Propionibacterium Fermentation Medium Optimization Based On HPLC Analysis

Propionibacterium shermanii is a commonly used VB12production strain byanaerobic fermentation. Amino acids, as very important organic nitrogens and growthfactors in the P. shermanii fermentation process, can effectively promote the growth ofmicroorganisms and biosynthesis of VB12. Using high performance liquid chromatographymethods, the contents of free amino acids, organic acids and VB12were determinated inthe P. shermanii VB12fermentation process. By analysing the changes of these substances,several key amino acids affecting VB12yield were found. In addition, adding exprimentsof amino acids were determined by the utilization of amino acids in the fermentationprocess. Finally, the yields of VB12were increased.The main research results are as follows:(1) Using DNFB as pre-column derivatization reagent, a reversed-phase highperformance liquid chromatography method was established to determine free amino acidsin P. shermanii fermentation broth. When the chromatographic conditions were optimized19kinds of amino acids were separated well within30minutes. Free amino acids in the P.shermanii fermentation broth by this method were determined, and fine results indicatedthat the method was accurate, reliable and met analytical need.(2) Using cationic exchange resin to pretreat fermentation broth, a HPLC method wasestablished to detect several organic acids. Resin pretreatment effect was remarkable, andmiscellaneous peak numbers were significantly reduced. Furthermore, treated sampleswere directly detected by HPLC. Acetic acid, propionic acid, succinic acid could bedetected simultaneously and these organic acid components had no loss basically.(3) A direct method of determinating adenosylcobalamin in the fermentation brothwas established by liquid chromatography. The intracellular adenosylcobalamin wasextracted by solvent extraction, and the extracts could be directly determined by HPLCwithout transformating cyanocobalamin.(4) Based on HPLC, the contents of amino acids, organic acids, VB12in thefermentation process were determinated. The whole fermentation process was analyseddeep by the determination results and indicated that several amino acids in thefermentation process were used in priority. One-factor experiment showed that the yieldsof VB12could be increased10%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 VB12yield were Tyr＞Asn＞Asp＞Met. Finally, VB12yield was increased by17%than that of the control group and addedamounts of the four amino acids Tyr, Asn, Met, and Asp were600μg/mL,400μg/mL,600μg/mL and400μg/mL respectively.