| Pulsed electric field(PEF)technology,an emerging non-thermal technique,can effectively inactivate bacteria and avoid additional food-quality losses.Generally,the efficacy of PEF can be enhanced by increasing the electric field strength,treatment time and combination with other technical approaches.Increasing the electric field strength and treatment time will increase the input of energy and even lead to high temperature,causing the thermal effect.Relatively,the synergistic treatment of natural antibacterial components and PEF can increase the hurdle factors so better inactivation effect can be achieved.Eugenol(4-allyl-2-methoxyphenol)is a natural component from essential oil and shows antibacterial activity against various bacteria strains,but the antibacterial mechanism is not fully understood.In addition,the study on the synergistic inactivation effect of PEF with eugenol has rarely been reported.Therefore,the aim of the study was to investigate the synergistic inactivation mechanism of PEF with eugenol based on cell membrane characteristics of Escherichia coli(E.coli).The main results were as following:1.The methods of gas chromatography and raman spectroscopy were used to investigate the changes of membrane fatty acid composition and fluidity of E.coli cells at the stationary phase cultivated with eugenol(< 0.5 MIC).Results indicated that increase of eugenol concentration resulted in increase of unsaturated fatty acid content and decrease of cyclic fatty acid content so that membrane fluidity increased.Accordingly,the efficacy of PEF increased,indicating that membrane fluidity was the dominant factor for synergistic inactivation effect of PEF with eugenol on E.coli.2.Quantitative real-time PCR(qPCR)analysis was conducted to investigate the transcription levels of fatty acid biosynthesis-associated genes(fabA、fabD、fabI and cfa)and rpos gene of E.coli cells at the stationary phase,aiming to explore the synergistic inactivation mechanism of PEF with eugenol(< 0.5 MIC).Results indicated that cfa gene was down-regulated and fabA gene was up-regulated when cells were cultivated at relatively higher eugenol concentrations,which altered the membrane fatty acid composition,contributing to the increase of membrane fluidity.However,rpos gene was not related to the efficacy of PEF.3.The antibacterial mechanism of eugenol was studied based on the changes of cell membrane and DNA of E.coli with eugenol(≥ 0.5 MIC)treatment.Eugenol was used to treat normal E.coli cells at the stationary phase.The results of relative conductivity analysis,cell constituents’ release assays and scanning electron microscope observation showed that eugenol destroyed cell membrane,causing destruction of ion homeostasis and the losses of intracellular nucleic acids and proteins,which finally resulted in the bacterial death.Besides,the results of fluorescence spectroscopy and atomic force microscopy indicated that eugenol binded DNA via groove-binding,perturbed the DNA secondary structure and even induced DNA molecules to be aggregated.4.The synergistic inactivation effect of eugenol(≥ 0.5 MIC)with PEF on E.coli was assessed using different electric filed strengths and eugenol concentrations.The results indicated that the combination of PEF with eugenol produced a synergistic effect against E.coli.The synergistic effect was reinforced as the electric field strength or the eugenol concentration increased.The possible reasons are as following: Eugenol can destroy cell membrane structure and function so promote the effect on cell membrane of PEF and lead to cell death.Besides,PEF can influence cell membrane structure so it may facilitate the access of eugenol to the cell membrane and allow entry of eugenol into the cells to interact directly with DNA so that synergistic effect was generated.This paper reveals the synergistic inactivation mechanism of eugenol with PEF.On one hand,it would strengthen the application of PEF in the food industry.On the other hand,it would open a new route for the application of eugenol in the food industry. |
