| Escherichia coli O157:H7 is a common foodborne pathogen,which poses a serious threat to food safety due to its strong pathogenicity and quick transmission.Moreover,the emergence of "Superbug" carrying new Delhi metal β-lactamase(NDM)makes E.coli O157:H7 treatment more difficult.Antimicrobial peptides(AMPs)have broad-spectrum antibacterial activity and the unique multi-target mechanism,which enable them to effectively inhibit multi-drug resistant bacteria and not to easily cause drug resistance.However,due to their cost and toxicity,antimicrobial peptides are limited in the wide applications of food.Therefore,our study attempts to reduce the dosage of antimicrobial peptidesby using them in a cooperative manner to achieve a balance between antibacterial activity and cytotoxicity.First,we screened highly selective antimicrobial peptides using NDM E.coli O157:H7 and several strains of E.coli O157:H7 with different pathogenic characteristics,and then studied the synergistic antibacterial activity and membrane mechanism of antimicrobial peptides,and then evaluated stability to determine their application scopes.Finally,they were applied to the preservation of lettuces and cucumbers to verify their antibacterial abilities and effects on food sensory quality.The main results are as follows:(1)Thanatin and BF-15a3 with the highest selection index were chosen from 12 antimicrobial peptides.The geometric mean values of the minimum inhibitory concentrations against three strains of E.coli O157:H7 were 4 and 10 μg/m L,respectively,and their hemolysis rate is less than 5% at 128 μg/m L.(2)The antibacterial activity of thanatin and BF-15a3 against 44 strains of E.coli O157:H7 was hardly affected by their pathogenic characteristics,and the MIC range was 2-8 μg/m L and 4-32 μg/m L,respectively.The 93.18% of the 44 strains of E.coli O157:H7 tested carried stx2(also carried at least one of eae or hly).They belong to ST11,ST4429 and ST1423 or 19 kinds of PT types.ST11 is the main dominant clone with significant polymorphism.Among them,36 strains showed resistance to at least one of nine antibiotics,with severe resistance.(3)In the checkerboard antibacterial test,thanatin and BF-15a3 showed synergistic bacteriostatic effect on E.coli O157:H7,and the MIC of the combination was reduced to 1/4 or 1/8 of that of the single use.Combined use will not lead to the enhancement of hemolytic activity and cytotoxicity,and it is not easy to induce bacterial drug resistance.(4)The results of sterilization kinetics showed that the combination of thanatin and BF-15a3 at lower concentration could quickly kill E.coli O157:H7.The membrane mechanism of synergistic action of antimicrobial peptides on E.coli was studied.The results showed that the combination of thanatin and BF-15a3 could destroy the integrity of cell membrane.They synergistically increased the permeability of outer membrane and inner membrane of E.coli,resulting in the leakage of intracellular ions and cytoplasm,the increase of ROS content,and finally the death of bacteria.(5)The stability test results showed that the antibacterial activity of thanatin and BF-15a3 was easily inhibited by serum,simulated gastrointestinal fluid and high concentration salt,while they had strong tolerance to high temperature,physiological concentration of salt,neutral and alkaline environment.They were suitable for longterm storage at 4 ℃ or-20 ℃.(6)AMPs combination treatment can significantly inhibit the growth of E.coli O157:H7 on the surface of fresh cut lettuces and cucumbers during storage,and will not affect the sensory quality of food. |
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