Synergetic Antibacterial Efficacy Of Cold Plasma And Clove Oil Loaded Nanofilms Against Escherichia Coli O157:H7 Biofilms On Vegetables

Vegetables are indispensable nourishment sources in our daily life.But vegetables are liable to be contaminated by pathogens during the circulating,which threatens human's health.Escherichia coli O157:H7 is one of the common pathogens in vegetables and it can form biofilms on the surfaces of vegetables.Biofilms are difficult to be eradicated by traditional methods,because they are protected by the extracellular polymer matrix.In this study,two kinds of cold sterilization technology,the cold plasma and the plant essential oil,were combined to treat biofilms synergistically.Clove oil were encapsulated in chitosan nanoparticles,and obtained clove oil loaded chitosan nanoparticles(CO@CNPs).Then,the nanoparticles were incorporated within nanofibers by electrospinning technique.The biofilms were initially damaged by the cold nitrogen plasma.Subsequently,the biofilms were further destroyed by sustained-release of CO@CNPs nanofilms.Eradicating biofilms on vegatables were achieved by the above synergistic effect.The main research contents and results of this paper are as follows:(1)The antibacterial properties and mechanism research of the cold nitrogen plasma.The antibiofilm effect was obviously enhanced after increasing the operating power from 300 W to 400 W.Between 400 and 600 W,it approximately had no further effect.With extending treatment duration from 1 min to 4 min,the antimicrobial activity was further enhanced accordingly.According to the analysis of CLSM and SEM,the plasma could damage the Escherichia coli O157:H7 biofilm morphology,making dense biofilms scattered to single bacteria.Moreover,the antibacterial mechanism was concluded as that the plasma could damage the structural integrity of bacteria,leading to the formation of perforations and notches in cell walls and cell membranes.As a result,the cell membrane penetrability was increased.Furthermore,the plasma induced the leakage of intracellular constituents,such as DNA and ATP,and eventually made bacteria cracking and death.(2)The preparation and antibacterial performance study of CO@CNPs.Clove oil had strong antimicrobial properties against Escherichia coli O157:H7.The MIC,MBIC were both 0.5 mg/mL,and the MBC,MBEC were both 1 mg/mL.Based on the ionic crosslinking method,the optimal CO@CNPs with uniform particle size distribution,great stability and high entrapment efficiency were produced.The average particle size was 154.9 nm,PDI value was 0.297,Zeta potential was 44.3 mV,and the entrapment efficiency was 39.5%.Antibacterial tests showed that CO@CNPs could effectively eliminate Escherichia coli O157:H7 biofilms.After 8 h treatment of 40%(w/v)and 50%(w/v)CO@CNPs,almost 99.981% and 99.999% reductions in populations were achieved,respectively.(3)The preparation and antibacterial test of CO@CNPs electrospinning nanofilms.CO@CNPs were successfully embedded within polyethylene oxide nanofibers by electrospinning.SEM images showed that the diameter distribution of fibers was uniform and no broken wires existed,indicating their great stability.According to the results of colony counting method,CLSM and SEM studies,CO@CNPs nanofilms could effectively eliminate Escherichia coli O157:H7 biofilms.(4)The application of plasma and CO@CNPs nanofilms in combination on vegetables.Compared with the respective treatment,the antibiofilm activity was significantly enhanced by the synergetic treatment.When cucumbers,green peppers,carrots and tomato cherries were treated by plasma(400 W)for 2 min and packaged with CO@CNPs nanofilms for 4 d,almost 99.9990%,99.9998%,99.9999% and 99.9996% reductions in populations were detected,respectively.Color analysis and sensory evaluation results showed that the combined treatment had no impact on the color and flavor of tested vegetables after 4 d.