The Degradation And Utilization Study Of Agarose,κ-carrageenan And Their Oligosaccharides By Human Gut Microbiota

Degradation and utilization of agarose (AP), agaro-oligosaccharides (AO),κ-carrageenan and κ-carra-oligosaccharides (KCO) by human intestine microbiotafrom Chinese individuals were investigated in the current thesis and bacterial strainsthat can hydrolyze those substrates were isolated. Bacteroides uniformis L8wasidentified to be able to degrade AP and AO, whereas Bacteroides xylanisolvens andDialister sp. were the primary degraders for KCO, which was reported firstly. Theglycosidases produced by B. uniformis L8, B. xylanisolvens and Dialister sp. werereported. A cross-feeding interaction in the process of degradation of AO and KCOwas also observed for the first time. Animal models of mono-associated mice with B.xylanisolvens and E. coli were established. A relatively serious inflammation wasdetected in a section of colon after the mono-associated mice were fed with KCO.These results suggested that human intestine microbiota may play the role in theproinflammatory effect of KCO.Initially, we screened the utilization ability of AP and AO by human gutmicrobiota in batch fermentation systems and found that AP and AO were degraded atdifferent extents and patterns. B. uniformis L8isolated from one of the human fecalslurries showed a pronounced ability to degrade AO and generated galactose as itsfinal end product. A synergistic strain, here classified as E. coli B2, was identified toutilize the galactose as a growth substrate. The cross-feeding interaction between B.uniformisL8and E. coli B2led to the exhaustion of AO supply. TLC associated withgrowth curves indicated that B. uniformis L8could degrade AO exclusively. Incontrast, KCO, guluronic acid oligosaccharides, and mannuronic acidoligosaccharides could not be hydrolyzed by B. uniformis L8. Current results indicatethat B. uniformis L8is a special degrader of AO in the human gut microbiota. Theintermediate of AP degraded by B. uniformis L8was indentified as neoagaro-oligosaccharides by MS and NMR, indicating the glycosidases produced byB. uniformis L8was β-agarase.In the second part of thesis, the degradation of κ-carrageenan with differentmolecular weight by human gut microbiota was investigated. Results from batchfermentation demonstrated that κ-carrageenan containing sulfate was more difficult todegrade than agarose, particularly those with molecular weights over100kDa.However, KCO with a molecular weight of4.5kDa or less could be hydrolyzed withvarious rates by human fecal samples.Two groups of synergistic strains, which wereDialister sp. accompanied by E. coli and B. xylanisolvens associated with E. coli wereisolated from the fecal sample under the selective pressure of antibiotics treatment ornot. In the process of purifying these bacteria, the cross-feeding phenomenon was alsoobserved. According to the TLC analysis, KCO was more extensively deleted than ifeither species was grown on this substrate alone. The fragments generated by B.xylanisolvens and Dialister sp. from KCO degradation were identified asκ-carraheptaose (DP=7), κ-carrapentaose (DP=5), κ-carratriose(DP=3)4-sulfate-Galactose, a minor of neo-κ-carratetraose (DP=4) and neo-κ-carrabiose(DP=2) based on the analysis by HPLC and MS. The glycosidase secreted by B.xylanisolvens was classified as κ-carrageenase by analyzing the sequences of productsgenerated from degradation of carra-oligosaccharides.κ-carrageenan is a food additive widely used as a thickener in various types offood. However, there is still a safety concern because a mass of animal studiesrevealed that κ-carrageenanhas potential proinflammatory influence in the colon ofanimals. To evaluate the inflammatory impact of KCO, germ-free mice that weremono-associated with B. xylanisolvens and E. coli were administrated KCO in thedrinking water. According to the histopathological results from HE stain, theinflammation was detected in a section of colon from the group of micemono-associated by B. xylanisolvens and E. coli and fed with KCO. In contrast, thegroups of germ-free mice fed with KCO and germfree mice colonized solely by B.xylanisolvens and E. coli exhibited less inflammation. The animal feedingexperiments suggested that KCO degrading bacteria from human gut may be involved in the proinflammatory effects of KCO.In summary, the current thesis investigated the degradation of AP, AO,κ-carrageenan and KCO by human intestine microbiota. In addition, the bacteria thatcan utilize marine carbohydrates have been isolated, and the glycosidases produced byB. uniformis L8, B. xylanisolvens and Dialister sp. were reported. Cross-feedinginteractions in the process of degradation of carbohydrates suggested that thedegradation and metabolism of marine carbohydrates are complex processes. Animalexperiments with mono-associated mice suggested the potential harmful impact ofKCO as a food additive. Furthermore, this is the first report identifying special gutbacterial strains which may play a significant role in the etiology of κ-carrageenanand κ-carra-oligosaccharides. Further study is being undertaking to investigate theimmune responses of proinflammatory impacts of KCO.