苜蓿青贮中梭菌多样性及其诱发丁酸发酵机理研究

As one of the highest quality forages, alfalfa is viewed as a difficult crop to ensile, primarily because of its high buffering capacity, low concentration of water soluble carbohydrates and vulnerability to the undesirable butyric acid fermentation, especially when ensiled at high moisture concentration. Of the many factors that can affect the silage fermentation, the type of micro-organisms that dominate the process often dictates the quality of the silage produced. However, sufficient data are not yet available on the clostridial community dynamics associated with the development of the butyric acid fermentation, and the mechanism by which the butyric acid fermentation develops during the ensiling of alfalfa has not yet been identified clearly. The objective of this study is to describe the clostridial community dynamics during the ensiling of alfalfa by the combined methods of high throughput sequencing and traditional culture-based techniques. Evaluation at species and subspecies level will be carried out using DNA-DNA homology analysis to determine the distribution of the different Clostridia. In addition, we will provide data on the fermentation characteristics and microbial counts of alfalfa silage. The results obtained by investigating the effect of fermentation products, LAB and their metabolites and ensiling conditions on the clostridial community diversity may aid to elucidate the mechanism by which the butyric acid fermentation develops during the ensiling of alfalfa. New technology to depress the butyric acid fermentation, such as screening bacteriocin-producing LAB strains, will be also proposed by taking advantage of morphological and physiological properties of representative Clostridia strains. Results of this study will contribute to target-based regulation methods to achieve safe and high-quality alfalfa silage production.

苜蓿是奶牛等草食动物的重要优质饲草，青贮可有效避免晾晒过程中不良气候条件造成的损失，但由于水分含量高，可溶性碳水化合物含量低，缓冲能较高，青贮过程中极易发生由梭菌引起的丁酸发酵，导致饲草品质下降。然而目前，参与苜蓿青贮发酵的梭菌种类、数量及种群演替规律尚不清楚，导致梭菌生长、诱发丁酸发酵的机理尚不明确。本项目通过传统分离培养与高通量测序相结合的方法，对苜蓿青贮中的梭菌进行分离、鉴定，以期明确苜蓿青贮中梭菌种群的构成；结合DNA-DNA同源性分析等分子生物学技术手段，将梭菌鉴定到种或亚种，进一步明确苜蓿青贮中梭菌多样性及种群演替规律；综合分析梭菌多样性与贮藏条件、发酵成分、乳酸菌及其代谢产物等因素的相关性，阐明苜蓿青贮过程中导致梭菌生长、诱发丁酸发酵的机理；针对参与苜蓿青贮发酵梭菌的生理生化特性，筛选有效抑制梭菌生长的新型乳酸菌，为构建安全、优质的苜蓿青贮饲料调制体系提供科学理论依据。

新鲜苜蓿直接青贮极易发生由梭菌引起的丁酸发酵，导致饲草品质下降。针对参与苜蓿青贮发酵的梭菌种类、数量及种群演替规律不清楚，导致梭菌生长、诱发丁酸发酵的机理尚不明确等问题，本项目通过传统分离培养与高通量测序相结合，发现在苜蓿原料上的梭菌菌群中Clostridium（43.2%）是最丰富的梭菌属，而Romboutsia timonensis（20.4%）、Terrisporobacter pertrolearius（17.0%）、C. celatum（16.7%）和C. tyrobutyricum（13.2%）是优势梭菌种。新鲜苜蓿直接青贮后，梭菌菌群中，Clostridium是最丰富的梭菌属，青贮过程中相对丰度持续增加，到发酵56d时达到99.0%；在种水平上，C. butyricum（6.07%）和C. perfringens（18.6%）分别在发酵第7 d和第14 d时相对丰度达到最大值，之后迅速下降，而C. tyrobutyricum在发酵7 d后相对丰度持续增加直到发酵56d（98.0%），明确了高水分苜蓿青贮中梭菌多样性及种群演替规律；相关关系分析表明Clostridium、Enterobacter和Enterococcus的相对丰度与青贮乳酸含量之间呈显著的负相关、与青贮pH值呈显著的正相关，丁酸和NH3-N含量与Clostridium呈显著的正相关；阐明了苜蓿青贮过程中导致梭菌生长、诱发丁酸发酵的机理，即融合魏斯氏菌（Weissella confusa）、短乳杆菌（Lactobacillus brevis）和Enterobacrter sp.在苜蓿青贮发酵前3天迅速增殖并主导细菌菌群，短乳杆菌和魏斯氏菌均属于异型发酵乳酸菌，导致青贮酸化效率缓慢，肠杆菌科细菌与乳酸菌竞争利用WSC，进一步降低青贮酸化效率。此时，产期荚膜梭菌（C. perfringens）、丁酸梭菌（C.butyricum）等梭菌芽孢开始萌发增殖并启动丁酸发酵，随着发酵的进行和青贮pH的下降，酪丁酸梭菌（C. tyrobutyricum）快速增殖并成为新的优势梭菌种主导丁酸发酵；针对参与苜蓿青贮发酵梭菌的生理生化特性，筛选到了有效抑制梭菌生长的新型乳酸菌Lactobacillus plantarum, LP31菌株，为构建安全、优质的苜蓿青贮饲料调制体系提供科学理论依据。