Propionibacterium Fermentation Process Analysis And Optimization Of Fermentation Medium

Vitamin B12, a water soluble vitamin, is necessary to maintain normal metabolism, and it is obtained by bacterial fermentation method. The main bacterial strains of producing Vitamin B12 are Propionibacterium shermanii, Propionibacterium frudenreichii and Pseudomonas denitrificans at present. In the P. shermanii VB12 fermentation process, the synthesis of vitamin B12 requires a variety of amino acids. As important precursors, amino acids can promote cell growth and biosynthesis of vitamin B12.But different kinds and concentrations of amino acids have different effect on P. shermanii, so it is significant to promote the products by optimizing the additive strategy of amino acids in the fermentation process.Using high performance liquid chromatography methods, the contents of free amino acids, organic acids and VB12 were determinated in the P. shermanii VB12 fermentation process. By analysing the changes of these substances, several key amino acids affecting VB12 yield were found. In addition, adding expriments of amino acids were determined by the utilization of amino acids in the fermentation process. Finally, the yields of VB12 were increased.The main research results are summarized as follows:(1) Using DNFB as pre-column derivatization reagent, a reversed-phase high performance liquid chromatography method was established to determine free amino acids in P. shermanii fermentation broth. When the chromatographic conditions were optimized 19 kinds of amino acids were separated well within 30 minutes. Free amino acids in the P. shermanii fermentation broth by this method were determined, and fine results indicated that the method was accurate, reliable and met analytical need.(2) Using cationic exchange resin to pretreat fermentation broth, a HPLC method was established to detect several organic acids. Resin pretreatment effect was remarkable, and miscellaneous peak numbers were significantly reduced. Furthermore, treated samples were directly detected by HPLC. Acetic acid, propionic acid, succinic acid could be detected simultaneously and these organic acid components had no loss basically.(3) A direct method of determinating adenosylcobalamin in the fermentation broth was established by liquid chromatography. The intracellular adenosylcobalamin was extracted by solvent extraction. This method was simple, accurate and reliable, and could be applied to analyze the yield changes of vitamin B12 in the P. shermanii fermentation broth.(4) Based on HPLC, the contents of amino acids, organic acids, vitamin B12 in the fermentation process were determinated. The whole fermentation process was analysed deep by the determination results and indicated that several amino acids in the fermentation process were used in priority. One-factor experiment showed that the yields of vitamin B12 could be increased 10% by adding alone four amino acids Tyr, Asn, Asp, and Met. An orthogonal experiment was desighed by the results of the one-factor experiment and indicated that effect sizes of the four amino acids to vitamin B12 yield were Tyr>Asn>Asp>Met. Finally, vitamin B12 yield was increased by 17% than that of the control group and added amounts of the four amino acids Tyr, Asn, Met, and Asp were 600?g/mL,400?g/mL,600?g/mL and 400?g/mL respectively.