Study Of The Mechanism Of Substantial Stimulating Effects Of Fructooligosaccharides On The Growth Of Bifidobacterium In Mice

Fructooligosaccharides(FOS)are a well-known class of prebiotic and are considered to selectively stimulate the growth of bifidobacteria in the gut.Recent studies have shown that60 species of intestinal bacteria can ferment FOS.Previous studies showed that FOS selectively promoted the proliferation of bifidobacteria both in vitro and in vivo by traditional plate counting,real-time quantitative polymerase chain reaction or metagenomic sequencing.However,there are no further investigations on the bifidobacterial composition and the mechanism of significant proliferation of bifidobacteira.In this study mice were fed with FOS in different doses(0%,5%,and 25%)for 4 weeks.The composition of fecal microbiota and the distribution of intestinal bacteria in mucosa and contents,in particular Bifidobacterium,were analyzed by sequencing the V3-V4 region and the groEL gene on the MiSeq platform,respectively.Finally,the properties of bacteria using FOS were detected by high performance liquid chromatography(HPLC),and genome sketches analysis of FOS-utilizing bacteria was performed to reveal the mechanism of significant proliferation of bifidobacteria by FOS.16S rDNA amplicon sequencing technique was used to analyze the composition of fecal microbiota in mice.The intake of high-dose FOS increased the relative abundance of Actinobacteria at the phylum level and Bifidobacterium exhibited the greatest relative abundance at the genus level(about 20%).14 species / subspecies were detected in Bifidobacterium through groEL gene sequencing.After FOS intervention,B.pseudolongum almost became the only Bifidobacterium specie,and the relative abundance was over 7.33%,indicating that FOS significantly promoted the proliferation of B.pseudolongum.The microflora analysis of mucosa and contents showed that the relative abundance of Bifidobacterium was significantly increased after high-dose FOS intake,and Coprococcus and Enterococcus also showed a significant increase in mucosa.After FOS intervention,B.pseudolongum became almost the only Bifidobacterium specie in the contents,indicating that high doses of FOS promoted the growth of B.pseudolongum in the intestine.As reported some bacteria in Bifidobacterium,Enterococcus and Lactobacillus were able to utilize FOS and the three genera were detected in different parts of cecum and colon,suggesting that there was no clear boundary among bacteria in the gut and the FOS-utilzing gut bacteria had the opportunity to contact FOS.Enterococcus hirae,Enterococcus faecalis,Lactobacillus rhamnosus,Lactobacillus murines and B.pseudolongum were isolated from mice feces and could utilize FOS.HPLC analysis of the growth of five species showed that B.pseudolongum can use both GF2 and GF3 in FOS,while other four species can only use GF2.Therefore,B.pseudolongum wasmore advantageous when in cofermention with other four species.This might be one potential mechanism for the significant proliferation of bifidobacteria by FOS in mice.Sucrose and fructose were detected in the supernatant of the culture of B.pseudolongum,suggesting that there may be extracellular hydrolases in the supernatant of B.pseudolongum.According to the result of genome annotation,B.pseudolongum had more species of FOS hydrolases than other four species,which may help B.pseudolongum to utilize FOS compared with other four species.