青贮辣木叶水解单宁降解乳酸菌筛选及作用机理研究

Tannins, usually divided into hydrolysable tannins and condensed tannins, are polyphenols that are widely distributed in plants. Tannins in feed are considered as antinutritional compounds due to their adverse effects on digestion and absorption of nutrients, especially for monogastric and aquatic livestock. Moringa oleifera leaves are highly nutritious, being a good source of protein, but the content of hydrolysable tannins is relatively high. Hydrolysable tannins-degrading microorganisms are isolated from materials that contain high levels of hydrolysable tannins. Isolation of hydrolysable tannins-degrading lactic acid bacteria from silage and degradation mechanism have not yet been reported. In this project, lactic acid bacteria with ability to degrade hydrolysable tannins will be isolated and screened from silages with high hydrolysable tannins content, then the strains will be identified. Tannase gene from the obtained lactic acid bacteria strains will be identified, cloned and expressed. To clarify the action mechanisms of these stains during ensiling, the isolated strains will be added and the expression of tannase gene, tannase activity, hydrolysable tannins content and hydrolysate will be analyzed in MRS broth medium and Moringa oleifera leaves silage. The study will provide a good technical support and theoretical basis for the degradation of hydrolysable tannins in silage, the preservation and utilization of the quality feed resources contain hydrolysable tannins like Moringa oleifera leaves.

单宁是广泛存在于植物体内的多酚类化合物，一般可分为水解单宁和缩合单宁。饲料中的单宁是一种抗营养因子，它会阻碍畜禽对营养物质的消化和吸收，尤其是对于单胃和水产动物。辣木（Moringa oleifera）叶营养丰富，是一种优质的蛋白质饲料资源，但水解单宁含量较高。科学家们已经从很多水解单宁含量高的材料中筛选出能降解水解单宁的微生物。但有关青贮饲料中具有降解水解单宁功能的乳酸菌筛选及其作用机制，尚未见报道。本项目从水解单宁含量较高的青贮饲料中分离、筛选和鉴定具有降解水解单宁能力的乳酸菌，并进行单宁酶基因的鉴定、克隆和表达。将筛选到的菌株添加到MRS肉汤培养基和辣木叶青贮饲料中，分析其单宁酶基因的表达，单宁酶活性，水解单宁含量及酶解产物，揭示其在青贮发酵过程中降解水解单宁的作用机理。为降低青贮饲料中水解单宁含量，有效贮藏、开发和利用辣木叶等含水解单宁高的优质饲料资源提供良好的技术支撑和理论基础。

辣木（Moringa oleifera）叶是一种营养丰富的优质蛋白质饲料资源，但其中抗营养因子水解单宁含量较高，阻碍畜禽对营养物质的消化和吸收。本项目筛选鉴定具有降解单宁能力的乳酸菌，并将其添加到辣木叶青贮中，研究其对发酵品质和单宁含量的影响。截止项目结题时，研究成果发表SCI论文8篇，中文核心期刊1篇；培养了研究生2名，基本完成了项目既定目标。研究结果如下：从香蕉叶、辣木叶、黄梁木叶等水解单宁含量较高的青贮饲料中初步分离具有降解水解单宁能力的乳酸菌9株，经过16S rDNA测序鉴定和生理生化鉴定，发现这9株菌均为植物乳杆菌Lactobacillus plantarum。经过单宁酶基因的克隆发现5株乳酸菌携带单宁酶基因。将这些菌株添加到香蕉叶中进行青贮，结果表明，这些菌株可以明显改善香蕉叶的发酵品质。其中菌株4和8降低了香蕉叶青贮中的单宁含量。将上述进一步筛选的乳酸菌添加到不同含水量的辣木叶中进行青贮。结果发现，辣木叶直接青贮品质较好，添加植物乳酸菌LP对辣木叶青贮品质影响不大，适当晾晒可以增加（P < 0.05）丙酸含量。微生物菌群分析结果表明，添加植物乳酸菌LP及晾晒处理对辣木叶青贮饲料的微生物菌群结构影响较小。测定单宁及酚类物质发现，添加植物乳酸菌LP对辣木叶青贮中多酚和简单酚含量无显著影响，在晾晒条件下，添加植物乳酸菌LP可以使辣木叶青贮饲料中水解单宁降低28.9%。因此，本课题的研究结果可以为降低青贮饲料中水解单宁含量，有效贮藏、开发和利用辣木叶等含水解单宁高的优质饲料资源提供良好的理论依据和可行方案。