The spoilage potential of Shewanella putrefaciens and S. baltica isolated from spoiled refrigerated Litopenaeus vannamei was evaluated by in vitro assays for trimethylamine oxide reduction, extracellular hydrolytic enzymes and biofilm formation, and in vivo inoculation into surface-sterilised shrimp followed by microbial, biochemical and sensory analyses during storage for 5 days at 4°C. S. baltica displayed higher spoilage potential than S. putrefaciens both in vitro and in vivo. Shrimp co-inoculated with them had one-day shorter shelf life than those mono-inoculated, based on the results of bacterial density, volatile base nitrogen, trimethylamine, volatile organic compounds and sensory analysis, which strongly suggests cooperation of Shewanella species in shrimp spoilage. Exogenous cyclo-(L-Pro-L-Leu) boosted bacterial growth, extracellular protease and collagenase activities, and biofilm formation of S. putrefaciens and S. baltica at least before they entered the stationary phase, indicating that cyclo-(L-Pro-L-Leu)-dependent quorum sensing, a recently suggested communication mechanism between them, contributes to the cooperation. ). We previously discovered three types of autoinducers, i.e. AHLs, AI-2 and DKPs, in refrigerated Litopenaeus vannamei; nevertheless, QS circuits based on AHLs and AI-2 for the SSO (i.e. S. baltica and S. putrefaciens) have been found to be incomplete (Zhu et al., 2015). The SSO can sense AHLs, but are incapable of producing these molecules. The exact reverse is the case for AI-2. Cyclo-(L-Pro-L-Leu), the most abundant DKP detected in refrigerated shrimp, can be secreted by S. baltica, and responded by both S. baltica and S. putrefaciens in quite distinct ways. Cyclo-(L-Pro-L-Leu)-dependent QS thus seems to be the interspecies communication mechanism between S. baltica and S. putrefaciens in refrigerated Litopenaeus vannamei. The current research evaluated cooperation of the SSO in shrimp spoilage by inoculating S. putrefaciens and S. baltica into surface-sterilised shrimp, and to examine contribution of QS-based communication to the cooperation, the effects of exogenous cyclo-(L-Pro-L-Leu) on the spoilage potential of S. putrefaciens and S. baltica were also determined.
Materials and methodsCooperation and QS in shrimp spoilage S. Zhu et al. Figure 1 (a) TVB-N and (b) TMA levels in shrimp with or without inoculation.S. putrefaciens + S. balticarepresents co-inoculationat the CFU ratio of 1. The results are presented as the mean AE standard deviation (n = 3). Different letters indicate significant differences in mean values (P < 0.05).