Effects Of Early Administration Of Lactobacillus Reuteri Isolated From Yaks On Host Intestinal Microbiome

The gut microbiota and its metabolic activity are essential for maintaining the dynamic balance of gut microecology and host health,of which the role of probiotics has been emphasized.The host microbiota is a large microbial assemblage that can be influenced by various factors,including diet,survival environment,and disease status.As a unique economic species,yak lives on the Tibetan plateau in China,facing a high incidence of intestinal diseases due to poor gut microbial development or other factors during the"window"of disease susceptibility.Early feeding management to promote the development of the digestive system,improve digestive function and reduce the susceptibility to intestinal diseases is an important foundation for the successful development of the yak industry.The current study screened one Lactobacillus reuteri strain LR601-1 with good probiotic potential and further emphasized its physiological benefits on host early development of growth and microbiota/metabolic profiles.The details experiments and main results are as follows:1.Biological characterization of Lactobacillus isolated from yaksA total of 10 strains of Lactobacillus,including 2 strains of Lactobacillus mucosus,2strains of Lactobacillus mucosae,3 strains of Leuconostoc pseudomesenteroides and 3strains of Lactobacillus reuteri,were isolated from the intestine of healthy yaks in the study.Next,Lactobacillus reuteri LR601-1 with good probiotic potential was screened by a series of tests,including antibacterial property test,acid and bile salt tolerance,antibiotic susceptibility,hemolysis,hydrophobicity,self-agglutination,and Caco-2 cell adhesion.Further,the strain was subsequently evaluated for safety by oral toxicity test in mice model,showed a safe and effective approach with good probiotic characteristics.2.Whole genomic sequencing to analyze the genetic information of Lactobacillus reuteri LR601-1The experiment analyzed the probiotic potential and biological properties of Lactobacillus reuteri LR601-1 by whole genome sequencing.We annotated five major classes of carbohydrate-active enzymes in the genomic information of LR601-1,i.e.,glycoside hydrolases（GHs）,glycosyltransferases（GTs）,carbohydrate esterases（CEs）,auxiliary oxidoreductases（AAs）and carbohydrate-binding domains（CBMs）.In addition,abundant enzymes belonging to these five major classes of carbohydrate-active enzymes were extensively annotated,which was important for a yak to utilize scarce forage resources efficiently.The genes encoding lysozyme enzymes,as well as enzyme activities,were detected.The wide KEGG function had been annotated in the LR601-1 genome,including amino acid biosynthesis,carbohydrate metabolism,glycolysis/gluconeogenesis,and pyruvate metabolism,and their role in promoting physiological benefits to the host.In addition,the genome of LR601-1 had no antibiotic resistance genes,and there was no risk of antibiotic resistance or the transmission of resistance genes.In conclusion,the LR601-1 genome had an ability as a potential probiotic characteristic with many genetic bases that are beneficial to the host’s life.3.Effects of early supplementation of LR601-1 on intestinal development in neonatal mice modelThe in vitro tests and genomic information demonstrated the good probiotic potential of LR601-1.So,we hypothesized LR601-1 as an excellent probiotic potential that may contribute to physiological benefits of the host intestine.Hence,we designed the following experiments to evaluate the physiological benefits of early supplementation of LR601-1 on the growth performance of neonatal mice,i.e.,the development of intestinal tissues,the proliferation and differentiation levels of intestinal epithelial cells,the expression levels of intestinal tight junction proteins,the production of secretory immunoglobulin A（s Ig A）and the maturation of the intestinal microbiota.The results showed that early supplementation with LR601-1 significantly increased weight gain,regulated intestinal s Ig A concentration,and improved serum antioxidant levels in developing mice from 2^ndweek of their age.In addition,it promoted the proliferation and differentiation of intestinal tissue by regulating the Wnt/β-catenin signaling pathway and significantly increased intestinal villus and crypt length.Furthermore,it increased the species richness and species diversity of intestinal microorganisms,improved the enrichment of beneficial intestinal bacteria,and further modulated the phenotype of the functional microbiome.4.Effects of early supplementation with LR601-1 on susceptibility to intestinal disease in adult mice modelGiven the physiological benefits of early LR601-1 supplementation on neonatal mice and the known knowledge that early microbial exposure on the shaping of the host’s gut microbiota later in life,we hypothesized that the physiological benefits and protective effects of early LR601-1 supplementation on the host could persist into later life.Therefore,further experiments were designed to study the effects of early supplementation of LR601-1 on the susceptibility of adult mice to intestinal damage,gut microbiota,and disruption of the gut metabolic profile under intestinal pathogens challenge.The results revealed that early supplementation with LR601-1 significantly inhibited decreased body weight,antioxidant capacity,fecal Lactobacillus viable count,and s Ig A concentration caused by pathogens challenge in adult mice（p<0.05 or p<0.01）.After the E.coli challenge,the disruption of the normal structure of the lamina propria and intestinal villi,and the infiltration of inflammatory cells accompanied by a significant decrease in the expression levels of tight junction proteins were observed in the small intestine.Meanwhile,early supplementation with LR601-1 alleviated these pathological changes to some degree.In addition,the LR601-1 maintained the enrichment of beneficial bacteria that promote intestinal health,competitively reduced the high abundance of pathogenic bacteria,and improved gut metabolic patterns.These results supported the effectiveness of early supplementation with LR601-1 to promote functional maturation and long-term health consequences for adults.5.Effects of early supplementation with LR601-1 on growth performance and gut microbial development in yaks’calvesThe physiological benefits of early supplementation with LR601-1 detected in model animals（mice）motivated us to perform field trials.The current study delves into the performance of LR601-1 as a beneficial managemental strategy,further highlighting their impact on growth performance,serologic investigation,gut microbiota,and metabolic profiling in yaks’calves.The results showed that supplementation with LR601-1positively affected the growth performance of yak calves with an increase in body weight of 3.16 kg（p<0.01）compared to the control groups.In addition,it could potentially improve serum antioxidant capacity and biochemical properties.Although all yaks were healthy,health-threatening microorganisms were enriched in the gut of yaks in the control groups.Furthermore,beneficial microorganisms gradually colonized the gut,which assembled into a more mature microbiota driven by the early supplementation strategy.The early supplementation with LR601-1 also resulted in significant changes in the intestinal metabolic profile,i.e.,metabolite concentrations and metabolic patterns,which include protein digestion and absorption,vitamin digestion and absorption,and biosynthesis of secondary metabolites.In conclusion,the Lactobacillus reuteri LR601-1 has strong antimicrobial activity and good probiotic potential,proving safe in vivo and in vitro trials.The early supplementation strategy with LR601-1 showed good physiological benefits in both model animals（mice）and yak calves,which could be used as a candidate strain with good probiotic potential for further clinical practices.The current study could contribute to early efficient and healthy feeding management strategies for the yak industry.