Study On Stable Butyric Acid-producing Bacterial Preparation And Its Characteristics

Butyrate-producing bacteria can metabolize butyrate to maintain animal intestinal health,which is one of the hotspots in the field of microbiology in recent years.Microecological agents can replace antibiotics in aquaculture and livestock breeding to avoid drug residues and improve the environment.At present,there are many types of microecological preparations in the market,but there are many shortcomings such as poor stability and short shelf life.Therefore,it is of great significance for the development of microecological preparations to screen out a strain of bacteria with high stability and high yield of butyric acid and explore the effects of different drying processes on its protection.In this paper,Clostridium butyricum TK520 was screened from 20 samples,and its growth characteristics and tolerance were studied.The spore rate was 77.1%and the butyric acid content in the fermentation broth was 4.97 g/L.The optimized fermentation process was as follows:fermentation time 48 h,peptone 3%,soluble starch 2%,glucose 1%,potassium dihydrogen phosphate 0.05%,manganese sulfate 0.02%,magnesium sulfate0.02%,calcium carbonate 1%as the medium,the spore rate reached 95%,and the number of viable bacteria was 6.92×10~8 CFU/ml.Secondly,protective agents were added to reduce the damage of living bacteria in the drying process and to prolong the shelf life of the agents.Through the response surface experiment,the optimal vacuum freeze-drying protective agent was determined as follows:composite protective agent(corn starch:skim milk powder:sorbitol=10:1:1)16%,arabic gum 1.5%,calcium lactate 1%.After freeze-drying,the viable count of bacterial powder was 4.79×10~9 CFU/g,and the viable count was 4.13×10~9 CFU/g after storage at 37℃for28 days.The freeze-dried survival rate was 67.5%,and the storage survival rate was 86.21%,which was close to the predicted results.When C.butyricum TK520 was prepared by spray drying method,the response surface experiment results showed that the optimum formula of spray drying protective agent was as follows:(corn starch:skim milk powder:calcium lactate:sorbitol=5:5:1:1)12%,sodium alginate concentration 0.2%,the number of viable bacteria after spray drying was 2.88×10~9 CFU/g,and the number of viable bacteria after storage at 37℃for 28 days was 1.89×10~9 CFU/g.It was proved that the drying survival rate of spray drying bacteria was 49.86%,and the storage survival rate was66.02%,which was close to the predicted value.Finally,the differentially expressed genes of the spray-dried inoculants stored at different temperatures were analyzed by using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes.The results showed that the differentially expressed genes were mainly enriched in nucleotide,enzyme activity,carbohydrate,cofactor and vitamin metabolism pathways.In summary,the stability of C.butyricum TK520 preparation by vacuum freeze-drying method was better than that by spray drying method.This study solved the problems of short shelf life and inconvenient transportation of bacterial liquid,and provided more scientific basis for the development of related microecological preparations in the future.