Resistant starch (RS) is the major energy source for microbial growth in the human large intestine and the fermentation of RS in the gut has a potential role in maintaining host health. Molecular profiling of the complex microbial communities in the human gut has revealed a remarkable inter-individual variation of their composition, which could be linked to variations in substrate fermentation in different individuals. In this project, significant inter-individual variation was seen in the composition of the human gut microbial communities and in the response to changes in dietary intake, using a range of qPCR primer sets. The range of qPCR primer sets was expanded in this study, for the assessment of an uncultured bacterial group related to Oscillibacter and Oscillospira spp. This group of bacteria showed a significant increase in obese subjects on a high RS diet and weight loss diet in samples from a previous human dietary intervention study. The group showing the greatest stimulation by RS, however, was related to Ruminococcus bromii. Furthermore, two out of 14 volunteers who showed very low R. bromii numbers also showed incomplete RS3 fermentation in the study of Walker et al. 2011. It was therefore decided to investigate the activity of human colonic bacteria against RS in vitro. Anaerobic pure cultures and defined co-incubations were performed to compare the abilities to degrade RS by four of the most abundant amylolytic species present in the human colon, which were Ruminococcus bromii, Eubacterium rectale, Bifidobacterium adolescentis and Bacteroides thetaiotaomicron. The four species showed the highest utilization of RS2 and RS3 (47-77%) when the RS was autoclaved in the medium, however, the utilization by E. rectale and B. thetaiotaomicron was limited for boiled RS (7-40%) and for raw RS (0.25-18%). Pairwise co-incubation of the four species revealed that utilization of boiled RS2 and RS3 by the other three amylolytic species was significantly increased in the presence of R. bromii, even in a medium that does not permit growth of R. bromii itself. The growth of Anaerostipes hadrus, which is a nonstarch utilizer, was also promoted in a co-incubation with R. bromii. Assays of soluble reducing sugar revealed that the bacterial growth stimulated by R. bromii relies on utilization of breakdown products released from RS by R. bromii amylases. Moreover, addition of R. bromii, but not other three amylolytic species, restored RS3 fermentation in vitro by the faecal bacterial community from one of the volunteers who had previously XVIII shown low starch fermentability in vivo. These results therefore suggest that R. bromii is a „keystone‟ species for the degradation of RS in the human large intestine. Finally, the starch-degrading enzyme system of R. bromii was investigated. Genome mining revealed 15 GH13 (glycoside hydrolases family 13) amylases and 8 SBDs (starchbinding domains), as well as many structural modules of cohesins and dockerins that are known to be involved in protein-protein interactions in related Ruminococcus species. The major extracellular amylases active against RS were identified by zymogram analysis together with peptide sequencing of the excised active bands. The gene products responsible were then identified from draft genome sequence information and found to include three large proteins with dockerin modules, one of which also contained a cohesin. An additional gene product encoding a cohesin domain was identified from the genome sequence that was considered a candidate for cell-surface attachement. The two cohesin domains were therefore over-expressed in order to investigate protein-protein interactions. Western-blotting using the over-expressed cohesins as probes demonstrated cohesindockerin interactions between certain proteins. Possible models for the organization of a R. bromii multienzyme amylase system were then proposed based on these computerbased analysis and on the results generated from protein-protein interaction studies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:582738 |
| Date | January 2013 |
| Creators | Ze, Xiaolei |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=201744 |
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