Characterization Of Gastrointestinal Bacterial Community And The Probiotic Properties Of Lactobacillus In Yaks

The gastrointestinal tract of animals is a complex micro-ecosystem with the various microbiome which is essential for the host's healthy development.The gastrointestinal systems of animals are affected by environmental and other factors.Yak is a unique specie of ruminants in the Qinghai-Tibet Plateau,China.It has excellent characteristics such as diseases resistance,roughage-resistance,with remarkable adaptability.Its microbiome plays an important role in maintaining intestinal homeostasis.This study revealed the yak characteristics of gastrointestinal bacterial community,and the analysis of Lactobacillus potential with the mechanism of alleviating intestinal inflammation.Our study provide the theoretical basis for the clinical application of the Lactobacillus.The major studies are:1 Characterization the bacterial community composition of the yak's gastrointestinal tractThis study was conducted to investigate bacterial community composition and their functions in different gastrointestinal segments of yaks by Illumina Mi Seq high-throughput sequencing.The results showed that the forestomach's diversity is significantly higher than the intestinal tract(p<0.05).At the phylum level,the relative abundance of Firmicutes and Bacteroidetes was dominant(p<0.05).While at the genus level,the relative abundance of unidentified?Ruminococcaceae and unidentified?Prevotellaceae in the forestomach was significantly higher(p<0.05);the relative abundance of unidentified?Enterobacteriaceae in the jejunum intestine was significantly higher than other segments(p<0.05);the relative abundance of unclassified?Clostridiales in the jejunum and ileum was significantly higher(p<0.05).The relative abundance of Alloprevotella and Bacterodies in the large intestinal were significantly higher(p<0.05).The relative abundance of the genes involved in carbohydrate metabolism,amino acid metabolism,and energy metabolism was higher in the forestomach(p<0.05).These finding suggested a significant difference in the bacterial community composition and function in yak's gastrointestinal tract.2 Isolation,identification,and probiotic characteristics of yak's Lactobacillus The gastrointestinal tract of yaks was selected for better probiotic strains.Out of 61 strains of Lactobacillus in the gastrointestinal tract,15 strains screened out as a candidate strain through acid and bile salts tolerance,artificial stimulation of gastrointestinal fluid,adhesion,hydrophobic,antibacterial,medicinal sensitivity,antioxidants,hemolytic activity,and the safety evaluation.Lactobacillus reuteri YLR001 and Lactobacillus fermentum YLF016 were screened out with good probiotic properties and safety.The results showed that the two strains could withstand the gastrointestinal environment with a good adhere ability to the intestinal mucosa.Also,their probiotic characteristics were superior to as compared to other strains.3.Complete genomic analysis of L.fermentum and L.reuteri from yaksThe genomic characteristics and potential functional genes of YLR001 and YLF016 were analyzed by whole-genome sequencing.The results showed that the YLF016 genome contained 1 chromosome(length 2094354 bp),containing 2130 coding sequences,58 t RNA genes and 15 r RNA operon.The YLR001 genome contains one chromosome(length 2242943bp)and three plasmids,containing 2463 coding genes,77 t RNA,18 r RNA operon and 1 s RNA.There were acid-tolerance,bile-tolerance,antioxidant,and adhesive-related genes in the lactobacillus genome that make those strains more resistant and adaptable.4.The alleviating effect of Lactobacillus on intestinal inflammation induced by LPS in miceTo study the properties of Lactobacillus in intestinal inflammation and mucosal injury.We compared the prevention effect of single strain and multi-strain mixture by lipopolysaccharide in order to create an intestinal inflammation model.The results showed that YLR001 and YLF016 have preventive effect on LPS-induced intestinal inflammation and reduced pro-inflammatory cytokines gene expression(IL-1?,IL-6 and TNF-?).Western blot results further showed that yaks Lactobacillus could significantly inhibit the up-regulation of TLR4 and the phosphorylation levels of I?B? and NF-?Bp65by LPS stimulation with activation of Nrf2 signaling pathway.It also increased the expression levels of antioxidant proteins(HO-1 and NQO1).Simultaneously,it increased the expression of intestinal tight junction proteins(Occludin,Claudin-1 and ZO-1).These results indicated that yaks Lactobacillus could inhibited the activation of TLR4-NF-?B,and may reduce the expression of pro-inflammatory cytokines.Thereby it activates Nrf2 antioxidant pathways to protect intestines from inflammatory injuries.Overall,the above results indicate that the Lactobacillus mixture-strain was more effective in alleviating intestinal inflammation and mucosal damage than single strain.5.Metabolomics study of Lactobacillus mixture in the intervention of LPS intestinal inflammation modelUntargeted metabolomics technology was used to determine the metabolic pathways related to intestinal inflammation.Combined with multivariate statistical analysis,the differential metabolites and potential metabolic pathways of mix-Lactobacillus alleviating intestinal inflammation in mice were studied.For the control and LPS model groups,PLS-DA pattern recognition showed that the samples of the two groups were significantly separated,indicate the significant change in model group.The results showed 71 metabolites between Con and LPS.Out of these,38 were up-regulated and,33 were down-regulated.While,between LPS and PLPS,58 were up-regulated,and 33 were down-regulated.Moreover,metabolic pathway analysis found that different metabolites mainly involved in Pantothenate and Co A biosynthesis,Sphingolipid,and Glycerophospholipid metabolism pathways.On the basis of the above study,we selected two better strains(Lactobacillus fermentum YLF016 and Lactobacillus reuteri YLR001)through screening of probiotics and whole genome sequencing that proved safe and strong resistant.Overall,this study revealed the regulation mechanism of Lactobacillus in the alleviation of LPS induced inflammatory diseases.Our study provides theoretical basis for utilization and the development of probiotic.