| Aging is an irreversible process,and aging is considered to be the cause of tissue and organ function decline with age.It is also associated with the development of age-related diseases,such as cardiovascular disease,diabetes,and cancer,so aging has become a hot topic in modern medical research.Most of the mechanisms of aging focus on oxidative stress and free radical theory,inflammation-induced aging,immune aging,intestinal flora imbalance,etc.Oxidative stress in the body leads to a large accumulation of free radicals,cell function decline,and proliferation,leading to increased cell damage and death.At the same time,the long-term chronic inflammatory response caused by the increase of pro-inflammatory cytokines,the gradual decline of immune system function and the occurrence of various diseases,inflammatory aging and immune dysfunction are closely related to the changes of intestinal flora.Therefore,aging is not the result of a single mechanism.The development of anti-aging drugs and natural products for these mechanisms and theories has also become a research hotspot.Objective:Due to the demand for natural,safe anti-aging substances.Lactic acid bacteria(LAB)with strong oxidizing properties were screened from traditional fermented food“Jiangshui”,pickles in Shaanxi and Sichuan and preserved LAB isolated from feces,and their physiological characteristics and safety were evaluated.At the same time,for exploring whether the combination of traditional Chinese medicine(TCM)and LAB can enhance the antioxidant effect in vivo,through the co-culture experiments of antioxidant TCM and LAB in vitro,the TCM that has no inhibitory effect on LAB or promotes its growth and has antioxidant effect was selected.D-galactose was used to construct an aging mice model,and mice were given TCM(mulberry),LAB(J2-5,9)and TCM combined with LAB to determine the differences in anti-aging effects of single and combined drug treatment strategies.Method:(1)experimental strains selection:The monoclonal strains in fermented foods were isolated by gradient dilution,selected acid-producing colonies by bromocresol violet medium,we detected whether these are Grgram-positive bacteria.Then DPPH scavenging test,hydroxyl scavenging test,ferrous ion chelation test,superoxide anion removal test and hydrogen peroxide resistance test were performed for selecting the strong antioxidant strains.Hydrophobicity experiments and auto-aggregation experiments to determine their intestinal adhesion ability in vitro.The safety of the strains was analyzed based on their minimum inhibitory concentration and hemolysis,and 16S rRNA was used for molecular biological identification.Antimicrobial activity tests showed them probiotic function.The cell-free supernatant of selected strains were used to explore the protective effect against oxidative damage cells.(2)Six TCM with antioxidant component,licorice,atractylodes,Licorice,atractylodes,mulberries,raspberries,astragalus,chinensis,were co-cultured with LAB as concentration of 5 mg/mL,10 mg/mL,20 mg/mL in anaerobic environment for 24 h,determine the influence of TCM on probiotics growth in vitro,select the most suitable TCM for in vivo experiments.(3)D-galactose as the concentration of 100 mg/kg,0.3 m L was injected in C57BL/6J mice by subcutaneous for 6 weeks.The negative control group(Neg Con)was injected subcutanenous and intragastric administration daily with 0.3 m L,0.2 m Lof 0.9%Na Cl solution,respectively.TCM group,J2-5,9 group and TCM+J2-5,9 group were intragastric administration with TCM solution,bacterial suspension,TCM solution mixed with bacterial suspension,respectively,the concentration of TCM was 2.0 g/kg,0.3 m L,and the concentration of bacterial suspension was 2×10~9 CFU.During this period,the weight was weighed every week,finally,the content of liver SOD,POD,MDA and CAT were calculated,the relative contents of IL-2,IL-4,IL-6,IL-10,and IFN-γin the spleen were measured,HE staining of colon section tissue was performed to determine the tissue damage degree caused by inflammatory aging.(4)Intestinal flora of feces from five groups were detected by 16S rRNA sequencing.The changes of intestinal flora after treatments were observed.Results:(1)Five strains J2-4,J2-5,J2-9,YP-1 and W-4 were screened by antioxidant experiments,and the hydrophobicity was 23.07±1.38%93.53±1.15%,85.42±0.80%,79.86±0.90%,and 82.12±0.65%,respectively.The auto-aggregation rate was 90.13±0.43%,90.37±0.44%,92.42±0.35%,82.87±0.1%,90.5±0.187%,respectively.The results of 16S rRNA showed that J2-4,J2-5 and J2-9 were Lactobacillus fermentum,YP-1 and W-4 were Lactobacillus paracasei.In the MIC test,Lactobacillus fermentum J2-4,J2-5 and J2-9 showed moderate resistance to polypeptides and tetracycline antibiotics,while Lactobacillus paracasei YP-1 and W-4 showed resistance toβ-amides and polypeptide antibiotics.All strains were sensitive to chloramphenicol and aminoglycosides.Hemolysis experiments showed that Lactobacillus fermentum J2-4,J2-5,J2-9 wereγ-hemolytic activity(non-hemolytic).Lactobacillus paracasei(W-4 in feces and YP-1 in pickles)showedα-hemolysis.After identifying the strains by 16S rRNA,hemolysis experiment,MIC resistance experiment,and determination of virulence genes.The antioxidant properties and probiotics of the selected strains were evaluated by protective experiment against oxidative damage cells and the antibacterial experiments.Finally,J2-5 and J2-9 were selected for the next exiperiment.(2)Except TCM-mulberry,five kinds of TCM showed the different degrees of inhibition on J2-5 and J2-9.(3)The results of visceral weight ratio showed that the ratio of LAB group in the spleen was the highest,and that in the TCM group was the lowest.In the kidney ratio,TCM group was the lowest,and there was no significant difference in other groups.In the the liver ratio of the aging group was the lowest,and there was no significant difference between the other groups.In the detection of free radicals and antioxidant enzymes,there was no significant difference in SOD activity between the TCM group and the aging group.The difference between J2-5,9 and TCM+J2-5,9 was significant,J2-5,9 was highest.TCM group,J2-5,9 group and TCM+J2-5,9 group,the difference was significant,P values were 0.0227,0.0028,0.0048,especially J2-5,9 group and TCM+J2-5,9 group.J2-5,9 group had the lowest MDA.The POD value of each group showed no significant difference in statistics.The POD activity of the TCM group,the J2-5,9 group,and the TCM+J2-5,9 group were similar.The POD activity of the TCM group was lower,than the J2-5,9 group.The content of CAT in J2-5,9 group was highest.Detection of pro-inflammatory factors and anti-inflammatory factors by q PCR:The content of IL-2in the aging group was higher than that in the other three groups.There was no significant difference between TCM group,J2-5,9 group(P<0.0001)and the TCM+J2-5,9 group(P=0.0096).The level of IL-6 in all treatment groups were higher than that in the aging group.There was a significant difference between the J2-5,9 group and the TCM+J2-5,9 group compared with the Con group.J2-5,9 and the TCM+J2-5,9 and TCM showed a significant up-regulation of IL-4.IFN-γin TCM group and TCM+J2-5,9 group was significantly decreased.HE staining of the mice colon showed that there was inflammatory cell infiltration in the colon tissue of the aging group,and the three treatment groups had an alleviating effect on inflammation and injury,especially the J2-5,9 bacterial group.The intestinal flora of each group of mice was measured to observe the effects of the experiment on the intestinal flora of normal mice,aging mice and mice in each treatment group.(4)After high-throughput sequencing of intestinal flora,compared with the aging model group,the abundance of Alpha diversity and Beta diversity in the J2-5,9 group was significantly increased,especially in the J2-5,9 group.The abundance of intestinal flora in aging group and different treatment groups changed differently.Conclusion:(1)Lactobacillus fermentum J2-5 and J2-9 screened by in vitro antioxidant test,colonization ability in vitro test and safety test,which can be used in food and medicine.(2)In animal experiments,the J2-5,9 bacteria group showed the strongest anti-aging effect;the antioxidant effect of TCM+J2-5,9 group was slightly lower than that of J2-5,9 group,and the TCM group was not good.The richness of intestinal flora in probiotics J2-5,9 group showed that probiotics J2-5,9 played a strong antioxidant function in the body.(3)At the same time,the treatment groups had different degrees improved the richness of intestinal flora.According to the comprehensive results,J2-5,9 showed the strongest therapeutic effect,improved the inflammation caused by aging,improved and enriched the intestinal flora,etc.The effect of TCM+J2-5,9 was slightly lower than that of single bacteria J2-5,9 group,and the effect of TCM group was not good.The concentration gradient of traditional Chinese medicine should be explored whether the effect is based on concentration. |
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