Evaluation Of Antioxidative Abilities Of Lactobacillus Plantarum And The Antioxidative Mechanisms

Lactic acid bacteria(LAB)with excellent antioxidative properties have significant application in enhancing the body’s ability to alleviate oxidative damage and preventing degenerative diseases.The evaluation of the antioxidative capacity of LAB relies on in vivo trials and in vitro tests.Compared with human and animal trials,antioxidative evaluation methods in vitro have low cost and high operability.However,there are many in vitro antioxidant assays with different principles,and their strengths and weaknesses are difficult to distinguish.Whether these assays can actually reflect the ability of LAB to relieve oxidative damage in vivo remains to be verified.All tests in vivo and in vitro have shown that the antioxidant capacity of LAB differs in among different species and different strains.A comprehensive evaluation model was established for assessing the antioxidant ability of LAB to alleviate oxidative damage by measuring antioxidant parameters in vitro.The LAB with different antioxidative properties were compared and analyzed for the intrinsic mechanisms.It is not only to provide a guidance for mining LAB with excellent antioxidant properties,but also to deepen the understanding of the antioxidant mechanism of LAB,enrich the theoretical study of LAB,lay the foundation for the application of LAB in health.The main research contents of this thesis are as follows:1.To globally evaluate the effects of LAB administration on oxidative damage,a metaanalysis was conducted.The results showed that the effects of LAB on serum superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-PX)activity,and malondialdehyde(MDA)content were significant(P < 0.05),indicating that LAB treatment could alleviate oxidative stress.The sub-group analyses showed that the LAB intervention significantly improved these three serum parameters in the D-galactose-induced oxidative stress model(P < 0.05),whereas for the high-fat diet model,it was only significant for the GSH-PX level.It suggested that the effects of LAB intervention on serum antioxidative parameters might be related to the oxidative stress models.In addition,a meta-analysis of the effects of LAB on hepatic SOD,GSH-PX levels and MDA yielded similar results.The model subgroup analyses revealed differences between models.In the D-galactose model,the ability of LAB to alleviate oxidative damage was correlated with the involved animal strain.The meta-analyses demonstrated that LAB intervention could significantly alleviate oxidative damage and there were differences between oxidative injury models.2.To establish a comprehensive evaluation model for the antioxidant capacity of LAB,the data of in vivo and in vitro antioxidant capacity of LAB in the literature were extracted.A microcystin-exposed mouse model was used to testify the analysis results.The antioxidative properties of 27 strains of Lactobacillus plantarum were determined with 2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl radicals and superoxide radical scavenging ability,reducing activity,resistance to hydrogen peroxide and ferrous chelating ability in vitro.Fuzzy synthetic evaluation models were used to evaluate the overall antioxidative capacity of the LAB.As a result,it was found that the increase in SOD levels induced by LAB administration was positively correlated with the hydroxyl radical scavenging capacity of LAB(P < 0.05).The decrease in MDA content was positively correlated with their DPPH radical scavenging ability,hydroxyl radical scavenging ability and reducing activity(P < 0.05),indicating that the determination of the antioxidative capacity of LAB in vitro could indeed reflect their abilities to alleviate oxidative damage in a certain degree,which is compatible with the ability of mixed LAB to alleviate oxidative damage in microcystin-LR-exposed mice model.Although there were some subtle differences between the overall antioxidant ranks of fuzzy synthetic evaluation with an analytic hierarchy process and with entropy theory,the high scoring strain(CCFM10),middle scoring strain(CCFM242)and low scoring strain(RS15-3)were obtained in both models.D-galactose-induced model was used to testify the antioxidaive activities of CCFM10,CCFM242 and RS15-3.Comparing the model group,the serum GSH,CAT,SOD and TOC levels in CCFM10 and CCFM242 groups were significantly improved(P < 0.05).The antioxdative activity of CCFM10 was higher than CCFM242,and RS15-3 had almost no effect on these parameters,which was consistent with their antioxidant capacity in vitro.3.To clarify the mechanism of L.plantarum alleviating oxidative damage,effects of L.plantarum administration on hepatic antioxidative parameters,expression of antioxidative enzyme gene and intestinal microflora structure were investigated.CCFM10 and RS15-3 administration significantly increased the level of total antioxidant capacity(TOC)and the content of glutathione(GSH).The level of catalase(CAT)activity of CCFM10 was similar to that of the control group,and RS15-3 had no effect on the decrease of CAT level.Compared with the control group,D-galactose exposure induced a marked increase in hepatic peroxiredoxin,glutathione peroxidase,glutathione reductase and thioredoxin reductase mRNA expression(P < 0.05),which were 1.71,3.43,3.36 and 12.55 times of the control group,respectively.The strain CCFM10 had a higher ability to down-regulate the oxidative gene than RS15-3,particularly for peroxiredoxin and glutathione peroxidase.Administration of Dgalactose for 8 consecutive weeks resulted in an increased proportion of Firmicutes(Dgalactose vs control,59.5% vs 51.09%)and a decrease in proportion of Bacteroidetes(Dgalactose vs control,31.66% vs 38.05%).L.plantarum treatment reversed the D-galactoseinduced changes in ratio of Firmicutes and Bacteroidetes.At the genus level,D-galactose caused a relative abundance of the specific genus of Clostridiales in the fecal flora of the mice to be twice that of the control group(37.57% vs 18.06%),and the relative abundance of the beneficial bacteria Lactobacillus was significantly reduced(24.44% vs 10.73%)(P < 0.05).The L.plantarum administration reversed these changes,resulting in a decrease in the Clostridiales abundance and an increase in the abundance of Lactobacillus,and the ability of CCFM10 to restore the bacterial community structure was higher than that of RS15-3.From the above,it seemed that the antioxidant capacity of L.plantarum strains might be related to their effect on host intestinal flora structure.4.To investigate the antioxidative metabolic molecular basis of L.plantarum,18 differential metabolites in the natural growth state of L.plantarum CCFM10 and RS15-3 were identified by gas chromatography-mass spectrometry method.18 and 13 important metabolites between the state of hydrogen peroxide stress and natural growth were for the strains CCFM10 and RS15-3,respectively.Of which,organic acids and amino acids were the major differential metabolites.Enrichment pathway analysis showed that there were differences in metabolic pathways such as energy metabolism,lipid metabolism,amino acid metabolism,coenzyme factor and vitamin metabolism under natural growth conditions between CCFM10 and RS15-3.Under the stress of hydrogen peroxide,the anabolic pathways of valine,leucine and isoleucine,and the metabolic pathway of pantothenate and CoA biosynthesis were the two main differential metabolic pathways of the two strains.It was speculated that these two pathways and the difference in antioxidant capacity of strains were related.