Sterilization Mechanism Of High Hydrostatic Pressure On Cronobacter Sakazakii

Cronobacter sakazakii(C.sakazakii)is a gram-negative non-bacilli bacterium,mainly found in infant formula milk powder,which is highly resistant to high temperature,dry and acidic environments,and could cause diseases with a fatality rate of 40-80%such as neonatal meningitis and colitis in infants and young babies.Special processing techniques(storage,homogenization,pasteurization,concentration,spray drying,packaging)are the main ways of microbial contamination in milk powder.While raw milk and pasteurized milk were contaminated by microorganisms during storage and transportation.The traditional thermal sterilization technology has the shortcoming of short shelf life,easy to destroy nutrients and flavor,but the high hydrostatic pressure(HHP)can solve them.In this paper,HHP technology was used as a means of sterilization,referring to the results of transcriptome sequencing,the cpxA deletion strain(?cpxA)was constructed,and the sterilization of the wild strain and?cpxA by HHP was studied from macroscopic,microscopic,and molecular levels.To explore the effect of cpxA on the ability of C.sakazakii to withstand HHP,the following results are obtained:(1)Taking C.sakazakii under HHP as the research object,the effects of C.sakazakii by HHP were investigated through experiments such as plate count,scanning electron microscope(SEM),propidium iodide(PI)fluorescence staining,and detection of intracellular substance leakage to explore changes in bacterial morphology,cell membrane integrity under HHP.The results showed that the lethality rate of C.sakazakii after 400MPa treatment could reach 99.98%.HHP led to the imbalance of pressure between vivo and vitro,which made the cell surface wrinkle in different degrees.With the change of the integrity and permeability of cell membrane,the fluorescence intensity of PI in cell after 400 MPa treatment was about 5 times that of the control group.The concentrations of extracellular nucleic acid,protein and K⁺were 1.13,4.25 and 1.12 times those in the control group,respectively.In addition,the biofilm-forming of C.sakazakii treated by 400 MPa after 24 h cultured was only 40.1%of that of the control group,indicating that the HHP treatment inhibited the biofilm-forming ability of C.sakazakii.(2)Based on the above results,the Illumina HiSeq sequencing platform was used to analyze the effects of initial reaction pressure(50 MPa)and high lethal pressure condition(400 MPa)on the genomics of C.sakazakii.The results showed that under the conditions of 50 MPa and 400 MPa,C.sakazakii up-regulated differential genes were 481 and 363,respectively,down-regulated differential genes were 154 and 395,respectively,and had84 differential genes in total.It involves Fe³⁺ABC substrate-transport ATP binding protein,RNA polymerase factor,amino acid ABC substrate-transport substrate binding protein,amino acid ABC transport substrate binding protein,etc.Combined the phenotypic effects of HHP on C.sakazakii and the results of KEGG enrichment analysis,the genes related to flagellum formation,peptidoglycan synthesis,and cell periplasmic protein were selected for q-PCR validation.The results showed that bacteria resist the stress of HHP by up-regulating the transcription of flagella-related genes(fliC,fliK,flgI,flgK,fliH),and down-regulate the transcription of the signal sensor cpxA of the two-component regulatory system of C.sakazakii,so it interferes with the normal formation of bacterial cell walls(amiA,amiC,ycfS).Up-regulate the gene(degP,dsbA,ppiA)related to folding of intracellular periplasmic proteins and the release degradation factors to degrade protein aggregation caused by adverse environments,and protect cells from misfolded proteins to reduce cell damage.The expression of genes related to the Acr A/B-Tol C pumping system has been up-regulated.(3)The?cpxA was constructed based on the result of transcriptome sequencing,and the role of histidine kinase CpxA in the Cpxtwo-component regulatory system of gram-negative bacteria in the resistance of C.sakazakii to HHP was analyzed.The plate count result showed that under the same 50 MPa condition(critical lethal condition),the number of viable bacteria of?cpxA was about 9%higher than that of the wild strain,and the fluorescence intensity was 1.15 times that of the wild strain.The leakage of nucleic acid,protein and K⁺in cells was significantly different(0.001<P<0.01).The amount of biofilm formation was significantly reduced in the first 12 h(0.01<P<0.05).On this basis,q-PCR results showed that the deletion of cpxA resulted in the inability of C.sakazakii to normally regulate the formation of flagella(fliC,fliK,fliI,flgK,fliH)and cell walls(amiA,amiC,ycfS)to resist the adverse environment under HHP.Moreover,periplasmic protein folding and degrading factors(degP,dsbA,ppiA)lost their anti-stress properties under HHP treatment,and could not protect cells from the damage caused by sedimentation proteins and denatured proteins.This study showed that HHP affected the cpxA of C.sakazakii,which was characterized by cell membrane integrity,bacterial membrane formation,flagellum composition,and periplasmic protein folding,and its downstream resistance pathway,providing a molecular theoretical basis for the HHP sterilization mechanism.