| Nowadays,the pursuit of fresh food is more and more popular in society.Although fresh food has advantages in flavor and nutritional value,it has potential risks in the aspect of foodborne microorganisms,thus promoting the research on cold sterilization technology.As a new cold sterilization technology,cold plasma has been drawing more and more attention.Plasma is a mixture of positive and negative particles composed of equal amounts of electrons,ions,and metastable particles.The gas produced has low temperature.Plasma has advantages of low equipment operation cost,high sterilization efficiency and no residue after reaction.It has broad prospects not only in the field of food as well as the sterilization of medical devices,water sterilization and air sterilization.Cold plasma can produce more than 400 kinds of particles.It contains a large number of highly oxidizing active substances,which act on different parts of microorganisms,for example,the oxidation of lipids in cell membranes,the cross-linking of proteins and the loss of genetic material and eventually lead to the death of microorganisms.The type or quantity of these bactericidal active substances produced by plasma is regulated by plasma excitation conditions,such as power supply voltage,power supply frequency,working gas,etc.In addition,the target of the plasma and the state of the sample also affect the bactericidal efficiency of the plasma.The multiple selectivity of the plasma to the target of action makes the plasma inactivation complex and changeable,which makes the research on the plasma more complicated.The purpose of this project is to study the influence of cold plasma excitation parameters,sample state and the generated active substance on the bactericidal effect of Salmonella typhimurium and Staphylococcus aureus.In this study,we explored the germicidal mechanism of cold plasma on Gram-negative bacteria and Gram-positive bacteria and its application in food inactivation and preservation in order to provide experimental basis and theoretical guidance for plasma application.The specific research content and results are as follows:1.The influence of generation parameters of cold plasma on the inactivation effectStudying the influence of power supply voltage,relative humidity and oxygen content in the package,standing time on dielectric barrier discharge(DBD)plasma inactivation performance,the best germicidal kinetic model was selected by the coefficient of determination(R2)and root mean square error(RMSE).SRIM simulation software was used to analyze the particle distribution in the cold plasma generator chamber.The results showed that in the range of 50 kV-90 kV,the inactivation rate of cold plasma increased with the increase of power supply voltage.When the relative humidity in the package is 50-80%,the inactivation rate of cold plasma is increased.And the relative humidity(80-90%)in the package is continuously increased,while the inactivation rate of cold plasma is decreased(P>0.05).The oxygen concentration in the package has a significant effect on the inactivation rate of cold plasma(P<0.05),showing a trend of first rising to the maximum value(10-30%)and then stabilizing(30-90%).The inactivation rate of low cold e plasma increased with the increase of standing time(P>0.05).The research found that the Log-linear+Shoulder+Tailer model had the highest fitting degree of inactivation curve for this equipment.The bactericidal curve of Salmonella typhimurium(S.typhimurium)and Staphylococcus aureus(S.aureus)was studied under this model,and it was found by dynamic that S.typhimurium was more sensitive to cold plasma parameter.SRIM simulation software showed that ionization of gas mainly occurred in the second half of the plasma chamber.Excimers are distributed outside and high energy electrons/ions are distributed inside in each microdischarge microfilament.2.Effects of bacterial environment on inactivation performance of cold plasmaThe effects of cold plasma on total colonies and sublethal damages of S.typhimurium and S.aureus were studied based on the Log-linear+Shoulder+Tailer model using variant conditions of osmotic pressure(0.9%NaCl and 2%NaCl),pH(pH=6 and pH=7.3),nutrient(1%BSA and 2%BSA)and surface(glass and agar).The results showed that when the cells are in a floating state in a liquid environment,although the D value is larger(55.02-106.03 s),the inactivation effect is thorough after a long period of cold plasma treatment.When the cells were inoculated onto a solid surface,the D value was small(9.02-23.66 s),but the inactivation was not complete.The bactericidal effect of cold plasma was more significant under conditions of high osmotic pressure(2%NaCl),low pH(pH=6)or low nutrition(1%BSA).The total number of colonies of S.typhimurium and S.aureus decreased by 8.09,6.05,3.93 log CFU/g and 7.89,3.57,2.95 log CFU/g,respectively.Under low osmotic pressure(0.9%NaCl),high pH(pH=7.3)or high nutrient(12%BSA),the total number of colonies decreased by 8.07,4.72,2.87 log CFU/g and 7.87,2.94,1.25 log CFU/g,respectively.When S.typhimurium and S.aureus were inoculated on the surface of agar,the maximum Sublethal Injury of microbes caused by cold plasma was significantly higher than that of(88.36 and 69.71%)inoculated on slides(42.36 and 56.78%).When the cells are in a suitable liquid environment,although the cold plasma inactivation effect is lowered,the maximum sublethal injury of microorganisms is remarkably improved.3.The content of cold plasma RONS and its influence on bactericidal effectThis research explored the influence of different reactive oxygen species and reactive oxygen species(RONS)in cold plasma on bactericidal performance through active matter removal experiment and bactericidal kinetic model,and used fluorescence reagent to detect its content.Finally,in vitro simulation was performed to verify the bactericidal performance of the RONS.The results showed that the RONS significantly affect S.typhimurium in cold plasma were in the following order.Hydroxyl radical was more than hydrogen peroxide,hydrogen peroxide was more than nitrogen oxide,nitrogen oxide was more than singlet oxygen.The RONS significantly affecting S.aureus were in the following order.Hydroxyl radical was more than nitrogen oxide,nitrogen oxide was more than hydrogen peroxide,hydrogen peroxide was more than singlet oxygen.The content of hydroxyl radical,hydrogen peroxide,singlet oxygen and nitrite increased with the increase of plasma treatment time.It was found in vitro experiments that:single hydroxyl radical,nitric oxide and hydrogen peroxide had no significant bactericidal effect on S.typhimurium and S.aureus(P>0.05).While the mixture of the three had significant bactericidal effect(P<0.05),and the bactericidal effect increased with the increase of the concentration of the mixture.4.Study on inactivation mechanism of cold plasmaThe effects of cold plasma on cell membrane permeability,cell morphology,proton motive force,DNA and intracellular ROS were studied.Gram-negative bacteria and Gram-positive bacteria were studied using S.typhimurium and S.aureus as raw materials,and the effects of cold plasma on microbial cell apoptosis were observed and verified.The results showed that cold plasma had significant effects(P<0.05)on cell membrane permeability,proton motive force and DNA oxidation of S.typhimurium and S.aureus.The cell lysis of S.typhimurium and the little morphological change of S.aureus were observed by scanning electron microscope.Intracellular ROS fluorescence intensity was higher in S.aureus.Intracellular free radical scavenger N-acetyl-L-cysteine had no significant protective effect on S.typhimurium,and extracellular free radical scavenger sodium pyruvate significantly improved the resistance of S.aureus and S.typhimurium to cold plasma(P<0.05).Annexin V fluorescence assay showed that treatment by cold plasma caused the apoptosis of S.aureus cells,and the apoptosis inhibitor Z-VDA-FMK significantly increased the total number of S.aureus colonies(P<0.05),but had no significant effect on S.typhimurium(P>0.05).5.Effect of cold plasma and modified atmosphere packaging on pork qualityThe longissimus of pig was used as the object to study the effects of cold plasma on total colony counts,color,lipid oxidation and protein oxidation under different modified atmosphere packaging(MAP)conditions during storage.It indicated that the inactivation rate of 60%MAP+DBD was much higher than that of 20%MAP+DBD(P<0.05).When treated with 60%MAP+DBD,TBARS and carbonyl values were significantly higher at the late time points(day 8 and day 12)compared to the meat treated with 20%MAP+DBD(P<0.05).In addition,there was no significant differences on total colony count between 40%MAP+DBD group and 60%MAP+ DBD group,but TBARS and carbonyl values were significantly higher in the group treated with 60%MAP+DBD(P<0.05).Although low temperature plasma can effectively reduce the total number of colonies in pork during storage,however,cold plasma treatment may increase the chances of color change,lipid oxidation and carbonyl formation in protein oxidation. |
PDF Full Text Request