| Post-harvest loss of fruits and vegetables is a global problem,the most common and serious losses which is caused by microbial infestation.The plant source preservatives have attracted great concern worldwide,but there are some problems,such as high cost,low efficiency and unclear targeting.Therefore,it is necessary to conduct in-depth research and development.The application of essential oils as the main plant source preservative is also limited in food due to their low aroma threshold and microbial resistance.The quorum sensing inhibition(QSI)effect can control the microbial decay process by inhibiting the signaling system produced by the spoilage characteristics,at a concentration far below the minimum inhibitory concentration(MIC),without killing the microorganism.In this paper,aiming at the main spoilage microorganisms in vegetables which were Erwinia carotovora(E.carotovora)and Pseudomonas fluorescens(P.fluorescens),the essential oil(EO)components with quorum sensing inhibition(QSI)effect were screened.The inhibitory effects of EO components on the QSI related characteristics,including motility,biofilm-formation,exopolysaccharide production and quorum sensing signal molecules of spoilage bacteria,were systematically studied,which provided basic data for postharvest preservation of vegetables.Four parts were included in the main research contents of these thesis: 1.Screening of quorum sensing inhibitory activity of essential oil componentsThe effect of 10 EO components on QSI indicator Chromobacterium violaceum CV026 were studied,the results showed that all tested EO components showed QSI activity at sub-minimal inhibitory concentration(MIC)of 0.1 mg/mL.The inhibitory effect of 10 EO components on the motilities(swimming,swarming,twitching),biofilm formation,exopolysaccharide production of E.carotovora and P.fluorescens were further investigated.Biofilm formation was inhibited by all EO components by inhibiting motilities and exopolysaccharide production.Salicylic acid and thymol showed the best inhibitory effect on biofilm formation of P.fluorescens(37.61%)and E.carotovora(47.24%),respectively.Hexanal as QS inhibitor on biofilm formation was first reported,with inhibition ratio of 25.86% and 20.18% on E.carotovora and P.fluorescens,respectively,which was confirmed by scanning electron microscope(SEM)and inversed fluorescent microscope(IFM).The violacein production in CV026 was significantly inhibited by different sub-MICs(1/10 MIC,1/5 MIC,1/2 MIC)of hexanal with a positive correlation(r = 0.9674).2.The quorum sensing inhibitory effect and dose-dependence of hexanal on E.carotovora and P.fluorescensThe motilities,exopolysaccharide production,and biofilm formation of E.carotovora and P.fluorescens were significantly inhibited by hexanal(P < 0.05)with no effect on growth curves of spoliage bacteria.A positive dose-dependent relationship(r = 0.8167 ? 0.9998)between the inhibition ratio and concentrations of hexanal,as well as a negative relationship(r =-0.8168 ?-0.9998)between the inhibition ratio and culture time during the critical biofilmformation stage(1–3 d),was observed.The biomass,average thickness and maximum thickness of biofilm were decreased(P < 0.05)observed by confocal laser scanning microscopy(CLSM)and SEM,showed good linear relationship with culture time and the concentration of hexanal,respectively(r = 0.8124 ? 0.9998,r =-0.8677 ?-0.9940).The extracellular enzyme activity associated with spoilage characteristics was decreased with the increase of hexanal concentration(P < 0.05),including cellulase,xylanase,pectate lyase,polygalacturonase,and protease.Chinese cabbage soft rot and lettuce leaf scorch could be significantly inhibited by hexanal without any phytotoxicity effect.1/2 MIC of hexanal showed the best inhibitory effect on biofilm formation and extracellular enzyme activity.3.The inhibitory effect and dose-dependence of hexanal on quorum sensing signal molecule AI-2 of E.carotovora and P.fluorescensThe reporter strain Vibrio harveyi BB170 was used to semi-quantitatively detect the signal molecule AI-2,the result showed that AI-2 content produced by E.carotovora was much higher than that of P.fluorescens.UPLC-MS/MS was used to detect AI-2 precursor of DPD.DPD production of E.carotovora and P.fluorescens were significantly inhibited by hexanal(P < 0.05),especially the DPD inhibition rate of hexanal against P.fluorescens was close to 100%.Under the inhibition of hexanal,different concentrations of DPD were added exogenously for 24 h,the biofilm formation of E.carotovora and P.fluorescens could be significantly restored(P < 0.05),and there was a positive correlation between exogenously added DPD concentration and biofilm formation recovery,respectively(r = 0.9934,r = 0.9942).It was confirmed that the QSI effect of hexanal can inhibit the DPD production to achieve the effect of inhibiting biofilm formation.However,there was no significant restoration effect on extracellular enzyme activity(pectate lyase,polygalacturonase,cellulase,xylanase and protease)(P < 0.05),indicating the QSI effect of hexanal on extracellular enzyme activity may be independent of DPD or AI-2 signal molecule in stable growth period.4.The inhibitory effect and dose-dependence of hexanal on quorum sensing signal molecule AHLs of E.carotovora and P.fluorescens?-galactosidase activities of E.carotovora and P.fluorescens which reflected AHL production,were significantly inhibited by hexanal,its inhibitory effect was concentration-dependent under MIC(r =-0.9656 ?-0.9968,r =-0.9147 ?-0.9984)during 0-24 h.UPLC-MS/MS was used to track the signal molecule AHLs.The main AHL of E.carotovora was identified as 3-oxo-C6-HSL,and the main AHL of P.fluorescens was C8-HSL.3-oxo-C6-HSL and C8-HSL were significantly inhibited by sub-MICs of hexanal(P<0.05)which showed a good dose-effect relationship,respectively(r = 0.9545,r = 0.9914).Particularly,1/2 MIC hexanal had a high inhibition rate for AHLs of E.carotovora and P.fluorescens,which were 76.27% and 60.78%,respectively.Under the inhibition of hexanal,different concentrations of AHLs were added exogenously for 24 h.There was a positive correlation between the concentration of exogenously added 3-oxo-C6-HSL and biofilm formation recovery of E.carotovora(r = 0.8866),and there was a positive correlation between the concentration of exogenously added C8-HSL and biofilm formation recovery of P.fluorescens(r = 0.9876).With the increase of 3-oxo-C6-HSL concentration,The activities of five extracellular enzymes in E.carotovora(pectate lyase,polygalacturonase,cellulase,xylanase and protease)were gradually restored and showed a positive correlation(r = 0.9398 ? 0.9945).After the addition of C8-HSL to P.fluorescens,protease activity was restored significantly and positively correlated(r = 0.9738),which indicated that the QSI effect of hexanal on biofilm may due to its inhibition of 3-oxo-C6-HSL and C8-HSL production.The QSI effect of hexanal on the activity of extracellular enzyme was associated with signal molecule of 3-oxo-C6-HSL and C8-HSL.AHLs production may be inhibited by hexanal by inhibiting the relative expression of ecbI and pcoI in E.carotovora and P.fluorescens.Furthermore,the effects of hexanal on the QS related indexes of these two spoilage bacteria were systematically analyzed,and it was found that they all had good correlation,indicating that the QSI effects of hexanal were mutually influential,and were all related to the suppression of signal molecules production in QS system.Therefore,this study was based on the anticorrosion effect of QSI,and provided basic data support for improving post-harvest preservation of vegetables. |
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