| Large yellow croaker is a high-quality source of animal protein which is highly susceptible to spoilage during storage.Low temperature is an important factor limiting microbial growth and plays a vital role in maintaining the quality of aquatic products.With the development of cold chain logistics,the cold chain transportation of aquatic products has been realized,at the same time,the spoilage potential of psychrotrophic bacteria has gradually become prominent.The psychrotrophic bacteria can be the dominant bacteria in aquatic products since the high growth rate at low temperatures.Studies have shown that Shewanella baltica is a specific spoilage organism(SSO)in refrigerated large yellow croaker.As a typical psychrotrophic bacteria,research of the correlation between its cold-adaptation mechanism and spoilage potential is of great significance for the preservation of refrigerated large yellow croaker.In this study,we used S.baltica isolated and identified in refrigerated large yellow croaker as the research object,and screened strains representative of cold-adaptation and spoilage ability.In view of differential proteomics analysis of strains at different culture temperatures,the differential proteins related to the synthesis of unsaturated fatty acids(UFA)were concerned;then the key regulatory proteins were overexpressed to explore the relationship of S.baltica cold-adaptation mechanism and spoilage potential.The main results are as follows:The yields of total volatile basic nitrogen(TVB-N)and trimethylamine(TMA)in 8 different S.baltica in 4 ? sterile fish juice were determined.The significant difference of TVB-N and TMA accumulation in the fish juice inoculated with different strains was found which indicated the difference of the spoilage ability of different strains.Among them,S.baltica 12 and 73 were the weakest and strongest strains,respectively.The differences in membrane fluidity and fatty acid composition of 8 S.baltica at 4 ? and 30 ? were determined by fluorescence polarization and gas chromatography-mass spectrometry(GC-MS).This study found that the fluorescence anisotropy of S.baltica 12 cultured at 4 ?.is 1.42-fold than at 30 ?,while S.baltica 73 is substantially the same.Compared with the variation range of different strains,the variation of S.baltica 12 was the largest,while S.baltica 73 was the smallest,and it was significantly different from other strains(P<0.05).Microviscosity has the same trend of change.Analysis of cell membrane fatty acid unsaturation revealed that S.baltica 73 increased the UFA content by 12%,while S.baltica 12 increased by only 2%.The content of palmitoleic acid(C16:1)which the highest proportion in UFA was also increased by 17%and 7%,respectively,which is consistent with the results of fluidity.The results showed that S.baltica 73 had the strongest cold-adaptation,while S.baltica 12 was the weakest.It is speculated from the above results that S.baltica has a positive correlation between cold-adaptation and spoilage ability.The proteomics analysis of the strains cultured at different temperatures showed that the proteins expressed at 4 ? was significantly different from that at 30 ?.A total of 651 differential proteins were identified by TMT,among which 262 were up-regulated and 389 were down-regulated.They affect many metabolic pathways in S.baltica such as nucleic acid metabolism,amino acid metabolism,flagella assembly,bacterial chemotaxis,and participate in many biological processes such as biological metabolism,cell growth,and biological regulation,and undertake some molecular functions such as nucleic acid binding and catalytic activity.A variety of UFA synthesis-related proteins such as iron-containing desaturase Des and 3-hydroxyalkyl-ACP dehydratase FabA were significantly up-regulated,promoting the synthesis of UFA in S.baltica,and improving the cell membrane fluidity of S.baltica,thereby the cold-adaptation ability were enhanced.The FadR was significantly up-regulated by 1.5-fold and the FabR was significantly down-regulated by 0.62-fold,which were the important regulatory proteins in UFA synthesis.There was no significant change in the protein involved in trimethylamine synthesis.The results of real-time PCR showed that the expression levels of UFA and TMA-associated genes were consistent with the corresponding proteins by quantitative proteomic,indicating that the proteomic results were reliable.The FabR and FadR overexpression strains of S.baltica 12,73 were constructed,and the results of spoilage and cold-adaptation phenotype indicate that the ability to produce TVB-N,TMA,membrane fluidity and UFA content significantly decreased in FabR overexpression strains compared with the original strain,indicating that FabR is is a negative regulator of c cold-adaptation.On the contrary,both spoilage and cold-adaptation phenotype were significantly up-regulated in the overexpression strains of FadR,which showed that FadR plays a negative regulatory role in the spoilage potential and cold-adaptation.The results of real-time PCR showed that FabR and FadR inhibited and promoted the expression of TMA and UFA synthesis related genes,respectively,which was consistent with the phenotypic results.The variation of TMA synthesis related genes was small,probably due to FabR and FadR changed the membrane fluidity which influenced the cell exchange rate to inhibit or promote the expression of TMA synthesis related genes and phenotypic changes.In summary,the cold-adaptation and spoilage potential showed a positive correlation in S.baltica.FadR plays a positive regulatory role 'in cold-adaptation.At 4 ?,FadR can promote the expression of UFA synthase Des and FabA to increase cell membrane fluidity and spoilage potential of strains.While,FabR negatively regulates the cold-adaptation,its expression inhibits the expression of UFA synthase,weakens cell membrane fluidity,and reduces the spoilage potential of strains.At 4 ?,S.baltica promoted the cold-adaptation by up-regulating and down-regulating the expression of FadR and FabR,which are the regulators of the UFA synthesis pathways,respectively. |
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