Starch With Different Amylose/amylopectin Ratio Modulate Composition And Fermentation Of Microbiota In Porcine Gut

The microbiota in large intestine of human being is highly complex and comprises many hundreds of bacterial species(or phylotypes).Bacteria are counted achieving to 1011-12 cells per ml of colonic contents in adults.The microbiota and their metabolites provide an enhanced understanding of the interaction between diet and host.Starch is the main carbohydrate for growth of the intestinal microbiota,which is also the major energy source in diet of host animal.Chemically,the starch consists of 2 main structural components,the amylose and amylopectin.Animals or human being digest high amylose maize starch(HAMS)very slowly and incompletely in their small intestine.After reaching the large intestine,the RS fractions were fermented by the colonic flora,resulting in short-chain fatty acids(SCFA)production.In addition to providing energy,SCFAs also play a regulatory role in a variety of physiological processes.Current studies have revealed that SCFAs act as endogenous ligands for G protein-coupled receptor GPR41 and GPR43.Studies in GPR41 and GPR43 have provided evidence to clarify the mechanism of short chain fatty acids on the physiological regulation.Combined,GPR41 and GPR43 provide an enhanced understanding of the interaction between microbiota,SCFAs,and physiologic function.However,whether diets with different amylose/amylopectin ratio in the starch modulate microbiota composition and characteristics of in vitro fermentation,or induce the gene expression of the GPR41 and GPR43 in tissues of growing pigs,are not completely clear so far.In the experiments described in this article,we investigated the effects of starch with different amylose/amylopectin ratio on luminal SCFA concentrations,and the expression of GPR41 and GPR43 genes in tissue obtained from liver,spleen,ileum,colon and backfat.Combined in vitro fermentation,starch with different amylose/amylopectin ratio modulate composition and fermentation of microbiota in porcine faeces.This paper focuses on the following parts:1 Effects of dietary amylose/amylopectin ratio on in vitro fermentation characteristics of pig fecal microbiotaTo compare the fermentation characteristics of fecal microbiota of growing pigs feeding by diets with different amylose/amylopectin ratio,an in vitro fermentation was conducted by two factor completely randomized experiment design,diets with amylose/amylopectin ratio at 0.97 and 0.26 were used as substrates,feces of pigs fed with these two diets were used as inocula.For the fermentable character of diets,results showed that the total SCFA production after 72 h of fermentation,the half time of asymptotic gas production(T1/2),the maximum rate of gas production(Rmax)and the time occurrence of Rmax(Tmax)differed between substrates(P<0.001),comparing diets with high amylose/amylopectin ratio,the low amylose/amylopectin ratio diets could be degraded easily by the fecal microbiota.For fermentative ability of fecal inoculum,the fecal microbiota from pigs treated with the high amylose/amylopectin ratio diets could degrade the starch diets rapidly,Rmax(P<0.001).TVFA,acetate,propionate and butyrate productions(P<0.05)were significantly higher in 12 h and 24 h.An inverse relationship between acetate/TVFA and butyrate/TVFA was observed during 72 hours in vitro fermentation,it is probably that acetate can be converted to butyrate due to compositional changes of bacterial communities.The time evolution of the lactate concentration during in vitro fermentation was analyzed,and it increased significantly in 12 h after inoculating the fecal microbiota of pigs treated with the low amylose/amylopectin ratio diets,then decreased gradually,whereas the lactate concentration decreased during the 72 hours with the high amylose/amylopectin ratio fecal inocula.2 Effects of dietary amylose/amylopectin ratio on composition of pig fecal microbiota during in vitro fermentationThe aim of this study was to investigate the effects of substrates or inocula on microbiota composition during the 72 h in vitro fermentation,16S rRNA gene based denaturing gradient gel electrophoresis(DGGE)and real-time PCR were used for microbial analysis.DGGE similarity dendrograms showed overall clusters in different samples,the samples were seperated into two cluster,according to the inocula.Furthermore,the samples with same substrate were segregated in one cluster.The number of bands in DGGE profiles from fecal microbiota of pigs feeding high amylose/amylopectin ratio diets was higher than that in L group,consistent with the enriched bacteria diversity.Both quantitative and qualitative analysis indicated that the community composition of bacteria changed after feeding diets with different amylose/amylopectin ratio,the numbers of Bacteroidetes,Lactobacillus spp.and Bifidobacterium spp.were promoted by high amylose/amylopectin ratio starch(P<0.05).In fact,Bifidobacterium spp.and Lactobacillus spp.had higher specific growth rates(P<0.05)in the groups inoculating the fecal microbiota of pigs fed up with the high amylose/amylopectin ratio diets in 12 h,moreover the relative abundance of Lactobacillus spp.was significantly increased after 24 h in vitro fermentation(P<0.05).Starch with low amylose/amylopectin ratio promoted the fermentation,the data suggest that Bifidobacterium spp.may be more pronounced in 24 h and 72 h(P<0.05).In conclusion,Bifidobacterium spp.may have an impact on degrading starch with different amylose/amylopectin ratio.Different fermentation characteristics of diets with different amylose/amylopectin ratio by fecal microbiota of pigs probably associated with the variation of microbiota composition.3 Effects of dietary amylose/amylopectin ratio on SCFA concentrations and gene expression of the GPR41 and GPR43 in growing pigsSCFAs are the main products of microbial fermentation in the gastrointestinal tract and have recently been proposed to mediate the effect of regulating a variety of physiological processes.The aim of this study was to investigate the effects of a diet high in amylose/amylopectin ratio on luminal SCFA concentrations,and gene expression of the GPR41 and GPR43 in tissue obtained from spleen,backfat,liver,cecum and colon.Eleven 55-day-old crossbred barrows(Duroc×Landrace×Yorkshire)assigned to one of two dietary treatment groups on the basis of gender and live weight in a complete randomize block design.Diets were identical in composition except for the source of maize starch,which was either a conventional low amylose-to-amylopectin ratio(Am:Ap)corn starch(group L,Am:Ap 0.26)or high Am:Ap maize starches(group H,Am:Ap 0.97).SCFA concentrations were measured in the intestinal contents collected at section(5 h postprandially).Analysis of the array data revealed that,the high Am:Ap maize starch significantly(P<0.05)increased the average daily gain of body weight,and reduced the pH in luminal contents of colon and cecum.The concentrations of TVFA,acetate and valerate in colon were significantly(P<0.05)higher in HAMS-fed pigs compared with LAMS-fed pigs.TVFA,acetate,propionate and butyrate concentrations determined in carotid plasma collected 300 min after feeding were significantly higher with HAMS consumption compared with after LAMS consumption(P<0.05).No significant differences(P>0.05)were found between the treatment groups with respect to in vivo glucose responses.The genes expression of GPR41 and GPR43 were in a significant tissue-specific.GPR41 was most adequately expressed in the ileum and spleen,which had a significantly higher expression level than any other tested tissue.The highest mRNA level of GPR43 was in the spleen.The relative abundance level of GPR43 was comparable in the ileum,colon,and adipose tissue.The expression level of GPR41 and GPR43 among the liver,spleen,ileum,colon,and adipose tissue was not affected by dietary treatment(P>0.05).