| Elevated serum lipids’level is widely recognized as a primary risk factor for the development of cardiovascular diseases. Studies have shown that reduce serum cholesterol level can significantly decrease the risk of cardiovascular diseases. Drug treatment is the main way to treat cardiovascular diseases, which has side effects and high cost. The serum lipid levels can be improved by Lactic acid bacteria (LAB), which is one of the most important probiotics in human gut, and confers beneficial changes to micro ecological system in long term. However, the probiotics from human body have higher safety. Therefore, LAB with auxiliary hypolipidemic was screened from the gut of Bama longevity of Guangxi in China in vitro, and the ability of lower lipids level and its effects to gut microbiota were further studied in rats in this paper, and the possible mechanism of lipid-lowering of LAB in vivo was explored, which could provide theoretical basis for the gut microbiota as a target for nutrition interventions to treat diseases.1. Selective medium were used to isolate LAB from the gut of Bama longevity of Guangxi Province in China. The strong ability of lowering cholesterol and triglycerides were screened in vitro test, and then the simulation of artificial gastric juice, bile tolerance, antibacterial and antimicrobial resistance test were studied, the ability of lowering cholesterol of growing, thermal death and dormant strain of LAB were also studied in vitro. The results showed that 156 strains of LAB were isolated from 15 feces samples, the lowering rate of cholesterol and triglycerides of 16 strains conclude Lp2, S10, Lf7 and Hsryfm 1301 were higher than 30.35% and 5.33% respectively, in which the survival rate in pH3.0 artificial gastric juice of Lp2, S10, Lf7 and Hsryfm 1301 were higher than 63.00%, the survival rate in medium containing 0.1%,0.3% and 0.5% bile salt were higher than 15.00%; the antibacterial ability of Hsryfm 1301 was higher than Lp2, S10 and Lf7, the antibacterial circle diameter of Escherichia Coli, Salmonella and Staphylococcus aureus were 14.67mm,11.35mm and 10.67mm; the ability antimicrobial resistance of Hsryfm 1301 was lower than Lp2, S10 and Lf7, which was sensitive to cefazolin, cephalosporins, tetracycline, chloramphenicol, rifampicin, and was moderate sensitive to streptomycin, and was resistant to penicillin G, norfloxacin and ciprofloxacin; the ability of lowering cholesterol of Hsryfm 1301 in medium containing 0.1%,0.3% and 0.5% bile salt were higher than Lp2, S10 and Lf7, which were 56.25%,28.91% and 13.20%. The growing, thermal death and dormant strain of Hsryfm 1301, Lp2, S10 and Lf7 can lower the cholesterol in medium, and the cholesterol-lowering rate of growing Hsryfm 1301 was higher than Lp2, S10 and Lf7; the way of cholesterol-lowering of Hsryfm 1301 in vitro were absorption and coprecipitation, and accounted for 62.50% and 36.50% of the total cholesterol-lowering rate respectively; and was identified as Lactobacillus rhamnosus by API and 16S rDNA gene sequence.2. The hyperlipidemia rat model was established by feeding high-fat diet for 4 weeks, with hypolipidemic agent Xuezhikang as the positive control, and the effect of intervention bacteria suspension, thermal death bacteria suspension, broken bacteria suspension and fermented milk of L. rhamnosus hsryfm 1301 for 4 weeks on lowering lipids level in hyperlipidemia rat was studied. The results showed that the level of serum total cholesterol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) of model group was significantly higher than control group after feeding high-fat diet 4 weeks (P<0.05), which were 2.05 mmol/L,1.98 mmol/L and 0.28 mmol/L; after rats were intervened fermented milk for 4 weeks, the utilization efficiency of diet of rats was significantly high than model group, which were 29.46%(P<0.05); the rat live index and body fat index was 3.98 and 3.55 respectively after feeding high-fat diet for 8 weeks which were significantly higher than other groups (P<0.05).The level of TC and TG of serum were 1.50 mmol/L,1.30 mmol/L and 1.53 mmol/L,1.28 mmol/L in rats, which were significantly lower than model group after rats were administered intragastrically bacteria suspension and fermented milk for 4 weeks (P<0.05); the level of TC, high density lipoprotein cholesterol (HDL-C) and LDL-C of serum were 1.90 mmol/L,0.38 mmol/L and 0.19 mmol/L, which were significantly lower than model group after rats were intervened thermal death bacteria suspension (P<0.05); after rats were intervened broken bacteria suspension for 4 weeks, the level of TC, TG, HDL-C and LDL-C of serum were 1.78 mmol/L,1.68 mmol/L and 0.17 mmol/L, which was significantly lower than model group (P<0.05); the level of TC, TG, HDL-C and LDL-C of serum were 1.76 mmol/L,1.65 mmol/L, 0.39 mmol/L and 0.19 mmol/L, which was significantly lower than model group after rats were intervened Xuezhikang for 4 weeks (P<0.05).The Superoxide dismutase (SOD) content of rats serum was significantly higher than model group after rats were intervened by bacteria suspension, broken bacteria suspension and fermented milk (P<0.05), which were 74.96 U/mL,73.58 U/mL and 73.96 U/mL, the effect of thermal death bacteria suspension on SOD content was less. The content of cholesterol, triglycerides and bile acid in rats feces were 0.23μmol/g,4.25μmol/g,28.87μmol/g and 9.85 μmol/g,4.03μmol/g,27.13μmol/g respectively after rats were intervened bacteria suspension and fermented milk, which were significantly higher than model group (P<0.05); the moisture content in feces were 46.15%,45.35% and 46.85% respectively after rats were intervened bacteria suspension, broken bacteria suspension and fermented milk, which was significantly higher than model group (P<0.05). The bacteria suspension, broken bacteria suspension and fermented milk could prevent liver and heart tissue pathology in rats caused by high-fat diet by transmission electron microscopy observed.3. The effects of intervention normal diet, high-fat diet and bacteria suspension, thermal death bacteria suspension, broken bacteria suspension and fermented milk of L. rhamnosus hsryfm 1301 for 4 weeks on the intestinal microbiota of hyperlipidemia rats were researched by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The results showed that the number of Clostridium leptum subgroup PCR-DGGE bands was increased, while the number of V3 region of 16S rRNA genes, Bacteroides spp., Lactobacillus and Bifidobacterium spp. PCR-DGGE bands in rats gut were decreased after rats were fed high-fat diet for 4 weeks; and the treatments of L. rhamnosus hsryfm 1301 had opposite effect.The content of Klebsiella pneumoniae and Uncultured bacterium of intestinal in rats were decreased or disappear in rats gut after feeding normal diet and high-fat diet for 4 weeks, the species of Clostridium leptum were increased at the same time, which were Clostridium botulinum A str. Hall, Clostridium pasteurianum BC1, Ruminococcus champanellensis 18P13, Ruminococcus albus 7 and Ruminococcus bromii L2-63, the Uncultured Bacteroidetes bacterium TM1-40 in control group rats had disappeared. Pantoea vagans and Clostridium thermocellum appeared in rats gut after being administered intragastrically bacteria suspension; and Streptomyces coelicolor and Clostridiales genomo sp. also appeared in rats gut of control and model groups; the abundance of Clostridium botulinum A str. Hall in control group was higher than other groups; the content of Ruminococcus bromii L2-63 decreased or disappeared after rats were administered intragastrically thermal death bacteria suspension, broken bacteria suspension and fermented milk. The abundance of Lactobacillus amylovorus GRL, Lactobacillus buchneri NRRL B-30929 and Lactobacillus sp. was increased by above intervention, and the species of Lactobacillus was increased in rats gut, which were Lactobacillus murinus DLM 3108, Lactobacillus gasseri ATCC 33323 and Lactobacillus sakei subsp.4. The effects of normal diet, high-fat diet and L. rhamnosus hsryfm 1301 bacteria suspension, thermal death bacteria suspension, broken bacteria suspension and fermented milk on the gut microbiota of hyperlipidemia rats were studied by Real-time Polymerase Chain Reaction (RT-PCR), the association between intestinal microbiota and serum lipids level at 9 w was also analyzed. The results showed that the content of Enterococcus spp. and Bacteroides spp. were significantly increased in rats gut after feeding normal diet for 8 weeks (P<0.05); the content of Enterococcus spp., Enterobacter spp. and Clostridium leptum were increased significantly, while the content of Bacteroides spp., Bifdobacterium spp. and Lactobacillus were decreased significantly in rats gut after feeding high-fat diet for 8 weeks (P<0.05); after L. rhamnosus hsryfm 1301 treatments intervention for 4 weeks, the content of Lactobacillus and Bacteroides spp. increased significantly and the content of Clostridium leptum decreased significantly in rats gut by supplementation bacteria suspension (P<0.05); the content of Enterococcus and Bacteroides increased significantly and the content of Enterobacter spp. and Clostridium leptum decreased significantly in rats gut by supplementation thermal death bacteria suspension (P<0.05); the content of Bifdobacterium spp., Lactobacillus and Bacteroides spp. increased significantly and the content of Clostridium leptum decreased significantly in rats gut by supplementation the broken bacteria suspension (P<0.05); the content of Enterococcus spp. and Bacteroides spp. increased significantly and the content of Enterobacter spp. and Clostridium leptum decreased significantly after rats were administered intragastrically fermented milk (P<0.05). A positive correlation was observed between the Clostridium leptum content and the level of TC, TG, LDL-C and HDL-C of serum, and a negative correlation was observed between the Enterobacter spp. content and the Lactobacillus and Bifidobacterium spp. content.5. The effects of normal diet, high-fat diet and L. rhamnosus hsryfm 1301 bacteria suspension and fermented milk on the gut microbiota of hyperlipidemia rats were studied by Illumina MiSeq high-throughput sequencing, the association between intestinal microbiota and serum lipids level at 9 w was also analyzed. The results showed that the relative abundance of 22 genera of intestinal microflora in rats were significantly changed after feeding high-fat diet and L. rhamnosus hsryfm 1301 bacteria suspension and fermented milk intervention; the index of Chaol/ACE and Shannon decreased significantly and the index of Simpson increased significantly due to the abundance and diversity of intestinal microflora were decreased after rats feeding high-fat diet for 4 weeks and normal diet for 8 weeks (P<0.05), which were recovered by supplementation bacteria suspension and fermented milk; and the growth of probiotics was improved, harmful bacteria were inhibited. A positive correlation between the relative abundance of Ruminococcus spp. and the level of TG, the relative abundance of Dorea spp. and the level of TC and LDL-C, and the relative abundance of Enterococcus spp. and the level of HDL-C, while the relative abundance of Butyrivibrio spp. negatively correlated with the level of TC and LDL-C. |
PDF Full Text Request