Vitamin B12 (Cobalamin) Production by Fermentation - YB Study

Vitamin B12 (Cobalamin) Production by Fermentation

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).


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.


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.


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


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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