Safety Evaluation Of Enterococci From Healthy Chinese Infants As Well As Their Cholesterol-lowering Effect In Vitro And In Vivo

The cardiovascular diseases become a major public health concerning and clinical challenging.A variety of lactic acid bacteria(LAB)were found to have potential to reduce cholestrol level.However,Enterococcus spp.,the species playing important role on the physiological functions on host,presented less information on degradation of cholesterol,especially on their mechanisms.The aim of this study was to select probiotic isolates of Enterococcus which have the potential to reduce cholestrol,and further study the mechanism and evaluate their safety.The whole research work is described in five chapters.In Chapter 1,a background of research advances in cholestrol-lowering effect of probiotics and overview of Enterococcus spp.are summarized,including species and distribution of cholestrol-lowering probiotics and their degradation mechanism,the biological characteristics and classification as well as the probiotic/pathogenic dual nature of Enterococcus spp.Moreover,the development tendency for lowering cholesterol by Enterococcus spp.is predicted.In Chapter 2,a total of 108 strains were isolated from faeces of 34 healthy infants,therein 38 strains of Enterococcus spp.were categorized to 22 species of E.faecalis(10 of E.faecium,three of E.hirae,two of E.durans,and one of E.casseliflavus)by 16 S rDNA sequencing.For safety evaluation,it was determined for some indexs(virulence gene detection,haemolysin,gelatinase,biofilm production and antimicrobial susceptibility)of enterococci.Totally 10 strains(E.faecium strains WEFA23,WEFA24,WEFA26,WEFA28,WEFA30 and WEFA32;E.hire strains WEHI01 and WEHI02;E.durans WEDU02;E.casseliflavus WECA01),which are free of virulence determinants and sensitive to common antimicrobial agent(e.g.ampicillin and vancomycin),were further assessed for their probiotic properties.In Chapter 3,ten of Enterococcus strains,which were identified as safe,were evaluated for their probiotic properties in vitro,Enterococcus faecium WEFA23 was selected due to the best cholesterol removal ability(1.89 ± 0.07 mg/1010cfu),highest glycodeoxycholic acid-hydrolase activity(1.86 ± 0.01 U/mg),strong adhesion capacity to Caco-2 cells(17.90 ± 0.19%),well survival ability in mimic gastric fluid and intestinal tract.The safety of E.faecium WEFA23 was verified by acute oral administration on mice,and found to have no adverse effects on general health status,bacterial translocation and gut mucosal histology.In Chapter 4,the cholesterol lowering effects of E.faecium WEFA23 on rats fed with high fat diet were investigated,the results showed that WEFA23 significantly decreased the body weight,fat accumulation of viscera,serum lipid levels(total cholesterol,triacylglycerols,and low-density lipoprotein cholesterol),blood glucose level and insulin resistance in high fat diet rats.These results supported that E.faecium WEFA23 might exert cholesterol-lowering effect on rats.To further explore the mechanism,the transcription of related genes,the composition of bile salt,synthesis of TMAO and composition of gut microbiota were determined.The results indicated that E.faecium WEFA23 down-regulated the expression of Fas,SCD1 and HOM-COAR to decrease the synthesis of cholestrol.E.faecium WEFA23 was also found to increase bile acid metabolism and the cholestrol excretion.On the one hand,E.faecium WEFA23 hydrolyzed the conjugated bile acid into unconjugated bile acid,decreased the mRNA level of FGF15 and increased that of CYP7A1.On the other hand,WEFA23 down-regulated the expression of FMO3 and decreased the production of TMAO,which can reduce the inhibitory effect toward CYP7A1.Moreover,the gut microbiota was analyzed by the 16 S rRNA sequencing.The results showed that E.faecium WEFA23 improved intestinal microbial flora diversity in high fat diet rats,increased the ratio of Bacteroidetes to Firmicutes,restored the abundance of Rikenellaceae and decreased the abundance of Veillonellaceae.In Chapter 5,the cholestrol-lowering effect and mechanism of Enterococcus are summarized,and perspective research aspects are proposed.In conclusion,E.faecium WEFA23 selected from 38 isolates might be used as a candidate for cholesterol lowering products.The strain can decrease the synthesis of cholestrol and TMAO,and promote bile salt acid metabolism via remodeling of the gut microbiota in host.Our research innovatively disclosed the cholestrol-lowering mechanism of Enterococcus from the view of related genes and gut microbiota,and provided novel insights on Enterococcus as a strategy to decrease TMAO level.