降胆固醇乳酸菌的筛选、鉴定及降解机制的研究
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摘要
[目的]随着生活水平的不断提高,人们对食品营养的要求也越来越高,但随之而来的负面影响却是动脉粥样硬化、冠心病、高血压等心脑血管疾病的发病率不断提高。国内外大量临床试验证实,服用乳酸菌及其相关制品具有减少人体血清胆固醇含量,降低心血管疾病发病率的功效。本文通过筛选具有降胆固醇功能的乳酸菌株,并研究了它们的体外益生特性、体外降胆固醇机制和大鼠体内降胆固醇功能疗效,为推动功能性乳酸菌-降胆固醇乳酸菌的研究和应用提供原始菌株和基础研究支撑。
[方法]全文以中国传统食品泡菜、腊肠为材料,以碳酸钙-MRS(calcium carbonate-Man Rogosa and Sharp Medium)选择性培养基筛选乳酸菌,应用改良的胆固醇培养基筛选具有较高降胆固醇能力的乳酸菌；结合菌落形态学、接触酶反应、革兰氏染色、碳水化合物微量鉴定管及16SrRNA寡核苷酸碱基序列分析鉴定；研究其耐酸性,耐胆盐活性,生长曲线及产酸特性；采用药敏纸片法测定菌株对抗生素的敏感性,双层琼脂牛津杯法测定菌株对常见致病菌的抑制效果；比较胆盐存在与否、不同胆盐成分对菌株在培养基中降胆固醇活性的影响；研究胆固醇在体外降解后,其在细菌沉淀、胞内和胞外的分布情况,探讨菌株体外降胆固醇的机制；构建高脂血症大鼠模型,测定灌胃菌株前后大鼠血清主要指标的含量变化,研究降胆固醇乳酸菌在体内的降胆固醇活性。
[结果]筛选得到的两个菌株LpTl和LpT2其降解率分别达到49.11%和50.03%,经鉴定为植物乳杆菌；同时,植物乳杆菌LpTl和LpT2都呈现了较强的耐酸性耐胆盐活性,在pH 3.0条件下存活率都比较高,在含0.3%胆盐的培养基中两株菌的生长都受到了一定抑制；其中植物乳杆菌LpTl在pH3.0培养基中能至少存活8 h,在含0.1%胆盐的培养基中能存活至少8 h；植物乳杆菌LpT2在pH2.0的培养基中至少能存活6 h,在含0.1%胆盐的培养基中都能存活至少8h；植物乳杆菌LpT1在第14小时开始进入对数生长期,在第22小时进入生长稳定期,生长30 h后最低pH值4.2左右；植物乳杆菌LpT2在第12小时进入对数生长曲线,第20小时进入稳定期,生长30 h后者最低pH值在4.0左右；植物乳杆菌LpTl和LpT2对18种抗生素都比较敏感,抑菌圈基本都在20 mm以上；它们的发酵上清液对沙门氏菌、铜绿假单孢菌、金黄色葡萄球菌、大肠杆菌、金黄色葡萄球菌的抑制效果都比较明显。
植物乳杆菌LpTl和LpT2在含有结合型胆盐牛磺胆酸钠培养基中的降解胆固醇的活性与未含有牛磺胆酸钠培养基中的降胆固醇活性没有显著差异；植物乳杆菌LpT2在含有牛胆盐与未含有牛胆盐培养基中降胆固醇的活性存在显著差异,而植物乳杆菌LpTl没有显著差异；饲喂高脂饲料7 d后,成功构建出高脂血症大鼠模型；灌胃菌株LpT1 4周后,大鼠血清中总胆固醇含量下降极显著(p<0.01),甘油三脂下降显著(p<0.05),高密度脂蛋白胆固醇几乎没有变化,低密度脂蛋白胆固醇含量下降比较显著(p<0.05),动脉硬化指数明显下降,HDL-C/TC的比值明显升高；灌胃植物乳杆菌LpT2 4 w后,大鼠血清中总胆固醇的含量明显下降(p<0.05),低密度脂蛋白胆固醇也下降比较显著(p<0.05),甘油三脂出现下降但不显著,高密度脂蛋白胆固醇出现下降；灌胃植物乳杆菌和阳性药物的大鼠体重增加率明显低于灌胃蒸馏水的大鼠体重增加率。
[结论]筛选得到的植物乳杆菌LpT1和LpT2在体外具有较高的降胆固醇活性,在体内对高脂血症大鼠也具有一定的疗效,同时能减缓大鼠对高脂饲料的利用率,缓解体重的增加率；另外,菌体在体外降胆固醇的活性和胆盐的存在没有明显的相关性,胆盐的组成能影响降胆固醇的活性,除共沉淀和同化作用等非代谢途径之外,还存在着复杂的代谢降解机制；菌体在大鼠肠道定植后,能产生特定的代谢产物或酶系,直接的或间接的作用于肝脏,调节肝脏代谢脂类物质朝着正常化趋势发展。体外、体内的综合实验表明筛选得到的两株植物乳杆菌具有潜在的生产应用价值。
[Objective]Nowadays, with the perceptible improvement of living standard, people's demand for food nutrition is increasing. At the same time, some negative effects are accompanied, such as atherosclerosis, coronary heart disease, high blood pressure and other cardiovascular or cerebrovascular diseases. Many clinical trials confirmed that orally taking Lactic Acid Bacteria (LAB) and their related products could not only reduce the serum cholesterol concentration but also reduce the risk of suffering from cardiovascular disease. This study was going to screen several cholesterol-lowering lactic acid bacteria strains and investigate their probiotic properties. Cholesterol-lowering mechanism in vitro and vivo would also be examed. The cholesterol-lowering strains and research papers can promote the devepment of functional lactic acid bacteria and supply an application research support.
[Methods]Original LAB strains were screened based on the calcium carbonate-MRS medium (calcium carbonate-Man Rogosa and Sharp Medium) from Chinese tradition pickles and sausage. The cholesterol-lowering strains were confirmed by screening with in vitro cholesterol levels. They were identified by morphologic observation, catalase reaction, carbohydrate reaction and 16SrRNA sequencing. Their acid resistances, bile salts resistance activity, growth curve, acid producing properties, sensitivity to antibiotics, inhibitory effect on common pathogenic bacteria, degradation of cholesterol and co-precipitation of cholesterol action in vitro had been systemic studied. Hyperlipidemia rats were introduced by being fed of fat-rich diet and then be fed of strain everyday, and we traced the change in serum to access their cholesterol-lowering activity in vivo.
[Results]Two strains LpT1 and LpT2 were obtained and identified as Lactobacillus plantarum, their cholesterol degradation ratio were 49.11% and 50.03%, respectively. Lactobacillus plantarum LpT1 and LpT2 showed high acid resistance and bile salt tolerance. Lactobacillus plantarum LpT1 could survive at least for 8 hours under pH 3.0 and for 8 hours in the medium containing 0.3% bile salt, while Lactobacillus plantarum LpT2 could survive for 6 hours and 8 hours under the same condition. LpTl reached the logarithm phase of growth and stationary phase of growth at 14th hour and 22th hour after been cultivated, while LpT2 at 12th hour and 20th hour after been cultivated. The final pH values were 4.2 and 4.0 after cultivated for 30 hours. Both of them were sensitive to the 18 kinds of antibiotics and their diameters of inhibition zone were more than 20 mm. The fermentation supernatant could significantly inhibit Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.
There was no significant difference in cholesterol-reducing activity of Lactobacillus plantarum LpT1 and LpT2 whether the culture medium contained coalescent bile salt sodium taurocholate or not. There was a significant difference in cholesterol-lowering activity when Lactobacillus plantarum LpT2 liveed in the medium with bovine bile salt or without (p<0.05). Hyperlipidemic rat model was built successfully through being fed fat-rich diet for 7 days; After being fed strain LpT1 for 4 weeks, the total cholesterol of rats serum decreased great significantly (p<0.01), triglyceride decreased significantly (p<0.05), high-density lipoprotein cholesterol almost no change in relatively, low-density lipoprotein cholesterol decreased significantly (p<0.05), arteriosclerosis index decreased significantly and HDL-C/TC value increased obviously. After being fed strain LpT2 for 4 weeks, the total cholesterol of rats serum decreased significantly (p<0.05), low-density lipoprotein cholesterol decreased significantly (p<0.05), triglycerides declined indistinctively. The results showed that after being fed Lactobacillus plantarum and lovastatin, rats'weight gain rate was lower significantly than those being fed with distilled water.
[Conclusion]The results showed that both Lactobacillus plantarum LpT1 and LpT2 had a high cholesterol-lowering activity in vitro and a positive effect on hyperlipidemic rats in vivo. The strains also could slow down the utilization of fat-rich diet to alleviate the weight gain simultaneously. In addition, there was no obvious correlation between cholesterol-lowering activity and the existence of bile salts in medium or not. However, composition of bile salts could affect cholesterol-lowering activity. In case to co-precipitation and assimilation, there was some complicated metabolic degradation mechanism. After bacterial colonized in rats gut, it could produce specific metabolites or enzymes to act on liver through direct or indirect ways. In conclusion these two strains showed a potential applied value.
[方法]全文以中国传统食品泡菜、腊肠为材料,以碳酸钙-MRS(calcium carbonate-Man Rogosa and Sharp Medium)选择性培养基筛选乳酸菌,应用改良的胆固醇培养基筛选具有较高降胆固醇能力的乳酸菌；结合菌落形态学、接触酶反应、革兰氏染色、碳水化合物微量鉴定管及16SrRNA寡核苷酸碱基序列分析鉴定；研究其耐酸性,耐胆盐活性,生长曲线及产酸特性；采用药敏纸片法测定菌株对抗生素的敏感性,双层琼脂牛津杯法测定菌株对常见致病菌的抑制效果；比较胆盐存在与否、不同胆盐成分对菌株在培养基中降胆固醇活性的影响；研究胆固醇在体外降解后,其在细菌沉淀、胞内和胞外的分布情况,探讨菌株体外降胆固醇的机制；构建高脂血症大鼠模型,测定灌胃菌株前后大鼠血清主要指标的含量变化,研究降胆固醇乳酸菌在体内的降胆固醇活性。
[结果]筛选得到的两个菌株LpTl和LpT2其降解率分别达到49.11%和50.03%,经鉴定为植物乳杆菌；同时,植物乳杆菌LpTl和LpT2都呈现了较强的耐酸性耐胆盐活性,在pH 3.0条件下存活率都比较高,在含0.3%胆盐的培养基中两株菌的生长都受到了一定抑制；其中植物乳杆菌LpTl在pH3.0培养基中能至少存活8 h,在含0.1%胆盐的培养基中能存活至少8 h；植物乳杆菌LpT2在pH2.0的培养基中至少能存活6 h,在含0.1%胆盐的培养基中都能存活至少8h；植物乳杆菌LpT1在第14小时开始进入对数生长期,在第22小时进入生长稳定期,生长30 h后最低pH值4.2左右；植物乳杆菌LpT2在第12小时进入对数生长曲线,第20小时进入稳定期,生长30 h后者最低pH值在4.0左右；植物乳杆菌LpTl和LpT2对18种抗生素都比较敏感,抑菌圈基本都在20 mm以上；它们的发酵上清液对沙门氏菌、铜绿假单孢菌、金黄色葡萄球菌、大肠杆菌、金黄色葡萄球菌的抑制效果都比较明显。
植物乳杆菌LpTl和LpT2在含有结合型胆盐牛磺胆酸钠培养基中的降解胆固醇的活性与未含有牛磺胆酸钠培养基中的降胆固醇活性没有显著差异；植物乳杆菌LpT2在含有牛胆盐与未含有牛胆盐培养基中降胆固醇的活性存在显著差异,而植物乳杆菌LpTl没有显著差异；饲喂高脂饲料7 d后,成功构建出高脂血症大鼠模型；灌胃菌株LpT1 4周后,大鼠血清中总胆固醇含量下降极显著(p<0.01),甘油三脂下降显著(p<0.05),高密度脂蛋白胆固醇几乎没有变化,低密度脂蛋白胆固醇含量下降比较显著(p<0.05),动脉硬化指数明显下降,HDL-C/TC的比值明显升高；灌胃植物乳杆菌LpT2 4 w后,大鼠血清中总胆固醇的含量明显下降(p<0.05),低密度脂蛋白胆固醇也下降比较显著(p<0.05),甘油三脂出现下降但不显著,高密度脂蛋白胆固醇出现下降；灌胃植物乳杆菌和阳性药物的大鼠体重增加率明显低于灌胃蒸馏水的大鼠体重增加率。
[结论]筛选得到的植物乳杆菌LpT1和LpT2在体外具有较高的降胆固醇活性,在体内对高脂血症大鼠也具有一定的疗效,同时能减缓大鼠对高脂饲料的利用率,缓解体重的增加率；另外,菌体在体外降胆固醇的活性和胆盐的存在没有明显的相关性,胆盐的组成能影响降胆固醇的活性,除共沉淀和同化作用等非代谢途径之外,还存在着复杂的代谢降解机制；菌体在大鼠肠道定植后,能产生特定的代谢产物或酶系,直接的或间接的作用于肝脏,调节肝脏代谢脂类物质朝着正常化趋势发展。体外、体内的综合实验表明筛选得到的两株植物乳杆菌具有潜在的生产应用价值。
[Objective]Nowadays, with the perceptible improvement of living standard, people's demand for food nutrition is increasing. At the same time, some negative effects are accompanied, such as atherosclerosis, coronary heart disease, high blood pressure and other cardiovascular or cerebrovascular diseases. Many clinical trials confirmed that orally taking Lactic Acid Bacteria (LAB) and their related products could not only reduce the serum cholesterol concentration but also reduce the risk of suffering from cardiovascular disease. This study was going to screen several cholesterol-lowering lactic acid bacteria strains and investigate their probiotic properties. Cholesterol-lowering mechanism in vitro and vivo would also be examed. The cholesterol-lowering strains and research papers can promote the devepment of functional lactic acid bacteria and supply an application research support.
[Methods]Original LAB strains were screened based on the calcium carbonate-MRS medium (calcium carbonate-Man Rogosa and Sharp Medium) from Chinese tradition pickles and sausage. The cholesterol-lowering strains were confirmed by screening with in vitro cholesterol levels. They were identified by morphologic observation, catalase reaction, carbohydrate reaction and 16SrRNA sequencing. Their acid resistances, bile salts resistance activity, growth curve, acid producing properties, sensitivity to antibiotics, inhibitory effect on common pathogenic bacteria, degradation of cholesterol and co-precipitation of cholesterol action in vitro had been systemic studied. Hyperlipidemia rats were introduced by being fed of fat-rich diet and then be fed of strain everyday, and we traced the change in serum to access their cholesterol-lowering activity in vivo.
[Results]Two strains LpT1 and LpT2 were obtained and identified as Lactobacillus plantarum, their cholesterol degradation ratio were 49.11% and 50.03%, respectively. Lactobacillus plantarum LpT1 and LpT2 showed high acid resistance and bile salt tolerance. Lactobacillus plantarum LpT1 could survive at least for 8 hours under pH 3.0 and for 8 hours in the medium containing 0.3% bile salt, while Lactobacillus plantarum LpT2 could survive for 6 hours and 8 hours under the same condition. LpTl reached the logarithm phase of growth and stationary phase of growth at 14th hour and 22th hour after been cultivated, while LpT2 at 12th hour and 20th hour after been cultivated. The final pH values were 4.2 and 4.0 after cultivated for 30 hours. Both of them were sensitive to the 18 kinds of antibiotics and their diameters of inhibition zone were more than 20 mm. The fermentation supernatant could significantly inhibit Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.
There was no significant difference in cholesterol-reducing activity of Lactobacillus plantarum LpT1 and LpT2 whether the culture medium contained coalescent bile salt sodium taurocholate or not. There was a significant difference in cholesterol-lowering activity when Lactobacillus plantarum LpT2 liveed in the medium with bovine bile salt or without (p<0.05). Hyperlipidemic rat model was built successfully through being fed fat-rich diet for 7 days; After being fed strain LpT1 for 4 weeks, the total cholesterol of rats serum decreased great significantly (p<0.01), triglyceride decreased significantly (p<0.05), high-density lipoprotein cholesterol almost no change in relatively, low-density lipoprotein cholesterol decreased significantly (p<0.05), arteriosclerosis index decreased significantly and HDL-C/TC value increased obviously. After being fed strain LpT2 for 4 weeks, the total cholesterol of rats serum decreased significantly (p<0.05), low-density lipoprotein cholesterol decreased significantly (p<0.05), triglycerides declined indistinctively. The results showed that after being fed Lactobacillus plantarum and lovastatin, rats'weight gain rate was lower significantly than those being fed with distilled water.
[Conclusion]The results showed that both Lactobacillus plantarum LpT1 and LpT2 had a high cholesterol-lowering activity in vitro and a positive effect on hyperlipidemic rats in vivo. The strains also could slow down the utilization of fat-rich diet to alleviate the weight gain simultaneously. In addition, there was no obvious correlation between cholesterol-lowering activity and the existence of bile salts in medium or not. However, composition of bile salts could affect cholesterol-lowering activity. In case to co-precipitation and assimilation, there was some complicated metabolic degradation mechanism. After bacterial colonized in rats gut, it could produce specific metabolites or enzymes to act on liver through direct or indirect ways. In conclusion these two strains showed a potential applied value.
引文
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