Study On High-density Culture Of Bifidobacterium Animalis And Preparation Of Highly Survival Rate Lyophilized Bacteria Powder

Probiotics have the functions of regulating the body’s immunity,balancing intestinal flora and promoting intestinal nutrient absorption,and Bifidobacterium animalis,which is isolated from animal intestinal flora and breast milk,is an important source of probiotics.In view of the low yield of Bifidobacterium animalis and the high lethality rate of lyophilized bacteria powder in industrial fermentation production,this project optimizes the composition of Bifidobacterium animalis high-density fermentation medium,constant pH batch feeding culture and main inhibition factors to improve the bacterial body yield,and improves the survival rate of bacterial powder by optimizing the lyophilized protective agent.(1)Using MRS medium as the base medium and the number of viable bacteria as the index,change the composition and addition amount of the basal medium,and on this basis,optimize the composition of the medium by the response surface,and increase the density of Bifidobacterium animalis 111.The results showed that the optimized medium formula was as follows:beef powder 53.58 g/L,yeast impregnation powder 20.33 g/L,glucose 39.83 g/L,potassium dihydrogen phosphate 2.00 g/L,magnesium sulfate 0.20 g/L,manganese sulfate 0.12 g/L,Tween-80 1.00 mL/L,cysteine 1.00 g/L,under these conditions,the viable bacterial count reached 6.50 × 109 CFU/mL,compared with MRS basal medium viable bacteria 2.13×108 CFU/mL increased by 29.81.(2)Taking constant pH batch feeding culture and the number of viable bacteria as the index,the composition ratio of feed solution was studied,and the factors affecting proliferation were mainly inhibited,and the results showed that the optimal pH was 6.0,the feeding combination was glucose 20 g/L,yeast impregnation powder was 7.5 g/L,beef impregnation powder was 20 g/L,the optimal carbon-nitrogen feeding ratio was 1:1.5,and the bacterial body concentration reached 2.65×1010 CFU/mL;While increasing the bacterial density,it was found that the most important inhibitory factor under the condition of constant pH batch feeding culture was the accumulation of organic acids,which led to an increase in osmotic pressure and inhibited the proliferation of bacteria,which provided theoretical reference for the next step of improving bacteria.(3)In order to increase the viable number of Bifidobacterium animalis 111 in lyophilized bacterial powder,the survival rate of Bifidobacterium animalis in the bacterial powder was taken as the index,and the centrifugation conditions,the fusion ratio of sludge and protective agent,the pre-freezing and freeze-drying time were optimized according to orthogonalization,and the effects of the rehydration conditions and storage temperature and timing of the bacterial powder on the survival rate of Bifidobacterium animalis 111 bacterium powder were optimized.The results showed that the optimal centrifugation conditions were 6000 rpm for 20 min,the fusion ratio of sludge and protective agent was 2:3,prefrozen at-80℃ for 2 h,dried at40℃ for 24 h,the optimal protective agent combination was 5.00%maltodextrin,6.00%trehalose,0.05%ascorbic acid,0.50%sodium glutamate,1.00%glycerol,the viable number of lyophilized bacteria powder was 1.38×1012CFU/g,and the highest survival rate of bacterial powder could reach 97.36%.Compared to the optimization,it has improved by 41.28%.(4)In order to study the effect of organic acids on the activity of lyophilized bacterial powder during the preparation of bacterial suspension,the survival rate of Bifidobacterium animalis 111 in bacterial powder was taken as an index,and the effects of preacid stress treatment,bacterial suspension fusion time,pH of bacterial suspension and different treatments of bacterial suspension on the survival rate of Bifidobacterium animalis 111 powder were explored by single factors.The results showed that the fermentation broth was centrifuged at 8℃ for 5 h and then centrifuged,the pH of the bacterial suspension was 6.5,the bacterial sludge was washed twice with sterile water,and the bacterial suspension was fused at 4℃ for 30min,so that the activity of bifidobacterium animal 111 lyophilized bacteria powder could reach 98.60%.