Research On The Antioxidant Ability And Mechanism Of Lactobacillus Plantarum NJAU-01

Meat products are susceptible to attack by reactive oxygen species(ROS)during processing and storage,resulting in oxidation of lipids and proteins,which in turn reduces the nutritional quality of meat products and can cause food safety problems in severe cases.The use of chemically synthesized antioxidants can not only effectively solve the problem of oxidation of meat products,but long-term excessive use would cause potential harm to human.A large number of studies have shown that lactic acid bacteria have strong antioxidant capacity.Lactobacillus plantarum NJAU-01(NJAU-01)isolated from Jinhua ham shows high antioxidant activity and has been verified in fermented sausages,but its antioxidant mechanism is unclear.This paper studied the effect of high pressure processing on the antioxidant capacity of cell-free extracts from L.plantarum NJAU-01,evaluated the in vitro antioxidant activity of L.plantarum NJAU-01,and explores the intrinsic mechanism of the strain's antioxidant activity under hydrogen peroxide(H2O2)stress.This helps to enrich the theoretical research on antioxidants of functional lactic acid bacteria and provides technical support for the development of commercial lactic acid bacteria as antioxidant applications.The main results are as follows:1.Comparison of cell disrupting methods for antioxidant capacity of cell-free extracts from Lactobacillus plantarum NJAU-01.High-pressure crushing method,glycine addition method,repeated freeze-thaw method,and lysozyme-ultrasonic combined method were used to disrupt the 'cell wall of L.plantarum NJAU-01.The high-pressure crushing method can break 99.99%of the bacterial cells in the suspension of L.plantarum NJAU-01,release the bacterial cell protein to the greatest extent,and the protein concentration of the cell-free extract is 0.45 mg/mL.The superoxide dismutase(SOD)enzyme activity and total antioxidant capacity(T-AOC)of cell-free extracts crushed by high pressure were 6.01 U/mL and 0.25 U/mL,respectively,which were significantly higher than those of other treatment groups(P<0.05).The results show that the high pressure disrupt method can effectively break cell wall of the L.plantarum NJAU-01 and shows a high antioxidant capacity of cell-free extracts2.Antioxidant activity in vitro of L.plantarum NJAU-01.Commercialized L.rhamnosus LGG and L.plantarum JMZ-12 were used as positive controls and negative controls,respectively.Free radical scavenging ability in vitro of lactic acid bacteria were compared.Moreover,electrochemical assay was used to rapidly assess ability of lactic acid bacteria to alleviate oxidative damage of macrophages RAW264.7 induced by H2O2.The results showed that DPPH free radical scavenging rate,hydroxyl radical scavenging rate,superoxide anion radical scavenging rate and reducing power of 109 CFU/mL L.plantarum NJAU-01 cell-free extracts were 24.20%,29.03%,18.21%and 40.13 ?mol/L L-cysteine,respectively,and its antioxidant capacity in vitro was comparable to the positive control LGG(P>0.05).Electrochemical assay results displayed that the anti-oxidation capacity of RAW264.7 after cell-free extract of L.plantarum NJAU-01 was significantly higher than that of other treatment groups(P<0.05),and its intracellular reactive oxygen levels were significantly lower than those of other treatment groups(P<0.05).Our date suggest that L.plantarum NJAU-01 cell-free extract exhibited the most antioxidant capacity.It can be used as a research basis for further analysis of the antioxidant mechanism of lactic acid bacteria.3.Proteomic study of L.plantarum NJAU-01 cell-free extract under H2O2 stress.In order to explore the intrinsic mechanism of the antioxidant activity of L.plantarum NJAU-01,a proteomics study was performed on L.plantarum NJAU-01 at different growth stages under H2O2 stress.The results showed that L.plantarum NJAU-01 was able to metabolize H2O2 with a maximum concentration of 4 mM,and the reduced glutathione(GSH)content of cell-free extracts showed a trend of first decline and then increase.The identified differential protein bands contained molecular chaperone DnaK,heat stress protein Hsp20,Alcohol dehydrogenase(ALDH),pyruvate kinase(PK),elongation factor Tu and citrate lyase ?-Subunit(CLB).Proteomics analysis revealed that a total of 317 proteins were identified in samples from each period.The most differential proteins were detected at the end of the delay period,in which 48 protein abundances increased and 57 protein abundances decreased.L.plantarum NJAU-01 repaired the damaged protein by expressing a large amount of methionine sulfoxide reductase(Msr)and thioredoxin reductase(Trx R)under H2O2 stress.Significant metabolic pathways include two-component system and nucleotide excision repair pathways.This study can provide a theoretical basis for the study of the antioxidant properties of lactic acid bacteria,thereby laying a foundation for the utilization and promotion of L.plantarum NJAU-01 with independent intellectual property rights.