| The health problems caused by food-borne pathogens have attracted more and more people’s attention around the world.Food-borne pathogens not only lead to food spoilage and nutrient loss,but also directly endanger human health.Synthetic preservatives are widely used in food to inhibit the growth of pathogens and prolong its shelf life.However,synthetic preservatives are potentially harmful,so people prefer to choose natural antibacterial ingredients.Thymol is a single phenolic compound extracted from thyme essential oil.It has a variety of biological activities and exhibits strong broad-spectrum antibacterial properties.It has potential application in the food field as a natural antibacterial ingredient,and studies on thymol have gradually shifted from the inhibitory activity to its development and application.However,the research on its antibacterial mechanism is not comprehensive,and being a key scientific problem to be solved urgently for the effective use of this natural antibacterial agent.This study was aimed to investigate the antibacterial properties and mechanisms of thymol against four common food-borne pathogenic bacteria,namely Escherichia coli O157:H7 ATCC35150（E.coli）,Salmonella enteritidis ATCC 9120（S.enterica）,Staphylococcus aureus ATCC25923（S.aureus）and Listeria monocytogenes ATCC 19115（L.monocytogenes）by using the GC-MS technique,electron microscopy,metabolomics,and transcriptomics.The main research results are as follows:（1）Thyme essential oil was extracted by steam distillation,and the extraction rate was0.43%.Gas chromatography-mass spectrometry（HS-PEME-GC-MS）analyzed various components in thyme essential oil,the main components were thymol（30.54%）,p-cymene（14.18%）,carvacrol methyl ether（11.16%）,caryophyllene（7.87%）,terpineol（5.22%）,linalool（4.33%）,isobornyl acetate（4.21%）,camphene（3.66%）,camphor alcohol（3.03%）and carvacrol（2.46%）,the above components accounted for 86.66%.Thyme essential oil has certain antibacterial activity,but the antibacterial ability of the components of thyme essential oil against food-borne pathogens is quite different.The main component thymol has the best antibacterial effect.The minimum bactericidal concentrations of E.coli,S.enterica,S.aureus and L.monocytogenes were 0.25 g/L（1.67 m M）、0.125 g/L（0.83 m M）、0.25 g/L（1.67m M）和0.5 g/L（3.33 m M）,respectively.（2）Growth kinetics studies showed that the effects of thymol on the dynamic growth process of E.coli,S.enteritidis,S.aureus and L.monocytogenes were different due to different strains.In the concentration range of 0 to 0.83 m M,thymol hardly affected the growth of E.coli;however,when thymol was 0.83 m M,it resulted in decreased bacterial yield and growth retardation of S.aureus and L.monocytogenes;S.enterica was more sensitive to thymol,and its maximum growth rateμdecreased,lag phaseλand Tg time were significantly prolonged in the concentration range of 0 to 0.42 m M thymol.E.coli,S.enterica,S.aureus and L.monocytogenes have certain differences in cell membrane fatty acids.Gram-negative bacteria E.coli and S.enterica mainly contain saturated,unsaturated and cyclic fatty acids,while Gram-negative bacteria The positive bacteria S.aureus and L.monocytogenes are mainly branched-chain and straight-chain fatty acids.Bacterial cell membranes exposed to sublethal concentrations of thymol were all altered to some extent.For example,in the presence of thymol,the content of total saturated fatty acids in the cell membrane of S.enterica decreased,while the content of unsaturated fatty acids increased significantly;higher concentrations of thymol（1.0 and 2.0 MIC）directly caused cell membrane rupture,resulting in four foodborne pathogens The intracellular contents of the bacteria are dissolved,resulting in changes in the morphology of the bacterial cells.（3）The genomic DNAs of E.coli,S.enterica,S.aureus and L.monocytogenes were extracted,and the binding properties of thymol to bacterial genomic DNA were studied by fluorescence spectroscopy,UV-Vis absorption spectroscopy and circular dichroism.The results of fluorescence spectra showed that thymol could interact with bacterial genomic DNA,resulting in fluorescence quenching.The binding was spontaneous,and the hydrophobic force was the main driving force;the binding constant of thymol to bacterial genomic DNA was 104M-1 order of magnitude.When combined with the experimental results of thermal denaturation,salt effect,circular dichroism,and molecular docking,it shows that thymol can bind to the groove A-T enriched region of DNA and change the secondary structure of the DNA molecule to a certain extent.（4）1185 metabolites were detected from four food-borne pathogenic bacteria by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry（LC-MS/MS）,and metabolomic analysis showed that the most different metabolites of Gram-negative bacteria E.coli and S.enterica were the biofilm formation pathway.The intracellular level of c AMP,a precursor of biofilm formation,was significantly reduced after thymol treatment,which could directly inhibit Flh D（C）transcription,indicating that thymol significantly inhibited the biofilm formation of Gram-negative bacteria.In addition,lipid metabolism,amino acid metabolism,and energy metabolism in the metabolites of Gram-negative bacteria,,i.e.S.aureus and L.monocytogenes were also relatively different,indicating that there is a correlation between the pathways.The most difference of metabolites of Gram-positive bacteria were lipid metabolism,and the intracellular levels of myristic acid and lauric acid,the terminal products of bacterial fatty acid production pathway,decreased significantly after thymol treatment,indicating that thymol significantly inhibits fatty acid synthesis in Gram-positive bacteria.（5）On the basis of metabolomics,the genomes of food-borne pathogens were sequenced by RNA-seq technology for E.coli and S.aureus,and a transcriptome database was constructed and differentially expressed genes were screened,and then bioinformatics analysis was used.The KEGG metabolic pathway related to differentially expressed genes systematically revealed and verified the inhibitory mechanism of thymol on food-borne pathogens.GO and KEGG enrichment analysis screened out 76 and 35 genes that playing an important role in the response of E.coli and S.aureus cells to thymol stress,respectively,mainly related to membrane transport,lipid metabolism,energy metabolism,amino acid biosynthesis and metabolism,carbon metabolism,quorum sensing,two-component systems,and other metabolic pathways.Under thymol stress,E.coli cells elicited the quorum-sensing system to impair bacterial behavior and reduce flagellar motility,to increase the conformational changes required for proton transport,thereby making cells to be tolerant.S.aureus cells responded to the stress of thymol mainly by changing the fatty acid composition of the cell membrane,increasing the formation of capsule and down-regulating the expression level of the gene cluster of pyrimidine biosynthesis. |
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