| Microorganisms are the major cause of spoilage of most seafood products. However, only a few members of the microbial community, the specific spoilage organisms (SSOs), give rise to the offensive off-flavours and toxic compounds associated with seafood spoilage. During the periods of storage, SSOs often vary according to the different conditions. Generally, Shewanella and Pseudomonas spp. are the two major SSOs of fish stored at low-temperature. Shewanella species are Gram-negative, H2S-producing and motile rods which have risen more attention in recent years because of their important roles in spoilage of fish and fish products.Quorum-sensing (QS) signaling molecules are able to mediate specific gene expression in spoilage bacteria in response to population density, and thus implicated in food spoilage. The aim of this work was to characterize the SSO from Pseudosciaena crocea at low-temperature (4℃) storage, and evaluate the spoilage capabilities of individual SSO strains. Furthermore, the QS signal molecules were identified from the SSO using GC-MS method.This study included the following three parts:1. A total of102strains of spoilage microorganisms were isolated from cultured Pseudosciaena crocea stored in air at4℃, and of these,74strains (72.5%) were characterized as H2S-producing bacteria. After conventional phenotypic and 16S rDNA sequence analysis,60of the74strains (81.1%) were identified as Shewanella spp., and48of the strains (64.9%) were identified as S. baltica. A small portion of the14remaining strains were assigned to Aeromonas and Enterobacteriaceae. Thus, the main SSO of cultured Pseudosciaena crocea stored at low-temperature was considered as S. baltica with strain-specific spoilage capability.2. Forty eight different S. baltica strains were respectively inoculated into sterile fish juice in order to evaluate the spoilage capabilities which were determined by total volatile base nitrogen (TVBN) and sensory analysis (off-odours). All the above results showed that the spoilage potential of S. baltica025was much higher than that of all the other strains, and the spoilage potential of S. baltica003was the lowest, indicating the same S. baltica species may exhibit different spoilage capabilities during the storage time of cultured Pseudosciaena crocea. A follow-up study was needed to carry on whole genome sequencing of S. baltica003and S. baltica025in order to characterize their genomic differences in marine seafood spoilage. By16S rDNA sequence analysis, S. baltica003and S. baltica025were identified as S. baltica OS223and S. baltica OS185, respectively. By brief analysis of whole genomic of these two S. baltica isolates, we found that the genes encoding trimethylamine-N-oxide reductase, cysteine synthase and biogenic amines synthase showed some differences and mutants.3. Four cyclic dipeptides (Diketopiperazines, DKPs) functioned as QS signal molecules were isolated and characterized from the extracellular metabolites of S. baltica025which had the strongest spoilage activity by gas chromatography mass spectrometry (GC-MS). By supplementation of four synthesized DKPs, the spoilage capability of S. baltica could be significantly enhanced. The TVB-N values in groups treated with synthetic DKPs (cyclo-(L-Pro-L-Gly), cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe)) were all significantly higher (p<0.001) than that of the control group after5-day storage of fish muscle juice. The activity for enhancement of spoilage capability in the cyclo-(L-Pro-L-Phe) treated group was the highest, while that in the cyclo-(L-Pro-L-Leu) treated group was the lowest. After24-h storage, the TVB-N value in the cyclo-(L-Pro-L-Phe) treated group was significantly higher (p<0.001) than that in the other DKPs treated groups. So far, this was the first attempt to characterize the DKPs as the signaling molecules in QS of S. baltica. Our study may provide some evidence on the role of DKPs involved in microbial spoilage. |
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