Production of cyanocobalamin/vitamin B12

1. PRODUCTION OF
CYANOCOBALAMIN

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
1. Fat soluble vitamins :- includes Vitamin A, D, E and K
2. 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:- C63H88CoN14O14P
 Molecular weight:- 1355.4

8. Biosynthesis of Cobalamin

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)
H2O2 (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[(NH2)4HPO4] 2.0
Magnesium sulfate[MgSO4.7H2O] 1.0
Manganese sulfate[MnSO4.7H2O] 0.2
Zinc sulfate[ZnSO4.7H2O] 0.02
Sodium molybdate[Na2MoO4.2H2O] 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(NO3)2.6H2O] 0.188
Ammonium
Hypophosphate[(NH2)4HPO4]
5.0
Magnesium sulfate[MgSO4.7H2O] 3.0
Manganese sulfate[MnSO4.7H2O] 0.2
Zinc sulfate[ZnSO4.7H2O] 0.02
Sodium molybdate[Na2MoO4.2H2O] 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 290C
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[CoCl2.6H2O]
0.02
pH 7.0
The medium is incubated
for 4days at 300C.
The medium is incubated at 300C 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 300C 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 300C for
24hrs without aeration, pH is adjusted to 6.5.

21. Isolation of cyanocobalamin

22. 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

23. 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.

24. Thank you