| As a kind of food-borne pathogenic microorganisms,Escherichia coli(E.coli)can spread quickly through contaminated food,drinking water and even daily contacting,which is highly infectious and dangerous.At present,there are many sterilization methods,including heat treatment,microwave sterilization,antimicrobial agent sterilization,ultraviolet sterilization and so on,but these methods have a negative impact on the nutrition or qualities of food,which are not in consistent with people's pursuit of high-quality food.In recent years,non-thermal sterilization technologies have become one of the hot issues for food scholars,such as natural antimicrobial agents,pulsed electric field(PEF),ultra-high pressure sterilization technology and so on,which can well solve the problems caused by traditional sterilization methods.Cinnamaldehyde(CIN),as a natural antimicrobial agent,has strong bactericidal ability,but its specific bactericidal mechanism is still not clear.Therefore,in order to improve greatly the mechanism of CIN against E.coli and to provide a more efficient and energy-saving method combined with PEF,the sterilization mechanism of CIN against E.coli ATCC25922 was deeply explored based on cell membrane and DNA.Besides,the synergistic effects of CIN and PEF were analyzed.The specific research contents and results were as follows.1.GC-MS was used to analyze the changes of fatty acid compositions in E.coli cell membrane under different concentrations of CIN(0,1/8,1/4 and 3/8 MIC).The results showed that with the increase of CIN concentration,the content of saturated fatty acids(SFAs)in cell membrane increased while the content of unsaturated fatty acids(UFAs)decreased,which led to the decrease of cell membrane fluidity and enhanced the resistance of E.coli to CIN.2.The growth of E.coli was inhibited and the logarithmic phase was delayed under the low concentration of CIN(0,1/8,1/4 and 3/8 MIC).When treated with high concentration of CIN(1/2,1,2 MIC),the integrity of cell membrane of E.coli will be destroyed,leading to wrinkling,collapse and dissolution of cell membrane.3.The changes of oxidative indexes such as ROS,MDA and SOD of E.coli cells were detected by agent kits,and the expression of SOD genes were determined by fluorescence quantitative PCR.These results showed that the oxidative damage caused by CIN to E.coli was more serious with the increase of CIN concentration,which led to the increase of ROS and MDA content and induced SOD activity increasing.In particular,the expression of SODc gene was significantly consistent with the change of CIN concentration,suggesting that SODc played an important role in the process of CIN treatment,reducing the damage caused by CIN.4.Analysis of gel electrophoresis,fluorescence spectroscopy and circular dichroism(CD)showed that CIN can bind to DNA of E.coli and combine with DNA via hydrogen bonds in minor grooves of DNA.In addition,atomic force microscopy(AFM)observation indicated that CIN can cause minor damage to the secondary structure of DNA,and induce the aggregation of DNA molecules,interfering its normal life activities.5.The study of viable count of E.coli cells treated with different concentration of CIN,different intensity of PEF and the combination of the two methods was carried out.These results showed that both CIN and PEF had significant bactericidal effect,and can exert synergistic effect,which could be improved with the increase of electric field intensity of PEF or the treatment concentration of CIN.Besides,adding a small amount of CIN can effectively reduce the field strengths of PEF,which helped to reduce the energy input of equipment.Based on cell membrane and DNA,the sterilization mechanism of CIN on E.coli was elaborated in this paper.The synergistic effect of CIN and PEF was analyzed,which provided an efficient,green and energy-saving sterilization method for food industry. |
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