| Lactic acid bacteria(LAB)are the main strain source of probiotics.Currently,the evaluation of probiotic function of LAB mainly focuses on living bacteria.In view of gradual decrease of the number of live bacteria in the process of production and storage of probiotics,the development of postbiotics including dead bacteria bacteria and their metabolites as main components has attracted attention.However,few studies concern about how to improve probiotic function of probiotics.Here,antioxidant capacities of 5 LAB strains and their postbiotics were evaluated by free radical scavenging experiment and oxidative stress model of intestinal epithelial cells NCM460.Furthermore,functional genes of LAB with excellent antioxidant activity were further explored and different culture conditions were used to enhance the antioxidant capacity of Lactobacillusplantarum CGMCC6888.On this basis,the potential alleviating effect of strain CGMCC6888 on atopic dermatitis was evaluated,in order to provide a basis for the application and development of postbiotics of LAB.The research results were as follows:1.Scavenging of excellent antioxidant LABBacterial cells,lysates and cell-free supernatant(CFS)of 5 LAB strains(Streptococcus thermophius CGMCC7.179,SDMCC050254,Lb.plantarum CGMCC6888,Lb.rhamnosus SDMCC050211 and Lb.paracasei SDMC050465)in laboratory were detected against free radicals.The results showed that the bacterial cells of 5 LAB strains had different scavenging activities against DPPH,superoxide anion(O2-)and hydroxyl(·OH)radical,and 3 Lactobacillus strains had stronger scavenging abilities against DPPH and ·OH,2 S.thermophilus strains had stronger scavenging abilities against O2-.Different components of the same strain had different scavenging abilities against the above free radicals.The CFS had excellent scavenging abilities on DPPH,while bacterial cells had excellent scavenging abilities on O2and ·OH.Except for S.thermophilus SDMCC050254,lysates of tested strains had scavenging abilities against these free radicals.However,heat-treatment did not significantly change the scavenging abilities of each component.Therefore,the tested strain and its metabolites had antioxidant capacities.Based on the oxidative steress model of intestinal epithelial cells NCM460,it was found that the bacterial cells and lysates of 5 LAB strains all alleviated the oxidative pressure of cells,and the effect of Lb.plantarum CGMCC6888 was the most significant.After NCM460 cells pretreated with bacterial cells and lysates of CGMCC6888,the intracellular activities of CAT,GSH-px and SOD increased by 6.44～12.56、1.44～1.48 and 1.23～3.28 times,respectively.Moreover,the transcription levels of Nrf2,the regulatory factor of antioxidant pathway,and its mediated antioxidant oxidase genes(such as HO-1,GCLC and TXNRD1)were increased by 1.17-3.88 times.The ROS level in NCM460 cells was decreased,and the cell survival rate was increased by 36.15%～63.20%,which effectively alleviated the oxidative damage of intestinal epithelial cells.2.Stimulation of heme-dependent catalase enhanced the cytoprotective effect of Lb.plantarum CGMCC6888 against oxidative stressLAB are generally considered facultative anaerobic and catalase negative.Here,a catalase-activiated Lb.plantarum CGMCC6888(CatA+)was obtained by the exogenous added heme.The scavenging free radical abilities of the bacterial cells/lysates towards O2-and OH were increased by about 2 and 1.5 times compared with those of the CGMCC6888 absent of heme(CatA-),respectively.Subsequently,the bacterial cells of CatA-and CatA+ decreased intracellular ROS levels and increased cell survival rate by 18.54%and 53.96%,respectively.The lysates also showed consistent results with the bacterial cells.Further analysis showed that CatA+ bacterial cells or lysates had more significant effect on the transcription of Nrf2 gene and Nrf2-related antioxidant enzyme gene and tight junction proteins ZO-1,OCLN and JAM-1 genes in NCM460 cells.The above results proved that Lb.plantarum CGMCC6888 with activated catalase had a stronger protective effect on intestinal epithelial cells damaged by oxidation,which provided a new idea for improving the antioxidant capacity of Lb.plantarum.3.Antioxidant capacity of Lb.plantarum CGMCC6888 under aerobic respiration conditionsLb.plantarum is generally cultured in static conditions,and it has been reported that it could obtain higher cell density through respiration culture.However,the antioxidant capacity of bacterial cells and other components under respiration cnditions is to be explored.Here,the antioxidant activities of CGMCC6888 under static,aerobic and respiratory conditions were analyzed.Firstly,the growth characteristics of CGMCC6888 under the three kinds of culture conditions were compared.It was found that the biomass of aerobic or respiration conditions was higher than that of the static conditions.In the aerobic conditions,the number of viable bacteria increased while decreased rapidly.Under the respiration conditions,bacterial cells viability was the highest.The antioxidant capacities of bacterial cells,lysates and CFS harvested under three conditions was tested.The results showed that each component of respiration had stronger free radical scavenging ability and the lysates had the better effect.In the intestinal epithelial cell model,respiration showed the stronger cytoprotective effects against oxidative damage.4.Potential alleviation of Lb.plantarum CGMCC6888 on atopic dermatitisIn view of the strong antioxidant activity of Lb.plantarum CGMCC6888,this study analyzed the protective effect of the strain and its lysates on the skin barrier under different culture conditions by the epidermal keratinocyte HaCaT as the model.The results showed that the heat-treated bacterial cells,lysates and CFS harvested from static,aerobic,catalase activated and respiratory conditions were not toxic to the keratinocyte HaCaT.The lysates and CFS of CGMCC6888 could promote the transcription of physical barrier gene(HAS,FLG,IVL and LOR)and chemical barrier genes(HBD)of HaCaT cells,and inhibit the transcription of cytokines(IL-33,IL-6 and TNF-α)and chemokines(MDC and TARC).These results indicated that CGMCC6888 enhanced the barrier function and reduced inflammatory response of HaCaT cells,indicating that CGMCC6888 had a potential role in alleviating atopic dermatitis.Among the four conditions,catalase activation and respiratory condition had the best effect,which also laid the foundation for the preparation of skin care material of LAB.In this study,Lb.plantarum CGMCC6888 with excellent antioxidant activity was screened by using free radical scavenging ability and cell oxidative stress model,and the functional genome and physiological characteristics were explored to enhance the antioxidant capacity of the strain.It has potential application value in relieving intestinal oxidative stress and atopic dermatitis. |
