Study On Synergistic Spoilage Mechanism Of Pseudomonas Fluorescens And Shewanella Putrefaciens Based On AHLs-mediated Quorum Sensing System

Turbot?Scophthalmus maximus L.?is an important aquaculture fish in China with high economic value and good market prospects.And the spoilage potential of turbot caused by microorganisms is also very serious.Studies have shown that Pseudomonas fluorescens and Shewanella putrefaciens are specific spoilage bacteria in the process of low temperature storage of farmed fish.The spoilage bacteria's growth and characteristics are subjected to quorum sensing?QS?,in which N-acyl-homoserine lactones?AHLs?are the most common signal molecules in Gram-negative bacteria.Previous study had isolated P.fluorescens PF08 and S.putrefaciens SP01 from turbot,discovering that P.fluorescens could produce AHLs while S.putrefaciens couldn't.In view of this,the effects of different types of exogenous AHLs on the expression of QS signaling molecules in S.putrefaciens were compared and analyzed,and the effect of P.fluorescens on the AHL-QS system of S.putrefaciens was further studied.At the same time,through inoculating in sterile turbot fillets,the spoilage potential of S.putrefaciens and P.fluorescens with symbiosis was studied.The main results are as follows:1.The AHLs signaling molecules in S.putrefaciens were not detected by the biosensor method and Gas Chromatography-Mass Spectrometer?GC-MS?method.The results indicated that S.putrefaciens did not produce AHLs or its activity was weak.Furthermore,the quorum sensing regulatory genes and spoilage genes of S.putrefaciens were detected and verified,and the specific primers of luxR,csB and trxR genes were successfully designed and amplified.In addition,bioinformatics analysis of the luxR protein sequence showed that the protein has no signal peptide region,no transmembrane structure,and is a hydrophobic protein with typical structural characteristics of the luxR family of proteins.2.Changes of biofilm and motility before and after the exogenous addition of AHLs were used to study the utilization degree of different signal molecules by S.putrefaciens.The results showed that different types of exogenous AHLs signal molecules could promote the formation of biofilm and motility phenotypes of S.putrefaciens.In addition,the sensitivity of S.putrefaciens to the exogenous signaling molecules was explored by real-time PCR?RT-qPCR?and molecular docking techniques.The results showed that exogenous AHLs addition up-regulated the expression of luxR and trxR genes,while down-regulated the expression of the csB gene?except C₄-HSL?.The molecular docking results showed that the stronger binding ability between AHLs and the receptor protein luxR,the stronger the luxR gene expression ability.3.The addition of P.fluorescens had certain stimulated effects on the bacterial growth rate,proteinase activity,motility phenotypes and biofilm development of S.putrefaciens through the symbiosis P.fluorescens with S.putrefaciens.In addition,S.putrefaciens,which could not produce AHLs by themselves,could sense AHLs secreted by P.fluorescens resulting in a decrease in AHLs activity in the environment.RT-qPCR results showed that the addition of P.fluorescens and CS down-regulated the luxR genes expression of S.putrefaciens,while the CFS addition group up-regulated the luxR gene expression,which was 0.58 times of that of the control group.And the addition of P.fluorescens inhibited the expression of csB and trxR genes.In conclusion,the addition of P.fluorescens promoted the expression of the spoilage potential of S.putrefaciens.4.Through the changes of microorganisms indicator-total viable counts?TVC?and physical and chemical indicators,including TBA value,pH value and volatile components,the spoilage potential of P.fluorescens,S.putrefaciens and the mixed bacteria during refrigeration and partial freezing storage was investigated.The results showed that under refrigeration freezing storage conditions,the viable counts of P.fluorescens and S.putrefaciens in the co-culture group was significantly higher than that in the single inoculation group.At the same time,the TVC,TBA value and pH value of the co-culture group were higher than those of the single inoculation group.Compared with refrigeration freezing,partial freezing could better delay the increase of TVC,TBA value and pH value.Furthermore,the volatile components in the sterile fish pieces during refrigeration freezing storage were analyzed by GC-MS.The results showed that the alcohols and aldehydes and ketones were higher in fish and were the main volatile smells.After the inoculation treatment,the content of aldehydes and ketones and alcohols increased,and the poilage potential of co-cultivation of the bacteria group was more obvious,which may be due to the synergistic effect between the co-cultured strains,and then promote each other's growth and enhance their spoilage activity,and accelerate the corruption of sterile fillets.