【作者】 郭翔；

【导师】 郭本恒；

【作者基本信息】 江南大学 ， 食品科学， 2009， 博士

【摘要】 随着人们生活水平的提高,高胆固醇含量膳食的摄入也逐渐增多,过高的胆固醇摄入量容易引发高胆固醇血症。通常,血液中胆固醇浓度每上升1 %,心脑血管疾病的死亡率就会上升2 %。因此,保持人体内胆固醇含量在正常范围内对于预防心脑血管疾病是至关重要的。国外研究表明许多乳酸菌在体内和体外都有降低胆固醇的功效。本文通过对传统发酵食品中降胆固醇乳酸菌的筛选,鉴定,生物学活性,安全性,粘附和定殖及其降胆固醇的作用的研究,得出如下结论:1.本研究以高胆固醇源培养基MRSO-CHOL为模型,从传统发酵食品中分离纯化得到66株具有降低胆固醇能力的乳酸菌菌株,从中筛选出5株具有较好降低胆固醇能力的菌株;在模拟消化环境下比较5株菌株的耐酸性、胆汁耐受能力和胆盐降解（牛磺胆酸钠）能力,最终确定GUO菌株为筛选目标菌株。GUO菌株经传统微生物鉴定方法和API 50CH细菌鉴定系统鉴定,确定GUO菌株为植物乳杆菌（Lactobacilluss plantarum）。2. Lactobacillus plantarum GUO的生物学特性结果显示:该菌株的最高,最低和最适生长温度分别为43℃,12℃和30℃;最高,最低和最适起始生长pH分别为9.4,3.6和5.7。Lactobacillus plantarum GUO采用MRS培养,2 h进入对数生长期,11 h进入稳定期,生长比较旺盛;而采用SKM培养则生长比较缓慢,产酸能力也很弱。进一步研究发现,Lactobacillus plantarum GUO的MRS培养液4℃保藏条件下,S值为15.2 d,而在脱脂奶中的S值为116.3 d。3.益生菌的粘附和定殖是发挥其益生作用的第一步,也是最重要的一步。Lactobacillus plantarum GUO对Caco-2细胞的粘附结果表明:该菌株对Caco-2细胞的粘附在一定浓度和时间范围内存在量效关系,当加入细菌浓度达到108 cfu/mL、孵育2 h时,粘附趋于饱和;稳定期的Lactobacillus plantarum GUO对Caco-2细胞的粘附效果最好。pH对于粘附作用具有显著影响,在酸性范围粘附性最强;Ca²⁺、Mg²⁺并没有参与到Lactobacillus plantarum GUO对Caco-2细胞的粘附。因此,Lactobacillus plantarum GUO对Caco-2细胞的粘附是多种因素共同作用的结果,其中粘附素与受体的结合占主导地位。4.采用PCR-DGGE研究了Lactobacillus plantarum GUO在大鼠肠道内的定殖特性:结果表明:大鼠持续摄入Lactobacillus plantarum GUO发酵乳制品10 d后,Lactobacillus plantarum GUO在大鼠肠道的结肠、回肠、盲肠部位定殖,但在十二指肠和直肠部位未定殖。Lactobacillus plantarum GUO在肠道定殖结肠部位定殖时间最长,停止灌胃后11 d仍有少量定殖;在结肠部位定殖量最高,灌胃停止3 d后检测回肠定殖量达108 cfu/g;回肠其次,最高约为107 cfu/g;盲肠仅第3组以及回肠第2组有定殖,约105 cfu/g。5.进一步对Lactobacillus plantarum GUO的体外降胆固醇作用研究发现,Lactobacillus plantarum GUO主要是通过同化与共沉淀两种方式共同降低培养基中的胆固醇,其中同化作用占63.5 %;共沉淀占36.5 %。在此基础上,对Lactobacillusplantarum GUO可能存在的降胆固醇相关酶进行了研究。采用硫酸铵沉淀、Sephacryl S-100层析、SDS-PAGE电泳等分离纯化得到了降胆固醇相关的酶,该酶的分子量为38,000 Da,最适底物浓度为150μg/mL,最适ATP浓度为2.5 mg/mL,最适pH为7.0,最适温度为55℃;在60℃以下,pH 6.0～9.0,酶活在30 min内基本保持稳定。6.本研究采用耐药性试验、Ames试验,急性毒性（经口LD50）试验,90 d喂养试验和致畸试验评价了Lactobacillus plantarum GUO的安全性。试验结果表明,在实验条件下,Lactobacillus plantarum GUO对氟哌酸（NOR）和庆大霉素（GEN）两种抗生素不敏感;对头孢拉定（CEF）、四环素（TET）中度敏感;对氯霉素（CMP）和红霉素（ERY）高度敏感。Lactobacillus plantarum GUO的Ames试验结果为阴性,无致突变作用;无急性毒性,最大耐受剂量MTD均大于20000 mg/kg。90 d喂养试验结果表明:在实验条件下,试验动物生长期间体征指数正常;脏体比,血液学指标无显著差异;各剂量组动物脏器未发现明显组织学病变。致畸试验结果表明:在实验条件下,各剂量组试验动物未见有母体毒性,胚胎毒性和致畸性。这表明Lactobacillus plantarum GUO是安全的,可以作为益生菌进行深度的开发利用。7.由于Lactobacillus plantarum GUO具有良好的功能特性和生物学活性,为了更好地发挥其益生作用,将其应用于产品中具有更重要的现实意义。在单因素实验基础上设计并优化了Lactobacillus plantarum GUO的发酵乳配方Lactobacillus plantarum GUO发酵乳的基本配方为:15 %SKM,5 %葡萄糖,3 %发酵剂。Lactobacillus plantarum GUO发酵乳饮料的基本配方为:发酵基料30 %,蔗糖13 %,果胶0.3 %,柠檬酸钠0.1 %。8.为了了解Lactobacillus plantarum GUO在产品中的降胆固醇功效,参照保健食品功能性评价方法和程序,将Lactobacillus plantarum GUO发酵乳灌喂高血脂模型大鼠。结果表明:与对照组相比,含有活菌的Lactobacillus plantarum GUO发酵乳能够显著降低大鼠体内的TC和LDL-C;而灭活的Lactobacillus plantarum GUO发酵乳则没有此功能。这表明Lactobacillus plantarum GUO在大鼠体内也具有良好的降低胆固醇功效。更多还原

【Abstract】 Cardiovascular disease（CVD） is a leading cause of death in the world. Diet has been identified as a means of conrtolling serum cholesteorl concentrations,and many health conscious people are adopting measures to decrease risk by reducing blood serum cholesteorl.Several investigations have reported that a fermented milk product with lactic acid bacterria have a a action to decrease seurm cholesterol coneentration.1. 66 strains of bacteria were isolated from the traditional fermented foods. Cholesterol-enriched MRSO-CHOL medium was used to analyze their cholesterol-reducing ability. 5 strains were screened for their excellent cholesterol-reducing ability. Low pH and bile tolerance, deconjugation of sodium taurocholate were tested and compared with cholesterol-reducing ability of these 5 strains, respectively. Strain GUO was selected as the optimal strain for all its tested abilities were among the top three and its cholesterol-reducing ability was best. Strain GUO was identified as Lactobacilluss plantarum by using API identification system.2. The growth characteristics of strain GUO was studied. It grew at optimal temperature 30℃,and the minimum 12℃,maximum 43℃.As for initial pH value for cell growth, the optimal value was 5.7,as well the upper and lower limitations were 3.6 and 9.4, respectively. The growth of L. plantarum GUO culture was tested in MRS broth and 10 % SKM medium. It was found L. plantarum GUO began logarithmic stage and maximum growth at 2 h and 11h, respectively. But it grew slowly in 10 % SKM and produced little acid. Stored at 4℃in MRS （pH 3.8） and SKM （pH6.8）,it was found the death stage of 90 % living cells of L. plantarum GUO,as directed to value S, was 15.2 d and 116.3 d, respectively.3. The results showed that the adhesion was bacterial cell concentration and incubation time dependent. When the bacterial cell concentration reached 108CFU/mL, after 2h’s incubation, the mammal cell surface tended to be saturated by L. plantarum GUO. The adhesive ability of L. plantarum GUO to Caco-2 cells reached maximal when the bacteria cells were harvested in stationary phase. The pH value of the solution demonstrated significantly effect on the bacterial adhesive ability,of which acid pH was optimal for the adhesion among the serial pH values tested. Whereas no such phenomena was observed for addtion of Ca²⁺and Mg²⁺（100 mM）.These findings indicated the adhesion of L. plantarum GUO to Caco-2 cells could be affected by various factors, among which the effect of adhesin and receptor was predominant.4. Two methods, incubation and Denaturing Gradient Gel Electrophoresis were applied to assess L. plantarum GUO’s colonization in intestinal tract of Wistar rats. Results showed that L. plantarum GUO mostly colonized rats’colon and ileum, the amount reached separately 107cfu/g and 108cfu/g, only little L. plantarum GUO colonized the jejunum and the caecum, the amount was less than 105cfu/g, but it couldn’t colonize the rectum and the duodenum. L. plantarum GUO colonized the colon for about 10 d, the ileum for 7 d and the caecum for only 3 d. 5. More studies on the mechanism of cholesterol removal in vitro was carried out. The results showed that L. plantarum GUO strain was able to precipitate and assimilate cholesterols accounting for 36.5 % and 63.5% respectively, which displayed assimilation as the predominant one. The specific activity of the enzyme was increased 2.5-fold, 20-fold, 39.6-fold, respectively. But SDS-PAGE showed that the purified sample was not homogeneous. Through PAGE preparation electrophoresis we got the electrophoretic pure enzyme, calculated the molecular weight was about 38,000Da. Characterization of the enzyme exhibited the optimal concentration of ATP and the concentration of the substrates was 2.5 mg/mL and150μg/mL respectively; the optimum pH and temperature was 7.0 and 55℃respectively and kept stable after 30 min maintenance below 60℃from pH 6.0 to 9.0.6. In accordance with methods of our country for toxicological assessment on food safety, antibiotic sensitivity test, Ames test, the acute toxicity test, 90 d feeding and the teratogenicity test were used to assay the safety of L. plantarum GUO. Antibiotic sensitivity test found that L. plantarum GUO showed no sensitivity to Norfloxacin and Gentamycin, but was significantly sensitive to Cefradine, Tetracycline, Chloramphenicol and Erythromycin. L. plantarum GUO in Ames test and the acute toxicity test showed to negative. The 90 d feeding test showed that L. plantarum GUO had no effect on the growth and development, tissue pathology, blood system, liver function and kidney function of the rat. The result of the teratogenicity test showed that L. plantarum GUO had no effect on the toxicity of the mothers body, the toxicity of the embryo and teratogenicity. In short, L. plantarum GUO was safe and has the potential for development into functional foods.7. Due to the functionality and biological activity of L. plantarum GUO, it will have a important meaning that is applied to milk industry. On the basis of the single factors experiment, we design and optimize the formula of fermented milk:15 % SKM、5 % glucose、3 % starter; the formula of fermented milk drink of L. plantarum GUO:the fermented base materials 30 %,sucrose 13 %,pectin 0.3 %,sodium citrate 0.1 %.8. The effects of L. plantarum GUO on serum lipids were estimated in SD rats fed cholesterol-enriched diets. Living strain L. plantarum GUO significantly reduced TC and LDL-C, the ability increased as the dose increased, and also increased HDL-C. Also, dead L. plantarum GUO culture could significantly reduce LDL-C and increase HDL-C.更多还原