Studies On The Mechanism Of The Inactivation Of Listeria Monocytogenes Treated By Pulsed Magnetic Field

Listeria Monocytogenes is a food-borne pathogenic microorganism which is widely found in raw and cooked meat products,aquatic products,soy products,sauces and other foods.It can cause human and animal disease after infection.Pulsed magnetic field?PMF?sterilization has received extensive attention in the academic community because of its slight impact on food quality.In this study,,we firstly studied the inactivation effect of PMF on L.monocytogenes,and then analyzed the inactivation mechanism from the perspective of bacterial morphological structure and Ca²⁺transmembrane behavior,and further studied the effects of PMF on the expression of genes and proteins of L.monocytogenes to reveal the mechanism of inactivation at molecular level.The main conclusions of the paper are as follows:?1?The L.monocytogenes was treated with a strength of 2-8 T and a pulse number of 10-50 by PMF.The residual rate was reduced to 9.60%with a strength of8 T and a pulse number of 20.After observation by scanning electron microscopy and transmission electron microscopy,it was found that the cells were deformed,and the cell contents were not intact after PMF treatment.?2?The optimal loading conditions of Fluo-4 AM calcium ion fluorescent probe in L.monocytogenes was 37°C and 120 min.After PMF treatment,the fluorescence intensity was significantly negatively correlated with the total number of colonies?P<0.01?,indicating that the cells death was closely related to the increase of intracellular calcium concentration.By laser confocal microscopy observation,is was further proved that PMF treatment accelerated the transmembrane movement of calcium ions,enhanced the permeability of cell membrane,and the increase of intracellular calcium ion concentration eventually led to the death of L.monocytogenes.?3?The transcriptomics method was used to study the expression of genes of L.monocytogenes before and after PMF treatment.GO analysis of differential genes revealed that PMF treatment could destroy the chemotaxis and motility of cells,affected the expression of genes involved in metabolism and cell membrane components,the expression of genes involved in DNA damage and the reaction of a harmful substance,causing a stress response of the cell to stimulus.KEGG metabolic pathway analysis of differential genes revealed that differential genes are involvedinchemotaxisandmotility-relatedmetabolicpathways,carbohydrate-related metabolic pathways,ABC transporter-associated metabolic pathways and participated in quorum-sensing-related signaling pathways and two-component system-related signaling pathways.The Rt-PCR analysis of five target genes?lmo0229,lmo0796,lmo1475,lmo1557 and lmo2205?showed that the expression levels of lmo0229,lmo0796,lmo1475 and lmo1557 were up-regulated by4.3,2.37,2.38 and 2.52 times,respectively,and the expression of lmo2205 was down-regulated by 0.57.The results were consistent with the results of the transcriptomics analysis.?4?The proteomics method was used to study the expression of proteins of L.monocytogenes before and after PMF treatment.GO analysis of differentially expressed proteins revealed that PMF affected the metabolic processes such as carbohydrate metabolism,nucleotide metabolism and sulfur metabolism of L.monocytogenes,further regulated the metabolic processes of sugar,protein,etc.through the influence of intracellular coenzyme,affected the bacterial translation process and affected genetic material such as bacterial DNA.By KEGG metabolic pathway analysis,it was found that differential proteins are involved in metabolic pathways such as carbohydrates,nucleotides,and sulfur metabolism.In addition,a variety of amino acid metabolic pathways were also affected by PMF.Therefore,PMF affected the normal physiological activity of cells,resulting in the death of bacteria.