目的 通过体外降脂实验筛选潜在的功能性双歧杆菌菌株,并评估它们对小鼠高脂模型的降脂效果。方法 从健康志愿者粪便中分离获得14株双歧杆菌菌株;以鼠李糖乳杆菌LGG（Lactobacillus rhamnosus GG）作为对照菌株,体外实验检测所有菌株的胆固醇降解率、耐酸和耐胆盐耐受率以及胆盐水解酶（BSH）活性。将C57BL/6J小鼠随机分为7组,包括正常饮食（ND+PBS）对照组、高脂饮食（HFD+PBS）诱导的脂肪肝模型组、高脂饮食干预组（HFD+4株双歧杆菌）和干预对照组（HFD+LGG）,分别给予普通饲料、高脂饲料（60% kcal）,ND、HFD组每日每只小鼠按0.2ml PBS进行灌胃,其它干预组按2×108CFU菌量灌胃, 共14 w。观察小鼠体重变化,每2 w收集粪便一次,最后处死小鼠并解剖,收集血液、肝脏、粪便等样品,检测脂肪指数、血清生化、肝脏血脂、肝脏病理以及肠道微生物组成和结构等指标。结果 依据体外实验筛选结果,筛选出4株双歧杆菌菌株（Bbr1、BA6、BL6、BB15）,其体外胆固醇降解率和BSH活性均高于LGG对照菌株（P<0.05）,且具有一定的耐酸耐胆盐活性。动物试验结果表明,与高脂饮食组相比,7~14 w内4株双歧杆菌干预小鼠体重显著下降（P<0.05）,BA6和BL6菌株干预后使肝脏胆固醇、甘油三酯浓度降低,肝脏病理结果显示脂肪空泡数量减少、肝脏病理评分显著降低（P<0.05）;灌胃BA6和BL6菌株后小鼠肠道微生物群组成结构发生改变,降低了有害菌如拟普雷沃氏菌属（Alloprevotella）的丰度,同时有益菌毛螺菌科（Lachnospiraceae）的丰度增加。结论 通过体外胆固醇降解和体内动物模型评价,青春双歧杆菌BA6和长双歧杆菌BL6具有改善高脂喂养小鼠脏器指数、肝脏病理及肠道微生物群等指标的作用,为未来开发降脂或缓解脂肪肝的益生菌产品提供了功能性基础实验研究数据。
Abstract
Objective To screen potential functional Bifidobacterium strains through in vitro lipid-lowering reduction experiments and evaluate their lipid-lowering effects in a high-fat diet fed mice. Methods Fourteen strains of Bifidobacterium were isolated from the feces of healthy volunteers. The well-accepted probiotics, Lactobacillus rhamnosus GG (LGG) were used as the control strain throughout this study. In vitro experiments were performed to detect cholesterol reduction rate, tolerance to acid and bile salt, and activity of bile salt hydrolase (BSH) of all tested strains. Those strains selected based on in vitro experiments were subject to the following animal experiments for verification in vivo. C57BL/6J mice were randomly divided into seven groups, including a normal diet control group (ND), a high-fat diet group (HFD), four high-fat diet intervention groups (gavaged with four Bifidobacterium strains,respectively, HFD+ Bifidobacterium), and a intervention control group (gavaged with LGG, HFD+LGG). Mice were fed normal diet or high-fat diet（60% kcal）and daily gavaged for 14 weeks with 0.2 ml PBS with or without 2×108 CFU of bacterial strains. The changes of body weight were measured weekly, and the feces were collected once two weeks. All mice were sacrificed to collect bloods, livers, stools and other samples. Some parameters including fat index, serum biochemistry, hepatic lipids, liver pathology, and intestinal microbiota were analyzed. Results Based on the in vitro experiments, 4 of 14 Bifidobacterium strains（Bbr1、BA6、BL6、BB15）were selected for the animal experiments. The in vitro cholesterol degradation rates and BSH activity of the four Bifidobacterium strains were all higher than those of the LGG control strain (P<0.05). Besides, the four strains had a certain degree of acid and bile salt resistance activity. Animal experimental results showed that the body weights of the mice in the four Bifidobacterium intervention groups decreased during weeks 7-14 as compared with the HFD group. A significant reduction in hepatic cholesterol and hepatic triglyceride concentrations was observed after the intervention of BA6 and BL6. Liver pathological examination showed a decrease in the number of fatty vacuoles leading to the reduced liver pathology scores (P<0.05). Fecal microbiome analysis indicated that the compositions of the intestinal microbiota were changed after the gavage of BA6 and BL6. Interestingly, the abundance of harmful bacteria (e.g. Alloprevotella) was found decreased and that of beneficial bacteria (e.g. Lachnospiraceae) increased. Conclusion Based on the results of in vitro and in vivo experiments, two bacterial strains, Bifidobacterium adolescentis BA6 and Bifidobacterium longum BL6, play a potentail role in alleviating fatty liver, by improving organ’s indices, liver pathology, and gut microbiota composition. Functional experimental research data are further needed for the future development of probiotic products for lipid-lowering and fatty liver alleviation.
关键词
双歧杆菌 /
益生菌 /
降胆固醇 /
脂肪肝 /
胆盐水解酶
Key words
Bifidobacterium /
probiotics /
cholesterol reduction /
fatty liver /
bile salt hydrolase
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基金
国家自然科学基金重大项目（No.32394054）
