| Human gastrointestinal tract harbors a diverse and complex microbiota whose composition and activities play important roles in host health and disease. Prebiotics are non-digestible food ingredients that modulate the structure and metabolism of gut microbiota by being fermented by colonic bacteria and thus improve host health. Fructo-oligosaccharides (FOS) are one type of prebiotics which is widely applied in food industry. Many studies demonstrated FOS stimulated the growth of bifidobacteria, but few studies systematically investigated its effects on the composition and activities of gut microflora, as well as the host metabolism. Studying prebiotic effects directly on human beings may have such difficulties as standardizing the diets of volunteers, collecting biopsy samples, and ethical issues etc.; as a consequence, Human Flora-Associated (HFA) mice/rat models are widely used to produce useful results, but the anatomy and physiology of rodent digestive tracts are significantly different from those of human, so the HFA rodent models have low relevance to human biology. Compared to rodents, pigs share more similarities with human in anatomical and physiological characteristics of the digestive system, so they can be better candidates for developing HFA model. Our lab have established HFA piglet model by demonstrating that the human-originated gut microbiota can colonize in the gut of ex-germfree piglets. In this dissertation, using HFA piglet model, we assessed the impacts of FOS on the structure of gut microbiota with microbial ecological techniques, and investigated the influences of FOS on the metabolism of the gut microbiota and the host with NMR based metabonomic methods.Before we studied the effects of FOS on the composition of gut microflora, a group-specific PCR-based denaturant gradient gel electrophoresis (DGGE) method was established and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (239 bp in length) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. DGGE patterns of fecal amplicons from 11 human individuals showed host-specific profiles; patterns of fecal samples collected from a child during 3 years demonstrated the structural succession of the population in the first two years and its stability in the third year. A clone library was constructed with 100 clones, consisting of 1,143 bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward bacterial universal primer and one reverse group-specific primer. Eighty-six clones produced the 239 bp C. leptum cluster-specific amplicons and the remaining 14 clones did not but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were from Faecalibacterium prausnitzii, 6% from Subdoligranulum variabile, 2% from the butyrate-producing bacterium A2-207 and 28% from unknown species. The identity of most bands in the DGGE profiles of the same adult was determined via comigration analysis with the 86 clones which included the 239 bp group specific fragments. These results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of C. leptum cluster in human gut flora.To study the influences of FOS on the structure of gut microbiota, fecal suspension was prepared from the fresh fecal sample collected from one healthy 27-year-old male adult donor and inoculated to ex-germfree piglets to produce HFA piglets. 10 newly born HFA piglets were evenly divided into 2 groups: 5 in control group being fed with basal diets, and 5 in prebiotic group having FOS added to the basal diets daily at the dose of 0.5 g/kg body weight/day. The feeding experiment lasted from day 1 to day 37 after the birth of piglets. Fecal samples on day 12, 17, 25 and 37 were collected from each piglet and fecal DNA was extracted. With group-specific PCR-DGGE and real-time PCR, the composition and amounts of Bacteroides genus, Bifidobcterium genus and C.leptum subgroup were compared between control and FOS-fed piglets. FOS did not change the composition or the amount of bifidobacteria, and the possible reason is the number of bifidobacteria in the gut of our HFA piglets was quite high, and thus it was difficult for FOS to produce significant bifidogenic effect. However, FOS mainly affected other bacteria: on day 12, two unknown bacteroides species were stimulated; on day 25, S. variabile was stimulated, and one unknown species from C.leptum subgroup was suppressed; on day 37, the amount of Bacteroides genus was decreased. Our work demonstrated that FOS can influence bacteria other than bifidobacteria in gut microbiota. We found for the first time that FOS stimulated the growth of S. variabile, which was revealed to actively interact with host metabolism, so it is necessary to isolate the bacteria in the future and study its relationship with prebiotics and host health.We compared the biochemical composition of contents in different regions of the large intestine of HFA piglets with NMR based metabonomic techniques, and studied the spatial distribution of the gut microbiota metabolism in different regions along the large intestine. Metabolites present in aqueous extracts of contents from the caecum, proximal colon, distal colon and faeces of 10 HFA piglets were profiled by 1H NMR spectroscopy. Many compounds originating from bacterial activities were detected, including short chain fatty acids (SCFAs), organic acids, phenolic compounds, amino acids, and amines; besides, nucleobases, bile acids, choline and glycosides were also identified in profiles. Principle Component Analysis (PCA), Partial Least Square Discrimination Analysis (PLS-DA) and Orthogonal Projection on Latent Structure (O-PLS) analysis were used to differentiate NMR profiles of different regions and revealed that the biochemical composition in gut contents changed significantly in different regions of the large intestine of HFA piglets. The levels of SCFAs decreased distally from cecum to feces; phenylacetate and 3-hydroxyphenylpropionate predominated in cecum and proximal colon but not in distal colon and feces; the amounts of branch chain amino acids and glycosides increased distally along the large intestine. Our results suggest that the endogenous microbiota metabolize differently in different compartments of the large bowel due to varied environmental factors along the large bowel, such as substrate availability and host gut physiology, etc. The patterns of biochemical changes along the large intestine of HFA piglets showed to be similar to those in human colon as reported in the literature, so our HFA piglets potentially can become an effective in vivo model for studies on the metabolism of human gut microbiota. The 1H NMR-based metabonomic techniques has a great potential to be used to monitor the changes in the metabolic activities of colonic microbiota in response to interventions such as prebiotics, probiotics, antibiotics and diseases. Besides, the biochemical composition of feces is different from that of large intestinal contents, so it is necessary to analyze the metabolites in colonic content samples when study the effects of prebiotics on the metabolism of gut microbiota at next step.In the last part, the influences of FOS on the metabolism of the gut microbiota and the host were studied with NMR-based metabonomic techniques. We collected feces from 5 control piglets and 5 FOS-fed piglets on day 12, 17, 25 and 37 after birth, as well as urine samples, and contents of proximal and distal colon from each animal on day 37. 1H NMR spectroscopy was applied to profile the metabolic composition of urine samples and aqueous extracts from feces and colonic contents. The Chemometric method, O-PLS was used to characterize the effects of FOS on the metabolism of the gut microflora and the host by comparing the NMR spectra of control and FOS-fed piglets. There is no statistically significant difference in fecal metabolite composition between two groups of piglets. In the proximal colon, FOS-fed piglets had higher levels of oligosaccharides, glucose, acetate, propionate and nucleobases than control animals. More marked differences between two groups were observed in distal colon: besides oligosaccharides, glucose, acetate, nucleobases, the quantities of lactate and amino acids were also increased by FOS treatment; the amount of bile acids was decreased. In the urine, FOS-fed piglets excreted more alanine, glycine, creatine and phosphocreatine, lactate, citrate, but less urea and allantoin; besides, FOS treatment also affected the quantities of some bacteria-origined metabolites in the urine such as dimethylamine, trimethylamine, trimethylamine N-oxide, phenylacetylglycine, hippurate and 4- hydroxyphenyl-lactate. As indicated by these results, FOS was fermented by the gut microbiota mainly in distal colon with the production of acetate and lactate, and the acids can be absorbed and utilized by the host as energy sources, and thus affected the energy metabolism of the host; FOS also modulated the amino acid metabolism of gut microbiota, and thus influenced the amino acid metabolism of the host and improved its N-balance; FOS was indicated to have a preventative effect on colon cancer by decreasing the amounts of bile acids in distal colon. The mechanism for the influence of FOS on the nucleic acid metabolism of the gut microbiota/host need to be further studied. The 1H NMR-based metabonomic techniques can be used to monitor the metabolic responses of colonic microbiota and the host to interventions such as prebiotics, probiotics, antibiotics and diseases. HFA piglet model was utilized in our study to systematically study the effects of the prebiotic ingredient FOS on the gut microbiota and host metabolism. Our results will provide helpful insight and guidance to understanding the relationship among the composition and metabolism of the gut microbiota, and the metabolism of host.
|
PDF Full Text Request