Fermentation Characteristics Of Butyric-FOS And Its Effects On DSS-induced Inflammatory Bowel Disease In Mice

Inflammatory bowel disease(IBD)is a chronic idiopathic intestinal inflammatory disease with unclear etiology.With the change of lifestyle,irregular incidence of sleep and diet has made the incidence rate of IBD increasing year by year.IBD has a long course,difficult to cure,and many side effects of drug treatment,which brings a great burden to patients.Therefore,it is urgent to explore a new way of dietary intervention and carry out targeted nutritional intervention.Fructo oligosaccharide(FOS)is a common prebiotic that is not easy to be digested by the upper gastrointestinal tract.FOS can be utilized by the gut microbiota of the lower gastrointestinal tract to exert many health benefits,including immune regulation.Butyrate is one of the metabolites of dietary fiber degraded by gut microbiota.It can regulate the structure of gut microbiota and maintain intestinal barrier.In this paper,two novel prebiotics Butyric-FOS(B-FOS)and n-Butyric anhydride-FOS(A-FOS)were formed by combining butyrate with FOS by enzymatic synthesis and anhydride method.The structure and fermentation characteristics of B-FOS and A-FOS were studied by using healthy human feces to simulate intestinal fermentation system in vitro.The IBD mouse model induced by dextran sulfate sodium(DSS)was used to verify the improvement effect and mechanism of B-FOS on enteritis symptoms in vivo.It is expected to provide new ideas for the development and research of new prebiotics,realize the accurate regulation of intestinal microecology,and provide reference for dietary intervention to alleviate IBD.The specific research contents are as follows:(1)B-FOS was successfully synthesized by using TLIM enzyme to catalyze the reaction of vinyl butyrate and FOS.In addition,n-Butyric anhydride reacted with FOS to synthesize A-FOS,which with higher degree of esterification.The structure of B-FOS and A-FOS were identified by Fourier transformed infrared spectroscopy(FT-IR),Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)and ~1Hydrogen-nuclear magnetic resonance(~1H NMR).The results showed that butyrate combined with FOS through ester bond(C=O).MALDI-TOF MS results showed that B-FOS had the corresponding molecular ion peaks of GF3-B,GF4-B,GF3-2B,GF2-B,GF5-B,GF4-2B,GF2-2B and GF5-2B,and their proportions in the sample were 43.59%,25.41%,8.57%,7.86%,6.59%,5.29%,1.50%and 1.19%respectively.A-FOS has higher esterification degree and more complex structure and its main components are GF3-10B,GF3-11B and GF3-9B.B-FOS was analyzed by ~1H NMR to determine the binding site of butyrate on FOS.(2)The fermentation characteristics and prebiotic activities of B-FOS and A-FOS were studied by in vitro fermentation system.The results showed that B-FOS had good fermentability,and 74.47%of the total sugar in the fermentation broth was degraded at 36h.But A-FOS could not be well utilized by gut microbiota.The composition of B-FOS fermentation broth was analyzed by MALDI-TOF MS.It was found that the gut microbiota would preferentially use monoesters(i.e.,GF3-B and GF4-B),and they were more inclined to degrade and utilize the sugar without butyrate than directly degrading the ester bond to obtain butyrate.After the determination and analysis of short chain fatty acids(SCFAs)by gas chromatography,it was found that B-FOS could significantly increase the production of butyrate in fermentation broth(p<0.05),and increased steadily with the extension of fermentation time.16S r RNA sequencing results of fermentation broth showed that B-FOS could increase the diversity and richness of gut microbiota and significantly increase the proportion of dominant bacteria Bacteroidota and Firmicutes(p<0.05),and reduce the relative abundance of Proteobacteria and Desulfobacterota,which containing more pathogenic bacteria in fermentation broth(p<0.05).At the genus level,B-FOS can significantly reduce the relative abundance of harmful bacteria Escherichia Shigella and Sutterella in the fermentation broth(p<0.05),and significantly increase the relative abundance of common probiotics Bifidobacterium and several butyrate-producing bacteria Ruminococcus,Faecalibacterium and Collinella(p<0.05),so as to improve the composition and structure of gut microbiota and change it to a trend conducive to human health.(3)Further explore the growth promoting effect of B-FOS on probiotics by using the bacteria culture system.It is found that B-FOS can promote the growth curve of Bifidobacterium lactis BB-12 and Lactobacillus casei strain Shirota.So,B-FOS may have potential effect on maintaining gut health.(4)The improvement effect and mechanism of B-FOS on colitis was using by IBD animal model.2.0%and 2.3%DSS aqueous solution to model colitis in mice for two cycles(8 days/cycle,4-day remission period,a total of 20 days).It was found that B-FOS improved the weight loss of mice caused by DSS,significantly reduced the disease activity index(p<0.05),and significantly alleviated the shortening of colon length of IBD mice(p<0.05).The results of H&E staining and Alcian blue staining of colon sections showed that the dietary intervention of B-FOS could alleviate the incomplete mucosal layer,inflammatory infiltration,decrease in the number of goblet cells and tissue edema of IBD mice.The relative m RNA expression levels of inflammatory factors and tight junctions protein showed that B-FOS down regulated the proinflammatory factor TNF-α,IL-18 and up regulate ZO-2 to improve the inflammatory response and enhance the function of intestinal barrier.The results of 16S r RNA sequencing of colonic contents showed that B-FOS could increase the diversity and richness of gut microbiota of IBD mice,inhibit the relative abundance of Proteobacteria and increase butyrate-producing bacterium Faecalibaculum in the colon,which was conducive to the further production and utilization of butyrate in the gut,so as to control inflammatory reaction,strengthen intestinal barrier and finally alleviate the symptoms of IBD mice.In conclusion,the new prebiotic B-FOS has no adverse effects on mice by the above indexes,and has good fermentation characteristics.It can stably produce a large amount of butyrate under the action of gut microbiota,so as to alleviate colitis in mice.B-FOS may promote gut health by adjusting the expression of inflammatory factors,protecting intestinal barrier and changing the composition and structure of gut microbiota.