猪后肠产甲烷菌群对不同类型膳食纤维的动态响应机制研究

Methanogens in the hindgut of monogastric animals play a key role in the metabolism of dietary fibers. Limited researches focus on the community structure of gut methanogens rather than their function during microbial fermentation of fibers. It has been proved that there are multiple responses of intestinal microflora to different dietary fibers. Our study showed that the diversity of colonic methanogens was significantly improved by β-glucan during in vitro fermentation, but the underlying mechanism remained unclear. Therefore, the objectives of current researche are: 1) to study the dynamic response of methanogens to soluble (β-glucan) and insoluble (microcrystalline cellulose) dietary fiber in the hindgut of pig in vivo with a cross-over trial; 2) to reveal dominant bacterial groups associated with the dynamic change of methanogens with molecular fingerprint technology; 3) to prove that gut methanogens may response to different types of dietary fibers through the interaction with symbiotic bacteria, and thus to help host utilize corresponding fibers efficiently through the functional alteration of microflora. Results of present study will help entirely understand the function of intestinal methanogens during the degradation of dietary fibers, and in turn, to promote the scientific utilization of different types of dietary fibers and studies on their prebiotic mechanisms.

单胃动物后肠产甲烷菌在膳食纤维代谢过程中起关键作用，现有研究多集中于对其群落结构的探讨，缺乏对其功能的系统研究。前人研究表明肠道细菌群落对不同类型膳食纤维的响应有较大差异。前期体外研究表明β-葡聚糖能显著改变猪肠道产甲烷菌群落多样性，但其影响机制尚不清楚。本项目拟从群落结构、代谢物及微生物蛋白组学层面上，采用典型可溶性膳食纤维（β-葡聚糖）与不可溶膳食纤维（微晶纤维素）的在体交叉试验结合分子生态学和宏蛋白质组学技术：1）研究猪后肠产甲烷菌群落对纤维类型转变的动态响应机制；2）揭示与产甲烷菌动态变化密切相关的细菌群落；3）初步证明产甲烷菌可能通过与共生细菌互作来对不同类型膳食纤维做出响应，实现微生物区系纤维降解功能的转变，从而帮助宿主有效利用不同类型的膳食纤维。本研究为全面了解后肠产甲烷菌在纤维降解中的作用机制提供理论依据，也为科学利用不同类型膳食纤维和其益生机理研究提供借鉴。

本研究考察了典型可溶纤维（SDF）燕麦β-葡聚糖和典型不可溶纤维（IDF）微晶纤维素及天然纤维（豌豆纤维）对单胃动物（猪、小鼠）后肠细菌和产甲烷菌组成及功能的影响；探索了猪后肠产甲烷菌和细菌分别对SDF和IDF的动态响应过程和可能的互作关系。结果发现：1）SDF和IDF均降低小鼠和猪采食量，但SDF显著提高两种动物阶段日增重（P < 0.05）。在饲粮营养水平一致的情况下，与含同等浓度的SDF饲粮相比，含5% MCC（IDF）饲粮促进猪的采食（P < 0.05），但前者粗纤维和ADF消化率显著高于后者（P < 0.05），暗示SDF在猪后肠的微生物发酵程度较高。两种纤维的混合饲喂促进小鼠脂肪沉积；2）猪和小鼠后肠均存在特异性利用两种纤维的核心菌群（如燕麦β-葡聚糖和豌豆纤维提高Bacteroides spp.相对丰度）。SDF上调结肠微生物与能量和碳水化合物代谢相关的KEGG通路，但不影响其潜在致病性；3）猪后肠细菌对SDF和IDF的实时动态响应过程呈现相反趋势。产甲烷菌和细菌均对饲粮纤维由IDF转变为SDF的响应更敏感。IDF较SDF更倾向于提高猪后肠产甲烷菌多样性。从饲粮中撤去外源纤维后，SDF和IDF对后肠微生物区系的影响均有持续效应；4）Methanobrevibacter与Methanosphaera是受纤维类型影响最敏感的产甲烷菌属，纤维缺乏引起vadinCA11属丰度增加。Methanobacteriaceae科产甲烷菌对IDF和/或SDF有特异性响应（豌豆纤维试验证实了此结果）；5）饲粮SDF转变为IDF引起Actinobacteria和Firmicutes的丰度变化，IDF转变为SDF时引起Bacteroidetes的丰度变化。缺乏SDF可能导致微生物（如Desulfovibrio spp.）对宿主肠道粘膜的侵蚀；6）饲粮SDF转变为IDF使猪后肠SCFAs浓度升高，与细菌多样性增加有关。乙酸和丙酸比例与产甲烷菌群落结构和活性有关；7）产甲烷菌对纤维类型转变的响应晚于细菌，暗示在此过程中产甲烷菌和细菌的复杂互作关系。产甲烷菌群落结构的改变及其与细菌（特别是氢利用菌，如SRB）的互作是保证纤维后肠发酵效率的关键因素。研究结果为动物生产上科学利用不同类型日粮纤维提供参考。

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