Gut Microbial Metabolism Of Oat ?-glucan And Its Effect On Intestinal Inflammation In Mice

Oat?-glucan is an important polysaccharide of the plant cell wall,which has been demonstrated to be able to reduce serum total cholesterol concentration and blood glucose level,alleviate constipation and improve cardiovascular health.The digestion and absorption together constitute one of the most important prerequisites for oat?-glucan to maintain the physiological health.However,whether oat?-glucan is degraded in stomach and small intestine has been unclear.Moreover,the interplay between oat?-glucan and gut microbiota are not clarified.Based on these,the present thesis primarily aims to(i)study the digestion and degradation of oat?-glucan in stomach,small intestine,and large intestine,(ii)reveal the potential molecular mechanism of oat?-glucan metabolism in gut,and(iii)clarify the effect of oat?-glucan metabolism by gut microbiota on gut health.The findings showed that oat?-glucan was mainly metabolized by gut microbiota in the large intestine,and the metabolic processes were primarily associated with GH1,GH3 and GH16 in mice and human gut.Moreover,oat?-glucan could alleviate the colitis by modulating gut microbial metabolism both in cell and animal experiments.The present work thus provides a comprehensive understanding of in vivo?-glucan metabolism,and provides reference values for mining of physiological functions and potential for clinical applications.The detailed information was shown below:Firstly,in vitro gastrointestinal tract digestion model and gut microbiota fermentation model were constructed to study the changes of relative molecular weight and spatial structure of oat?-glucan in simulated stomach and small intestine,and examine the degradation of oat?-glucan in simulated gut microenvironment and the effect of oat?-glucan on gut microbiota.The results showed that the relative molecular weight of oat?-glucan was decreased from469983 to 388504 in simulated gastric phase and to 383577 in simulated small intestinal phase,respectively.Moreover,the triple-stranded helix structure of oat?-glucan was also influenced.The in vitro simulated colonic fermentation showed that oat?-glucan could be fermented in the simulated mice and human gut.The oat?-glucan content(10 mg/m L)could be reduced to 3.29 mg/m L in the simulated mice gut and 1.39 mg/m L in the simulated human gut within 3 h of fermentation,and almost completely degraded within 6 h of fermentation.The produced final metabolites of oat?-glucan fermentation in mice and human gut were both short-chain fatty acids(SCFAs),mainly acetate,propionate,and butyrate.It is noteworthy that oat?-glucan could be degraded more quickly in simulated human gut than in simulated mice gut and thus mainly produced propionate while butyrate was especially enriched in simulated mice gut.Secondly,in vitro fermentation was performed by inoculation with fresh feces from different batches of mice and different subjects of human beings,to find out stable responders to oat?-glucan under different microenvironments and systematically analyze the metabolic outcomes and involved metabolic pathways of oat?-glucan in simulated gut.The result showed that,at the genus level,Lactobacillus is the main microbe that is stably enriched by oat?-glucan in simulated mice gut,while Bacteroides is the primary microbe stably enriched by oat?-glucan in simulated human gut.The analysis of metabolic outcomes and relevant metabolic pathways of oat?-glucan fermentation showed that oat?-glucan degradation in simulated mice and human gut was both accompanied by amino acid metabolism and fatty acid biosynthesis.Next,the universal screening method and specific screening method were used to isolate and purify these microorganisms that primarily metabolize oat?-glucan in mice and human gut.These microorganisms were then identified and identified microorganism were inoculated in medium containing oat?-glucan to confirm the metabolic capacity and explore the potential molecular mechanism of oat?-glucan degradation.The results showed that 4 species including 63 strains of Lactobacillus that could metabolize oat?-glucan were obtained from mice gut microbiota,and 9 species including 19 strains of Bacteroides probably having ability to metabolize oat?-glucan were obtained from human gut microbiota.The 4 species of Lactobacillus from mice gut microbiota were all able to degrade oat?-glucan and produce small amounts of reducing sugars.There was no obvious difference in metabolic capacities.The 9 species of Bacteroides from human gut microbiota all possessed the ability to degrade oat?-glucan into reducing sugars,of which Bacteroides xylanisolvens Bac02 and Bacteroides koreensis Bac08 displayed the strongest metabolic capacities.Whole-genome sequencing and genome-wide functional annotation revealed that 2 families of GH1 in mice-derived Lactobacillus and 1 family of GH3 together with 1 family of GH16 in human-derived Bacteroides were responsible for oat?-glucan metabolism.The 4 enzymes and oat?-glucan were combined by hydrogen bonding.Sequence alignment further revealed that the 4carbohydrate hydrolases in the present study were all firstly reported and they were named Lm GH1₁₅₄₂,Lm GH1₁₅₄₉,Bk GH3₄₄₇₉,and Bk GH16₃₂₆₃,respectively.Then,the downstream metabolic pathways of gut microbial metabolism of oat?-glucan were examined and the effect of polysaccharide structures on SCFAs biosynthesis was explored.After in vitro polysaccharides fermentation,the content of SCFAs and organic acids were measured,and the relationship between SCFAs and organic acids was analyzed to further describe the biosynthesis pathways of SCFAs.Then the biosynthesis pathways of SCFAs formation by various fiber polysaccharides fermentation were compared to clarify the effect of polysaccharides structures on SCFAs biosynthesis.16S r RNA sequencing was adopted to study microbial community before and after the fermentation,and correlation analysis was performed to determine the role of microbiota in SCFAs biosynthesis.The results showed that 7 organic acids including G6P,F6P,PGA,pyruvate,KGA,malate,and succinate were involved in the biosynthesis of SCFAs.The SCFAs biosynthesis pathways were not only related to the types of monosaccharides but related to the chain structures of fiber polysaccharides,including glycosidic linkages and oligosaccharide units.Moreover,gut microbiota mediated the SCFAs biosynthesis.Finally,the effect of oat?-glucan metabolism on intestinal inflammation was evaluated.LPS-induced macrophage inflammatory model was established to assess the effect of the metabolites produced by gut microbial metabolism of oat?-glucan on inflammation in Raw264.7 cells.DSS-induced colitis mice model was further established to study the effect of oat?-glucan on colitis in mice.The levels of proinflammatory cytokines and tight junction proteins,gut microbial community structure,SCFAs contents,gut metabolic profiles were examined and correlation analysis was performed to assess the role of gut microbial metabolism of oat?-glucan in colitis remission.The results showed that the metabolites produced by 4 species of Lactobacillus probably inhibited LPS-induced inflammatory responses and the metabolites produced by 9 species of Bacteroides all significantly suppressed LPS-induced inflammatory responses.Intragastric administration of oat?-glucan(800 mg/kg/day)remarkably ameliorated clinic symptoms for colitis,colon tissue damage,cell apoptosis,inflammatory activation,and mucosal barrier function in colon tissue,regulated gut microbial community structure,and significantly increased SCFAs contents in colitis mice,especially acetate,propionate,and butyrate.Simultaneously,intragastric administration of oat?-glucan also caused changes in gut metabolic profiles and pathways in colitis mice.Remarkably,the levels of acetate and propionate were significantly related to colitis amelioration,in contrast,other intestinal metabolites affected tight junction proteins levels via regulating the expression levels of inflammatory cytokines.