Fermentation Process of Vitamins

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BACKGROUND

Vitamins are the organic compounds which perform some specific functions for normal growth & maintenance of an organism.¹ The vitamins cannot be mixed by higher organisms because of which these are taken in small amounts. Vitamins are necessary for metabolism of all the living beings. Vitamins are differentiated as Fat Soluble Vitamins and Water Soluble Vitamins. Fat Soluble Vitamins are Vitamin A, E, D, K and Water Soluble Vitamins: Vitamin B, C, H, and P.² For Vitamins such as B12 fermentation process is carried out for the production of the same.³ Fermentation is a chemical breakdown of a substance by yeasts/ bacteria/ other microorganisms.

So the basic objective of the test is to carry the production of vitamin-B12 by the process of fermentation

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REQUIREMENTS

Chemicals:    Corn steep glucose

        Glucose

        Cobalt salt

        Corn oil/ Soyabean Oil (anti-foaming agent)

        Ammonium Hydroxide

        Ammonium Phosphate

Apparatus:    Fermenter

        Centrifuge

        Flask

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PROCEDURE

Vitamin B12

Vitamin B12 is a water soluble vitamin which plays an important role in functioning of nervous system normally. It metabolises every cell of human body.⁴ It is also known as cobalamin.

For fermentation process Streptomyces griseus, Pseudomonas denitrificans, Streptomyces olivaceus and Propionibacterium shermanii are used. Cyanocobalamin is the stable form of Cobalamin which is not found in nature.

Media preparation

Medium preparation is important as it involves the use of microorganism to achieve the products. For vitamin B12 fermentation glucose is used as carbon source (corn steep glucose) and for nitrogen source Ammonium Phosphate/ Ammonium Hydroxide is used.

Inoculation

From slant culture of S. olivaceus inoculation is done in 100-250 ml of inoculums medium contained in Flask. The flask is then incubated on a shaker to oxygenate the system. Thus this flask culture is often used to inoculate larger inoculums

Production medium

Cobalt salt is added to the medium as maximum amount of cobalamin is obtained.⁵ For conversion of other cobalamins cyanide is added into Vitamin B12.

Sterilization

The medium which is prepared is then sterilized by an autoclave with 15 lbs pressure at 121^oC. The medium which is thus sterilized is used for fermentation. Medium is sterilised at high heat and high pressure or directly sterilised together with the fermenter. Medium is then heated at 121°C. High temperature is used to minimise degradation of the components of media

Fermentation

Vitamin fermentation is basically done in the batch mode. Nutrients and inoculums are then added to the sterile Fermenter and are then left for some time.⁶ The anti-foaming agent is then added. After a good amount of product is present in the Fermenter the content is then taken out & then the product is extracted. In microbial cells the cyanocobalamine remains in the form natural substances such as 5,6-dimethyl benzimidazole cobalamide and pseudovitamin B12. The culture broth contains 10-15 mg Vitamin B12 / lt.

Centrifuge

The culture broth is then yield & centrifuged to get a concentrated amount of mass of cells.

Biomass removal

The impurities are separated from the culture medium. For removal of such impurities a vacuum filter is commonly used. The cell mass is treated with a dilute acid & the temperature is maintained at 10-30^oC. Then cyanide solution is added to separate 5,6-dimethyl benzimidazole cobalamide and pseudovitamin B12 as a result of which Vitamin B12 (cyanocobalamine) is then released in solution. Vitamin B12 which is in the liquid form is then separated by chromatography process (adsorption column chromatography).

Solvent addition

The adsorbed cyanocobalamine is then removed by using phenolic solvent.

Drying

The solvent portion is then evaporated by exposing to normal because of which crystals of cyanocobalamine is left in the vessel which is stored for further use.

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CONCLUSION

Crystals of cyanocobalamine are then stored and further used in medicinal form or as prescribed.

STRUCTURE OF VITAMIN B12 (SOURCE: WIKIPEDIA)

BATCH FERMENTER (SOURCE: WIKIPEDIA)

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REFERENCES

1. Miller A, Korem M, Almog R, Galboiz Y.;”Vitamin B12, demyelination, remyelination and repair in multiple sclerosis”. 2005: 233 (1-2);93-7
   
2. Fortmann SP, Burda BU, Senger CA, Lin JS “Vitamin and mineral supplements in the primary prevention of cardiovascular disease and cancer: An updated systematic evidence review for the U.S. Preventive services task force”. Annals of internal medicine: 2013: 159(12); 824-34
   
3. Vitamin and mineral supplement fact sheets vitamin B12: dietary supplements: 2011; retrieved on 03-08-2013.
   
4. Scott JM, Molloy AM ” The discovery of vitamin B12″. Annals of Nutrition & Metabolism: 2012. 61(3) 239-45.
   
5. Yamada k, Sigel H, Sigel RK. ” Cobalt:its role in health and disease:2013. 295-320
   
6. .Heinzle, Elmar, Arno P. Biwer and Charles L.;”development of sustainable bioprocesses:modelling and assessment.” 2007 Martin TL, Mesulam MM, Mufson EJ; “The light side if Horseradish peroxidise histochemistry” J Histochem. 1984: 32(7): 793.