The Isolation And Identification Of Shewanellabaltica And The Mechanism Study Of Differences Of Spoilage Potential In Shewanellas From Large Yellow Croaker

Large yellow croaker had rich nutrition, higher pH values and higher humidity, they were extremely spoilaged in the post-harvest storage and transportation process. There were studies have shown that the aquatic products of temperate waters specific spoilage organism were S. putrefaciens and P. aeruginosa. At present, the scholars of China on the SSO’s research mainly focused on the identification and aspects of shelf life prediction, as well as the research of spoilage potential on SSO was still not deep enough. So this paper would use that traditional identification combined with the16S rRNA method to screen and identify the SSO, select the Shewanella with different spoilage potential and from the respect of trimethylamine oxide reductase gene to study the difference of spoilage potential of Shewanella.1. H2S-producing bacteria isolated from the refrigerated large yellow croaker were identified using the physicochemical reactions and16s rRNA.22strains with differences spoilage potential were selected in the sterile fish juice and fish block. The results showed that all H2S-producing bacteria were identified to Shewanella, in which S. baltica, S. putrefaciens and S.hafniensis accounted for59.4%,36.1%and4.5%, respectively. Significant differences of the spoilage potential were in fish juice among Shewanella observed. The sensory score and TVB-N content of isolates S. baltica XH2and XH8were significantly higher than other strains (p<0.05), whereas the indicators of isolates S. putrefaciens XH14and S. baltica XH17were the lowest. The sample inoculated S. baltica XH2was found to produce off-odors after72h, higher than107cfu/g of TVC after48h, strong reduction ability of TMAO to TMA, high accumulation of TVB-N, cadaverine and putrescin, followed by S. baltica XH8, S. putrefaciens XH14and S. baltica XH17.2. The optimum temperature offour bacteria was25℃, they grew well at salt of0.3%, grew slowly at salt of3%and they had a tolerance to salt. The capacity TMAO reduction of XH2was0.174μg/100mL higher than XH17, the ability of form biofilms and the extracellular protease activity of XH2was41%and13.7%more than XH17respective, and the content of AI-2and DKPs signaling molecules of XH2was27.5times and2.2times than XH17respective. The results shew that there was a relationship between spoilage potential and these traits (TMAO reduction, the ability of form biofilms, the extracellular protease activity) of Shewanella, quorum sensing may be involved in regulation of spoilage potential.3. The2490bp fragment of tor A gene which coded TMAO reductase were amplified in four Shewanella isolates.The homology of the four sequence was98%and the expression of tor A gene of four isolates was positive correlated with the spoilage potential, in which S.baltica XH2exhibited the highest expression. The Tor A protein of four strains were secretory protein, there were difference between theoretical pI, the aliphatic index, gravy, the content of amino acid and other physical and chemical properties, but advanced structure of proteins are similar. Furthermore, the biggest molecular weight and aliphatic index of predicted TorA protein were observed, whereas theoretical pI and gravy were the lowest in S, baltica XH2. TMAO reductase gene cluster sequences from the specific spoilage S. baltica XH2was amplied, four bright bands were amplied, sequenced, spliced and analysed. The length of TMAO reductase gene cluster was9711bp. Bioinformatics analysis revealed that the sequence contained TMAO reductase promoter torECAD, and regulation system torRTS with7open reading frames (ORFs). The seven ORFs were torE, torC, torA, torD, torS, torT and torR, conresponding to nitrate reductase, C-type cytochrome, TMAO reductase, chaperones sensors, periplasmic proteins and response regulator respectiely. BLASTn analysis showed that the sequence of7ORFs had93%,99%,99%,99%,96%,97%,98%similarities to that of S. baltica OS185TMAO reductase gene cluster.The expression of torA gene from Shewanella with different spoilage potential were different which may be caused by differences of the regulation sequence of TMAO reductase gene cluster.