香蕉植株上乳酸菌的系统分类学研究及其在香蕉副产物及残次果饲料调制加工中的应用

Lactic acid bacteria (LAB) and its products were widely applied in industries such as light industry, food industry, pharmaceuticals industry, and feed industry. Located in tropical area, Hainan Island boasts of abundant of animal, plant and microorganism resources. A large numbers of LAB species were obtained from plant sources. In our previous experiments which could be predicted that a variety of wild LAB were living on surface layer of tropical fruits. However, the natural LAB composition and species diversity in banana plants have not been studied. The objective of this study was to screen isolate, and identify the LAB from fruit residues with particular interest in species that are most likely to play an important role in the fermentation process. Firstly, to isolate wild LAB from various species of banana plants by using modified medium with adding banana plants source extract, and to analyze these isolates characterization based on the phenotypic properties of morphology, physiology and biochemical, and fermentation characteristic. To identify these isolates at the molecular level, 16S rDNA sequence analysis and DNA-DNA hybridization experiments will be studied. To use the banana by-products (stems and leaves, stalk, peel, waste) and defective banana effectively for animal feed, their chemical composition and silage fermentation characteristics will also be studied.

乳酸菌及其产品以其独特的生物学功能，在轻工、食品、医药及饲料工业等行业有广泛应用。海南岛位于热带地区，其动植物和微生物资源非常丰富，我们在前期实验中从植物性分离源里成功获取了大量乳酸菌菌株，可预测在热带水果表层生息着多种多样的野生乳酸菌。但是，对热带水果乳酸菌的菌种构成和多样性的研究及其菌种库的构建未见报道。本项目从各品种的香蕉植株上用添加分离源抽出液改良培养基分离野生乳酸菌，首先用细胞形态观察，生理生化性状及碳源发酵特性等分类法对分离菌株进行分析的同时，用16S rDNA序列分析法及核糖体分型法解析乳酸菌的种群构成、多样性及分类系统，并采用DNA-DNA相同性实验法进行菌种水平鉴定，探索分布于各品种香蕉植株中特有的新种乳酸菌，挖掘具有产细菌素和高产乳酸等功能的新型益生乳酸菌资源，开发香蕉副产物（茎叶、果柄、废弃物等）及残次果的饲料调制加工技术，为乳酸菌分类学研究和海南畜牧业的发展奠定基础

海南香蕉种植面积达到100多万亩，生产香蕉的同时也产生了大量香蕉假茎、叶和皮等残次物，以及因台风、冷空气等自然灾害所产生的残次果和废弃物。长期以来，除小部分用作肥料外，大部分弃于野外，任其腐烂，既浪费资源又污染环境。本项目从各品种的香蕉植株上用添加分离源抽出液改良培养基分离野生乳酸菌，首先用细胞形态观察，生理生化性状及碳源发酵特性等分类法对分离菌株进行分析的同时，用16S rDNA序列分析法及核糖体分型法解析乳酸菌的种群构成、多样性及分类系统，并采用DNA-DNA相同性实验法进行菌种水平鉴定，探索分布于各品种香蕉植株中特有的新种乳酸菌，挖掘具有产细菌素和高产乳酸等功能的新型益生乳酸菌资源，开发香蕉副产物（茎叶、果柄、废弃物等）及残次果的饲料调制加工技术，为乳酸菌分类学研究和海南畜牧业的发展奠定基础。我们从海南各香蕉种植基地采集香牙蕉、龙牙蕉、芭蕉等植株和果实样品；并对其附着微生物进行分析，掌握了样品上附着微生物的规律和数量。将筛选到的200多株乳酸菌株进行多次分离纯化及保藏；用经典的乳酸菌分类法和16S rDNA序列分析法对所筛乳酸菌进行分类鉴定，经分类鉴定得知所筛选菌株为：Lactobacillus plantarum、L.brevis、L.fermentum、L.pentosus、L.perolens, Pediococcus pentosaceus，Enterococcus casseliflavus, E.gallinarum, E. casseliflavus；其中L.plantarum占55%。乳酸菌株的生物学特性测试表明：L.plantarum对E.coli、Salmonella和Listeria monocytogenes等致病菌均有抑菌效果。L. plantarum WL7-3 抑菌效果最好；L.plantarum WFr6-4、WFl5-3 和 CG1等菌株，其培养时间是12h-18 h，pH值约3.5，这些菌株具有产酸量高、产酸速度快、适应期短、可发酵多种碳源等优势，同时其还具备耐高温、耐干旱等海南热带特征。课题组将所选乳酸菌株应用在香蕉茎叶及残次果青贮饲料调制加工，取得了较好的结果。青贮试验证明了，所选乳酸菌株是香蕉茎叶及残次果青贮饲料调制加工的优良微生物发酵剂。