辅助降血脂益生乳酸菌的筛选及其对高血脂大鼠肠道菌群的影响

发布时间：2018-06-04 09:21

本文选题：鼠李糖乳杆菌 + 降血脂　；参考：《扬州大学》2015年博士论文

【摘要】：血清中的血脂水平过高是引起众多心血管疾病的主要原因。研究表明,降低血清中的血脂水平能够显著降低心血管疾病的死亡率。药物治疗是当前治疗心血管疾病的主要方式,但是药物治疗成本较高,并且具有较大的副作用。乳酸菌作为人体肠道中重要的益生菌,已被证实能够改善血清中的血脂水平,并使肠道微生态系统发生长期而有益的变化,而来源于人体自身的益生菌通常具有较高的安全性,因此本文从广西巴马长寿地区的人群肠道中分离筛选出体外辅助降血脂能力较强的乳酸菌,并研究其在高血脂大鼠体内的降血脂效果以及对高血脂大鼠肠道菌群的影响,从而探索乳酸菌在体内降血脂的可能机制,并为以肠道菌群为靶点进行的益生菌干预治疗提供理论依据。1.利用选择性培养基从广西巴马长寿地区人群采集的粪便样品中分离乳酸菌,通过体外试验筛选出降胆固醇及甘油三酯能力较强的乳酸菌,然后进行模拟人工胃液、胆盐耐受性、抑菌及耐药性试验；并研究其生长菌体、热致死菌体及休眠菌体的胆固醇降解率及其在体外的降胆固醇方式。结果表明,从15份粪便样品中分离获得156株乳酸菌,其中Lp2、S10、Lf7及Hsryfm 1301等16株乳酸菌的胆固醇及甘油三酯的降解率均分别大于30.35%和5.33%, Lp2、S10、Lf7及Hsryfm 1301在pH3.0的人工胃液中存活率均大于63.00%,在含0.1%、0.3%及0.5%胆盐的培养基中存活率均大于15.00%；Hsryfm 1301的抑菌能力较强于Lp2、S10及Lf7.对大肠杆菌、沙门氏菌及金黄色葡萄球菌的抑菌圈直径分别为14.67mm、11.35mm及10.67mm；Hsryfm 1301的耐药性低于Lp2、S10及Lf7,对头孢唑啉、头孢拉定、四环素、氯霉素、利福平素均敏感,对链霉素中度敏感,对青霉素G、诺氟沙星及环丙沙星均耐药;Hsryfm 1301在含0.1%、0.3%及0.5%胆盐的培养基中的降胆固醇能力较强于Lp2、S10及Lf7,降解率分别为56.25%、28.91%及13.20%；Lp2、S10、Lf7及Hsryfm 1301的生长菌体、热致死菌体及休眠菌体均能降低培养基中的胆固醇,Hsryfm 1301生长菌体的胆固醇降解率为61.90%,高于Lp2、S10及Lf7；Hsryfm 1301体外降胆固醇的主要方式为菌体吸收及共沉淀,分别占其总降解率的62.50%及36.50%。经API及16S rDNA测序将Hsryfm 1301鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。2.通过饲喂4周高脂饲料来建立高血脂大鼠模型,以降血脂药物血脂康作为阳性对照,研究L. rhamnosus hsryfm 1301的菌体悬浮液、热致死菌体悬浮液、菌体破碎液及其发酵乳在高血脂大鼠体内的降血脂效果。结果表明,饲喂4周高脂饲料,模型组大鼠血清中的胆固醇(Serum total cholesterol, TC)、甘油三酯(Triglycerides, TG)及低密度脂蛋白(Low density lipoprotein cholesterol, LDL-C)含量显著高于对照组(P0.05),分别为2.05mmol/L、1.98mmol/L、0.28mmol/L,表明高血脂大鼠模型建模成功；灌胃4周L. rhamnosus hsryfm 1301发酵乳后,大鼠对饲料的利用率显著高于模型组(P0.05),为29.46%；饲喂8周高脂饲料后,模型组大鼠的肝指数及体脂指数均显著高于对照组及L. rhamnosus hsryfm 1301各处理组(P0.05),分别为3.98和3.55。灌胃4周L. rhamnosus hsryfm 1301的各处理方式后,菌体悬浮液组及发酵乳组大鼠血清中TC、TG的含量显著低于模型组(P0.05),分别为1.50mmol/L、1.30mmol/L及1.53mmol/L、 1.28mmol/L;菌体热致死组大鼠血清中TC、高密度脂蛋白(High density lipoprotein cholesterol, HDL-C)及LDL-C的含量显著低于模型组(P0.05),分别为1.90mmol/L、 0.38mmol/L及0.19mmol/L;菌体破碎液组大鼠血清中TC、TG及LDL-C的含量显著低于模型组(P0.05),分别为1.78mmol/L、1.68mmol/L及0.17mmol/L;灌胃4周的血脂康后,人鼠血清中TC、TG、HDL-C及LDL-C的含量也显著低于模型组(P0.05),分别为1.76mmol/L、 1.65mmol/L、0.39mmol/L及0.19mmol/L。灌胃4周L. rhamnosus hsryfm 1301的菌体悬浮液、菌体破碲液及其发酵乳后,大鼠血清中氧化物歧化酶(Superoxide dismutase, SOD)浓度显著高于模型组(P0.05),分别为74.96U/mL.73.58U/mL及73.96U/mL,而热致死菌体对大鼠血清中的SOD浓度影响较小；菌体悬浮液组及发酵乳组大鼠粪便中胆固醇、甘油三酯和胆汁酸的含量显著高于模型组(P0.05),分别为10.23μmol/g、4.25μmol/g和28.87μmol/g及9.85μmol/g、4.03pμmol/g和27.13μmol/g;菌体悬浮液组、菌体破碎液组与发酵乳组大鼠粪便中的水分含量显著高于模型组(P0.05),分别为46.15%、45-35%及46.85%；通过透射电镜观察发现,L. rhamnosus hsryfm 1301菌体悬浮液、菌体破碎液及其发酵乳均能预防由高血脂引起的大鼠肝脏和心脏组织的病变。3.利用聚合酶链式反应一变性梯度凝胶电泳(Polymerase Chain Reaction-Denaturing gradient gelelectrophoresis, PCR-DGGE)技术研究基础饲料、高脂饲料及L. rhamnosus hsryfm1301的菌体悬浮液、热致死菌体悬浮液、菌体破碎液及其发酵乳对高血脂大鼠肠道菌群的影响。结果表明,饲喂4周高脂饲料增加了大鼠肠道中柔嫩梭菌属PCR-DGGE图谱的条带数,降低了肠道微生物16S rRNA基因V3区、拟杆菌属、乳酸菌及双歧杆菌属PCR-DGGE图谱的条带数,而L. rhamnosus hsryfm 1301的各处理方式对大鼠肠道微生物的影响正好与之相反。饲喂4周基础饲料及高脂饲料后,大鼠肠道中Klebsiella pneumoniae及Uncultured bacterium的丰度下降甚至消失,同时增加了大鼠肠道中柔嫩梭菌的种类,分别为Clostridiumbotulinum A str. Hall、Clostridium pasteurianum BC1、Ruminococcus champanellensis 18P13 、Ruminococcus albus 7及Ruminococcus bromii L2-63,而对照组大鼠肠道中的Uncultured Bacteroidetes bacterium TM1-40肖失。试验第9周,灌胃4周L. rhamnosus hsryfm 1301菌体悬浮液后,大鼠肠道中出现了Pantoea vagans及Clostridium thermocellum,对照组及模型组大鼠肠道中出现了Streptomyces coelicolor及Clostridiales genomo sp.,对照组大鼠肠道中Clostridium botulinum A str. Hall的丰度高于其它组；热致死菌体悬浮液、菌体破碎液及其发酵乳使大鼠肠道中Ruminococcus bromii L2-63的丰度下降或消失,菌体悬浮液增加了大鼠肠道中Lactobacillus amylovorus GRL的丰度,菌体破碎液增加了大鼠肠道中Lactobacillus buchneri NRRL B-30929的丰度,发酵乳则增加了大鼠肠道中Lactobacillus sp,的丰度；菌体悬浮液、菌体破碎液及发酵乳还增加了大鼠肠道中乳酸菌的种类,分别为Lactobacillus murinus DLM 3108、Lactobacillus gasseri ATCC 33323及Lactobacillus sakei subsp.。4.利用实时荧光定量聚合酶链式反应(Real-time ploymerase chain reaction, RT-PCR)的方法研究基础饲料、高脂饲料及L. rhamnosus hsryfm 1301的菌体悬浮液、热致死菌体悬浮液、菌体破碎液及其发酵乳对高血脂大鼠肠道菌群的影响,并分析高血脂大鼠的肠道菌群与其血清血脂水平的相关性。结果表明,饲喂8周基础饲料显著增加了大鼠肠道中拟杆菌和肠球菌的数量(P0.05),饲喂8周高脂脂饲料显著增加了大鼠肠道中柔嫩梭菌、肠球菌以及肠杆菌的数量(P0.05),同时显著降低了拟杆菌、乳酸菌及双歧杆菌的数量(P0.05);L. rhamnosus hsryfm 1301各处理方式干预4周后,菌体悬浮液显著增加了大鼠肠道中拟杆菌及乳酸菌的数量(P0.05),并显著降低了柔嫩梭菌的数量(P0.05)；热致死菌体悬浮液显著增加了大鼠肠道中拟杆菌及肠球菌的数量(P0.05),并显著降低了柔嫩梭菌及肠杆菌的数量(P0.05)；菌体破碎液显著增加了大鼠肠道中拟杆菌、乳酸菌及双歧杆菌的数量(P0.05),并显著降低了柔嫩梭菌的数量(P0.05)；发酵乳显著增加了大鼠肠道内拟杆菌及肠球菌的含量(P0.05),同时显著降低了柔嫩梭菌及肠杆菌的数量(P0.05)；高血脂大鼠肠道内柔嫩梭菌的数量同其血清中TC、TG、HDL-C及LDL-C的含量呈显著正相关(P0.05),肠道内乳酸菌及双歧杆菌的数量同肠杆菌的数量呈显著负相关(P0.05)。5.利用高通量测序的方法研究基础饲料、高脂饲料及L. rhamnosus hsryfm 1301菌体悬浮液及其发酵乳对高血脂大鼠肠道菌群的影响,并分析高血脂大鼠的肠道菌群与其血清血脂水平的相关性。结果表明,高脂饲料、L. rhamnosus hsryfm 1301菌体悬浮液及其发酵乳显著改变了大鼠肠道中柯林斯菌属、拟杆菌属、普氏菌属、乳杆菌属、肠球菌属、瘤胃球菌属等22个属的微生物的相对丰度(P0.05)；饲喂8周基础饲料及4周高脂饲料降低了大鼠肠道中微生物物种的丰度及多样性,使得大鼠肠道菌群的Chaol/ACE署Shannon指数显著降低(P0.05),Simpson指数显著增加(P0.05)；而L. rhamnosus hsryfm 1301菌体悬浮液及其发酵乳的干预恢复了大鼠肠道中微生物物种的丰度及多样性,显著增加了大鼠肠道菌群的Chaol/ACE和Shannon旨数(P0.05),并降低了Simpson旨数(P0.05)；同时促进了大鼠肠道中乳杆菌属的繁殖,并抑制了大肠杆菌／志贺氏菌的生长。大鼠肠道中瘤胃球菌属的相对丰度与血清中TG的含量、Dorea spp的相对丰度与血清中TC及LDL-C的含量、肠球菌属的相对丰度与血清中HDL-C的含量均呈显著正相关(P0.05),而丁酸弧菌属的相对丰度与血清中TC及LDL-C的含量呈显著负相关(P0.05)。
[Abstract]:The high level of blood lipids in the serum is the main cause of many cardiovascular diseases. Studies have shown that reducing blood lipid levels in the serum can significantly reduce the mortality of cardiovascular diseases. Drug treatment is the main treatment of cardiovascular disease at present, but the cost of drug treatment is higher, and it has a larger side effect. As an important probiotic in human intestinal tract, it has been proved to be able to improve blood lipid levels in the serum and to make long-term and beneficial changes in the intestinal microecosystem, while the probiotics derived from the human body often have high safety. In order to explore the effect of blood lipid lowering in hyperlipidemic rats and the effect on the intestinal flora of hyperlipidemia rats, the possible mechanism of lactic acid bacteria in blood lipid reduction in the body was explored, and the theory of probiotic intervention therapy based on the intestinal microflora as the target for the treatment was based on the.1. selection medium. Lactic acid bacteria were isolated from the fecal samples collected from the longevity region of the West Bama, and the cholesterol and triglyceride lactobacillus with strong ability to reduce cholesterol and triglycerides were screened in vitro, and then simulated artificial gastric juice, bile salt tolerance, bacteriostasis and drug resistance test, and the cholesterol degradation rate of the growing mycelium, heat lethal and dormant bodies and the rate of cholesterol degradation were studied. The results showed that 156 strains of lactic acid bacteria were isolated from 15 fecal samples, of which the cholesterol and triglyceride degradation rates of 16 strains of lactic acid bacteria such as Lp2, S10, Lf7 and Hsryfm 1301 were respectively greater than 30.35% and 5.33%, Lp2, S10, Lf7 and Hsryfm 1301 were more than 63% in pH3.0, with 0 in the artificial gastric juice. The survival rates of.1%, 0.3% and 0.5% of bile salts were greater than 15%, Hsryfm 1301 was stronger than Lp2, S10 and Lf7. were 14.67mm, 11.35mm and 10.67mm for Escherichia coli, Salmonella and Staphylococcus aureus, and Hsryfm 1301 was lower than Lp2, S10 and Lf7, and cefazolin, Cefradine, and four rings. It was sensitive to chloramphenicol and rifampicin, moderately sensitive to streptomycin, resistant to penicillin G, norfloxacin and ciprofloxacin, and Hsryfm 1301 in 0.1%, 0.3%, and 0.5% bile salts was stronger than Lp2, S10 and Lf7, and the degradation rates were 56.25%, 28.91% and 13.20%, Lp2, S10, Lf7 and Hsryfm 1301. Both lethal and dormant bacteria could reduce the cholesterol in the medium. The cholesterol degradation rate of Hsryfm 1301 growth bacteria was 61.90%, higher than Lp2, S10 and Lf7. The main way to reduce cholesterol in Hsryfm 1301 was bacterial absorption and co precipitation, accounting for 62.50% of the total degradation rate and 36.50%. by API and 16S rDNA sequencing to identify Hsryfm 1301. A hyperlipidemic rat model was established for Lactobacillus rhamnosus.2. by feeding a high fat diet for 4 weeks. A blood lipid lowering drug xuexuekang was used as a positive control to study the bacterial suspension of L. rhamnosus hsryfm 1301, heat lethal mycelium suspension, bacterial breakage and its fermented milk in hyperlipidemia rats. The results showed that the levels of serum cholesterol (Serum total cholesterol, TC), triglyceride (Triglycerides, TG) and low density lipoprotein (Low density lipoprotein cholesterol, LDL-C) in the model group were significantly higher than those of the control group (P0.05). Rat model was successfully modeled. After 4 weeks of L. rhamnosus hsryfm 1301 fermented milk, the utilization rate of rat feed was significantly higher than that of model group (P0.05), which was 29.46%. After feeding for 8 weeks high fat feed, the liver index and body fat index of the model group were significantly higher than that of the control group and L. rhamnosus hsryfm 1301 treatment group (P0.05), 3.98 and 3.55. respectively. After the treatment of L. rhamnosus hsryfm 1301 for 4 weeks, the content of TC and TG in the serum of the mycelium suspension group and the fermented milk group was significantly lower than that of the model group (P0.05), which were 1.50mmol/L, 1.30mmol/L and 1.53mmol/L, 1.28mmol/L, and the serum of the rats in the body heat death group was TC, and the high-density lipoprotein (High) The content of -C) and LDL-C was significantly lower than that of the model group (P0.05), which were 1.90mmol/L, 0.38mmol/L and 0.19mmol/L, respectively. The content of TC, TG and LDL-C in the serum of the strain group was significantly lower than that of the model group (P0.05). The concentration of oxide dismutase (Superoxide dismutase, SOD) in serum was significantly higher than that in model group (P0.05), 1.76mmol/L, 1.65mmol/L, 0.39mmol/L and 0.19mmol/L. for 4 weeks L. rhamnosus hsryfm 1301, and the concentration of Superoxide dismutase, SOD) in the serum of the rats was significantly higher than that of the model group (P0.05). 6U/mL, and the effect of heat lethal mycelium on the concentration of SOD in the serum of rats was less. The content of cholesterol, triglyceride and bile acid in the feces of the mycelium suspension group and the fermented milk group was significantly higher than that of the model group (P0.05), 10.23 mu mol/g, 4.25 mol/g and 28.87 u mol/g and 9.85 mu mol/g, 4.03p u mol/g and 27.13 micron mol/g, and the bacterial suspension group, The water content in the feces of the mycelial breakage group and the fermented milk group was significantly higher than that of the model group (P0.05), 46.15%, 45-35% and 46.85%, respectively. Through transmission electron microscopy, it was found that the L. rhamnosus hsryfm 1301 mycelium suspension, the mycelial breakage and the fermented milk could prevent the pathological changes of the liver and heart tissues of rats induced by hyperlipidemia.3. Using polymerase chain reaction (Polymerase Chain Reaction-Denaturing gradient gelelectrophoresis, PCR-DGGE) to study the basic feed, high fat feed and L. rhamnosus hsryfm1301 bacterial suspension, thermally lethal mycelium suspension, bacterial breakage and fermented milk for the intestinal flora of hyperlipidemia rats The results showed that 4 weeks of high fat feed increased the number of bands in the PCR-DGGE map of the intestinal tract of the genus Clostridium tenx in rats, and reduced the number of bands of the 16S rRNA gene in the intestinal microbes, the bacillus, Lactobacillus and Bifidobacterium in the PCR-DGGE map of the genus 16S, and the treatment of L. rhamnosus hsryfm 1301 on the intestinal microorganism in rats. On the contrary, the abundances of Klebsiella pneumoniae and Uncultured bacterium in the intestines of rats were reduced or even disappeared after feeding for 4 weeks of basal diet and high fat diet, and the species of Clostridium tenx in the intestines of rats were increased, respectively, Clostridiumbotulinum A str. Hall, Clostridium pasteurianum BC1, Ruminococcus Ruminococcus. Is 18P13, Ruminococcus albus 7 and Ruminococcus bromii L2-63, and Uncultured Bacteroidetes bacterium TM1-40 Shaw loss in the intestinal tract of the control group. After ninth weeks of trial, the intestine of the rat was 4 weeks of L.. Streptomyces coelicolor and Clostridiales genomo sp. were found in the intestinal tract, and the abundance of Clostridium botulinum A str. Hall in the intestinal tract of the control group was higher than that of the other groups; the bacterial suspension and the fermentation milk made the Ruminococcus bromii abundances in the intestine of the rats decreased or disappeared, and the suspension of the bacterial body increased. The abundance of Lactobacillus amylovorus GRL in the intestine of rats increased the abundance of Lactobacillus buchneri NRRL B-30929 in the intestinal tract of the rat, and the fermented milk increased the abundance of Lactobacillus SP in the intestine of the rat, and the bacterial suspension, the mycelium breakage and the fermented milk also increased the species of lactic acid bacteria in the rat intestine. Lactobacillus murinus DLM 3108, Lactobacillus gasseri ATCC 33323 and Lactobacillus sakei subsp..4. use real time fluorescent quantitative polymerase chain reaction (Real-time ploymerase chain) to study base feed, high fat feed and 1301 bacterial suspension, heat lethal bacterial suspension The effect of liquid, mycelium breakage and its fermented milk on the intestinal flora of hyperlipidemia rats and the correlation between the intestinal flora of hyperlipidemia rats and the serum lipid levels were analyzed. The results showed that the number of bacilli and Enterococcus in the intestines of rats was significantly increased by feeding 8 weeks (P0.05), and the rats fed with high fat fat diet for 8 weeks significantly increased the rats. The number of Clostridium tenx, Enterococcus and Enterobacteriaceae (P0.05) significantly decreased the number of bacteriobacteria, lactic acid bacteria and bifidobacteria (P0.05), and the number of bacteriobacteria and lactic acid bacteria (P0.05) in the intestinal tract was significantly increased after 4 weeks of intervention by L. rhamnosus hsryfm 1301, and the tender shuttle was significantly reduced. The number of bacteria (P0.05); the number of bacteriobacteria and Enterococcus (P0.05) in the intestinal tract was significantly increased by the heat lethal bacterial suspension, and the number of Clostridium tenosium and Enterobacter cloacae (P0.05) was significantly reduced. The bacterial breakage significantly increased the number of bacteriobacteria, lactic acid bacteria and bifidobacteria (P0.05) in the intestines of rats (P0.05), and significantly reduced the tender shuttle. The number of bacteria (P0.05); fermented milk significantly increased the content of bacteriobacterium and Enterococcus (P0.05) in the intestines of rats, and significantly decreased the number of Clostridium tenoides and Enterobacteriaceae (P0.05). The number of Clostridium tenosi in the intestinal tract of hyperlipidemia rats was significantly positively correlated with the content of TC, TG, HDL-C and LDL-C in the serum (P0.05), and the lactic acid bacteria in the intestinal tract and the intestinal Lactobacillus in the intestinal tract. The number of bifidobacteria was negatively correlated with the number of Enterobacteriaceae (P0.05).5. using high throughput sequencing method to study the effects of high fat feed and L. rhamnosus hsryfm 1301 suspension and its fermented milk on the intestinal flora of hyperlipidemic rats, and to analyze the phase of intestinal flora and serum lipid levels in hyperlipidemic rats. The results showed that high fat feed, L. rhamnosus hsryfm 1301 mycelium suspension and its fermented milk significantly changed the relative abundance of 22 microbes in the intestinal tract of Collins, pridella, Lactobacillus, Enterococcus and rumen, and 8 weeks of basal feed and 4 weeks of high fat diet were reduced. The abundance and diversity of microbial species in the intestinal tract of rats greatly reduced the Shannon index of Chaol/ACE in the intestinal flora of rats (P0.05), and the Simpson index increased significantly (P0.05), while the intervention of L. rhamnosus hsryfm 1301 suspension and its fermented milk restored the abundance and diversity of the microorganism species in the rat intestinal tract, and increased significantly. The Chaol/ACE and Shannon decree (P0.05) of the intestinal flora and the number of Simpson (P0.05) were reduced, and the reproduction of Lactobacillus in the intestinal tract of rats was promoted and the growth of Escherichia coli / Shigella was inhibited. The relative abundance of the rumen and the content of TG in the sera, the relative abundance of Dorea spp and the T in the serum in the intestine of rats. The relative abundance of C and LDL-C showed significant positive correlation with the content of HDL-C in serum (P0.05), while the relative abundance of Vibrio butyrate was significantly negatively correlated with the content of TC and LDL-C in serum (P0.05).
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R151.1

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