| Quorum sensing (QS) is a cell-to-cell communication mechanism used by bacteria to regulate their collective behaviors in a cell density-dependent manner. In recent years, an increasing number of empirical evidences demonstrate that bacterial food spoilage is regulated by quorum sensing (QS). The objective of this paper was to study the role of QS in the spoilage process of refrigerated Litopenaeus vannamei and the potential of QS inhibitors in shrimp preservation. Details and results of the work are shown as follows:1. Autoinducer activities in refrigerated Litopenaeus vannameiWe examined the presence of autoinducers in shrimp during refrigerated storage using bioreporter (.Agrobacterium tumefaciens A136, Vibrio harveyi BB170) assays, thin-layer chromatography and gas chromatography-mass spectrometry. The results revealed the presence of three types of autoinducers including acetylated homoserine lactones (AHLs) (i.e., N-hexanoyl-homoserine lactone, N-oxohexanoyl-homoserine lactone and N-octanoyl-homoserine lactone), autoinducer-2, and cyclic dipeptides (i.e., cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe)). Exogenous N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) shortened the lag time and increased the growth rates of bacteria in shrimp samples under refrigerated storage, while exogenous 4,5-dihydroxy-2,3-pentanedione did not. However, according to the levels of total volatile basic nitrogen (TVB-N) and the volatile organic components in the shrimp samples, exogenous 4,5-dihydroxy-2,3-pentanedione (DPD, the autoinducer-2 precursor) did not accelerate the shrimp spoilage process as N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) did. In summary, our results suggest that quorum sensing involves the spoilage of refrigerated Litopenaeus vannamei.2. Study on the specific spoilage organisms (SSO) of refrigerated Litopenaeus vannameiDominant spoilage bacteria of Litopenaeus vannamei stored at 4°C was determined by 16SrDNA. The results showed that the dominant bacteria in fresh shrimp were Aeromonas spp., Photobacterium spp., Shewanella spp., and Chromobacterium spp., while those at the end of shelf life were Shewanella spp., Pseudomonas spp., Acinetobacter spp., and Neisseria spp. Spoilage-bacteria-inoculated sterile Litopenaeus vannamei meat stored at 4°C was assayed to evaluate the spoilage ability of Shewanella spp., Pseudomonas spp., and Acinetobacter spp. The result showed that yield factors (YTVBN/CFU) of Shewanella spp., Pseudomonas spp., and Acinetobacter spp. was 26.4x10-9,13.7×10-9 and 8.9x10-9, respectively. The results indicated that the spoilage potential of Shewanella spp. was significantly higher than that of Pseudomonas spp., and Acinetobacter spp., so Shewanella spp. was regarded as the SSO of refrigerated Litopenaeus vannamei. Exogenous N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) shortened the lag time of total bacteria in shrimp samples inoculated with the SSO, while DPD displayed no significant effect. C6-HSL, cyclo-(L-Pro-L-Leu) and exogenous C6-HSL, cyclo-(L-Pro-L-Leu) and DPD significantly increased the percentage of SSO from 40.6±8.9% to 67.1±3.6%,72.5±9.9%, and 53.3±6.2% respectively at storage for 5 d.3. The quorum sensing system of SSOAutoinducer-2, rather than any AHL, was detected in extracts from pure cultures of the SSO, i.e., Shewanella putrefaciens (SS01) and Shewanella baltica (SA02). As for the cyclic peptides, only SA02 was determined to produce cyclo-(L-Pro-L-Leu). According to the transcription levels of LuxR (the master quorum-sensing regulator) in the SSO in response to exogenous autoinducers, the SSO could sense AHLs and cyclo-(L-Leu-L-Leu), rather than autoinducer-2, cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe). In accordance with the results of LuxR expression, the production of biofilm matrixes and extracellular proteases in the SSO was regulated by exogenous AHLs and cyclo-(L-Pro-L-Leu), rather than DPD, cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe). Exogenous N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) increased the growth rates and population percentages of the SSO in shrimp samples under refrigerated storage, and interestingly, exogenous DPD also increased the population percentages of the SSO in vivo by inhibiting the growth of the competing bacteria.4. Effects of Acyl-homoserine lactone-dependent eavesdropping on the metabolism and spoilage potential of SA02 and Acinetobacter sp. (ACT)SA02 and ACI are the domain spoilage bacteria of refrigerated Litopenaeus vannamei. SA02 could eavesdrop C4-HSL, O-C6-HSL and O-C8-HSL produced by ACI. The cell density of SA02 was enhanced by 40-fold when it is cocultured with ACI in 100-fold diluted tryptic soy broth medium. The confocal laser scanning microscopy and spectrophotometric analysis revealed that SA02 regulated the biofilm formation of ACI via modulating the concentrations of remained AHLs in the environment. The spoilage of shrimp samples co-inoculated with SA02 and ACI was accelerated compared to samples mono-inoculated with either of the strains. There were richer and more diverse off-odours, higher TVB-N levels, and higer total bacterial counts in the co-inoculated samples, so eavesdropping of AHLs by SA02 seemed promoted the spoilage potential of both strains.5. Effects of cinnamaldehyde on the QS and spoilage potential of SA02 and ACISubinhibitory concentrations of cinnamaldehyde could inhibit the AHLs activity of ACI. According to the results of real-time quantitative polymerase chain reaction (RT-qPCR), cinnamaldehyde showed no effects on the luxR expression of SA02. The AHL inhibitory effect was directly proportional to the concentrations of cinnamaldehyde. Cinnamaldehyde could significantly decrease the AHL activity in shrimp samples co-inoculated with SA02 and ACI, which indicated cinnamaldehyde was able of blocking the eavesdropping of AHLs by SA02. Cinnamaldehyde also reduced the levels of total bacteria count and total volatile basic nitrogen in shrimp samples co-inoculated with SA02 and ACI, and prolonged the shelf life of the samples. |
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