vitamin B12


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Applied microbiology

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vitamin B12

  1. 1. Dr.K.Gajalakshmi Assistant professor, PSGR krishnammal college for Women, Coimbatore
  2. 2. • vitamin B12, vitamin B12 or vitamin B-12, also called cobalamin, 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 human body, especially affecting DNA synthesis and regulation, but also fatty acid synthesis and energy production. • Vitamin B12 is important for the way the body works, and people who don't have enough of it may feel tired or have a lack of energy.
  3. 3. • Vitamin B12 helps in the production of healthy red blood cells that carry oxygen around the body. • Not having enough vitamin B12 is called vitamin B12 deficiency anaemia. • This condition makes the body produce larger than normal red blood cells, described as megaloblastic or macrocytic, which don't do their job as well. • Once diagnosed, vitamin B12 deficiency can usually be treated successfully with B12 injections and sometimes with B12 tablets.
  4. 4. Structure of Vitamin B12
  5. 5. • Species from the following genera are known to synthesize B12: Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Lactobacillus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas. • Industrial production of B12 is through fermentation of selected microorganisms. Streptomyces griseus, a bacterium once thought to be a yeast, was the commercial source of vitamin B12 for many years.[48][49] The species Pseudomonas denitrificans and Propionibacterium shermanii are more commonly used today
  6. 6. • • • • Methylcobalamin (shown) is a form of vitamin . B12. Physically it resembles the other forms of vitamin B12, occurring as dark red crystals that freely form cherry-colored transparent solutions in water Minot and Murphy -reported -liver juice, in 1926. Rickets and Smith -isolated from liver cells of animals in 1942. Rikes - Microbial source of Cyancobalamine demonstrated in 1948. – Streptomyces griesus
  7. 7. Steps involved in Microbial Cyanocobalamine production Step-6
  8. 8. For Media preparation • • • • Carbon source- as 1.Corn steep glucose 2. Beet molasses 3.Soyabean meal/Glucose • Nitrogen source-as • 1.Ammonium phosphate • 2. Ammonium hydroxide
  9. 9. Step-1 Formulation of medium Medium+Cobalt salt Medium+ Cobalt salt Sterilization Starter culture of Propioni bacterium shermanii o Inoculati n Anaerobic fermentation for 3 days Aerobic fermentation for 4 days Centrifugation Harvested Broth Cell Harvest Acid treatment and heating Released Pseudo-vitamin B12 Bakers coenzyme
  10. 10. Released Pseudo-vitamin B12 Bakers coenzyme Addition of cyanide solution Cyanocobalamine in liquid Adsorption chromatography Adsorption on IRC-50 resin Elution with a phenolic compound Cyanocobalamine in solvent Evaporation Cyanocobalamine crystals
  11. 11. 2. Sterilization • Prepared medium is sterilized by autoclaving • The sterilized medium is then used for fermentation.
  12. 12. 3.Making Starter culture • • The following microbes were suitable for Industrial fermentation of cyanocobalamine. 1.Bacillus megaterium 2. Streptomyces olivaceous • 3.Propionibacterium shermanii • 4. Pseudomonas denitrificans 5.Rhodopseudomonas palustris
  13. 13. Inoculum Wild strains Mutant strains of--- Inoculum Improved strain- produce 50,000 times more vitamin B12 than wild strain .This is the great boon for biotechnologists to produce vit B12.
  14. 14. Batch fermentation • Most fermentations are batch processes • Nutrients and the inoculum are added to the sterile fermenter and left to get on with it! • Anti-foaming agent may be added. • Once the desired amount of product is present in the fermenter the contents are drained off and the product is extracted. • After emptying, the tank is cleaned & prepared for a new batch.
  15. 15. Continuous fermentation • Some products are made by a continuous culture system. • Sterile medium is added to the fermentation with a balancing withdrawal of broth for product extraction.
  16. 16. • UPSTREAM PROCESSING • Upstream processing encompasses any technology that leads to the synthesis of a product. Upstream includes the exploration, development and production. • DOWNSTREAM PROCESSING The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing
  17. 17. Aerobic Fermentation • It is a Batch fermentation • Continuous fermentation also found to be effective. • Sterilized medium is filled in stirred tank fermenter 1% of inoculum (Starter culture) is added in to the fermenter. • Anaerobic condition is maintained to encourage the production of 5,6-dimethyl benzimidazole cobalamide (DBC) by P.shermanii
  18. 18. Aerobic Fermentation • Anaerobic Fermentation is over sterile air is pumped in to the fermenter. • The culture is stirred well for proper aeration • Aerobic fermentation is performed for 4 days. • During this process some amount of DBC and pseudo vitaminB12 (adeninyl cobalamine) are produced. • DBC and pseudovitaminB12 (adeninyl cobalamine) are immediate precursors of cyanocobalamine
  19. 19. Recovery of Cyanocobalamine • Inside the microbial cells the cyanocobalamine exits in the form of natural substances such as DBC and pseudovitaminB12 . • The cultured broth contains 10-23mg vitB12 per liter. • It is harvested and centrifuged at high speed to get a concentrated mass of cells. • The cell mass is treated with a dilute acid and heat stock at 10-30o C. • During this treatment precursors of pseudovitaminB12 are released free.
  20. 20. • Then it is treated with Cyanide solution to split the DBC and pseudovitamin B12 . • As a result cyanocobalamine (Vit B12) is released free in the solution. • Cyanocobalamine in the liquid is seperated by using an adsorption column chromatography with IRC-50 resin. The adsorbed cyanocobalamine is then eluted out of the column using phenolic solvent. • The solvent fraction is evaporated by exposing it to atmospheric air. • As result crystals of cyanocobalamine is let in the vessel. It is stored for future use.
  21. 21. USES • It is a food preservative • It is a co-factor • It is a protective medicine