甲烷菌不同类群与细菌的共生策略对猪后肠纤维发酵模式的影响机制

Methanogens, mainly colonized in the colon of pigs, can utilize the metabolites of bacteria to produce methane, and in turn, to decrease the hydrogen partial pressure and maintain the fermentation of microbes in the hindgut. Studies associated with the function of methanogens in the gut of pigs are very limited. Our previous researches showed that methanogens and certain bacterial species particularly responded to soluble (SDF) and/or insoluble (IDF) dietary fibers. Due to the substrates preference of different methanogenic species, the alteration of community of methanogens may change their ability to remove the bacterial metabolites, which finally may probably influence the fermentation efficiency of dietary fibers in the hindgut, as well as the growth of pigs. Therefore in current study, to investigate the symbiotic relationship between different methanogenic species and bacteria during the degradation of dietary fibers, three species of methanogens with different metabolic pathways, polysaccharide utilizing bacteria and hydrogenogenic bacterium will be co-cultured in vitro with oat-derived β-glucan (SDF) or microcrystalline cellulose (MCC, IDF) as the unique carbon substrate, respectively. Then the exclusive inhibitor of methanogens, bromochloromethane, will be used to establish in vitro and in vivo “methanogen-removed” models, and the three methanogens mentioned above will be re-inoculated into the models to compared the metabolic pathways of methanogens, and bacterial metabolites as well as the associated energy metabolism pathways in colonic and hepatic tissues of the animals. Furthermore, combing the growth performance and the nutrients digestibility of the pigs, the underlying mechanism of symbiotic strategy between methanogens and bacteria influencing the fermentation efficiency of the two fibers will be revealed on the species/strain, group and the whole levels. Results will provide new reference for the efficient utilization of different fiber sources by targeting the specific microorganisms in the hindgut of pigs.

猪肠道甲烷菌数量稀少，主要定植于结肠，可利用细菌代谢物产甲烷降低氢分压，维持细菌的正常发酵，但相关研究极少。前期研究表明猪后肠甲烷菌和特定细菌对可溶（SDF）和不可溶纤维（IDF）有特异性响应。鉴于甲烷菌有底物偏好性，其群落变化可改变对发酵终产物的移除能力，最终影响纤维发酵效率和动物生长。故本项目将首先以燕麦β-葡聚糖（SDF）或微晶纤维素（IDF）为唯一碳源底物，通过3种不同代谢类型的甲烷菌、多糖利用菌和氢营养细菌的体外共培养，探明甲烷菌不同类群与细菌的共生模式；其次通过添加抑制剂溴氯甲烷构建体外和在体“去甲烷菌”模型，再引入上述3种甲烷菌，比较分析发酵体系或后肠甲烷菌代谢途径，和细菌代谢产物及相关能量代谢通路，结合动物生长性能和消化率，从菌种/株、菌（类）群、整体三个层面探明甲烷菌与细菌共生策略对上述两种纤维发酵模式和效率的影响机制，为靶向调控特定微生物以高效利用不同纤维源提供参考。

古菌是区别于真核生物和细菌的一类单细胞微生物，在单胃动物肠道内的数量仅占微生物总量的1.5%，远少于细菌，是名副其实的“少数派”。猪肠道内的古菌可以利用H2、CO2、有机酸或甲基化合物产生甲烷，也被称为甲烷菌，主要定植于后肠，对降低肠道气体分压，保证细菌发酵效率极为重要。日粮纤维对猪肠道健康和肠道功能的维持至关重要，主要依靠后肠细菌进行发酵利用。目前关于猪肠道甲烷菌功能的研究极少，探索其与细菌的互作对纤维利用效率的关系，可以为猪生产上科学选择不同纤维源提供参考。本项目在前期研究基础上，分别通过体外和体内试验，初步探明了猪后肠甲烷菌活性对不同类型纤维利用效率的影响，并解析了在此过程中甲烷菌与细菌的共生模式。结果表明：1）在猪后肠模拟发酵系统中添加浓度为20 μM的溴氯甲烷（BCM）可以显著抑制甲烷菌活性；口服10 g/100 kg体重的BCM 7 d即可有效抑制生长肥育猪后肠甲烷菌活性。2）猪后肠甲烷菌活性显著影响纤维的体外发酵模式，且其对以菊粉为代表的可溶性纤维（SDF）发酵模式的影响大于以小麦麸纤维为代表的不可溶性纤维（IDF）。3）相对于IDF饲粮，肥育猪采食SDF饲粮可明显提高其后肠短链脂肪酸浓度；在甲烷菌活性被抑制时，采食IDF饲粮的猪对粗纤维的消化率高于采食SDF饲粮的猪，其原因很可能是甲烷菌活性对猪后肠SDF发酵能力的影响大于IDF。4）猪后肠甲烷菌活性显著影响细菌组成，其中多糖利用菌和硫酸盐还原菌受影响程度大于乙酸产生菌。甲烷菌活性对SDF发酵效率的影响大于IDF。当先抑制甲烷菌活性再恢复时，对饲喂SDF饲粮的肥育猪养分消化率和生长性能有潜在改善作用。总之，本研究成功构建了猪的体外和在体抑甲烷菌模型，深入阐明了猪后肠甲烷菌对不同类型日粮纤维的利用效率和养分消化率的影响机理，研究结果为特定微生物种群的营养调控，实现对不同纤维源的合理、高效利用提供了参考，同时也为完善猪“后肠营养”理论提供了重要数据。