Screening Of Yak-derived Lactobacillus And Study On Lactobacillus And Inulin On Intestinal Flora And Metabolomics Of Mice

Lactobacillus are part of the natural intestinal flora.Due to their extensive biological function,Lactobacillus have always been the focus of research.Yak is a special species in high altitude areas and has excellent adaptability to extreme environment.However,it is easy to suffer from various diseases caused by intestinal flora and metabolism disorders due to the long-term lack of adequate nutrition.With the high incidence of antibiotic resistance,it is particularly important to find new feed additives that can replace antibiotics.Therefore,Lactobacillus were isolated from yak feces in this study.The probiotics,antibacterial activity,antibiotic sensitivity and antioxidant capacity of the isolated Lactobacillus were evaluated in vitro,and the safety of the isolates to mice was also verified.In addition,the optimal compound ratio of strain L4 and inulin was determined and made into L4-inulin compound,and the effects of the L4 and inulin compound on intestinal microflora and metabolomics of mice were studied by high-throughput sequencing and metabonomics.The results of the research were shown as follows:1.Isolation,identification and in vitro stability of Lactobacillus from yaksFour strains of Lactobacillus sakei,Enterococcus hirae,Pediococcus acidilactici and Weissella confusa were isolated from feces of yaks and named as L4,E5,P7 and W8,respectively.The results of tolerance to acid,bile salt,enzyme and temperature showed that P7 was highly tolerant to acid,bile salt and digestive enzyme,while E5 was more resistant to temperature.The four strains could inhibit the growth of Pasteurella multocida,Escherichia coli(C83902),Staphylococcus aureus(BNCC186335)and Salmonella enteritidis(NTNC L3349),among which L4 had the best inhibitory effect on S.aureus,E5,P7 and W8 also had strong inhibitory effect on E.coli.In addition,the four strains mainly produced organic acids and bacteriocin production to inhibit common intestinal pathogens.The four strains were highly sensitive to seven commonly used clinically antibiotics such as doxycycline,and had no hemolytic activity.2.In vitro antioxidant capacityThe four strains showed certain hydroxyl radical and DPPH scavenging ability,but the scavenging ability was weak,and the scavenging ability of cell suspension was stronger than that of cell-free extracts.The ability of the isolated strains against lipid peroxidation was similar to that of scavenging free radicals with a lower inhibition rate.Besides that,All the four strains had strong reducing activity and showed strong tolerance to hydrogen peroxide.3.Safety evaluation of isolates in miceAfter 14 days of intragastric administration of four strains of Lactobacillus,no diarrhea,death or other disease symptoms were found in the mice,and no obvious pathological changes were observed in the eyes at autopsy.The duodenal section showed that the intestinal villi were intact,the epithelial cells were arranged neatly,and the length of intestinal villi was significantly increased(P <0.01).4.Effect of L4 and inulin compound on intestinal microflora of miceThe growth promoting effect on L4 was most obvious(P < 0.05)when inulin was added at a concentration of 0.8%,so this concentration of inulin was chosen to complex with L4.Based on the high-throughput sequencing results,supplementation of the L4 and L4-inulin compound significantly altered the intestinal microbial composition of mice and increased the diversity of the bacterial community.The intake of L4-inulin compound increased the abundance of beneficial bacteria such as Actinobacterota,Acidobacteriota and Bacteroidota in the intestines of mice.Whereas intake of L4 alone also increased the relative abundance of beneficial bacteria such as Marvinbryantia,Ruminococcaceae,Actinobacteriota and Bifidobacterium in the duodenum.Therefore,the addition of L4 and L4-inulin compound can promote the transformation of intestinal microflora to a beneficial direction in mice.5.Effect of L4 and inulin compound on material metabolism in miceUntargeted LC-MS metabolomics indicated that intake of L4 and L4-inulin compound altered gut metabolites in mice.The expression levels of some fatty acids,glycerophospholipids,amino acids and other metabolites were significantly up-regulated.These metabolites were associated with metabolic pathways involved in Isoflavonoid biosynthesis,Systemic lupus erythematosus,Glycosaminoglycan biosynthesis-chondroitin sulfate / dermatan sulfate,Leishmaniasis,Choline metabolism in cancer and Glycerophospholipid metabolism.The L4 and L4-inulin compound can regulate the levels of Genistein,Glycinol,Phosphatidylserine,chondroitin,and 1-acyl-GPC,thereby enhancing the protective effect on mice.In conclusion,four Lactobacillus strains with probiotic properties,safe and non-toxic,were isolated from yak feces in this study,which could significantly increase the length of duodenal villus,enhance the digestion and absorption capacity,and improve the growth performance of mice.The supplementation of strain L4 and L4-inulin compound could significantly increase the intestinal microflora diversity of mice and transform them into beneficial microflora.In addition,it also promoted the production of beneficial intestinal metabolites such as fatty acids,glycerolipids,and amino acids.Therefore,this study is expected to provide theoretical and experimental gist for the application of yak feed additives in the future.