内蒙古天然牧草青贮饲料中产纤维降解酶乳酸菌筛选及作用机理研究

Inner Mongolian grassland has rich biodiversity and specific species due to its complex and diverse environment and climate conditions. Recently, the lactic acid bacteria germ plasm in Inner Mongolian grassland has been widely focused by the scientists both from native and overseas. Based on the existing research results, the project will conduct to ensile native grass from meadow steppe, typical steppe, desert steppe and steppe desert, isolate and screening lactic acid bacteria producing cellulase and hemicellulase according to the degradation rate of cellulose hemicellulose in native grass silages, understand the kinds of the enzyme. At the same time, the enzyme-producing lactic acid bacteria strains with merits and advances in fermentation will be characterized and identified, and using these stains to making silage. By doing this, to select optimal lactic acid bacteria strains specialized at accelerating fermentation process and making sure good quality of ensiled forage, and to study the action mechanisms of these stains during fermentation,the effect on the fermentation quality, nutritive value and Microflora in native grass silage and the synergistic action of the lactic acid bacteria. The above research are supposed to set up primarily the databases on biodiversity of and germ plasm of enzyme-producing lactic acid bacteria of Inner Mongolian grassland, and to provide important database and information of lactic acid bacteria for further finding out and utilization of Inner Mongolian grassland lactic acid bacteria.

内蒙古草原复杂多样的地理环境和气候条件，孕育了丰富并且特有的生物种质资源，近年来内蒙古草原乳酸菌种质资源引起了国内外学者的极大关注。本项目根据现有的研究基础，拟通过制作内蒙古不同类型草原区（草甸草原、典型草原、荒漠草原和草原化荒漠）天然牧草青贮饲料，以青贮饲料中纤维素和半纤维素降解率为线索，分离筛选出具有产纤维素酶和半纤维素酶功能的乳酸菌，明确乳酸菌的产酶种类；同时，筛选出温度适应阈值宽、发酵速度快、适应性能和酶活性强的优良产酶乳酸菌株，并研究其在天然牧草青贮发酵过程中的作用机理，对青贮饲料发酵品质、营养价值、微生物群落的影响及复合添加产酶乳酸菌之间的协同作用。通过上述研究将初步建立内蒙古草原天然牧草青贮饲料中功能型乳酸菌种质资源库，同时也可为发掘和利用内蒙古草原的优良乳酸菌种质资源提供科技支撑。

内蒙古草原复杂多样的地理环境和气候条件，孕育了丰富并且特有的生物种质资源，近年来内蒙古草原乳酸菌种质资源引起了国内外学者的极大关注。项目组从内蒙古草甸草原、典型草原和荒漠草原80余个样点采集天然牧草青贮饲料150余份，从中分离出乳酸菌924株，涵盖6个属30个种。筛选出产纤维素酶乳酸菌10株，产半纤维素酶乳酸菌9株；通过测定每株乳酸菌菌液的酶活，选出3株产β-葡萄糖苷酶活性较高（79.25 mU/mL~95.32 mU/mL）的乳酸菌，分别为植物乳杆菌（CE4）、戊糖片球菌（CE15）和粪肠球菌（CE20）；两株产阿魏酸醋酶活性较高（8.59 mU/mL、9.99 mU/mL）的乳酸菌，分别为植物乳杆菌（HCE3）和肠膜明串珠菌肠膜亚种（HCE20）；通过分析上述5株乳酸菌的生理生化指标，发现两株植物乳杆菌（CE4、HCE3）的产酸能力、生长速率和耐酸能力优于其他菌株。将CE4和HCE3分别添加至天然牧草、芨芨草、玉米秸秆等高纤维组分含量的原料中调制青贮饲料，两株乳酸菌处理组的青贮饲料pH、乙酸含量和氨态氮占总氮量显著低于对照组（p < 0.05），乳酸含量显著高于对照组（p < 0.05），表明两株乳酸菌可改善青贮饲料的发酵品质；与对照组相比，CE4处理组纤维素含量降低5.48%~8.48%，HCE3处理组半纤维素含量降低5.45%~8.89%，CE4+HCE3处理组纤维素和半纤维素含量分别降低5.59%~7.94%和4.68%~7.48%，表明两株乳酸菌可降低青贮饲料的纤维组分。通过分析大针茅青贮饲料厌氧和有氧发酵过程中微生物数量和多样性，发现整个发酵过程未出现变形菌门与厚壁菌门转换现象，肠杆菌科细菌演替主导发酵过程，乳酸菌在大针茅青贮饲料中为小众菌群。通过上述研究已初步建立内蒙古草原天然牧草青贮饲料乳酸菌种质资源库和功能型乳酸菌种质资源数据库，也为发掘和利用内蒙古草原的优良乳酸菌种质资源提供科技支撑；同时，大针茅青贮饲料微生物演替规律研究，为天然牧草青贮饲料发酵过程中微生物群落演替机制及其调控技术研究提供理论基础。