Vitamin B12 (Cobalamin) Production by Fermentation

Vitamin B12 is crucial for maintaining healthy blood and nerve cells and for creating DNA, the genetic material in all cells. It also helps prevent megaloblastic anemia, a condition that causes tiredness and weakness.

Vitamin B12 is produced on an industrial scale using fermentation with specific microorganisms. For many years, the bacterium Streptomyces griseus, once mistakenly thought to be a fungus, was the main source of commercial vitamin B12. Today, species like Pseudomonas denitrificans and Propionibacterium freudenreichii are used for large-scale production.

Industrial Production of Vitamin B12 | Process, Applications

What is vitamin B12?

  • Vitamin B12 is a water-soluble essential vitamin containing cobalt elements. 
  • The molecular structure of cobalamin is composed of a cobalt ion-centered corrin ring and 5,6-dimethylbenzimidazole as a base. 
  • Vitamin B12 is also known as cobalamin or cyanocobalamin.
  • There are four common derivatives of vitamin B12, namely cyanocobalamin, methylcobalamin, hydroxocobalamin, and adenosylcobalamin. 
  • It is red due to cobalt content due to this it is called a red vitamin and is one of the few colored vitamins.
  • Vitamin B12 is synthesized by microorganisms, and higher animals and plants cannot manufacture vitamin B12.
  • Vitamin B12 is the only vitamin that requires the help of an intestinal secretion (endogenous factor) to be absorbed.
  • It is relatively stable in neutral and weakly acidic solutions and is easily destroyed or decomposed in alkaline solutions and sunlight.
  • B12 is mainly found in animal food such as meat, fish, and dairy products. soybeans and some herbs also contain B12. 
  • Vitamin B12 is essential for promoting growth, keeping nerve tissue healthy, and forming normal red blood cells (erythrocytes).

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Discovery of vitamin B12

  • Vitamin B12 is composed of cobalt-containing porphyrin compounds. 
  • It was initially found that taking the whole liver can control the symptoms of pernicious anemia. 
  • After 20 years of research, a red crystalline substance with the effect of controlling pernicious anemia was isolated from the liver in 1948, named vitamin B12. 
  • Its structural formula was determined in 1963. 
  • Synthesis was completed in 1973. 
  • Vitamin B12 is one of the latest B vitamins discovered so far.

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Microorganisms used for Vitamin B12 Production

  • Different types of microorganisms have different abilities to produce vitamin B12. Therefore, different microorganisms can be used to produce vitamin  B12. 
  • Today, vitamin B12 fermentation technology plays a huge role in the food, Pharma, and Medical industry. 
  • To produce vitamin B12 large number of micro-organisms are used like Yeast, bacteria, and fungi. 
  • Vitamin B12 is mainly produced by submerged fermentation using Propionibacterium shermanii and Pseudomonas denitrificans from different sources.
  • Commercial production of Vitamin B12 is done by Bacillus megatherium, Pseudomonas denitrificans, Propionibacterium shermanii, or Sinorhizobium meliloti.
  • The main advantages of using this microorganism are: that it is easier to obtain and easy to handle its ease of handling. It gives maximum yield and can ferment a variety of cheap raw materials like molasses and starch.

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Process of Industrial Production of Vitamin B12

Step involved in Industrial production of Vitamin B12:

  1. Upstream Process: It involves the Preparation and Sterilization of the medium, Preparation of Inoculum, Making starter culture, etc.
  2. Fermentation Process
  3. Downstream Processing/ Recovery of Products

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Preparation Of Inoculum

  • To obtain a culture of vitamin B12-producing microorganisms the slant culture of S. olivaceus is inoculated in 100-250ml of inoculum medium, contained in an Erlenmeyer flask. 
  • The Seeded flask is incubated on the platform of a mechanical shaker to aerate the system. 
  • This flask culture is then subsequently used to inoculate (2 or 3 successive transfers are made to obtain the required amount of inoculum cultures.)
  • Generally to prepare inoculum Bennett’s agar medium is used. 
  • Component of Bennett’s Agar medium is (g/L) Yeast extract 1.0 Beef extract 1.0, N-Z-Amine A (Enzymatic hydrolase of casein) 2.0, Glucose 10.0, Agar 15.0, D/W 1000 mL, pH 7.3.

 

Preparation and sterilization of Production Medium

  • The production medium consists of carbohydrates, proteinaceous material, and a source of cobalt and other salts. 
  • It is necessary to add cobalt to the medium for maximum yield of cobalamin. 
  • Cyanide is added for the conversion of another cobalamin to vitamin B12. 
  • Sterilization can be done batch-wise or continuously. 
  • In batch sterilization, the medium is heated at 250°F for 1 hour. 
  • In continuous sterilization, the medium is heated at 330°F for 13 min by mixing with live steam.

 

 Fermentation

  • During the fermentation process must maintain the following factors Temperature, pH, Aeration, and Agitation. 
  • Maintaining a temperature of 80°F in the production tank is satisfactory during fermentation.
  • At the beginning of the fermentation process, pH falls due to rapid consumption of sugar, then rises after 2 to 4 due to lysis of mycelium. 
  • pH 5 is maintained with H2SO4 and reducing agent Na2SO4. 
  • Maintain the Optimum rate of aeration is 0.5 volume air/volume medium/min.
  • Add Antifoam agents or Defoaming agents like corn oil, soyabean oil and silicones can be used.
  • During the process maintain aseptic conditions to produce the maximum product. 
  • All the equipment must be sterile and all transfers are carried out under aseptic conditions.

 

Downstream process/Recovery of Vitamin B12

  • During fermentation, most cobalamin is associated with the mycelium present in a bioreactor.
  • This boiling mixture of mycelium at pH 5 liberates the cobalamin quantitatively. 
  • Fermentation broth containing cobalamin is subjected to further process to obtain crystalline B12.
  • Filtration of broth to remove mycelium. The filtered broth is treated with cyanide to bring the conversion of cobalamin to cyanocobalamin. 
  • Adsorption of cyanocobalamin from the solution is done by passing it through adsorbing agents packed in a column. 
  • Cyanocobalamin is then eluted from the adsorbent by the use of an aqueous solution of organic bases or solutions of Na-Cyanide and Na-thiocyanate. 
  • Extraction is carried out by countercurrent distribution between cresol, amyl phenol, or benzyl alcohol and water, or a single extraction into an organic solvent (e.g. Phenol) is carried out. 
  • Chromatography on alumina and final crystallization complete the process.
  • Lastly, we get vitamin B12.

 

Functions of Vitamin B12

The main functions and functions of vitamin B12 are as follows:

  • Promote the development and maturation of red blood cells and prevent pernicious anemia.
  • Vitamin B12 Plays an important role function and development of brain and nerve cells.
  • It helps in the production of the body’s genetic materials DNA and RNA.
  • It also helps to produce energy by converting carbohydrates into glucose.
  • Increase the utilization of folic acid and promote the metabolism of carbohydrates, fats, and proteins.
  • It can promote the synthesis of protein, which plays an important role in the growth and development of infants and young children.
  • Eliminate restlessness, concentrate, and enhance memory and balance.
  • It is an indispensable vitamin for the function of the nervous system and participates in the formation of lipoprotein in the nervous tissue.
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