Functional Evaluation And Compatibility Research Of Prebiotics And Probiotics Based On Human Gut Microbiota In Vitro Fermentation Model

The balance of gut microbiota is closely related to human health,prebiotics,probiotics and synbiotics,known as functional food dietary supplements,have been proved to modulate the structure of gut microbiota and be beneficial to the host,however,different animal models used in different studies are inconsistent,which makes it difficult to compare the evaluation results of prebiotics in different studies,so there is no unified evaluation method for the prebiotic effect of different prebiotics.At the same time,the results of animal models or human studies are affected to some extent due to the significant individual differences of hosts.The in vitro fermentation model of gut microbiota is not an effective limitation of the sample size and avoids individual differences in animals,so it is an effective supplement to animal models and human experiments.Besides,although synbiotics have higher modulating effects than products containing only probiotics or prebiotics in some cases,the current compatibility of prebiotics and probiotics still lacks basic information and only a few compatibility combinations are rated as synbiotics.Therefore,the establishment of an efficient probiotic / prebiotic evaluation system has important basic value for the development of synbiotic products.In this study,an in vitro fermentation model of human gut microbiota was established and evaluated,and the prebiotic effects of eleven prebiotics(Galacto-oligosaccharide(GOS),Xylo-oligosaccharide(XOS),Fructo-oligosaccharide(FOS),Isomalto-oligosaccharide(IMOS),Inulin,Konjac flour(KF),Mannan-oligosaccharide(MOS),Alginate-oligosaccharide(AOS),Chitosan-oligosaccharide(COS),?-glucan and Resistant dextrin(RD))were compared using the in vitro fermentation model,then the eleven prebiotics were pairwise combined with six probiotics(Lactobacillus reuteri(RE),Lactobacillus paracasei(PA),Lactobacillus casei(C),Lactobacillus rhamnosus(R),Lactobacillus plantarum(PL),Bifidobacterium animalis(A)),preliminary screening to obtain synbiotic combinations that could significantly stimulate the growth of probiotics,and used the in vitro fermentation model to evaluate its potential prebiotic effect.The results showd that:Although the number of microbial species cultured in the single-phase model was greatly decreased,to some extent,it could restore the changes of gut microbiota in animals and provided a good in vitro model for the study of the effect of dietary components on gut microbiota.However,the high-throughput based on this single-phase chemostat model was relatively low.In this study,the single-phase chemostat model was combined with a batch culture fermentation model.The stability of the system was detected by the method of 16S-rDNA-PCR-DGGE.The gut microbiota cultured by the chemostat model could be gradually stabilized with the extension of fermentation time,and the 48 h in vitro batch culture fermentation couldd maintain the stable gut microbiota structure continuously.All eleven prebiotics could modulate the overall structure of the human gut microbiota and cause specific changes in microorganisms at the "phylum","genus" and "species" levels,all of which could increased the content of short-chain fatty acids(SCFAs),but different prebiotics had different accumulations of acetate,propionate,butyrate and valerate,and XOS,KF and ?-glucan showed the highest accumulation of acetate,propionate and butyrate,respectively.Besides,based on the OTU abundance Bray-Curtis distance matrix,eleven prebiotics were initially classified into three groups(A: IMOS,Inulin,COS,MOS,FOS,and ?-glucan;B: KF,RD,and AOS;C: GOS and XOS),and then 13 differential species were found by pairwise comparison,and most of which belonged to Firmicutes.The ten synbiotics with remarkable proliferation effects were obtained by screening eleven prebiotics and six probiotics in pairwise combination according to the effect of prebiotics on the proliferation of probiotics,namely Inulin+PA,FOS+PA,GOS+PA,FOS+PL,AOS+PL,Inulin+R,FOS+A,GOS+A,AOS+A,IMOS+A.All ten synbiotics could modulate the overall structure of human gut microbiota,and cluster analysis based on the OTU abundance Bray-Curtis distance matrix showed that the flora structure of AOS+A,Inulin+PA,Inulin+R,FOS+A were similar,AOS+PL,GOS+C,FOS+PL,FOS+PA,IMOS+A,GOS+A groups were similar.At the "phylum","genus" and "species" levels,the ten synbiotics all showed positive regulatory effects on beneficial bacteria,but the modulation was specific to the genus / species,like IMOS+A,GOS+C and FOS+PL could increase the relative abundance of Faecalibacterium,Inulin+PA,AOS+PL,GOS+C,FOS+PA and IMOS+A could increase the relative abundance of Lactobacillus.At the same time,it also proved that prebiotics-probiotics combination that were reasonably compatible could modulate the beneficial bacteria in the gut microbiota more actively than supplementing prebiotics alone.Tax4 Fun predicted the function of the gut microbiota showing that FOS+A,Inulin+PA,Inulin+R,AOS+A synbiotic intervention could enhance the ability of carbohydrate metabolism of gut microbiota,while the FOS+PL group was the opposite.By detecting changes in the concentrations of metabolites SCFAs,it was found that all synbiotic groups except FOS+A and AOS+PL significantly increased the SCFA concentrations.Among them,AOS+A,Inulin+R,Inulin+PA,and IMOS+A significantly increased acetate,propionate,butyrate and valerate,while GOS+C only significantly increased the concentrations of propionate and butyrate,GOS+A,FOS+PA and FOS+PL significantly increased the concentrations of butyrate.