Effect Mechanism Of Lotus Seed Resistant Starch On The Biotransformation Of Taurocholic Acid By Fecal Bacteria In Vitro Fermentation System

RS is not easy to digest and absorb in the small intestine,but it can be used by the intestinal flora to regulate the intestinal microecological balance,promote the production of short-chain fatty acids and regulate the metabolism of bile acids.BAs are a class of important metabolites in the body,which are derived from cholesterol catabolism in the liver and can be transformed by intestinal microorganisms.BAs are widely distributed in the liver and intestine and are essential for the health of the body.Previous studies from our team have found that LRS can regulate the community structure of gut microbiota and promote the conversion of primary to secondary BAs.In order to further explore the mechanism of LRS regulating intestinal flora to affect the biotransformation of BAs,this study studied the effects of LRS and TCA on the fecal flora of rats under in vitro fermentation system using GLU and HAMS as controls.The effects of fecal flora and BAs on the structural characteristics of LRS were studied from the aspects of apparent structure,molecular structure and crystal structure.The changes in the content of BAs and the activities of BAs metabolic enzymes were clarified,the biotransformation pathway of TCA was constructed,and the mechanism of the effect of LRS on the biotransformation of TCA during the in vitro fermentation was revealed.The results help to understand the changes of functional carbohydrates on the biotransformation of BAs and the effects of these changes on the metabolism and health of the body,and provide certain scientific guidance for individualized precision nutrition design.Findings were as follows:（1）Effects of LRS and TCA on fecal microflora of ratsWe used LRS,GLU and HAMS as carbon sources,and cultured rat fecal microflora in taurocholic acid-containing medium for 0,8,16,24,32h,respectively.The growth of fecal microflora and p H value of the medium were measured at different fermentation times.16S r RNA high-throughput sequencing technology was used.The effects of LRS and TCA on the flora structure at different growth stages were studied from the aspects of flora diversity,taxa,differential flora and functional prediction.The results showed that carbon source had an impact on the structure of the bacterial community at the early stage of fermentation,and at the late stage of fermentation,fermentation time had an impact on the structure of the bacterial community,and the effect of carbon source on the structure of the bacterial community was greater than that of fermentation time.At the phylum level,compared with unfermented,fermentation of LRS and GLU can promote Firmicutes and inhibit Proteobacteria growth to varying degrees,while fermentation of HAMS can promote Proteobacteria growth.In genus level,in vitro fermentation the LRS and GLU promotes Enterococcus,Bacillus,Lactobacillus and unclassified＿c＿Bacilli growth,inhibition of Escherichia-Shigella of the genus proliferation.While HAMS in vitro fermentation can promote the proliferation of Escherichia-Shigella and Proteus,and inhibit the growth of Lactobacillus and Enterococcus.According to the significance test of different microflora between groups,different carbon sources mainly affected the relative abundance of Enterococcus,Escherichia-Shigella,Bacillus,Proteus and Lactobacillus during in vitro fermentation of rat fecal microflora,and all had significant differences.（2）Effect of structure changes of LRS during microbial fermentationThe changes of structural characteristics of LRS and HAMS during fermentation were studied to explore the direct adsorption between starch and BAs,and the correlation between starch structural characteristics and dominant bacteria was constructed.The results showed that the apparent structure of LRS changed significantly after microbial fermentation.The structure of starch particles became rougher and hollow-out,with a large number of spherical and rod-shaped bacteria attached.However,after HAMS were fermented by bacterial flora,the sticky aggregation of particles was aggravated,the surface crystals increased,and the surface of particles was damaged.The results of crystal and molecular structure showed that during the fermentation of the microbial group for 32 h,the microbial group did not change the crystal type of LRS and HAMS,but the fermentation of the microbial group increased the crystallite degree,the proportion of microcrystalline region,the degree of ordered structure and the proportion of double helix structure of different carbon sources,and reduced the proportion of subcrystalline region and amorphous region.This indicated that during the fermentation of the microbial group,The flora first utilized the amorphous and subcrystalline regions of LRS and HAMS.The results of adsorption capacity of starch to BAs showed that fermentation of bacterial flora promoted changes in the structure of starch substrate,which was conducive to the adsorption of BAs by starch,and LRS was more conducive to adsorption of BAs than HAMS.In addition,RC of LRS was dominated by Bacillus and unclassified＿c＿Bacilli,while R_995/1022 and R_1047/1022 were dominated by Enterococcus and Lactobacillus.The RC of HAMS during fermentation was dominated by Proteus and Bacillus,while the R_995/1022 and R_1047/1022 were dominated by Escherichia-Shigella and Bacillus.（3）Effect mechanism of LRS on taurocholic acids bioconversion during in vitro fermentationThe contents of BAs and the activities of bile acid metabolizing enzymes were measured to establish the relationship between the dominant genus and BAs metabolism.On this basis,the correlation between structural features,dominant genus and BAs was established to clarify the effect of LRS and HAMS on BAs transformation by regulating the dominant genus,and the TCA biotransformation pathway was constructed.The results showed that LRS,HAMS and GLU could promote the reduction of primary BAs and conjugated BAs,increase of secondary BAs and free BAs,and effectively reduce the contents of total BAs during in vitro fermentation.Among the effects of LRS group were the most obvious.According to the analysis of TCA metabolite content changes,LRS,HAMS and GLU could promote the reduction of TCA content and increase the contents of CA,DCA,3β-CA,UCA and 12-oxo-CDCA,among which LRS had a more significant effect.According to TCA metabolic enzyme activity analysis,LRS,HAMS and GLU could promote the activities of BSH,BAL,BACT,bile acid 7α-dehydratase,3α/β-HSDH,7α/β-HSDH and 12α-HSDH with the extension of fermentation time,among which LRS tended to promote the dehydroxyl pathway.However,HAMS tends to promote the epimerization pathway.Based on the structural characteristics,the correlation between dominant bacteria and BAs,Changes in the crystal and molecular structures of LRS and HAMS were associated with Enterococcus,Bacillus,Escherichia-Shigella,Proteus and Lactobacillus and other bacterial genera have certain relationships.The structural characteristics of LRS were positively correlated with the dominant genus of Firmicutes,while the structural characteristics of HAMS were positively correlated with the dominant genus of Proteobacteria during the fermentation process.In addition,LRS and GLU mainly stimulate the secretion of metabolic enzymes in the dehydroxyl pathway by Enterococcus,Bacillus,unclassified＿c＿Bacilli and Lactobacillus.Including BSH,BAL,bile acid 7α-dehydratase,which promote TCA dissociation and CA dihydroxylation.And HAMS mainly stimulate the secretion of metabolic enzymes in the epimerization pathway by Escherichia-Shigella,Enterococcus,Bacillus,Proteus and Lactobacillus.Enzymes such as BSH,3α/β-HSDH,7α/β-HSDH,etc.,thus facilitate TCA dissociation and CA epimerization.