Effects Of Anoxybacillus Flavithermus From Bovine Colostrum On Bacterial Structure And Immune Function In Intestine Of Newborn Rats

Studies have found that milk contains a large number of microorganisms,including probiotics and pathogenic bacteria.In order to reduce the harm of pathogenic bacteria to young animals,milk is generally pasteurized and then fed to calves.However,the beneficial bacteria in colostrum are an important source of intestinal microorganisms in newborn animals,and the pasteurization process is not conducive to the early establishment of intestinal flora and the early development of intestinal immune system and intestinal digestive system in young animals and may affect its growth and development and intestinal health.Our previous results showed that there were common core bacteria in different cow colostrum,including Anoxybacillus flavithermus with high abundance.these bacteria may play an important role in the early establishment of intestinal flora and the early development of immune system in calves.In order to verify this hypothesis,this paper first analyzed the effect of pasteurization on the structure of milk bacteria,and on the basis of isolation and identification of Anoxybacillus flavithermus from milk,the effects of the bacteria added to milk on the intestinal flora structure and intestinal immune indexes of young rats were studied in order to provide a theoretical basis for the development of new calf beneficial bacteria.Experiment 1 effect of pasteurization on microbial diversity in ordinary milkIn order to explore the effect of pasteurization on the microbial structure of ordinary milk,fresh milk samples were collected,3 samples were not treated,and the other 3 samples were pasteurized.16SrDNA high-throughput sequencing was used to detect the changes of microbial structure and diversity before and after pasteurization.The results showed that pasteurization reduced the number of living bacteria in milk,but had little change in the microbial structure of milk.Experiment 2 Isolation and identification of Anoxybacillus flavithermus from bovine colostrumIn the previous experiment,it was found that Anoxybacillus flavithermus was one of the core microorganisms in bovine colostrum.In order to explore the biological function of the bacteria,we isolated and cultured Bacillus strains from colostrum by traditional isolation and pure culture techniques,and isolated and identified Anoxybacillus flavithermus from colostrum by morphological identification,specific primer amplification and 16S rDNA sequencing.Experiment 3 Effects of Anoxybacillus flavithermus on intestinal microorganisms and immunity in neonatal ratsIn this experiment,the newborn rats were fed with milk at first,the newborn rats were isolated and fed independently every day.It was found that the rats on the 12^th day could be artificially fed away from the mother and grew well.So the follow-up experiments all choose the offspring that are more than 12 d old.In the experiment,14 d old rats and mice were randomly divided into two groups according to the same body weight.One group was supplemented with 8×10⁶CFU Anoxybacillus flavithermus every day,and the other group was blank control group.The daily nutrition of offspring was supplied by pasteurized normal milk.The feeding experiment lasted for 7 d.After feeding,cecal microbial structure,the length of intestinal villi and crypt depth,ileal immune gene expression,ileal mucosal s IgA and total antioxidant function were measured.It was found that the short-term addition of Anoxybacillus flavithermus increased the relative abundance of beneficial bacteria Faecalibacterium and Lactobacillus Royale in the caecum contents of offspring（P=0.025,0.062）,and decreased the relative abundance of pathogenic bacteria Elizabethkingia（P=0.062）.Anoxybacillus flavithermus had no significant effect on the villi and crypt of duodenum and ileum,and the expression of My D88,LYZ,IFN-β,IL-6 and MUC2 genes in the duodenum and ileum of newborn mice（P=0.138,0.374,0.374,0.322,0.278）,and had no significant effect on s IgA secretion of ileal mucosa and total antioxidation function of intestinal mucosa（P=0.167,0.272）,and no significant effect of Anoxybacillus flavithermus on the villi and crypt of duodenum and ileum of newborn mice.The results showed that the addition of Anoxybacillus flavithermus had no adverse effect on the offspring,but the short-term addition could optimize the intestinal flora structure of the offspring..