【作者】 郭春锋；

【导师】 张兰威；

【作者基本信息】 哈尔滨工业大学 ， 化学工程与技术， 2011， 博士

【摘要】 目前,心脑血管疾病已成为许多国家居民的首要死因,而引发心脑血管疾病的重要原因之一就是高胆固醇血症。药物治疗一直是临床处理高胆固醇血症的主要方式,然而降胆固醇药物往往存在着一定的副作用。益生菌是一类能够对人体健康起到促进作用的活体微生物,通常具有较高的安全性,因此使用益生菌预防和治疗高胆固醇血症具有重要意义。在本研究中,从27份人类粪便样品中分离出了150株乳酸菌,以该150株乳酸菌为受试菌株,对潜在的降胆固醇益生菌进行了筛选,同时并对筛选出的菌株的体外降胆固醇的机制、功能特性的影响因素及体内降胆固醇功能等进行了研究。基于胆盐解离途径对潜在的降胆固醇益生菌进行了筛选,最终筛选出干酪乳杆菌F0822、干酪乳杆菌F0422、屎肠球菌F0511和屎肠球菌IN7.12等4株乳酸菌;基于DCA脱除途径对潜在的降胆固醇益生菌进行了筛选,干酪乳杆菌F0822被最终筛选出来。干酪乳杆菌F0822是同时具有较强胆盐解离能力和脱氧胆酸(DCA)脱除能力的潜在降胆固醇益生菌。对上述4株乳酸菌的胆盐解离特性进行了研究,获得了如下发现:⑴胆盐直接影响菌株胆盐水解酶(BSH)的分布。在无胆盐的培养条件下,上述4株乳酸菌的BSH几乎全部集中在细胞内,而在含胆盐的培养条件下,胞内的BSH有部分转移到了细胞外;⑵细菌细胞膜阻碍了胞内的BSH对底物胆盐的解离作用,因而裂解细胞比活细胞具有更强的胆盐解离能力;⑶干酪乳杆菌F0822和干酪乳杆菌F0422活细胞对甘氨酸结合胆盐具有较强的解离能力,而屎肠球菌F0511和屎肠球菌IN7.12则对牛磺酸结合胆盐具有较强的解离能力。对干酪乳杆菌F0822脱除DCA的机制进行了研究,发现干酪乳杆菌F0822脱除DCA的机制应该主要归结为细胞壁的S层蛋白对DCA的吸附作用。该S层蛋白对DCA的吸附作用是在疏水作用推动作用下发生的氢键吸附。DCA的羧基是吸附在S层蛋白上的主要基团。采用现代仪器手段对该S层蛋白的理化特性进行了表征,发现该S层蛋白的分子量为43 kDa、等电点为9.7、热变性温度为78℃,含40.8%的疏水性氨基酸,不含半胱氨酸。在体外研究了吐温80对干酪乳杆菌F0822和干酪乳杆菌F0422功能特性的影响,发现0.2%(v/v)的吐温80能显著(P<0.05)增强两株干酪乳杆菌的胆盐解离能力和胆盐耐受能力。吐温80增强该两株菌胆盐解离能力的机制与吐温80增强菌株胞外的BSH的活性有关,而吐温80增强该两株菌胆盐耐受能力的途径与吐温80降低菌株胞内物质泄漏程度和增加菌株细胞内16:0游离脂肪酸的含量有关。在体外研究了氯化钙对干酪乳杆菌F0822和干酪乳杆菌F0422功能特性的影响,发现0.3%(m/v)的氯化钙能显著(P<0.05)增强该两株菌的黏附能力。对于干酪乳杆菌F0822,氯化钙增强菌株黏附能力的途径与钙离子介导S层蛋白与上皮细胞间的黏附作用有关;而对于干酪乳杆菌F0422,氯化钙增强菌株黏附能力的途径与钙离子介导脂磷壁酸与上皮细胞间的黏附作用有关。采用大鼠对干酪乳杆菌F0822和干酪乳杆菌F0422的体内降胆固醇功能进行了评价,发现干酪乳杆菌F0822在低剂量(1×108 CFU/mL饮用水)下就能显著(P<0.05)降低大鼠血清TC、LDL-C及肝脏TC水平,而干酪乳杆菌F0422只有在高剂量(1×109 CFU/mL饮用水)下才能发挥如此作用。增加大鼠每日粪便中总胆汁酸的排泄量是该两株菌在体内发挥降胆固醇功能的主要机制。在大鼠体内研究了0.6%(v/v)吐温80和0.1%氯化钙(m/v)的混合物对干酪乳杆菌F0822和干酪乳杆菌F0422降胆固醇功能的影响,发现该混合物可显著(P<0.05)增强干酪乳杆菌F0822和干酪乳杆菌F0422降低大鼠血清TC和LDL-C水平的能力。这提示该混合物可以用作干酪乳杆菌F0822和干酪乳杆菌F0422在体内发挥降胆固醇功能的促进剂。更多还原

【Abstract】 Cardiovascular and cerebrovascular diseases are presently the leading causes of death in many countries, and hypercholesterolemia is a major reason leading to such diseases. Medications are the main approach for managing hypercholesterolemia. However, there are intractable side effects of the medication. Probiotics are viable microorganisms that have a beneficial effect to human heath when ingested. Probiotics are generally regarded as safe for human consumption, and therefore, use of probiotics to prevent or control hypercholesterolemia has important significance. In this study, 150 lactic acid bacteria (LAB) were isolated from 27 human fecal samples. The probiotics with potential cholesterol-lowering function were screened from the 150 LAB, and subsequently the in vitro cholesterol-lowering mechanisms, influencing factor of functional properties and in vivo cholesterol-lowering effects of the selected strains were investigated.Based on bile salt deconjugation pathway, 4 LAB (Lactobacillus casei F0822, Lactobacillus casei F0422, Enterococcus faecium F0511 and Enterococcus faecium IN7.12) were screened out. Based on deoxycholic acid (DCA) removal pathway, L. casei F0822 was screened out. L. casei F0822 was a potential cholesterol-lowering probiotic that had stronger bile deconjugation ability as well as a stronger DCA removal ability.The 4 LAB as mentioned above were investigated for the bile salt deconjugation property. The following conclusions were drawn: (1) Bile salts markedly affected bile salt hydrolase (BSH) location of the strains. In the absence of bile salts, BSH of the strains was located in intercellular fractions almost entirely, and in the presence of bile salts, a portion of intercellular BSH was transfered to extracellular broth. (2) Cellular membranes blocked the deconjugation action of intercellular BSH against conjugated bile salts, and thus resulted in that the lysed cells of the strains had stronger bile salt deconjugation ability than the whole cells. (3) The viable cells from L. casei F0822 and L. casei F0422 showed stronger deconjugation activity against glyco-conjugated bile salts, and the viable cells from E. faecium F0511 and E. faecium IN7.12 showed stronger deconjugation activity against tauro-conjugated bile salts.The mechanisms on DCA removal for L. casei F0822 were investigated. It was concluded that the DCA removal action for L. casei F0822 should be attributed to binding of the S-layer protein on DCA. The binding action belongs to hydrogen-bond binding that occurs under hydrophobic action of DCA. Carboxyl of DCA was the main group bound to the S-layer protein. The physicochemical properties of the S-layer protein were determined using modern instruments, and results demonstrated that molecular weight, isoelectric point and thermal denaturation temperature of the S-layer protein were 43 kDa, 9.7 and 78℃, respectively. Moreover, the S-layer protein contained 40.8% hydrophobic amino acids and are absent of cysteine.The effects of tween 80 on functional properties of L. casei F0822 and L.casei F0422 were investigated in vitro. It was found that 0.2% (v/v) tween 80 could significantly (P<0.05) enhance bile salt dedonjugation ability and bile salt tolerance ability of the strains. The mechanism on the tween 80 enhanced bile salt deconjugation ability of the strains was related to the tween 80 enhanced activity of extracellular BSH of the strains. The mechanism on the tween 80 enhanced bile salt tolerance ability of the strains was related to the tween 80 reduction in the degree of intercellular material leakage and enhanced the percentage of intercellular 16:0 free fatty acid to total intercellular free fatty acids.The effects of CaCl2 on functional properties of L. casei F0822 and L. casei F0422 were investigated in vitro. It was found that 0.2% (m/v) tween 80 could significantly (P<0.05) enhance adhesive ability of the strains. For L. casei F0422, such phenomenon was related to the Ca ion mediated adhesive action between lipteichoic acid and the epithelial cell. For L. casei F0422, such phenomenon was related to the Ca ion mediated adhesive action between the S-layer protein and the epithelial cell. The in vivo cholesterol-lowering effects of L. casei F0822 and L. casei F0422were investigated in rats. The results demonstrated that L. casei F0822 could significantly (P<0.05) decrease serum TC, LDL-C and liver TC levels of rats at a low dose(1×108 CFU/mL driking water), while L. casei F0422 could significantly (P<0.05) decrease serum TC, LDL-C and liver TC levels of rats only under a high dose(1×109 CFU/mL driking water). It was the main cholesterol-lowering mechanism for the two strains to increase daily total fecal bile acid excresion of rats.The mixture of 0.6% (v/v) tween 80 and 0.1% (m/v) CaCl2 could significantly (P<0.05) enhance the potential capability of L. casei F0822 and L. casei F0422 to decrease serum TC and LDL-C levels of rats. This finding suggested that such a mixture might have potential as a facilitating agent that helps L. casei F0822 and L. casei F0422 to exert a cholesterol-lowering function.更多还原