| Probiotics have been demonstrated to have several health benefits,such as modulation of gut microbiota and immunity,anti-oxidation,regulate inflammation,inhibition of cancer and prolongment of lifespan.There are plenty of longevity areas in China with abundant probiotics strains,update description of special functional lactic acid bacteria is scarce,especially for the exploration of molecular mechanisms to extend lifespan.It is quite common for residents of Gaotian Village,Liuyang City,Hunan Province to live longer than 90 years old,which is much higher than the average life expectancy in China.So far there is no research on the longevity of residents in this area.Therefore,based on the systematic analysis of the structure of the intestinal microbiota of residents in Gaotian village,in this study LAB strains were isolated and screened out for properties of probiotics e.g.antioxidant.Thereof one strain of L.plantarum was focused on exploring its function and mechanism of its anti-oxidant,protection of epicellular barrier and prolongment of lifespan in mice.16S r DNA high-throughput sequencing was used to ascertain the difference of intestinal microbial community between Gaotian and other region,and the results showed that the diversity of species of intestinal flora of Gaotian villagers was higher than that of control group.OTU cluster analysis and Venn diagram showed that the special intestinal microbial OTU of Gaotian villagers was 248 and control group was 60.Remarkably,some special species,i.e.,Methanobacterium,Butyricimonas,Deinococcus,and Streptococcaceae,have been found in Gaotian villagers.To quantitatively compare the main flora constitution in all samples,real-time PCR was performed,and the results showed that the biomass of Lactobacillus was generally significantly 40 times higher than that of control group.Ninety isolates were isolated from the gastrointestinal tracts of 21 elderly individual of longevity by traditional methods.Thereof 15,15,25,32,16,1 and 1 isolates were classified as 15 Lactobacillus plantarum,L.fermentum,L.mucosae,L.salivarius,Enterococcus hirae and Enterococcus faecalis,respectively.Those isolates were systematically evaluated for survival capacity under stress conditions(hydrogen peroxide,simulated gastric fluid and simulated intestinal juice)and antioxidant ability.Three strains(i.e.,Lactobacillus plantarum FLPL05,LPL0902 and LPL0302)were selected as candidates for testing their antioxidants in a model of D-galactose induced aging mice.D-galactose induced aging mice was used to evaluate the safety and probiotic properties of L.plantarum.The results showed that the level of ALT and AST of L.plantarum FLPL05 deceased significantly,and no differences were found in the index glucose,lipid metabolism of serum and renal function.A significant increase was found in the activities of glutathione peroxidase(GSH-PX)in serum and total superoxide dismutase(T-SOD)in the liver,and a decrease in the level of malondialdehyde in serum.Regarding m RNA expression level detected quantitatively by real-time PCR,the results showed that L.plantarum FLPL05 significantly upregulated antioxidant genes(i.e.,MT1 A and MT1 M in HT-29 and Caco-2)and those genes(i.e.,MT1,MT2,GPx1,and GPx2)in the intestinal tract of the model mice.Natural aging mice were used to evaluate the effect on lifespan and change of intestinal microbiota of mice via oral administration of probiotic L.plantarum FLPL05 and FLPL0302.The results showed that L.plantarum FLPL05 significantly prolonged the lifespan of 30% of natural ageing mice.The analysis of intestinal microbiota revealed that L.plantarum FLPL05 increased the relative abundance of Firmicutes and decreased the abundance of Bacteroides,accompanied by increased proportions of Lactobacillus,Desulfovibrio and Akkermansiain intestinal microbiota as well as reduced proportions of Facklamia,Clostridium and Ruminococcus.The molecular mechanism of delaying aging and promoting health in mice was explored through isobaric tags(IBT).Results showed that after administration of L.plantarum FLPL05,there were 197 and 124 of proteins in the intestine were upregulated and down-regulated,respectively.Those proteins were classified by using GO database.Results showed that proteins in FLPL05 were 1982 terms according to biological process,365 terms according to cellar component and 341 terms according to molecules function.Following biological pathways were classified using KEGG database.Results showed that 236 terms in L.plantarum FLPL05,including immune system,transport and catabolism.It was found that L.plantarum FLPL05 inhibited the increase of inflammation and intestinal permeability due to aging of mice.Results also indicated that L.plantarum FLPL05 protected the intestinal barrier through promotion of the expression of tight junction proteins(ZO-1,claudin-4 and occludin)and reduction of the apoptosis of intestinal cells,thereby maintaining health.RT-q PCR results showed that antioxidant genes and TERT were up-regulated by L.plantarum FLPL05.The genetic background of L.plantarum FLPL05 was related to prolonging lifespan and protecting the integrity of the intestinal barrier.Results of genomic analysis shown that,L.plantarum FLPL05 was genetically linked to its antioxidant system,i.e.,glutathione and thioredoxin involved in global regulation of defense against hydrogen peroxide challenge.L.plantarum FLPL05 contained a complete gene cluster including 22 loci for production of bacteriocin,which helps it antagonize pathogenic bacteria and protect the intestinal barrier.L.plantarum FLPL05 exhibited 287 pan genes(229 genes unknown function,58 annotated genes)compared with that of L.plantarum strains ATCC14917,ATCC8014,and WCFS1,i.e.,exopolysaccharide biosynthesis protein transport protein and CRISPR-Cas system.In conclusion,high-throughput sequencing technology was used to analyze the structure of the intestinal microbiota of the elderly with longevity,the traditional methods were used to isolate the beneficial lactic acid bacteria,and to explore the mechanism of anti-aging.This study provides ideas and methods for exploring the resources of probiotics with Chinese characteristics and exploring their probiotic mechanisms,thereby laying a technical foundation for the application of probiotics. |
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