Sterilization Mechanism Of High Power Pulsed Microwave On Biofilms-forming Spoilage Bacteria And Its Application In The Preservation Of Crab Meat

Listeria monocytogenes(L.monocytogenes,G~+)and Pseudomonas aeruginosa(P.aeruginosa,G~-)are typical spoilage bacteria in aquatic products.They often adhere to the surface of aquatic products and processing equipment in the form of biofilm,leading to product spoilage and deterioration.High power pulsed microwave(HPPM)is a new physical sterilization technology that can achieve low-temperature sterilization.In this paper,we investigated the inactivation effect of HPPM on L.monocytogenes and P.aeruginosa and analyzed the mechanism of cell membrane disruption at the cellular level.Proteomics techniques analyzed the expression of differential proteins at the molecular level.It was eventually applied to the preservation of crab meat to provide a theoretical basis for the application of this technology.The main results are as follows.(1)The sterilization effect of HPPM was significantly enhanced with the with increasing frequency and pulse duration(P<0.05).At the optimal sterilization parameters(200 Hz,9 min),the total colony counts of L.monocytogenes and P.aeruginosa decreased by 5.09 and 4.81 lg CFU/m L,respectively.Confocal laser imaging(CLSM)observed a significant increase in the number of dead or damaged cells,showing red fluorescence.Scanning electron microscopy(SEM)showed that the cells in the treatment group were deformed seriously,and the cell membrane boundary became blurred or even broken.(2)The formation of biofilm was inhibited by HPPM treatment followed by incubation,with an overall inhibition rate of 33.62%-63.96%.The inhibition rate was positively correlated with the treatment time.After treatment at 200 Hz for 9 min and incubation for 24 h,optical microscopy,SEM and CLSM observed that the biofilm in the control group formed dense and highly adherent clumps on the covering layer,and the biofilm in the treatment group was dispersed and sparsely colonized on the surface of the sliver,and some bacterial cells showed a collapsed appearance.(3)After treatment at 200 Hz for 9 min,transmission electron microscopy(TEM)observed that the cell wall boundary of the treatment group was damaged or even broken,nuclear chromatin and protein were damaged,and a large area of blank area with low electron density appeared in the cell center.The protein and nucleic acid contents in the supernatant were increased,and whole cell protein were consistent with the above trends of protein leakage.Compared with the control group,Na~+-K~+ATPase activity was decreased by 73.95%and 53.64%,and intracellular ATP concentration was decreased by 80.10%and 99.27%,respectively.Intracellular reactive oxygen species may not be a major pathway of cell membrane damage by HPPM.(4)After treatment at 200 Hz for 9 min,the differently expressed proteins of L.monocytogenes and P.aeruginosa were 204 and 452,respectively(difference value>1.2 or<0.83,P<0.05).Go analysis showed that there were significant differences in cell process,cell structure and catalytic activity pathway between pre-treatment and post-treatment.KEGG analysis showed that the main species of differential proteins are related to cell membrane structure and metabolic functions,including cell membrane composition,energy metabolism,amino acid metabolism and transport,DNA replication and protein translation,and signal transduction.To gain a deeper understanding of the functions of the identified proteins and the targets of HPPM,the biologically relevant properties of key differentially expressed proteins involved in significant enrichment pathways were analyzed.In summary,the response of L.monocytogenes and P.aeruginosa to HPPM stress is likely to be a complex phenomenon,involving a combination of different metabolic pathways.The molecular mechanism of inactivation of bacteria by HPPM was verified by RT-q PCR,which indicated that the proteomic results were reliable.(5)The total colony counts,TVB-N values and TBARS of crab meat at 4,15 and 25°C slowly increased and sensory scores slowly decreased with increasing storage time.A predictive kinetic model was established to predict the storage quality changes at 4,15 and 25℃.According to the change of total number of colonies,TBV-N and TBARS,the shelf-life prediction equation was ln Ct-ln C0=3.54×107exp(-46650/RT)t,ln Ct-ln C0=2.03×107exp(-42750/RT)t,ln Ct-ln C0=3.46×107exp(-10811/RT)t.Because of the time interval between colony counts and quality,the colony counts prediction equation with low bias is used to accurately monitor the quality and shelf life of crab meat in real time during storage.