Gut Microbiota Structure Analysis And Study On The Isolation,Identification And Biological Characteristics Of Probiotics From Tibetan Sheep

Tibetan sheep is one of the important livestock in the Qinghai Tibet Plateau.Its good excellent adaptability is closely related to gut microbes.Therefore,in order to study the structure and functional characteristics of Tibetan sheep intestinal microflora and obtain probiotic strains from Tibetan sheep.16 S rDNA amplicon sequencing technology and macrogenomic sequencing technology were used to analyze the diversity,structure and functional characteristics of microorganisms in the duodenum,ileum,cecum,colon and rectum of Tibetan sheep.At the same time,the dominant strains of intestinal tract were screened by selective medium,and the biological characteristics were studied.The results are as follows:1.16 S rDNA amplicon sequencing showed that 51 phyla and 571 genera were annotated in the intestine of Tibetan sheep.The dominant bacterial phyla are Firmicutes,Bacteroides and Proteobacteria,and the dominant species are unidentified＿ruminococcaceae,methanobrevibacter and Bacteroides.At the phylum level,the relative abundance of Bacteroidetes and spirochetes in large intestine was higher;At the genus level,the relative abundance of Candidatus＿saccharimonas in the small intestine increased significantly;The relative abundance of unidentified＿prevotellaceae,Bacteroidetes,unidentified＿Spirochaetaceae and alistipes in large intestine increased significantly.2.A total of 120 631.63 Mbp of raw data were obtained by macrogenome sequencing.After de redundancy,1 951 147 ORFs were used for subsequent analysis.According to the similarity of intestinal microbial structure and function,the intestinal samples were divided into two groups: duodenum and ileum were small intestine group(SI),cecum,colon and rectum were large intestine group(LI).At the phylum level,the dominant bacteria in the small intestine are Firmicutes and Euryarchaeota,and the large intestine is mainly Bacteroides and Firmicutes.At the genus level,Clostridium and methanobrevibacter are the dominant bacteria in the small intestine,and Bacteroides and Clostridium are the main bacteria in the large intestine.At the same time,the gene abundance annotated to metabolism pathway is the highest,replication,recommendation and repair are the dominant functions,and abundant resistance genes such as glycoside hydrolase and tetracycline antibiotics are annotated.3.Nine dominant strains were isolated by traditional culture method,including Lactobacillus sakei,Lactobacillus mucosae,Lactobacillus curvatus,Bacillus subtilis,Bacillus velezensis,Bacillus safensis,Enterococcus Faecium,Enterococcus hirae and Enterococcus faecalis,which have good biological characteristics.Among them,The diameter of the inhibition circle of Bacillus velezensis on Clostridium perfringens was 34.8mm.Isolated strains were sensitive to common antibiotics such as cefoperazone,amikacin and erythromycin and had no hemolytic activity.The safety test results showed that intragastric administration of the isolated strain had no significant effect on the hematology,serum biochemical indexes and organ index of mice,and the isolated strain had no pathogenicity.In conclusion,this experiment provides a theoretical basis for the ecological breeding of Tibetan sheep and provides candidate strains for the research and development of Tibetan sheep probiotics.