Optimization Of Culture Medium And Fermentation Processes For Two Kinds Of Fish Bacterial Vaccines

Vibrio scophthalmi and Edwardsiella piscicida are two extremely harmful marine fish pathogens,which have caused huge losses to the marine fish farming industry for a long time.The use of fish vaccines as a substitute for antibiotics in the prevention and control of fish diseases has gradually been valued by the industry.Our laboratory has developed inactivated V scophthalmi vaccine and live attenuated E.piscicida vaccine against the two pathogens in previous researches,but the low cell density in the fermentation of the two pathogens affects the promotion and use of the vaccines.In this study,two optimized culture mediums and fermentation process were obtained by optimizing the culture medium and fermentation process,which increased the amount of bacteria but didn’t affect the immunogenicity,thus solved the demands for vaccine preparation.Firstly,the culture medium and fermentation process of Vibrio scophthalmi MAVS001 were optimized,a suitable medium for cell culture was selected as the basal medium from a variety of mediums in the laboratory.Subsequently,with the basal medium,the optimal type and concentration of carbon source and nitrogen source were determined through a single factor experiment,and a substance that was beneficial to cell growth was added to the medium through the additive screening experiment,in addition,its optimal concentration was determined.Then,through the Plackett-Burman design,the steepest ascent method,and the response surface methodology,an optimized medium for Vibrio scophthalmi was finally obtained.The formula was:3.2 g/L glucose,16 g/L yeast extract,11.4 g/L tryptone,20 g/L NaCl,1 g/L MgSO4·7H2O,6.8 g/L KH2PO4,1.35 g/LNaOH.The optimization of fermentation conditions showed that 20%liquid volume,3%inoculum volume,28℃,200 r/min could cultivate V.scophthalmi MAVS001 better,reaching the highest OD600 of 6.2 after 10～12 hours of cultivation.Furthermore,the fermentation process of Vibrio scophthalmi MAVS001 was optimized in a 3 L bioreactor.Through the optimizations of feed components and feed rate,a short and simple control process was established,in which the feeding formula is 400 g/L glucose+50 g/L yeast extract+30 g/L tryptone,the initial rate is 15 ml/h,and the rate was increased by 5 ml/h every 1 h,and maintained after 30 ml/h.During the cultivation process,the pH was maintained at about 6.8 by ammonia water and phosphoric acid,the dissolved oxygen was controlled at about 30%by the linkage of the rotating speed and ventilation,and the temperature was maintained at about 28℃.Using this control process to culture Vibrio scophthalmi MAVS001 in a 3 L bioreactor,the cell density could reach up to OD600 of 29.7,which is 51.8%higher than the original constant-rate carbon source feeding process and 381%higher than the shake flask fermentation.Subsequently,referring to the optimized route of V.scophthalmi,the medium and fermentation process were optimized for the live attenuated E.piscicida strain WED.Since the culture effect of the screened wed basal medium has been significantly improved compared with the traditional TSB medium,the complex components of the medium were simplified and optimized,and the final component formula is:7.5 g/L glycerol,12 g/L yeast extract,20 g/L tryptone,8.2 g/L Na2HPO4·12H2O,3 g/L KH2PO4,2 g/L MgSO4·7H2O.The optimized conditions showed that 20%liquid volume,3%inoculum volume,24℃,200 r/min culture for 22～24 h could cultivate the live attenuated E.piscicida strain WED better.The highest OD600 value could reach 13.5,which is approximately 150%higher than that of the traditional TSB medium.Furthermore,in the 3 L bioreactor,through the optimization of feed composition and feed rate,a short and simple operation control process for cultivating live attenuated E.piscicida strain WED was obtained:the feed formula was 200 g/L glycerol+36 g/L yeast powder+60 g/L peptone,the initial rate is 20 ml/h,and the rate was increased by 10 ml/h every 2 h,and maintaind after 60 ml/h.During the cultivation process,the pH was maintained at about 6.8 through ammonia water and phosphoric acid,the dissolved oxygen was controlled at about 30%by the linkage of the rotating speed and ventilation,and the temperature was maintained at about 24℃.Using this control process to cultivate the live attenuated E.pscicida strain WED in a 3 L bioreactor,the cell density could reach up to OD600 of 28.0,which is 83.2%higher than that in the original constant-rate carbon source feeding process and 124%higher than that in the shake flask fermentation.Finally,we evaluated the immune effect of the bacterial vaccine produced by the optimized culture medium and fermentation process,in order to investigate whether the change of culture process will affect the core quality index of the vaccine,two kinds of pathogenic bacteria cultured in a shake flask and a bioreactor were used to prepare the vaccine and conduct an immune challenge experiment on turbot.The results showed that the relative percentage survival(RPS)of inactivated V.scophthalmi vaccine that prepared using the bacteria in the shake flask and the bioreactor were 81.5%and 86.4%,respectively,and there was no significant difference between them.At the same time,the relative percentage survival of live attenuated E.piscicida strain WED were 82.2%and 85.6%,respectively.Therefore,the culture medium and fermentation process developed for V scophthalmi and live attenuated E.piscicida strain WED can not only improve the cell yield,but also maintain the immune protection effect of the vaccine,and achieve the specified quality index.