【作者】 钱程；

【导师】 霍贵成；

【作者基本信息】 东北农业大学 ， 食品科学与工程， 2005， 硕士

【摘要】 本试验以青春双歧杆菌和嗜酸乳杆菌为对象,对益生菌在机体肠道内的粘附定植以及提高免疫功能的机理进行了研究,目的在于寻找出益生菌产生粘附作用的物质,以及在肠道细胞上的粘附素受体,以及益生菌产生免疫促进作用成分。 双歧杆菌和乳杆菌都属于革兰氏阳性菌。革兰氏阳性菌菌体细胞壁表面主要由肽聚糖(Peptidoglycan,PG)、多糖(Polysaccharide,Ps)和表层蛋白质(S-层蛋白,Surface-Layerprotein,S-layerprotein,SLP)组成,这些物质在益生菌菌体与宿主肠道细胞的粘附过程中直接或间接的起到不同程度的作用。 本实验对益生菌粘附机理的研究主要集中在于菌体代谢产物和细胞表层成分,即肽聚糖和表层的蛋白质。实验中首先用TritonX-100结合胰蛋白酶、DNA酶、RNA酶和蛋白酶E的方法,对青春双歧杆菌菌体细胞壁所含的完整肽聚糖进行提取;采用十二烷基硫酸钠(sDS)结合胰蛋白酶、三氯乙酸处理的方法提取嗜酸乳杆菌菌体细胞壁所含肽聚糖,并确定了最适的提取条件为用质量浓度为80g/L的十二烷基硫酸钠,沸水浴处理10min,然后用胰蛋白酶和三氯乙酸连续处理;同时对提取物进行蛋白质含量的测定和氨基酸分析,以确定提取过程是否真正有效的去除了受试菌株菌体内含有的蛋白质。 通过不同方法分别对受试菌株代谢产物、菌体表面成分以及肠道细胞表面物质在粘附过程中的作用进行了研究。确定青春双歧杆菌的粘附素为菌体分泌到培养基中的一种蛋白质,肠道上皮细胞对青春双歧的粘附素受体为细胞表面的一种糖蛋白;嗜酸乳杆菌的粘附素是菌体细胞壁上的一种蛋白质,其在肠道上皮细胞上的粘附受体为蛋白。 实验中还对灭活青春双歧杆菌和嗜酸乳杆菌菌株的粘附能力进行了初步研究,发现经热处理失活后的青春双歧杆菌依然具有粘附能力,而且并未受到明显影响;而嗜酸乳杆菌在经过热处理后,则基本丧失粘附能力。 提高机体免疫力是益生菌的主要功效之一。本实验以昆明小鼠为对象进行动物实验,检测青春双歧杆菌与嗜酸乳杆菌细胞壁肽聚糖促进免疫功能的作用。结果显示,与对照样本相比,接受菌液注射和肽聚糖注射都会对受试动物的免疫指标产生影响,其血清中自介素IL-6水平、血清溶菌酶活力、腹腔巨噬细胞数量、巨噬细胞吞噬能力都得到明显提高,说明细胞壁肽聚糖对机体免疫功能具有明显促进作用。更多还原

【Abstract】 The Bifidobacteria adolescent and Lactobacillus acidophilus were tested in this experiment to study the mechanism of adhesion and immunologic enhancement. The aim of this study is to research on the adhesin of probiotics and the receptor of adhesin. The components with the function of immuno-boost were also studied.Bifidobacteria adolescent was treated by TritonX-100, Trypsin, DNase, RNase and Proteinase E to extract the Peptidoglycan(PG).Bifidobacterium and Lactobacillus are Gram positive bacterium. The cell wall of Gram positive bacterium are composed of Peptidoglycan, Polysaccharide and Surface-Layerprotein and they may play different roles during the adhension between Protiotics and the intestinal epithelia of host.This study concentrated on the functions of cell wall Peptidoglycan and Surface-Layerprotein" in adhesion. The Lactobacillus acidophilus was treated by Sod.Dodecyl Sulfate(SDS), TrichloroaceticAcid(TCA) and Trypsin with the same purpose. The 80g/L SDS and heated with boiling water bath for 10 minutes was the optimal process condition to collect PG.The protein content and amino acid composition of PG extracted were tested to ensure the protein in bacteria cell wall was eliminated.Several different measures were adopted to determine the function of metabolite and the surface components of bacteria tested and the adhesin receptor of intestinal epithelia in adhension.The adhesin of Bifidobacteria adolescent was confirmed to be a certain kind of protein produced and excreted to the media by bacteria, and the receptor on intestinal epithelia should be some kind of glycoprotein. While the adhesin of L. acidopholus is protein on the surface of bacteria cell wall and the receptor is protein.The adhension ability of heat-inactived probiotics was also tested. The result showed that the inactived B. adolescent still can adhere to the intestinal epithelia while L. acidophilus cann’t. The result approved that the adhesin of them were different from each other.Mice were injected with bacteria suspension or PG and examed the influence on immunity. The Interleukin-6 level, Lysozyme level, numbers and phagocytic activity of Macrophage of mice injected with suspension or PG was improved significiently when compared with controls. The result showed that PG can boost the immunity of animals tested.更多还原