Study On Mechanism Of Aronia Melanocarpa Anthocyanins Inhibiting Escherichia Coli O157:H7 Based On Proteomics And Metabolomics

Aronia melanocarpa anthocyanins（AMAs）are secondary metabolites of higher plants and are classified as recognized safe（GRAS）food ingredients.It has the functions of clearing free radicals in the body,anti-inflammatory,anticancer,antibacterial,etc.Foodborne pathogenic bacteria not only cause changes in food flavor,but also lead to the production of toxic and harmful substances,leading to human food poisoning.Even low concentrations of pathogenic bacteria(100 CFU·m L^-1)pose a high risk to public health.Among numerous foodborne pathogens,Escherichia coli O157:H7 is one of the pathogens causing serious foodborne diseases worldwide.Escherichia coli O157:H7 can produce a large number of toxins,and can self regulate to adapt to various host environments.It has the characteristics of high incidence rate,strong pathogenicity and wide prevalence.It can be transmitted through animal based foods（including poultry,fish,and milk）,fruits,and vegetables,posing a serious threat to the health of food processors and consumers.Controlling this pathogen and preventing potential outbreaks are of great significance.Traditional antibiotic therapy leads to high bacterial resistance and immune response,resulting in a rapid decrease in the effectiveness of antibiotics.And its non-standard use violates the concept of environmentally friendly development.Therefore,it is urgent for researchers to develop effective new antibacterial agents to prevent and control the spread of Escherichia coli O157:H7.Many studies have focused on natural plant polyphenol extracts,which have the characteristics of being natural,low toxicity,and making it difficult for pathogenic bacteria to develop resistance,making them the most promising candidates for natural antibiotics.Therefore,this study explored the response of Escherichia coli O157:H7 cells to AMAs stress,clarified the mode of action of natural phenolic antibacterial agents,and determined the inhibitory mechanism of AMAs on Escherichia coli O157:H7 cells.The main research findings are as follows:（1）The antioxidant capacity of AMAs crude extract and AMAs was measured using three different methods:ABTS,DPPH,and FRAP.The results showed that the antioxidant capacity of AMAs was significantly higher than that of AMAs crude extract.The antibacterial activity of AMAs against five foodborne pathogens,including Escherichia coli O157:H7,Listeria monocytogenes,Staphylococcus aureus,etc,was tested by determining the minimum inhibitory concentration and the minimum bactericidal concentration.The test results showed that AMAs had the most obvious antibacterial effect against Escherichia coli O157:H7,with the minimum inhibitory concentration of 0.625 mg/m L and the minimum bactericidal concentration of 1.25 mg/m L.（2）Through the determination of the release of potassium ions and 260 nm absorption cell materials in Escherichia coli O157:H7 cells,as well as the observation of scanning electron microscopy（SEM）and transmission electron microscopy（TEM）,it is concluded that AMAs can destroy the integrity of the cell wall and membrane of Escherichia coli O157:H7,and increase the permeability of the cell membrane.Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）was used to analyze the changes of the total protein content of cells.It was found that with the increase of the concentration of AMAs treatment,the clarity of protein bands in Escherichia coli O157:H7 cells gradually decreased,and many protein bands disappeared after 2 MIC of AMAs treatment.In addition,agarose gel block and fluorescence spectrum tests were used to explore the interaction between AMAs and genomic DNA.It was concluded that after 2 MIC AMAs treatment of Escherichia coli O157:H7 cells,the complex of genomic DNA and AMAs almost remained in the pores without migration;The addition of cyanidin-3-galactoside（C3G）to EB-DNA complex led to obvious fluorescence quenching.C3G and EB had a competitive relationship in binding to DNA.Finally,the evaluation of the application of AMAs on fresh cut vegetables showed that the number of Escherichia coli O157:H7 cells in the AMAs treatment group decreased by 1.15 log CFU/g and 2.15 log CFU/g respectively on the first and fifth days,which could effectively inhibit the growth and reproduction of Escherichia coli O157:H7 on lettuce.（3）TMT（Tandem mass tag）quantitative proteomics technology was used to study the response of Escherichia coli O157:H7 cells under AMAs stress.The results showed that a total of 628 proteins changed significantly after exposure to AMAs,of which 366 proteins showed up-regulation and 262 proteins showed down-regulation.Through subcellular localization,43.53%of these differential proteins were located in the cytoplasm,7.06%in the outer membrane,2.94%in the cell membrane,32.94%in the inner membrane and 10.29%in the periplasm.According to the results of KEGG analysis,it was found that the differential proteins between the control group and the AMAs treatment group were related to 94 KEGG pathways,including oxidative stress,DNA replication and repair,protein biosynthesis,cell wall and cell membrane physiological functions,and nucleotide biosynthesis.At the same time,MRM validation was performed on the randomly selected differential proteins,and the results were consistent with the results of TMT proteomics.（4）The reaction of Escherichia coli O157:H7 to the anthocyanin treatment of Sorbus nigricans was studied by non-targeted metabonomics.The results showed that there were 1338metabolites of Escherichia coli O157:H7 treated with AMAs（624 differential metabolites in positive ion mode and 714 differential metabolites in negative ion mode）.According to bioinformatics analysis,glycolysis/glycogenesis,purine metabolism,pyrimidine metabolism,glutathione metabolism,alanine,aspartic acid and glutamic acid metabolism,galactose metabolism,citric acid cycle（TCA cycle）,starch and sucrose metabolism,and fructose and mannose metabolism are considered to be important pathways for Escherichia coli O157:H7cells to adapt to stress.Comprehensive proteomics and metabolomics analysis showed that the differentially expressed proteins and metabolites were mainly concentrated in pyruvate metabolism,glycolysis/gluconeogenesis,alanine,aspartic acid and glutamic acid metabolism,and pentose phosphate metabolism,indicating that AMAs can interfere with various metabolic pathways of Escherichia coli O157:H7 cells to achieve antibacterial effect.