| Quorum sensing (QS) system is a kind of information communication mechanism among bacterial cells, many specific functions of bacteria are mediated by QS system, such as bioluminescence, biofilm formation, toxins production, synthesis of extracellular enzyme. This paper aims to investigate the QS system and relatation of QS and spoilage in Shewanella baltica on Pseudosciaena crocea. The QS system in specific spoilage organism S. baltica from the Pseudosciaena crocea was investigated by using LC-MS/MS, GC-MS and reportor stains. The changes of quality deterioration and QS signal molecules at refrigerated storage were investigated, and QS on specific spoilage organism (SSO) was analyzed. Furthermore, tea polyphenols (TP) interfered with the QS-related spoilage of S. baltica was evaluated.To investigate the eleven kinds of quorum sensing signal molecules N-acylated-L-homoserine lactones (AHLs) secreted by6kinds of bacteria. AHLs were determined by biosensors C. violacceum CV026and A. tumefaciens A136. Furthermore, the method of LC-MS/MS detecting simultaneously the AHLs was developed. The results showed that two kinds of P. aeruginosa could secret AHLs by biosensors. Qualitative and quantitative analysis of11kinds of AHLs were attained by optimized LC-MS/MS parameters. High level of AHLs with3-oxo-Ci2-HSL were produced by P. aeruginosa. Low content of AHLs secreted by S. putrefaciens XH4were C4-HSL. However, low level of AHLs were detected in S. putrefaciens ATCC8071and S.baltica XH2.The GC-MS method was developed for the qunatifiation detection of the diketopiperazines (DKPs), by optimizing the chromatograph parameter, and the characteristic ion in scans mode. The results showed that calibration lines was prepared by using GC-MS-SIM (R2>0.998), the limit of detection were0.04to0.10mg·L-1, and quantification limit were0.12to0.18mg·L-1. The precision and accuracy were3.1%to10.2%and96.4%to107.8%, respectively. The recovery were among51.8%to88.5%, the RSD were1.4%to8.3%. The higher level of DKPs were detected, when CH3Cl served as extracting solvent and LB used as medium. The contents of DKPs secreted by P. aeruginosa and S. baltica were7.96and9.61mg·L-1, respectively. The main types of DKPs were cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe). The quality was deteriorated when Pseudosciaena crocea stored at4℃. The results indicated that microbial population in the refrigeratedfish significantly increased, and the5th day was the end of shelf-life. TheTVC, H2S producing-bacteria, CFC bacteria and VRGBA bacteriareached7.72,7.42,7.31and5.83lgcfu℉g-1respectively. The TVB-N andTMA were31.90mgN·100g-1and10.32mg·100g-1, respectively. TheAI-2activity was found in the initial storage of fish matrix, whichincreased with the growth of bacteria. Furthermore, production of AI-2and DKPs were observed in six Shewanella isolates. AI-2and DKPsactivities differed significantly among Shewanella strains, in which S.baltica XH2exhibited the highest peak of AI-2and DKPs on theexponential growth phase at9h and12h respectively. The anti-QS and spoilage inhibitory potentials of green TP against S.baltica were observed. TP at below inhibitory concentrations interferedwith AI-2and DKPs activity of S. baltica in the initial growth, especiallyAI-2signal molecular. The inhibition of AI-2by (0.25,0.50,0.75mg·mL-1) TP were27.7%to100%, the degradation rate were13.4%to75.2%.but no significantly effect on S. baltica at0.25mg·mL-1TP, lowinhibiton (13.2%and51.7%) was abserved by adding.50,0.75mg·mL-1TP.(revise) Moreover,(0.25,0.50,0.75mg·mL-1) TP inhibitedQS-associated biofilm development at a rate of20%to79%, inhibition of EPS with48%to88%Similarly, swimming motility was inhibited (87.7%to97.1%). And extracellular protease activity was totally inhibited with0.25mg·mL-1TP or more. The production of TMA in S. baltica in a concentration-dependent manner. Exposure to green TP was also found to be associated with down regulation of the transcriptions of tor A gene involved in the synthesis of TMAO reductase (inhibitory rate were17.4%to96.9%).The component of TP was analyzed by HPLC, the key components were gallic acid (GA), Epigallocatechin (EGC), Catechin (C), Epicatechin (EC), Epicatechin gallate (EGCG) and Epicatechin gallate (ECG), they were0.1%、10.2%、0.8%、5.2%、32.7%and9.3%, the EGCG was the highest. The inhibition of AI-2by EGCG at the concenration (1,5,10μg·mL-1) against S. baltica were2.9%-51.4%, degradation of AI-2were0%～23.7%. However, there was no effect on DKPs by EGCG.Our results indicated that high AI-2and DKPs activities were found rather than AHLs in the S. baltica. AI-2activity enhanced significantly after the quality deterioration in Pseudosciaena crocea stored at4. In vitro QS-inhibitory and anti-spoilage effects of green TP extract rich in catechins against a spoilage bacterium and the biofilm formation, motility, spoilage protease activity were significantly inhibited. It suggested that the spoilage factor of S.baltica could be mediated by QS system, which provided a new idea for resisting to spoilage in aquatic product. |
PDF Full Text Request