The Effect And Mechanism Of Autoinducer AHLs In Regulating Spoilage Ability Of Shewanella Baltica Isolated From Large Yellow Croaker

Microorganisms are the major cause of spoilage of most seafood products,particularly,large yellow croaker were extremely easy to spoil because of the high content of soluble protein and moisture,and the incompact structure.Pseudomonas spp.and Shewanella spp.are recognized as the specific spoilage organisms(SSOs)of iced marine fish and freshwater fish,respectively.The increasing evidence implicated that quorum sensing(QS)could be involved in bacterial food spoilage.N-acyl homoserine lactone(AHL)family in Gram-negative bacteria have been the most intensively studied autoinducers of QS signals,however,few reports have recently been published on the identification of AHLs and the regulatory molecular pathways of QS in the spoilage potential of Shewanella baltica.Therefore,In this article,we developed a reliable,rapid and sensitive analytical method to identify and quantify AHLs with an ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry(UPLC-QqQ-MS/MS).In this work we investigated the different spoilage ability and the function of exogenous supplement of synthesized AHLs in different S.baltica strains.This work also studied the regulatory molecular pathways of QS signals and the expression of spoilage related genes induced by exogenous AHLs.The main results are as follows:1.We developed a new method to determine AHLs accurately by UPLC-QqQ-MS/MS,which allows the quantification of all the 11 AHLs known as autoinducers within 5.6 min with excellent linearity(r2>0.99).AHLs were extracted thrice with acidified ethyl acetate(0.1%v/v formic acid)and the recovery was more than 88.4%.This method was applied to S.baltica and 9 out of 11 AHLs were detected and quantified which was never detected in that species before.Since C10-HSL was found in most of the strains with a relative higher concentration compared to other AHLs,we concluded it to be the most prominent AHL in S.baltica.The production of AHLs vary in kind,quantity and growth phase in different S.baltica strains.2.The addition of synthesized AHLs had no effect on the bacterial growth rate and quantity,but promoted biofilm development,TVB-N production and proteolytic activity.C10-HSL treated group grew denser everywhere and formed a homogenous layer with thicker biofilms,which significantly enhanced biofilm formation.The existence of carbonyl groups in AHL didn't show significant effect on biofilm formation.Different AHL analogues were responsible for the biofilm formation in different grow phases.Exogenous AHLs had significant effect on the production of TVB-N in both the highest and lowest spoilage potential S.baltica strains.TVB-N production was markedly increased when treated with C8-HSL,oxo-C8-HSL,C10-HSL and oxo-C0-HSL at 4? for 1d,but had no effect when extended to 4 days.C8-HSL and oxo-C12-HSL treated groups significantly enlarged the diameter of transparent zone,indicating the improvement of protease activity,while C7-HSL and C14-HSL treated groups presented opposite change.However,there was no direct relation between the change of spoilage potential induced by exogenous AHLs and the detected QS signals in same S.baltica strains.3.In this study,we investigated the AI-2 activity of S.baltica using harveyi BB170 strains bioassay,which showed that all S.baltica strains do produce AI-2.AI-2 activity peaked in the late exponential phase and greatly decreased during the stationary phase.qPCR results demonstrated that the transcriptional profile of pfs but not luxS was highly correlated with the level of AI-2 production.The transcriptional profile of pfs partly attributed to AI-2 activity,as AI-2 activity was regulated by the concentration of luxS substrate.This study also found that the maximum transcriptional level of luxR occurred at the mid to late exponential phase,consistent with the concentration of AHLs.Exogenous AHLs with a carbon chain length of 10 C or less greatly promoted the transcription of luxR.The spoilage potential of S.baltica was highly correlated with the transcriptional profile of torT and tor A,similarly to the production of TVB-N induced by the same AHLs.