| The microbial seed industry is the core strategic resource that guarantees the development of modern large-scale health industry and technological innovation.Among them,the human body,environment and food microbes are important components of the microbial seed industry resources.In response to the lack of international voice in the domestic microbial seed industry and the increasingly serious problem of global aging,in this study,samples of human body,environment and food were collected from "World's Longevity Town-Jiaoling,China",and based on pure culture technology,a microbial strain resource bank in healthy and longevity areas was constructed.Based on metagenomics technology,a metagenomics database of the gut microbiota of healthy and long-lived people was constructed.The distribution characteristics of resistance genes in the gut microbiota of different age groups in the longevity area and the correlation between the environment and the host in the healthy and longevity area were characterized.Screening of anti-oxidant target bacteria in vitro and research on its probiotic properties were carried out,and in vivo research was completed on the anti-aging mechanism of Lactobacillus plantarum(LP124)on the repair of oxidatively damaged mice.Therefore,this thesis was of great significance for studying the health functions of microorganisms on the human body.The main research content,results and conclusions were as follows:(1)Based on pure culture technology to isolate microorganisms from samples of human body(feces,body surface,saliva),environment(soil,water,air),and fermented food(pickles,sausages,soaked garlic,etc.),to construct microbial strain resources library in healthy and long-lived areas.The results showed that:the final establishment of a healthy and long-lived population microbial strain resource library containing 145 genera,515 species,and 4819 strains from different sources,including 89 genera and 286 species of 3958 strains of healthy and long-lived human-derived microbial strain resources library(32 genera,126 species,2055 strains from feces samples;28 genera,69 species,804 strains from body surface samples;29 genera,91 species,1099 strains from saliva samples);An environmental microbial strain resource bank of 770 strains in 45 genera and 138 species from different sources(16 genera,54 species,407 strains of soil samples isolated strains;13 genera,41 species,265 strains of water samples isolated strains;16 genera,43 species,98 strains of air samples isolated strains);11 genera,26 species,91 strains of food microbial strain resource library.(2)Based on the analysis of the characteristics of the gut microbiota of different age groups in the longevity area based on metagenomics,a metagenomics database of the gut microbiota of the healthy and long-lived population was constructed.The results showed that the composition of the gut microbiota of the population in the longevity area was mainly Firmicutes,Bacteroidetes,and Proteobacteria at the phylum level;The genus level was mainly Bacteroides,unidentified Prevotellaceae,Faecalibacterium,unidentified Lachnospiraceae,etc.;The diversity of the gut microbiota of the long-lived elderly was significantly higher than that of the young group;At the level of longevity-related strains,there were mainly Akkermansia,Methanobrevibacter,Alitipes,Lactobacillus,etc.;The health-related populations Akkermansia,Lactobacillus,Christensenellaceae with higher abundance in the gut microbiota of the long-lived elderly;The gut microbiota of the long-lived elderly had strong oxidoreductase activity and biodegradability of exogenous substances;Lactobacillus was significantly related to the biodegradation and metabolism of xenobiotics,the metabolism of terpenoids and polyketides,and superoxide dismutase;Akkermansia was significantly related to the biodegradation and metabolism of xenobiotics,the biosynthesis and metabolism of glycans,the biosynthesis of other secondary metabolites,superoxide dismutase and catalase.(3)Based on metagenomics,the cumulative effect of antibiotic resistance genes in the gut microbiota of long-lived people,and characterized the distribution characteristics of resistance genes in the gut microbiota of different age groups in longevity areas.The results showed that the older population had the highest abundance of antibiotic resistance genes,and the antibiotic resistance genes in the human gut microbiota accumulated with age and became more complex;The tetracycline resistance genotype tetQ was the most abundant antibiotic resistance gene in the gut microbiota;The main carrier of antibiotic resistance genes was Bacteroides;It was found that antibiotic efflux,inactivation and target changes were the main antibiotic resistance mechanisms;A comprehensive list of antibiotic resistance in healthy people including extremely longevity had been established,and potential recommendations for controlling the use of antibiotics had been provided.(4)Based on 16S rRNA genes amplicon high-throughput sequencing,the environmental microbial community characteristics analysis in the longevity area to characterized the correlation between the environment and the host in the healthy and longevity area.The results showed that in the environmental microbiota of the longevity area,the soil microbiota was composed mainly of Proteobacteria,Acidobacteria,Bacteroidetes,etc.at the phylum level;At the genus level,it was mainly Chujaibacter,Bryobacter,Lactobacillus,Lysobacter,etc.;The biomarkers with significant differences between groups were Lactobacillus,Muribaculaceae,Ruminococcaceae,Lachnospiraceae,etc.;The species with different contributions between groups,from high to low,were Chujaibacter,Acidipila,Lactobacillus,Rhodanobacter and so on.The composition of water microbiota,mainly Proteobacteria,Bacteroidetes,Firmicutes,etc.at the phylum level;At the genus level,they were mainly Ralstonia,Cupriavidus,Phyllobacterium,Sediminibacterium,etc.;The biomarkers with significant differences between groups were Lactobacillales,Psychrobacter,Bacteroides,Ruminococcaceae,Faecalibacterium,etc.;The species with different contributions between groups were ranked unidentified Cyanobacteria,Cupriavidus,Ralstonia,etc.from high to low.Nearly 50%of the gut microbiota came from the surrounding water environment,mainly Prevotella,Bacteroides,Faecalibacterium,etc.It was speculated that the diversity of water and its microorganisms played an important role in connecting the environment with health and longevity.(5)Based on the constructed strain library and omics database for healthy and long-lived people,the in vitro antioxidant screening index was used to screen probiotics with good antioxidant functions,and their probiotic properties and safety were verified.The results showed that a probiotic strain of Lactobacillus plantarum 124(LP124)with good antioxidant function was screened in vitro;LP124 had good acid tolerance,bile salt tolerance,and salt tolerance.The lowest pH was 3.5,the highest bile salt concentration was 0.1%,and the highest salt concentration was 6%;LP124 had good antibacterial properties and had certain inhibitory effects on Listeria monocytogenes,Staphylococcus aureus,Enterobacter sakazakii,and Pseudomonas aeruginosa.It had no inhibitory effect on Bacillus cereus,Bacill us subtilis,Escherichia coli,and Salmonella typhimurium;LP124 had good self-agglomerating ability,good mutual agglutination ability with pathogenic bacteria,and good surface hydrophobicity;LP124 had a large number of oxidoreductase-related genes in the body and had specific molecular targets.LP124 had good safety characteristics,no hemolysis,no drug resistance genes,no virulence genes,and sensitivity to antibiotics.(6)Through the research of LP 124 on the repair mechanism of D-galactose-induced oxidative damage in mice,and explored the anti-aging mechanism of probiotics.The results showed that LP124 significantly regulated the composition of the gut microbiota of oxidatively damaged mice,and increased the abundance of the beneficial bacteria Akkermansia and Bacteroides;LP124 had strong amino acid metabolism,lipid metabolism,exogenous biodegradation and metabolism,and carbohydrate metabolism;The derived small molecule L-ascorbic acid was used as an effective substance participating in the antioxidant reaction;L-ascorbic acid metabolites were related to metabolic pathways such as ascorbic acid and aldonic acid metabolism,vitamin digestion and absorption,and glutathione metabolism;Both LP124 and Akkermansia significantly up-regulated the levels of GSH-Px,SOD,TrxR and T-AOC in liver and kidney tissues to varying degrees;Significantly down-regulated the level of MDA and protein carbonyl in liver and kidney tissues,and significantly down-regulated the level of ET in plasma;Significantly up-regulated the relative expression levels of GSH-Px,SOD,TrxR and Nrf2 mRNA genes in the liver and kidneys to varying degrees;Reduced the pathological changes of cells in the liver,kidney and small intestine tissues of mice.In summary,this paper aimed at the backward status of my country's microbial seed industry,and constructed a healthy and long-lived regional characteristic microbial strain library and an omics database containing human,environmental and food samples with my country's independent intellectual property rights;Explained that longevity-related strains(beneficial bacteria Akkermansia,Lactobacillus,Christensenellaceae)could be used as biomarkers of health and longevity,and the antioxidant activity of the gut microbiota could promote health and longevity;A strain of LP124 with anti-aging function was selected.Its good probiotic properties and oxidative damage repair ability could be used as a regional longevity factor,and L-ascorbic acid could be used as a material basis for anti-aging. |
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