Effects Of Functional Oligosaccharides On The Gut Bacteria And The Mechanism

The human gut is colonized by over 1012 organisms, belonging to >1,000 species. Maintaining the balance of the microbial communities is crucial for host health, and perturbation of their composition may induce a range of diseases including obesity, diabetes, and colitis. Oligosaccharides in the diet are able to reach the large intestine without digestion and absorption, thus modifying the composition of gut microbiota by bacteria fermentation. Oligosaccharides are usually considered to favorably stimulate the growth of bifidobacteria and lactobacilli. There are increasing evidences that oligosaccharides are able to be utilized by many commensal gut bacteria, including Bacteroides, Clostridium, Escherichia and Streptococcus. In addition, side effects of oligosaccharides consumption were observed in vivo, such as increase in colonic permeability and the translocation of Salmonella and the development of intestinal discomfort. Therefore, the relationships between oligosaccharides and gut bacteria are more complex than the bifidogenic effects.In order to make clear of the relationships between oligosaccharides and gut bacteria, the oligosaccharides-utilizing species among the gut bacteria were first studied with the bioinformatics analysis and traditional culture method. The high throughput 16 S r DNA metagenomic sequencing techniques were then applied to investigate the effects of oligosaccharides on the composition of microbiota in fecal, content and mucus samples. Finally the effects of oligosaccharides utilization on the intestinal colonization were studied using the PCR techniques. The main results were shown as follows.83 genes responsible for the transport and 73 genes responsible for the catabolism of oligosaccharides were obtained through literature mining. BLASTP search results showed that the genes related to the transport and catabolism of oligosaccharides were widely distributed among the gut bacteria, indicating that oligosaccharides were not the specific substrates for bifidobacteria and lactobacilli. Lactulose- and fructooligosaccharides- utilizing bacteria were isolated from healthy adult feces. Combined with the BLASTP results, the in vitro pure culture experiments showed that 35 species and 26 species were proved to be capable of utilizing lactulose and fructooligosaccharides(FOS), respectively. Cronobacter sakazakii, Enterococcus faecium, Klebsiella pneumonia, and Pseudomonas putida were reported for the ?rst time to be capable of utilizing lactulose. Cronobacter sakazakii, Marvinbryantia formatexigens, Ruminococcus gnavus and Weissella paramesenteroides were found to be able to utilize FOS for the first time.The effects of low dose and high dose of lactulose and FOS on the composition of fecal microbiota in male C57BL/6J mice were investigated using the high throughput 16 S r DNA metagenomic sequencing techniques. The relative abundance of Actinobacteria signi?cantly increased and that of Bacteroidetes decreased after lactulose and FOS consumption. In addition, the abundance of Olsenella in fecal microbiota significantly increased and reached a comparable level with Bifidobacterium after high dose of FOS intervention. Olsenella strain Mou02 was isolated from mice feces and was con?rmed, for the ?rst time, to be capable of using FOS.In order to further reveal the mechanism of oligosaccharides effects on the intestinal microbiota, lactulose was chosen and its effects on the luminal and mucosal microbiota of cecum and colon were studied using the 16 S r DNA metagenomic analysis. The composition of luminal microbiota differed significantly from that of mucosal microbiota. In the luminal contents, the most abundant phylum was Firmicutes, while that was Proteobacteria in the mucus. After lactulose treatment(15%), the abundance of Actinobacteria and Verrucomicrobia significantly increased in the content and that of Proteobacteria notably increased in the mucus at the phylum level. The abundance of Bacteroidetes was decreased in both the content and mucus. Bifidobacterium, Akkermansia, Helicobacter and Alistipes were the most dominating genus in the phylum of Actinobacteria, Verrucomicrobia, Proteobacteria and Bacteroidetes, respectively. Lactulose can reach the distal colon in the high dose group and its effects on the luminal and mucal microbiota were speculated as the following mechanism. In the cecum and the three parts of colon, lactulose was fermented by Bifidobacterium belonging to Actinobacteria with the production of short chain fatty acids(SCFAs), which resulted in the decrease of p H. The production of SCFAs in the colon was supposed to promote the secretion of mucins, which increased the abundance of mucin-utilizing Akkermansia in the content and Helicobacter in the mucus. However, the growth of Alitipes may be inhibited, which lead to the decrease in the abundance of Bacteroidetes in both the content and mucus.To further develop the meaning of oligosaccharides utilization to the bacteria in the gut, the effects of FOS on the intestinal colonization of three isolated FOS-utilizing strains were studied using PCR techniques. During the growth on FOS, the generation time of Escherichia coli CCFM8415 was shorter than that of Klebsiella sp. CCFM8375 and that of Lactobacillus plantarum ST-III was the longest. The results indicated that FOS could promote the intestinal colonization of all the three strains, in which E. coli CCFM8415 and K. sp. CCFM8375 were able to better colonize in the mice gut for a longer time than L. plantarum ST-III. The strain L. plantarum ST-III was not capable of colonizing in the gut when the gene Sac A, encoding β-fructosidase, was knocked out(ΔSac A). Therefore, the ability of utilizing oligosaccharides is a competitive advantage for bactaria in the gut and can promote the intestinal colonization.