【作者】 王俊国；

【导师】 张和平；

【作者基本信息】 内蒙古农业大学 ， 畜产品安全生产， 2008， 博士

【摘要】 对采自我国新疆和蒙古国不同地区传统发酵乳制品(以酸马奶为主)中的61株乳杆菌进行了耐胃酸、耐胆盐及降胆固醇能力等潜在益生特性的研究,同时利用16S rDNA序列同源性分析的方法对部分益生特性好的菌株的鉴定结果进行了验证,并对这些菌株是否具有胆盐水解酶活性进行了定性及定量检测;通过体外试验筛选出对胆固醇有不同吸收、吸附作用及具有不同胆盐水解酶活性的九株乳杆菌,灌胃给高脂血症大鼠,观察它们对血脂的影响,为系统地了解乳酸菌降胆固醇的作用机理和体内外相关性提供了基础数据,也为进一步开发和利用传统发酵乳制品中的乳酸菌资源奠定了基础。通过对蒙古国及我国新疆牧区酸马奶等传统发酵乳制品中的降胆固醇益生乳杆菌的筛选及对其降胆固醇体内外相关性的研究,得出以下结论:在61株乳杆菌中有12株耐酸性、耐胆盐、粘附性及降胆固醇能力较好菌株;生长菌株对培养基中胆固醇的移除率依赖于其在含0.3%胆盐培养基中的生长情况,二者存在显著正相关性(P<0.01)。有3株菌的休眠菌株对胆固醇的移除率高于其生长菌株的,表明它们对胆固醇有较强的吸附能力。利用16S rDNA同源性分析的方法对13株乳杆菌进行了鉴定,其中有10株与传统生理生化方法鉴定的结果一致;另三株菌通过进一步的分析,确认符合16S rDNA同源性分析鉴定的结果。对12株具有较好益生特性的乳杆菌的胆盐水解酶活性进行了定性筛选和定量测定,有4株菌具有胆盐水解酶活性,通过乳酸菌在体外降胆固醇的试验,发现胆盐水解酶活性与菌体洗涤液中胆固醇含量呈极显著的正相关性(P<0.01)。将体外筛选出的九株对胆固醇有不同吸收、吸附作用及具有不同胆盐水解酶活性的菌株灌胃高脂血症大鼠,观察其体内外降胆固醇的相关性,发现灌胃乳杆菌组对大鼠血脂都有一定程度的降低作用,但菌株间差异显著(P<0.05)。在体外对胆固醇吸收或吸附能力强的菌株,在动物体内降胆固醇功能明显。灌胃含有胆盐水解酶活性的乳酸菌菌株,其粪便中游离胆酸含量高于模型组,随着菌株胆盐水解酶活性的减少粪便中游离胆酸含量依次降低,但对降血脂作用较弱。通过对试验大鼠的肝脏大体形态及肝脏、心血管组织切片的观察,发现在所有菌株中LIP-1的肝脏及心血管组织状态最接近正常对照组。更多还原

【Abstract】 A total of 61 Lactobacillus strains isolated from traditionally fermented dairy products(mainly isolated from home-made koumiss) in Mongolia and Xingjiang of China were assessed for potential probiotic properties. After their tolerance of bile salt and survival rates in artificial gastrointestinal juice at pH3.0 and ability to remove cholesterol from culture had been evaluated, 13 stains of Lactobacilli were further identified by 16S rDNA sequences homology analysis for their good probiotic properties. Potential Bile Salt Hydrolase activity were examined by high-performance liquid chromatography (HPLC) analysis. Based on these data,9 strains of Lactobacilli were selected for a feeding trial on account of their various assimilation of cholesterol abilities and binding of cholesterol abilities and BSH activity. The effects of nine strains on serum lipids and liver and fecal cholesterol and fecal total bile acids were estimated in rats fed a high lipid alone or supplemented with different Lactobacillus strains for 4 weeks.The results provides the basic data for systematically understanding possible mechanisms of cholesterol removal in-vitro and in-vivo by Lactobacillus, and gives useful information for further exploitation and utilization of lactic acid bacteria in traditional fermented milk products.Twelve strains of Lactobacilli were selected for potential probiotic properties on account of their strong bile-salt resistance and low pH tolerance and hydrophobicity properties. Our findings indicated that cholesterol removal and growth are closely related. Cholesterol assimilation in the presence of 0.3% bile showed good relationship with bile tolerance of the strains.Three strains showed stronger binding of cholesterol ability than others.Thirteen strains of Lactobacilli that had been identified by standard conventional identification methods were further identified by16S rDNA sequences homology analysis. The results showed ten strains had the same as results with traditional classification. Three strains which showed different results by conventional identification methods and 16S rDNA sequences homology analysis were further analyzed. The last results showed 16S rDNA sequences homology analysis can be deemed credible.Four strains of Lactobacilli were selected for their Bile Salt Hydrolase activity by HPLC analysis. Cholesterol concentration in the washing buffers showed greatly relationship with bile salt hydrolase activity of the strains.In order to evaluate the correlation of Lactobacillus strains cholesterol-lowering effects and possible mechanisms of cholesterol removal between in vitro and in vivo, 9 strains of Lactobacilli were selected for a feeding trial on account of their various assimilation of cholesterol abilities and binding of cholesterol abilities and BSH activity. The results showed that all of Lactobacillus strains were effective in reducing serum total cholesterol , triglyceride and low- density lipoprotein cholesterol, and liver cholesterol, while increasing the fecal excretion of total cholesterol, compared with the model group. Those strains which had strong assimilation and blinding of cholesterol abilities in vitro would excreted more cholesterol in fecal than the others. Those strains which had BSH activity would excreted more bile acid in fecal than the model group, and the amount of total bile acid in fecal was consistent with the activity of BSH in vitro. The hepatic tissue slice and the aortal tissue slice in rat fed a different diet were observed. The results showed fed LIP-1 can greatly alleviate pathological changes of the hepatic tissue and the aortal tissue. Our findings indicated those strains which had strong assimilation and blinding of cholesterol abilities in vitro would greatly reduce serum cholesterol in vivo.更多还原