牦牛瘤胃纤维素降解菌对提高暖季型牧草青贮发酵品质的调控机理

Tropical grass is difficult to ensile owing to its lower water-soluble carbohydrate and higher lignocellulosic contents. The exogenous fibrolytic enzyme has been explored to induce direct conversion of structural carbohydrates into soluble sugars for lactic acid fermentation during ensiling. However, high cost, instability and weakly acid resistance of commercial enzymes limit their widespread application in silage. Fortunately, cellulolytic bacteria possess many advantages, such as safe, low cost and wide adaptability. It also could effectively hydrolyze structural carbohydrates into mono-saccharides for lactic acid fermentation during ensiling. Therefore, this study is designed to screen and isolate facultative anaerobic cellulolytic bacteria with wide substrate specificity, high stability, and strong acid-resistance, and then further evaluate their effects on the structural carbohydrate degradation, soluble carbohydrate compositions, fermentation quality and microbial community of tropical grass during ensiling. Meanwhile, with the aid of scanning electron microscopy technology, the spatial and temporal changes in structural carbohydrate degradation would be intuitively compared and analyzed, the degradation mechanism of structural carbohydrates would be identified, and the action mechanism of cellulolytic bacteria to enhance the fermentation quality of tropical grass would be also revealed. Overall, adequately investigating the cellulolytic bacteria provides more insights into silage making of tropical grass and the development of new silage additives.

暖季型牧草粗纤维含量高，水溶性碳水化合物含量低，难以调制出优质青贮饲料。目前多数研究通过添加外源纤维酶达到改善青贮发酵品质的目的，但由于酶制剂成本较高、性能不稳定及pH适应范围较狭窄等因素限制了其在青贮中的广泛应用。纤维素降解菌具有安全、低成本、适应性广等优点，可降解牧草细胞壁成分中的结构性碳水化合物，将其转化为单糖进而被乳酸菌利用。为此本项目拟从西藏牦牛瘤胃中筛选具有广泛的底物特异性、稳定性和耐酸性的兼性厌氧纤维降解菌，研究其对暖季型牧草青贮过程中结构性碳水化合物、水溶性碳水化合物组分含量、发酵品质、微生物群落结构及多样性变化的影响，并结合扫描电镜技术，直观地比较和解析结构性碳水化合物降解的时空变化规律，明确纤维素降解菌对结构性碳水化合物的降解机理，揭示提高暖季型牧草青贮发酵品质的作用机制，为纤维素降解菌在暖季型牧草青贮饲料生产中的应用及新型青贮添加剂的研制提供科学依据。

随着草食畜牧业规模化、集约化的发展，优质饲草及草产品供给不足，因地制宜提高牧草青贮发酵品质是草牧业提质、增效的迫切需求。纤维素降解菌的酶解作用可促进青贮过程中结构性碳水化合物的降解，增加水溶性碳水化合物含量，是改善青贮发酵品质的有效策略。经富集、筛选、产酶及降解特性分析，从西藏牦牛瘤胃中获得具有广泛底物特异性、稳定性及耐酸性的兼性厌氧纤维素降解菌（系）。将纤维素降解菌单独或与植物乳杆菌（Lactobacillus plantarum）组合添加于象草青贮中，均不同程度改善了青贮发酵品质，其中组合添加加速了青贮饲料中结构性碳水化合物的降解，保留了相对较高的水溶性碳水化合物（WSC 20.58~31.98%），与阳性对照组（商品酶制剂和里氏木霉）相比，纤维素降解菌Enterococcus faecium Y83在牧草青贮中具有降解木质纤维素和促进乳酸发酵的双重作用。为了进一步明确纤维素降解菌的应用效果，通过比较水稻秸秆和甜高粱在不同青贮时间点发酵相关参数、碳水化合物组分、酶解效率及青贮饲料表面形态结构变化，构建了WSC发酵动力学模型，发现外源纤维素降解菌与乳酸菌协同作用加速了粗纤维的降解，改善了青贮发酵品质；同时也发现外源纤维素降解菌可定向调控青贮过程的微生物群落结构，促使青贮后期乳酸菌的相对丰度提高（Lactobacillus和Pediococcus）。本项目挖掘与利用青贮用瘤胃高效纤维素降解微生物资源，丰富了特色功能性微生物种质资源库，初步明确了兼性厌氧纤维素降解菌和乳酸菌对提高青贮发酵品质的协同作用机制及对结构性碳水化合物的降解机制。研究结果为兼性厌氧纤维素降解菌（系）在暖季型牧草及农作物秸秆青贮中的应用及新型青贮添加剂的研制提供了理论指导。