Vitamin B 12 (Cyanocobalamine ; Cobamide)

7.4. Vitamin B 12 (Cyanocobalamine ; Cobamide)

Vitamin B 12 is produced commercially by the aid of a direct fermentation procedure using Streptomyces species, for instance : Streptomyces olivaceus.

Rickes et al.* (1948) first and foremost recovered the active crystalline vitamine B 12 , as given below from a S. griseus culture (that also eventually produced the antibiotic resein).

* Rickes EL et al. Science, 108 : 634-635, 1948c.

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Prolonged intensive and extensive researches have adequately proved and revealed that at least ‘small quantum’ of vitamin B 12 could be synthesized by a host of microorganism variants belonging specifically to actinomycetes and bacteria ; and that relatively ‘large quantum’ was prevalent particu- larly amongst the microorganisms pertaining to the intestinal habitats.

In general, cobamides (vitamin B 12 ) essentially comprise of a ‘porphyrin nucleus’ to which the ribose and phsphate residues are attached strategically. Nevertheless, the cobamide variants do differ in their corresponding purine, benzimidazole on other base located in the nucleotide-like segment of the molecule ; and besides, in the chemical functional moiety duly attached to the Co atom.

The various steps involved in the production of Vitamin B 12 are enumerated sequentially as stated below :

(1) S. olivaceous is allowed to grow with adequate constant aeration at 27°C preferably in a

nutritionally rich crude-medium having glucose as a major source of carbon. (2) A potential source of cobalt (Co) between 2-10 ppm is duly incorporated into the above

medium in the form of its salt cobalt chloride [CoCl 2 . 6H 2 O] to serve as a precursor. Thus,

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the relevant organism meticulously scavenges low levels of cobalt from the prevailing me- dium, whereas the respective higher levels are proved to be toxic in nature.

(3) Total duration of the fermentative process lasts between 3 to 4 days or until such time when mycelium lysis commences to take place. In this manner, a major segment of the vitamin B 12 produced remains very much contained within the microbial cells until autolysis* comes into force ; and, therefore, the recovery of the vitamin from the ‘fermentation broth’ is tremendously simplified by actually initiating harvesting before autolysis has turned out to

be adequately serious i.e., a situation when vitamin B 12 is still lodged within the mycelium securedly.

(4) At harvesting stage, both the solids and the mycelium are duly filtered or centrifuged to separate them from the ensuing ‘fermentation broth’, and dried subsequently for an appro- priate usage in the form of either vitamin B 12 enriched animal-or poultry-feed supplements.

(5) Alternative Method of Recovery : In this specific instance, the vitamin B 12 is predomi- nantly released from the concerned cells by several tried and tested methodologies, namely : alcohol treatment, heat, acidification etc.

Example : The adequately completed ‘fermentation broth’ is first acidified, treated with sodium sulphite so as to protect the vitamin, and finally the admixture of culture and fermen- tation broth is subjected to careful heating by employing steam-heated coils with agitation or passing low-pressure steam slowly with proper agitation.

(6) The ‘solid residues’ as well as ‘spent mycelium’ are duly separated either by ‘filtration’ or ‘centrifugation’ , and the resulting clear fluid thus obtained is meticulously evaporated strictly

under superb vacuum facilities. Note :

(1) Main bulk of vitamin B 12 is used as such in solid dosage forms and liquid prepara- tions.

(2) Relatively small proportion of the vitamin B 12 is further purified and crystallized for the exclusive treatment of pernicious anemia** and other vital medicinal usage via the IV route of administration.

Latest Method of Production : The latest method adopted for the present-day commercial pro- duction of vitamin B 12 is usually carried out by adequately aerated submerged bacterial fermentations making use of strains of Propionibacterium or Pseudomonas with a beet-molasses based culture me- dium and the adequate supplementation with requisite amount of the cobalt-salts.

Vitamin B 12 may also be produced on a large-scale by using Bacillus megaterium, and the over- all recovery from this fermentative procedure almost approaches quite similar to those obtained from the Streptomyces organisms.

The details of commercial production from Propionibacterium shermanii and Pseudomonas denitrificans are described as under :

* The self-dissolution or self-digestion which occurs in cells by enzymes in the cells themselves. ** A chronic, macrocyclic anemia marked by achlorhydria. It mostly occurs in 40 to 80 year old northern

Europeans with fair skin, but has been reported in other races as well and ethanic groups. It is rare in blacks and Asians.

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