Study On Inhibition Of Quorum Sensing Of Pseudomonas Fluorescens PF-08 In Chilled Turbot By Carvacrol And Its Preservation

Turbot is an important aquaculture fish in China with high economic value and good prospects for development.Related studies showed that the Quorum-sensing(QS)phenomenon was closely related to the deterioration of aquatic products.Therefore,a new strategy for the preservation of aquatic products is provided by blocking the quorum sensing system of the spoilage microorganism to regulate the expression of the putrid factor.Chromobacterium violaceum CV026 was used in this paper to detect the quorum sensing inhibitory activity.By Pseudomonas fluorescens PF-08 as the research object,the regulation of carvacrol on the in vitro characteristics of rot were studied by mycetin,biofilm,extracellular protease,cluster and motility index.Effects of strain PF-08 producing AHLs type and carvacrol on the yield of AHLs were analyzed by GC-MS.Inhibitory effect of carvacrol on rhlI/R and flagellar gene flgA in Pseudomonas fluorescens PF-08 was studied by using RT-qPCR technology.Then,the inhibitory mechanism of carvacrol on strain PF-08 QS system were studied by molecular docking technology.Finally,combination of quorum sensing inhibitors carvacrol and traditional antistaling agent protamine,to verify the preservation of chilled turbot fillet.Based on the interference quorum sensing,this paper provided a new idea for the preservation strategy of aquatic products.1.Effect of quorum sensing and biofilm formation inhibition by carvacrol were studied by chromobacteria CV026 detection strain.Meanwhile,the inhibitory effect of different concentrations of carvacrol on Pseudomonas fluorescens PF-08 proteolytic activity and bacterial migration were examined.The results showed that carvacrol could effectively reduce the yield of chromobacteria CV026 without effecting the growth of bacteria.When the concentration of carvacrol was 0.125μL/mL,the purple pigment production decreased 53.16%,inhibition rate of Pseudomonas fluorescens PF-08 biofilm formation was 65.02%.After joining the carvacrol,The surface layer of the biofilm was thinner and the gap increased.At the same time,the carvacrol also reduced the activity of proteolytic,swarming and swimming motility of in a concentration-dependent manner.Thus,the inhibitory activity of carvacrol on quorum sensing activity was obvious.2.The effect of carvacrol on Pseudomonas fluorescens PF-08 AHLs was detected by GC-MS.The results showed that strain PF-08 produced mainly two kinds of signal molecules,C4-HSL and C8-HSL respectively,the production of C4-HSL and C8-HSL decreased significantly when carvacrol was added.When the incubation time of the bacteria was 36 h,the yield of AHLs in the treatment group reduced 41.57%and 36.22%,respectively,compared with the control group.The the effect of carvacrol on QS system was explored by using RT-qPCR technology,results showed that carvacrol had obvious inhibitory effect on rhlI/R gene and flagella gene flgA.When the concentration of carvacrol was 0.031 μL/mL,the rhll gene rhlR gene decreased 47.88%and 35.39%,respectively.In addition,when the concentration of carvacrol was 0.125 μL/mL,the maximum inhibitory rate of flagellum gene flgA was 41.76%.Finally,the mechanism of carvacrol on strain PF-08 QS system was further analyzed by molecular docking software SYBYL-X 2.0.The results found that the docking results of carvacrol and RhlR protein(5.31)was better than the natural ligand of C4-HSL(4.09),showed that carvacrol could compete C4-HSL combined with RhlR protein.The specific mechanism was that AHLs analogues were formed,and AHLs could competed with the RhlR receptor protein to block the QS system.3.The combination of quorum sensing inhibitors carvacrol and conventional preservative protamine,and the effect of compound preservative on the chilled turbot fillets were studied.The results showed that the treatment group could effectively delay the deterioration of sensory quality of turbot fillets,and total viable counts,TVB-N value,TBA and K values were all lower than those of control group.The compound preservative treatment could keep the turbot fillet hardness and remain good texture features,reduce the formation of free water,enhance the binding power of fish to water,control the water distribution in fish,delay fish flavor deterioration,and has little effect on its color.Thus,carvacrol could be used as QSI applied in the refrigerated storage of turbot,and conventional perservative and QSI compound used in preservation of aquatic products has a certain feasibility.Carvacrol treatment group could extend the shelf-life about 2-3 d compared with the control group,and the carvacrol and protamine compound treatment group could prolong the shelf-life about 6-7 d.