Mutation Breeding And Fermentation Technology Of Clostridium Butyricum Superior Strains

In this study,a Clostridium butyricum strain LXKJ-1 provided by Hubei Green Snow Technology Co.,Ltd.was selected as the research object,Using ARTP(atmosperic room temperature plasma)plasma mutagenesis technology,a mutant strain LXYB-2 of C.butyricum with strong ability to inhibit butyric acid and high fermentation level was obtained.In this study,single factor and response surface methodology were used to optimize the fermentation process of strain LXYB-2,and its application potential was simultaneously examined:The method of viable count was used to evaluate its stress resistance.At the same time,the antibacterial effect in vitro was determined by the Oxford cup method,and the acid production capacity of the fermented liquid was determined by gas chromatography.The research results of the project are as follows:1.Using ARTP plasma mutagenesis technology,8 strains of positive mutants were obtained by mutation breeding of Clostridium butyricum LXKJ-1.Among these mutations,the highest mutation rate was detected at 90s,reaching 15.46%.The positive mutant strain had the highest ability to withstand sodium butyrate up to 32 g/L,and its ability to resist sodium butyrate was greatly improved compared to the initial strain.Two strains of high-yield strains LXYB-2 and LXYB-9 were screened through further fermentation screening of sodium butyrate-resistant strains.The fermentation levels reached 8.3×10~8CFU/mL and 9.33×10~8 CFU/mL,respectively.Further,we studied the genetic stability of these two high-yield strains and found that there was no significant change in the number of viable cells in the 5th passage of LXYB-2.From this we obtained a strain LXYB-2 which is resistant to butyric acid,stable in passage,and high in viable cell count.2.The viable bacteria count method was used to evaluate the resistance of the two strains to high temperature,artificial gastric juice,artificial intestinal fluid and choline,and the effect of antibacterial effect in vitro was evaluated by Oxford cup method.The results showed that:The tolerance of the two strains at 90°C for 5 minutes was significantly different(p<0.05).There were no significant differences between the two strains in artificial gastric and intestinal fluid tolerance tests(p>0.05).In the bile salt tolerance experiment,when the bile salt mass fraction reached 0.4%,the bile salt tolerance was significantly different(p<0.05).In vitro antibacterial test:The two strains had good antibacterial activity against 9 intestinal pathogens,but the antibacterial activity results were significantly different(p<0.05).3.Based on the single factor experiment,this experiment used Design Expert software to conduct response surface analysis to determine the optimum fermentation medium and fermentation process conditions.The optimum fermentation medium:corn flour 4%,ammonium sulfate 2.49%,yeast extract0.5%,calcium carbonate 1%,sodium acetate 0.6%,dipotassium hydrogen phosphate 0.15%,magnesium sulfate 0.06%,manganese sulfate 0.05%;The optimum fermentation conditions were as follows:initial pH of fermentation:8.0,Finally,the number of viable cells of strain LXYB-2 reached 1.211×10~9 CFU/mL,an increase of 44%compared to the unoptimized.4.Gas chromatography was used to evaluate the acidogenic ability of the mutagenized strain LXYB-2.The results showed that the content of butyric acid in strain LXYB-2 reached 53.93 mmol/L,the content of acetic acid reached 33.09 mmol/L,the contents of butyric acid and acetic acid were increased by 108.3%and 117.4%respectively compared with the original strains.