Vitamin B12 fermentation

BACKGROUND

The cobalamin family of substances, which includes the vitamin B12 molecule cyanocobalamin, is made up of a corrinoid ring, an upper and a lower ligand. Adenosine, methyl, hydroxy, or a cyano group can all be used as the upper ligand. Prokaryotes produce vitamin B12, which prevents mammals from developing pernicious anaemia.

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In bacteria and archaea, respectively, the aerobic or anaerobic pathway is used for the microbial de novo production of vitamin B12. Certain strains can also produce cobalamin by utilizing a salvage pathway to take in corrinoids. From -aminolevulinate (ALA), tetrapyrrole compounds such as cobalamin, heme, and bacteriochlorophyll are produced.

Many bacterial species have complicated interdependent and interactional relationships with these tetrapyrrole compounds. A cobalamin riboswitch in the 5′ untranslated regions (UTR) of the relevant gene’s controls vitamin B12 production and transportation to keep levels steady.

Vitamin B12

In the 1920s, research into vitamin B12 was started in relation to pernicious anemia, a condition that was originally reported in 1824. The primary signs and symptoms of this condition were exhaustion, loss of appetite, headaches, and, in more serious cases, dementia, memory loss, muscle weakness, and peripheral neuropathy, which might be fatal if left untreated.

In 1926, Minot and Murphy showed that a specific diet containing significant portions of lightly cooked liver and muscle meat could help patients with pernicious anemia recover from the illness. They postulated that an unidentified “extrinsic component” present in animal livers was the reason the treatment worked.

Although it took more than 20 years for the so-called “extrinsic factor” to be discovered and purified, they were nonetheless given the Nobel Prize in Physiology or Medicine in 1934 for this discovery. In 1948, two pharmaceutical company research teams—Folkers at Merck, Sharp & Dohme, and Smith at Glaxo—isolated a cobalt chemical from animal livers that had the ability to treat pernicious anemia on its own.

A year later, the same substance could also be isolated from various bacterial cultures, milk, beef, and other sources. Vitamin B12 was the name of this red, octahedral, crystalline cobalt substance. Years later, it was interestingly discovered that this substance was actually cyanocobalamin (CNCbl), a synthetic, physiologically inactive version of cobalamin produced during the industrial process of extracting and isolating from the liver. In addition, Dorothy Hodgkin determined the structure of CNCbl, the first cobalamin isoform, in 1955.

REQUIREMENTS

Propionibacterium freudenreichii strain

Nutrient broth

Spectrophotometer

Centrifuge

Funnel

Filter paper

Potassium cyanide

Sodium nitrite

Zinc chloride

Tannic acid

Cresol

Production medium:

Polypeptone – 12.5 g/L

Casamino acids – 11.0 g/L

Yeast extract – 2.5 g/L

NaH2PO4.2H2O – 2.25 g/L

MgCl2.6H2O – 0.4 g/L

K3PO4 – 1.76 g/L

CoCl2.6H2O – 0.018 g/L

FeSO4.7H2O – 0.01 g/L

Ca-pantothenate – 4 mg/L

Biotin – 0.3 mg/L

Glucose – 40 g/L

Tween 80 – 1g/L

CoCl2.6H2O – 0.05 g/L

5,6-dimethybenzimidazole – 0.07 g/L

PROCEDURE

1. The Propionibacterium freudenreichii strain used for the vitamin B12 production. ^[1]

2. For inoculum of P. freudenreichii was transferred to 20 ml nutrient broth from stock culture, and incubate for 24 hr at 30-37˚C

3. Prepare B12 production medium, and sterilize it. ^[2]

4. Add P. freudenreichii aseptically to the sterilize B12 production medium, and incubate at 30-37˚C for 24-48 hr

5. Take small amount of sample after the periodic time period for the glucose concentration and B12 can determine by using the spectrophotometric analysis. ^[2]

6. For recovery of Vitamin B12, centrifuge the broth and separate the supernatant and pellet (cell mass concentrate). ^[1]

7. Take cell mass, mix in aqueous solution and heat it for cell lysis to get corrinoids.

8. Add potassium cyanide, in presence of sodium nitrite in the corrinoids and heat it.

9. Filter the solution, and treat filtrate with zinc chloride

10. Precipitate out B12 by adding tannic acid or cresol.

CONCLUSION

Vitamin B12 production done by using the Propionibacterium freudenreichii strain and assay was done by using the spectrophotometrically analysis.

REFERENCES

1. https://dl.tgxlink.eu.org/2079/1587535079Unit+III+Microbial+production+of+vitamins.pdf?hash=AgADOg
2. Ken-ichiro Miyano, Kaiming Ye, Kazuyuki Shimizu, “Improvement of vitamin B12 fermentation by reducing the inhibitory metabolites by cell recycle system and a mixed culture”, Biochemical Engineering Journal, Vol. 6, 2000.
3. Álvaro Calvillo, Teresa Pellicer, Marc Carnicer, Antoni Planas, “Bioprocess Strategies for Vitamin B12 Production by Microbial Fermentation and Its Market Applications”, bioengineering journal, 2022.
4. Huan Fang, Jie Kang and Dawei Zhang, “Microbial production of vitamin B12: a review and future perspectives”, Microbial Cell Factories, 2017.

Also read:

• Microbiological Examination of Water (Drinking Water, Supply Water and Pond Water)
• Methods for purification of enzymes
• Bacteriological Analysis of Water
• Isolation of industrially important microorganisms from soil
• Bioinsecticide /Biofertilizers isolation production purification and assay
• Microbial Enzyme production and its characterization
• Production of Organic acid by fermentation

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