葡萄渣缩合单宁调控苜蓿青贮乳酸菌特性及发酵品质的应用基础研究

CTs have antimicrobial properties as well as could inhibit protein degradation during forage ensiling and metabolism in rumen. The resources of grapes pomace which contained high level of condensed tannins (CTs) were abundant in Ningxia Helan mountain east foothill area, and have a positive effect on regulating the lactobacillus of alfalfa silage. At present, the model of effects of CTs on regulation of characteristics of lactobacillus in most of studies was the lactobacillus in the animal intestines. There was lack of theoretical support for the influence of CTs on properties of lactobacillus in the ensiling process. In the present study, the content of CTs in grape pomace and molecular weight characteristics of CTs will be determined, and then the effects of purified CTs and grape pomace on fermentation quality and nutritional value will be determined through ensiling test. The diversity of lactobacillus will be analyzed by degeneration gradient gel electrophoresis (DGGE). The lactobacillus in alfalfa silage will be identified by 16SrRNA after separation and purification. After cultivated in RPM， to screen and identify the significantly induced factors which affect the growth and metabolism of lactobacillus by techniques including comparative proteomics, mass spectrometry, and Western blot. Our research will systemically demonstrate the regulatory mechanisms of grapes pomace CTs on characteristics of lactobacillus in alfalfa silage and effects on the silage quality. The results will be enrich the theory of regulation of CTs on characteristics of lactobacillus in alfalfa silage and will provide scientific foundation for improving fermented quality of alfalfa silage.

缩合单宁( CTs)因具有抗菌特性及抑制青贮和瘤胃中蛋白质降解的作用而备受关注。宁夏贺兰山东麓富含CTs的葡萄渣资源丰富，应用于调控苜蓿青贮品质前景广阔。目前CTs调控乳酸菌特性的研究多以动物肠道内乳酸菌为模型，CTs对青贮过程中乳酸菌特性的作用机制尚缺乏理论支撑。本项目首先测定葡萄渣中CTs含量及CTs分子量特点，采用实验室青贮、营养价值评定及变性梯度凝胶电泳等方法，开展纯化CTs和葡萄渣调控苜蓿青贮发酵品质及对乳酸菌多样性影响的研究；在此基础上，利用16SrRNA将从苜蓿青贮中分离纯化的乳酸菌鉴定，鉴定后在含不同浓度CTs的RPM上培养，通过比较蛋白质组学、质谱及Western blot，筛选乳酸菌显著表达差异的生长代谢因子，阐明CTs对苜蓿青贮乳酸菌特性的调控机制。研究结果理论上将丰富CTs调控苜蓿青贮乳酸菌特性的认识，实践上能够为提高苜蓿青贮品质和拓展葡萄渣的利用途径提供科学依据。

宁夏贺兰山东麓富含缩合单宁（CTs）的酿酒葡萄渣资源丰富，CTs能够抑制苜蓿青贮和反刍家畜瘤胃中蛋白质降解，从而提高反刍家畜饲粮中粗蛋白质的有效利用效率。但CTs有一定的抗菌特性，CTs对苜蓿青贮过程中乳酸菌特性的作用机制尚缺乏理论支撑。本课题首先确定了宁夏贺兰山的东麓主栽酿酒葡萄渣单宁含量和分子量特性。研究表明，贺兰山东麓地区主栽酿酒葡萄渣中CTs含量在3.83 % ~ 5.18 %之间；通过预实验研究表明，水解单宁能够抑制青贮蛋白酶活性，降低青贮过程中蛋白水解程度，减少非蛋白氮和氨态氮含量，但水解单宁在不同添加水平下对青贮发酵产物影响甚微，总体评价对苜蓿青贮增益效果不大，而葡萄缩合单宁显著降低了苜蓿青贮过程中粗蛋白质的降解，改善了苜蓿青贮发酵品质和营养特性；我们进一步通过青贮试验和实验室检测分析，苜蓿青贮中添加贺兰山东麓地区主栽酿酒葡萄渣，能够增加苜蓿青贮微生物丰富度，尤其是实际微生物丰富度（Sobs），同样提高了Chao1指数和Ace指数；其中，马尔贝克葡萄渣和蛇龙珠葡萄渣处理组Shannon多样性指数较大，Simpson多样性指数较小；苜蓿青贮中肠球菌属（Enterococcus）比例升高，其中马尔贝克和美乐处理提高了魏斯氏菌（Weissella）丰度，蛇龙珠处理提高了肠球菌丰度；添加100 g/kg葡萄渣，苜蓿青贮质量等级由四级上升至二级，提高了苜蓿青贮品质，青贮蛋白酶活性、非蛋白氮和氨态氮含量下降，并延长了青贮有氧变质时间。本课题研究成果丰富了缩合单宁调控饲草青贮过程微生物特性的相关理论，研究成果能够为优化饲草加工调制提供实践参考。