Combined Inactivation Effect And Mechanism Of Protocatechuic Acid And High Hydrostatic Pressure Technology Against Escherichia Coli O157:H7

High hydrostatic pressure(HHP)non-thermal sterilization technology has the advantages of unsignificant temperature change,no damage to food flavor substances and heat sensitive nutrients,and environmental friendliness,which has been widely used in food industry.HHP would induce the production of sublethal microorganisms,which could be repaired in proper environment and therefore cause potential safety hazards.PCA has properties of antibacterial,antioxidant,anti-inflammaroty and other biological activities,which is a potential new multifunctional food additive.In order to deal with this problem,this study attempts to combine HHP with protocatechuic acid(PCA)to achieve better sterilizing effect.Therefore,this study established a sublethal Escherichia coli O157:H7 model induced by PCA,and the HHP stress was then added to study the combined bacterticidal effect,bactericidal mechanism(from cell level and molecular level),and the effects of different combined sequences on bactericidal effect and mechanism.The main results are shown below:1.The sublethal rate and the amount of sublethal cells were calculated by spread plate count method on non-selective and selective mediums,respectively.The optimal sublethal model was selected from samples treated with different PCA concentrations and treatment times.The sublethal rate was 99.99% when the PCA concentration was 1.25 mg/m L(1/2 minimum inhibitory concentration,MIC),and the treatment time was 60 min.The amount of sublethal cells was 20.08 ± 1.76%,which had the property of recovering in proper environment.2.The sublethal mechanism of PCA was initial exploration from the cellular level.It was found that the permeability of cell membrane increased,membrane depolarization,ATPase(which embedded in the cell membrane)activity severly inhibited,while the integrity was not seriously damaged after the sublethal treatment with PCA.Within the cell,SOD and CAT enzyme activities showed a same tendency of increasing first and then decreasing,the ATP content decreased,and the structure of intracellular macromolecules(protein and nucleic acid)changed.It could be inferred that the formation of sublethal E.coli induced by PCA might be related to the changes of cell morphology,membrane permeability,ion leakage,membrane potential,key enzyme activity,ATP content and macromolecular structure.3.Based on the sublethal model,HHP stress was add,and then the effects of different combination sequences(PCA+HHP and HHP+PCA)on the bactericidal effect were compared.It could be found that the combined treatment would enhance the sterilization effect,about 9 log10 CFU(colony forming units)were killed,and the treatment sequence will affect the final sterilization effect.The HHP+PCA sequence had a better sterilization effect compared to PCA+HHP sequence.When the pressure is 100 MPa,there is a synergistic effect,and when the pressure reaches 400 MPa,no visible colony could be detecte-d in HHP+PCA group.In PCA+HHP group,no visible colony could be detected only when the pressure reaches 500 MPa.4.From the cellular level,it was found that the bactericidal mechanism of combined treatment could be related to the membrane permeability and integrity,membrane potential,key enzyme activities,ion and protein leakage,and the change of protein and nucleic acid structures.Besides,according to the results of HPLC,the HHP+PCA group would cause PCA to enter the cell,while the PCA and PCA+HHP groups not.There was an interaction between PCA and E.coli DNA.Based on the results of melting point measurement and fluorescence quenching method,the binding between PCA and DNA is complex,which might be groove binding(low PCA concentration)and intercalation binding(high PCA concentration).5.The sublethal mechanism of PCA and the mechanism of HHP+PCA treatment from the molecular level were measured through the combined analysis of transcriptome and metabolomics.It was found that there were significant differences on both transcriptome and metabolome level between the untreated group and sublethal group and combined group.Sublethal treatment mainly affected the membrane related(membrane lipid and fatty acid metabolism),carbon metabolism,purine metabolism and amino acid metabolism pathways.The combined treatment mainly affected the pathways of genetic information processing(DNA replication,mismatch repair,homologous recombination),energy synthesis(oxidative phosphorylation),amino acid metabolism and membrane related(membrane lipids and peptidoglycan)metabolism.In summary,the synergy of PCA and HHP has the potential to become a new combined non-thermal sterilization method.On the premise of HHP treatment,it could help PCA enter the cell and combine with DNA to destroy the DNA structure.At the same time,it could improve the sterilization effect of HHP and avoid the potential food safety hazards caused by sublethal E.coli O157:H7.Through biochemical experiments and transcriptome-metabolome analysis,it could be found that the sterilization mechanism of PCA and HHP is muti-targeted,which mainly affects the cell membrane metabolism,cell wall peptidoglycan synthesis,nucleic acid structure and metabolism,energy metabolism and key enzyme activities.