Production of vitamin B12

Production of cyanocobalamin/vitamin B12

1. Production of Cyanocobalamin Prathap H m

2. Contents Introduction Cobalamin-still a chemical fortification Structure Biosynthesis of cyanocobalamin Sources and Occurrence Deficiency Dietary recommendations for humans Vitamin-B12 derivatives Organisms for the production of Vitamin-B12 Production of cyanocobalamin Uses of cyanocobalamin

3. Introduction Vitamins are micronutrients that are essential for the metabolism of all living organisms Vitamins are organic compounds that are essential in the diet in small amounts to promote and regulate body functions necessary for growth, reproduction and the maintenance of health Vitamins are essential for the normal growth and development of a multicellular organism 1912- Casimir funk- Polish biochemist- coined the term ‘Vitamin’ Casimir funk originally used the word ‘ vitamine ’, today we know that these vitamins are vital to life, but they are not all amines, so the ‘e’ has been dropped and called the term ‘Vitamin’ Humans are incapable of synthesizing most vitamins

4. Nomenclature and classification Vitamins can be broadly classified into two main categories Fat soluble vitamins :- includes Vitamin A, D, E and K Water soluble vitamins :-Nine water soluble vitamins are known, namely; Thiamin (Vitamin B1) Riboflavin (Vitamin B2) Niacin (Vitamin B3) Pantothenic acid (Vitamin B5) Pyridoxine (Vitamin B6) Biotin (Vitamin B7) Folic acid (Vitamin B9) Cobalamin (Vitamin B12) Vitamin C

5. Vitamin B12(Cobalamin) Is a complex biochemical structure, but is water soluble One of the most alluring and fascinating molecules in the world of science and medicine is Vitamin B12 Vitamin B12 is unique among the vitamins in being synthesized almost exclusively by Microorganisms 1912- Minot and Murphy- demonstrated that they were able to cure pernicious anemia, a disorder first described in 1835 1958- Barker et. al.- discovered and crystallized the first biologically active coenzyme forms of vitamin B12 1958- Smith and co workers- produced 14C enriched methyl cobalamin

6. Cobalamin: still chemical fortification Cobalamin or Vitamin B12- the largest and most complex of all the vitamins Cobalamin is the term generally used to refer to a group of cobalt containing compounds Cyanocobalamin, which is used in most supplements- readily converted to coenzyme forms of cobalamin in the human body Cobalamin is the only vitamin that is synthesized by microorganisms & only a few bacteria and archea Industrial production of vitamin B12 is usually performed by microbial fermentation processes

7. Structure of cobalamin Molecular formula:- C 63 H 88 CoN 14 O 14 P Molecular weight:- 1355.4

8. Biosynthesis of C obalamin

9. Sources and occurrence Foods   Content (µg/100g) Animal meats Mutton, cooked 2.6   Chicken, cooked 9.4 Cow’s milk   0.4 Eggs Chicken, cooked 1.3 Shellfish Oyster 28.1-46.3   Mussel 10.3-15.7   Short-necked dam 37.0-52.4 Fish meats Skip jack, dark muscle 158.5   Yellowfin tuna, dark muscle 52.9   Rainbow trout, cooked 4.9 Edible algae Purple laver 32.3-77.6   Green laver 31.8-63.6   Chlorella 200.9-211.6

10. Deficiency People are at risk of Vitamin B12 deficiency include The elderly Those who’ve had surgery that removes the part of the bowel that absorbs Vitamin B12 People on the drug metformin for diabetes People following a strict vegetarian diet Those taking long-term antacid drugs for heartburn Smokers, Alcoholics, those with anemia are at high risk of having deficiency Low levels of Vitamin B12 cause folate levels to drop

11. If having B12 deficiency, correcting low folate levels may simply mask the deficiency and fail to fix the underlying problem. Pale or Jaundiced skin Weakness and Fatigue Sensations of pins and needles Changes to Mobility Glossitis and Mouth Ulcers Breathlessness and Dizziness Disturbed Vision Mood Changes High Temperature Symptoms of deficiency

12. Causes for the deficiency Weight loss surgery Acid reducing medications Atrophic gastritis – Stomach lining has thinned Pernicious anemia – Hard for body to absorb vitamin B12 Crohn’s disease Celiac disease Grave’s disease – Immune system disorder Conditions affect Small Intestine

13. Rich Low Fiber Vitamin A Vitamin C Vitamin D3 Vitamin E Vitamin B12 Folate Iron Magnesium Cholesterol n-6 Polyunsaturated fatty acids n-3 Polyunsaturated fatty acids Carbohydrates Saturated fatty acids Nutrient imbalance in vegetarian diets.   Age Requirements in µg Infants Up to 6months 0.4 Babies 7-12 months 0.5 Kids 1-3 years 0.9 Children 4-8 years 1.2 Children 9-13 years 1.8 Teens 14-18 years 2.4 Adults 18 and above 2.6 Daily requirement for the human beings according to the age

14. Vitamin B12 derivatives Original name Ion or molecule coordinated Semi-systematic name Systematic name Vitamin B12 CN - Cyanocobalamin α-(5,6-dimethylbenzimidazolyl) cobamide cyanide or α-(5,6-dimethylbenzimidazolyl) cyanocobamide Vitamin B12α     Vitamin B12β OH - (alkaline solution)   H 2 O 2 (acid solution) Hydroxocobalamin     Aquocobalamin α-(5,6-dimethylbenzimidazolyl) hydroxocobamide α-(5,6-dimethylbenzimidazolyl) aquacobamide Vitamin B12е ONO -     SCN - Nitritocobalamin or nitrocobalamin   Thiocyanatocobalamin α-(5,6-dimethylbenzimidazolyl) cobamide nitrite α-(5,6-dimethylbenzimidazolyl) cobamide thiocyanate

15. Organisms for the production of Vitamin B12 Acetobacterium sps . Aerobacter aerogenes Agrobacterium sps . Ashbya gossipii Azotobacter sps . Bacillus subtilis Bacillus megatherium Clostridium butyricum Clostridium cochlearium Clostridium flabelliferum Clostridium tetanomorphum Corynebacterium sps . Eremothecium sps . Escherichia coli Flavobacterium sps . Lactobacillus arabinosis Lactobacillus casei Micromonospora sps . Mycobacterium phlei Mycobacterium smegmatis Mycobacterium tuberculosis Nocardia sps . Propionibacterium freudenreichii Propionibacterium sherrmanii Propionibacterium seae Protaminobacter sps . Proteus sps . Pseudomonas sps . Rhizobium sps . Salmonella sps . Serratia marcescens Staphylococcus aureus Streptococcus faecalis Streptomyces albidoflavus Streptomyces antibioticus Streptomyces aureofaciens Streptomyces colomiensis Streptomyces fradiae Streptomyces griseus Streptomyces olivaceus Streptomyces roseochromogenus Torula sps . Xanthomonas sps .

16. Production of Cyanocobalamin The production of Vitamin B12 is mainly to overcome the deficiency The industrial production of vitamin B12 mainly consists of Fermentation The fermentation process can be done by numerous microorganisms Because of their growth and their high productivity, Propionibacteria and Pseudomonas were selected for industrial purposes.

17. Steps involved in production

18. Laboratory scale Fermentation In laboratory scale fermentation process we mainly use Pseudomonas denitrificans Maintenance Medium: Contents g/L Beet molasses 60.0 Bakers yeast 1.0 Nitrazomonas amine 1.0 Ammonium Hypophosphate[(NH 2 ) 4 HPO 4 ] 2.0 Magnesium sulfate[MgSO 4 .7H 2 O] 1.0 Manganese sulfate[MnSO 4 .7H 2 O] 0.2 Zinc sulfate[ZnSO 4 .7H 2 O] 0.02 Sodium molybdate[Na 2 MoO 4 .2H 2 O] 0.510-3.0 Agar 25.0 pH 7.4 Production Medium: Contents g/L Beet molasses 100.0 Bakers yeast 2.0 Cobalt nitrate[Co(NO 3 ) 2 .6H 2 O] 0.188 Ammonium Hypophosphate[(NH 2 ) 4 HPO 4 ] 5.0 Magnesium sulfate[MgSO 4 .7H 2 O] 3.0 Manganese sulfate[MnSO 4 .7H 2 O] 0.2 Zinc sulfate[ZnSO 4 .7H 2 O] 0.02 Sodium molybdate[Na 2 MoO 4 .2H 2 O] 0.510-3.0 5,6-Dimethylbenzimidazole 0.025 pH 7.4 Fermentation:- The media inoculated with the seed culture is incubated for 90hrs at 29 C with agitation (420rpm) and aeration (1v/v/m)

19. Pilot scale fermentation Maintenance Medium: Production Medium: Contents g/L Tryptone 10.0 Yeast extract 10.0 Filtered tomato juice 200.0 Agar 10.0 pH 7.2 Contents g/L Corn steep liquor 40.0 Glucose 100.0 Cobalt chloride[CoCl 2 .6H 2 O] 0.02 pH 7.0 The medium is incubated for 4days at 30 C. The medium is incubated at 30 C for 24hrs without aeration . Seed culture medium:- This is of different types and prepared according to stages First stage medium:- It is similar to maintenance medium but is devoid of agar. It is incubated for 2days at 30 C without agitation. Second stage medium: - It includes Corn steep liquor-20g/L, Glucose-90g/L and pH is 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.

20. Isolation of cyanocobalamin

21. Uses of Vitamin B12 Vitamin B12 or cobalamin has a wide spectrum of benefits to human health. Cell repair and maintenance DNA formation Relieves Fatigue Treats Sickle cell disorder Reduces Cholesterol Levels Treats Alzheimer’s Disease Cures Anemia Prevents Breast Cancer

22. conclusion Microbial production is commercially feasible for vitamin B12, but, this is produced in low concentrations, even in optimized fermentation methods. In addition recovery and purification is a complex process and it involves several operational steps. Therefore a wide scope exists for extensive research in recovering vitamins from fermentation broth with minimum number of steps . By this production of vitamin B12 is very essential for the people who are suffering from diseases by its deficiency.

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