Research On The Antioxidant Ability And Mechanism Of Different Lactic Acid Bacteria

Oxidative stress is the basic reason of aging and age-related diseases, more than that, it may also cause many diseases, such as, atherosclerosis 、 hyperlipidemia 、 diabetes 、 arthritis 、neurodegenerative disease、cardiovascular and cerebrovascular diseases and so on. So prevention of oxidative stress becoming a research focus in recent years. A large number of studies have shown that, lactic acid bacteria(LAB) possess a series of probiotic function, such as, regulating the immune system 、 keeping balance of intestinal flora and antioxidant capacity, it can scavenging the reactive oxygen species(ROS) in the intestinal tract, and keeping the ROS in a stable state.We research on the four strains of LAB, they are Lactobacillius acidophilus NCFM, which is the most extensive researched in present; Lb. plantarum ATCC 14917, which is a type strain of Lactobacillus plantarum subsp. plantarum; Lactobacillus rhamnosus LGG, which possess a lot of function and widely used in all kinds of foods; Lactobacillius plantarum NDC 75017, which was isolated from traditional fermented dairy products of Tgo region in Inner Mongolia. At first we compare the free radical scavenging capacity of the four strains of LAB, and then investigate the protective effect of four strains of LAB against oxidative stress in Caco-2 cells, at last make a through inquiry of the mechanism of LAB antioxidant by digital gene expression(DGE).(1) We compared the radical scavenging ability of the four strains and their fermentation solution by measuring their hydroxyl radical、superoxide anion radical and DPPH radical scavenging activity. The results show that, in the aspect of hydroxyl radical scavenging activity, the LAB is better than the fermentation solution, in the aspect of superoxide anion radical and DPPH radical scavenging activity, the fermentation solution is better than the LAB. LGG possess the strongest hydroxyl radical scavenging activity, the fermentation solution of Lb. plantarum NDC 75017 possess the strongest DPPH radical scavenging activity, the fermentation solution of Lb. plantarum ATCC 14917 possess the strongest superoxide anion radical scavenging activity.(2) We investigate the protective effect of four strains against oxidative stress in Caco-2 cells, Caco-2 cells was exposed to the 500μM hydrogen peroxide(H2O2) for 30 min, co-culture the four strains and four inactivated bacteria with the oxidative stress Caco-2 cells for 4h, after that the activity of intracellular antioxidant enzymes, including superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) was measured. The results show that, compared to the normal cells, the activity of intracellular SOD in Caco-2 cells was significantly reduced and the activity of intracellular GSH-Px was markedly increased, all the four strains provided protection against H2O2-induced SOD activity reduction and GPx activity increased in different degrees, especially LGG and NDC 75017, but the effect of inactivated bacteria is not obvious.(3) Then we make a through inquiry of the mechanism of antioxidant ability of LGG and NDC 75017, after co-culture the two strains with the oxidative stress Caco-2 cells for 4h, extract the RNA of Caco-2 cells and DGE sequencing of the RNA, screening the significant difference genes, after GO enrichment and KEGG enrichment, clear and definite the signal pathway, which was regulated by LAB during the oxidative stresss. The results show that, compared with the normal Caco-2 cells, there are eight genes significant up-regulation in the injured Caco-2 cells, and they are mostly related to immunity、inflammation and aging signal pathways. After disposed with LGG and NDC 75017, there are six genes and three genes down-regulation respectively in the eight genes, it shows that the mechanism of the two strains can relieve oxidative stress by down-regulate the genes related to oxidative stress. Meanwhile, in the results of GO enrichment and KEGG enrichment, we found that the CASP3 ing TNF signal pathway was activated by the two strains, so we concluded that maybe the two strains also have some antitumous effect.(4) Real-time PCR was used to confirm the eight genes selected in DGE sequencing, The results show that, the level of gene expression in the Real-time PCR are similar with which in the DGE sequencing, it proved that the results of DGE was highly exact.Through this test, we compared the radical scavenging ability of the four strains and investigate the protective effect of four strains against oxidative stress in Caco-2 cells, selected two LAB which possess strong antioxidant capacity, and research the molecular mechanism of their antioxidant capacity by DGE sequencing. Provided a theoretical basis for screening LAB with antioxidant effect in the future, and also provided a new method to research the molecular mechanism of LAB.