Determination And Extraction Of Vitamin B₁₂ In Industrial Fermentation

Vitamin B₁₂ is widely used in medicine and food additive, which is produced mainly by fermentation. In this paper, a new method to determine vitamin B₁₂ in industrial fermentation was studied. VB₁₂ extraction from fermentation broth was also researched. The study in this paper was based on industry applications, which have special valuable reference to improvement of VB₁₂ fermentation and extraction.1. Method to determine VB₁₂ in fermentation broth directlyVitamin B₁₂ is produced by fermentation. Hydroxycobalamin deoxyadenosylcobalamin and methylcobalamin are mainly products by fermentation. In this paper, VB₁₂ was produced by Propionibacterium freudenreichii fermentation, cells were collected by centrifugation. Sample was analyzed by HPLC after pretreatment. Sample can be well separated by the method in 25min. The data all meet the needs of methodology. The method is simple, rapid and accurate, which can be used in the fermentation process of Vitamin B₁₂. The method can also provide help on directive breeding of. Propionibacterium freudenreichii.2. Research on cobalamins light stability and a rapid method to determine VB₁₂The light stability of cobalamins was studied. As a result, Hydroxycobalamin and cyanocobalamin are stable in experimental time. The decomposers of deoxyadenosylcobalamin and methylcobalamin is hydroxycobalamin, the percentage of molecule transform is up to 100%. Photolysis with different lamp-house was carried out. Photolysis velocity of deoxyadenosylcobalamin and methylcobalamin is proportionable with light intensity.On the base of the above method to direct determine VB₁₂ in fermentation broth using HPLC, A new determination method has been established. Sample was photolyzed after cell disruption, and then cobalamins in sample were transformed to hydroxycobalamin entirely. The concentration of VB₁₂ was obtained in form of hydroxycobalamin. The new determination method is simple and accurate, and do not need strict condition.3. Four methods to extract VB₁₂ from Propionibacterium freudenreichii by cell disruptionFour methods were applied to disrupt cell for extracting VB₁₂, including ultrasonication, boiling, microwave heating, high-pressure homogenization. The kinetics of cell disruptionwas studied. Experiment showed that methods of boiling and microwave heating can get high extraction ratio, and protein released from cell can be removed in operating process. The method of boiling is suitable for industry production, because it needs simple equipment and can be managed in large scale. The microwave heating method is rapid and efficient, can be used in HPLC determination process of Vitamin B12.4. Separation and purification hydroxycobalamin from fermentation broth by macroporous adsorption resinsCobalamins in fermentation broth transformed to hydroxycobalamin entirely by photolysis. Four different macroporous adsorption resins were studied to research the processes of static adsorption and desorption for hydroxycobalamin. Meanwhile influence of pH and temperature on adsorption and desorption were determined. The type of CAD45 resin has excellent performance in static adsorption and desorption for hydroxycobalamin. Optimal dynamic conditions were investigated for resin CAD45 in process of adsorption and desorption, and kinetic of which was discussed. As a result, a primary process for separation and purification hydroxycobalamin from fermentation broth using CAD45 resins was established.