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Modulating Effects of Fructo-oligosaccharides on Gut Microbiota and Host Metabolism in Human Flora-Associated Piglet Model

Author: ShenJian
Tutor: ZhaoLiPing；Sunil Kochhar
School: Shanghai Jiaotong University
Course: Microbiology
Keywords: Prebiotics Fructooligosaccharides Human flora of piglets Intestinal flora Taxa specific PCR Metabolomics NMR Multivariate statistical analysis
CLC: R151
Type: PhD thesis
Year: 2008
Downloads: 645
Quote: 2
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Abstract

The human gastrointestinal tract colonization with a very complex microbial flora, its structure and function have a very close relationship with a host of health and illness. Prebiotics are nondigestible food ingredients, and the structure and metabolism of the bacterial fermentation in the large intestine, the regulation of intestinal flora, and to improve the health of the host. Fructooligosaccharides is a universal application of prebiotics, many studies show that it can promote the growth of bifidobacteria, but has not been studied in the system level to investigate the structure and metabolism of fructooligosaccharides on intestinal flora and host metabolism . Directly to the man-made object to study, there is a lot of inconvenience to the diet of subjects, such as the difficulty of standardized sampling difficulties and ethical issues; many studies using rat / mouse model of the human source flora (Human Flora-associated, HFA) and made a lot of achievements, but the anatomy and physiology of the digestive tract of rodents features people significantly different results greater limitations. The physiological structure of the pig's digestive system with high similarity to human, more suitable to build an animal model of the human intestinal flora. Therefore, the laboratory was established in the early work of HFA piglet model to prove that the human intestinal flora can otherwise sterile piglets intestinal colonization. HFA piglet model of this thesis the oligo fructose molecular ecology research on the impact of the composition of the intestinal flora, and nuclear magnetic resonance (NMR)-based metabolomics methods investigation oligo fructose metabolism and host of the intestinal flora Metabolism. First, we established for the human intestinal flora, the most dominant bacterial populations one - Softening the Clostridium taxa taxa-specific PCR-DGGE and clone library method. Using a pair of soft Clostridium taxa specific primers to obtain the 239 bp of the 16S rRNA gene fragment can be DGGE well separated. 11 healthy people the stool soft Clostridium groups DGGE profiles showed that the bacterial composition of such groups to host specificity; 3 years of monitoring structure fluctuations in the first two years for a child's stool soft Clostridium groups using DGGE remain stable, while in the third year. An adult fecal DNA as a template with a bacteria common upstream primer and a soft Clostridium taxa specific downstream primer 1143 bp of the 16S rRNA gene fragment was amplified and cloned built library, 100 clones picked. Sequence analysis showed that 100 clones are soft Clostridium groups; 64% clone from Faecalibacterium prausnitzii 6% from Subdoligranulum variabile 2% production of butyric acid bacteria A2-207, 28% from the unknown bacteria. Position through efficient than cloning the 239 bp fragment migration position in DGGE DGGE profiles in the bar with the sequence of the vast majority of bands in the spectrum are determined. The results show that the soft Clostridium taxa specific DGGE and clone library analysis and monitoring human intestinal flora soft Clostridium taxa. For the research Fructooligosaccharide human intestinal microflora, we collected a healthy 27-year-old male fresh stool sample preparation bacterial suspension inoculated into the cesarean sterile piglets intestinal build HFA piglets. 10 newborn HFA piglets average assigned to the control group and the prebiotic group, the control group of piglets eating basic recipes, prebiotic group of piglets fed a certain amount of oligofructose daily on the basis of the basic recipe, so continuing to 37 days of age . In 12 days, 17 days, 25 days old and 37-day-old fecal samples collected each piglet and extracted fecal DNA respectively taxa-specific PCR-DGGE and real-time quantitative PCR method at different time points were compared between the two groups piglets, Bacteroides, the genus Bifidobacterium soft Clostridium taxa composition and quantity. At four time points are not observed oligofructose role in promoting bifidobacteria, which may be due to the higher the HFA piglets in this study Bifidobacterium abundance, prebiotics are not easy to produce significant their number increased role. Fructooligosaccharides affect other bacteria: at 12 days of age, the the two unknown Bacteroides level improved; 25 days of age, fructooligosaccharides improve the level of Subdoligranulum variabile inhibit an unknown bacteria belonging to the the tender Clostridium taxa ; 37 days of age, the proposed genus Bacillus quantity oligofructose significantly reduced. This work showed that oligofructose can affect the intestinal bacteria Bifidobacterium outside. Fructooligosaccharides can stimulate the growth of S. variabile, studies have shown that this bacteria has important physiological functions, so in the future work necessary to separate the functional analysis it Prebiotics and human health and the the relationship between the. Then, we used NMR-based metabolomics compared HFA piglets metabolites of the different regions of the contents of the large intestine, the law of the spatial distribution of large intestinal flora metabolism. Demonstrated by 1H NMR spectroscopy technique the HFA piglets of 10 and 37 days of age cecum, proximal colon, distal colon contents and feces biochemical composition found that different types of metabolites site contains similar, there are a lot of bacterial metabolites , such as short chain fatty acids, organic acids, phenolic acids, methylamines substances, various amino acids, etc.; also from food, host secreted substances or sources have not yet been identified, such as carbohydrates, bile acids and nucleic acid bases. Multivariate statistical methods, principal component analysis (Principle Component Analysis, PCA), small squares (Partial Least Square Discrimination Analysis, PLS-DA) and orthogonal small squares (Orthogonal Projection on Latent Structure, O-PLS) to distinguish large intestine contents of the different regions and fecal metabolites Spectrum found that the content of the short chain fatty acids from the cecum extension colorectal until feces gradually reduced, phenylacetic acid and 3 - hydroxy-propionic acid was significantly higher in the caecum and proximal colon from the cecum to the feces in the distal colon and feces, the branched-chain amino acid content of successively higher, the the macromolecule relative content of the glycoside is gradually increased from the cecum to the distal colon. This study shows that different physiological and ecological conditions of the large intestine in different parts of the metabolic activity of the intestinal flora are also differences. Contents in metabolites in the HFA piglets colorectal different section changes reported human colon contents biochemical composition similar trend, it has the potential to become a powerful model to study human intestinal flora metabolism. NMR-based metabolomics method can quickly show metabolites of the intestinal contents, reflect the general picture of the metabolic activity of the intestinal flora, can be used for monitoring the activities of the intestinal flora, study foreign intervention (such as prebiotics, probiotics etc.) flora metabolism. Metabolites in the feces is not exactly the same as the contents of the large intestine, so in the next step to study the impact of prebiotics on gut flora metabolism, you need to analyze the changes in the composition of the metabolites in the colon contents. Research fructooligosaccharides on intestinal flora and host metabolism, five control group and 5 prebiotics piglets at 12 days of age, 17-day-old, 25-day-old and 37-day-old feces, and 37-day-old proximal colon contents, the distal colon contents and urine collection. Biochemical composition of these samples with orthogonal small squares (Orthogonal Projection on Latent Structure, O-PLS) were compared between the two groups of piglets NMR spectra show by NMR. No significant differences in the biochemical composition of the two groups of piglets faeces of four time points. 37 days old, prebiotics piglets proximal colon contents containing a higher content of oligosaccharides, glucose, acetic acid, propionic acid, and nucleic acid bases; oligofructose principally affected HFA piglets distal colon contents metabolites, oligosaccharides, glucose, acetic acid, lactic acid, nucleic acid bases, and a variety of amino acid content and reduces the content of the bile acid; the prebiotic group piglets urine containing a higher level of alanine glycine, creatine, creatine phosphate, lactic acid, citric acid, allantoin and urea content is low, some of the metabolites produced by the intestinal flora, or by the intestinal flora and host of metabolic such as dimethylamine phenyl acetylglycine, trimethylamine, trimethylamine oxide, hippuric acid, and 4 - hydroxyphenyl lactic acid content in the difference in the two groups of piglets urine. Infer from these metabolic changes, fructooligosaccharides play a prebiotic effect mainly in the the HFA piglets distal colon within: fructooligosaccharides colonic microflora fermented to produce short chain fatty acids, thus affecting the energy metabolism of the host; fructooligosaccharides regulation flora in the metabolism of amino acids , affect a host of amino acid metabolism, improve the nitrogen balance of the host organism. Fructooligosaccharides reduce the phenomenon of bile acids in the colon that it has a certain role of the prevention of colon cancer. Fructooligosaccharides also have an impact on the colonic flora / host nucleic acid metabolism, but the mechanism needs further study. 1H NMR-based metabolomics technology can be used for monitoring the impact of nutritional interventions on the intestinal flora and host metabolism. In this study, the HFA piglets as a model, survey fructooligosaccharides on intestinal flora composition and metabolism, and host metabolism, further study of microbial community structure and flora features, flora metabolism and host metabolic relationship between useful ideas and clues.

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