). Several microorganisms like Clostridium acetobutylicum, Mycobacterium, Mycocandida riboflavin, Candida flareri, Eremothecium ashbyii and Ashbya gossypii are used in commercial production. Riboflavin is produced by chemical synthesis also. It is alloxazine derivative which consists of a ptreidine ring condensed to a benzene ring. The side chain consist of a C5-polyhydroxy group, a derivative of ribitol.
Basic medium consists of corn steep liquor 2.25%, commercial peptone 3.5%, soyabean oil 4.5% but it can be supplemented further by addition of different peptones, glycine, distiller's soluble, or yeast extracts. The glucose and inositol increase the production of riboflavin. The medium should be kept at 26-28°C for 4-5 days incubation after inoculation the submerged growth of Ashbya gossypi is supported by insufficient air supply. The excess air inhibits mycelia production and reduces the riboflavin yield. The fermentation progresses through three phases:
First Phase- In this phase rapid growth occurs with small quantity of riboflavin production. The utilization of glucose occurs resulting in to decrease in pH due to accumulation of pyruvate. By the end of this phase, the glucose is exhausted and growth ceases.
Second Phase- Sporulation occurs in this phase. The pyruvate decreases in concenteration. Ammonia accumulates because of an increase in deaminase activity. The pH reaches towards alkalinity.
Third Phase- There is a rapid synthesis of cell bound riboflavin (FMN and FAD). This phase is accompanied by rapid increase in catalase activity.
As the fermentation completes, the autolysis takes place with releases free riboflavin in to the medium as well as retained in the nucleotide form. The riboflavin is present both in solution and bound to the mycelium in the fermentation broth. The bound vitamin is released from the cells by heat treatment(1h, 120°C) and the mycelium is separated and discarded. The riboflavin is then further purified.
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