Idendification Of Quorum Sensing Signal Molecules From Sturgeon Specific Spoilage Organism And Biological Function Of AHLs In Regulating Spoilage

The specific spoilage bacteria of vacuum packed strugen stored at 4℃ and its quorum sensing signals AHLs were investigated,and the spoilage phenotype and spoilage mechanism regulated by AHLs were also studied.The results of this study will further our understanding of how QS regulate sturgeon spoilage and provide theoretical foundation for developing novel sturgeon preservative technology for maintaining sturgeon freshness by disturbing QS.The main research contents and results were summarized as follows:(1)Sturgeon was vacuum packed and stored at 4℃.Sturgeon muscle was analyzed to determine quality changes.The results showed that putrescine,cadaverine and Hx can be good biomarkers for quality assessment of vacuum-packed sturgeon stored at 4℃.Psychrotrophs,Enterobacteriaceae and Aeromonas were identified as the dominant spoilage bacteria.The spoilage abilities of the dominant spoilage bacteria were further studied,and Aeromonasveronii LP-11.Citrbactocer freundii LPJ-2 and Raoultella ornithinolytica LPC-3 were identified as the specific spoilage organism(SSO).(2)Agrobacterium tumefaciens KYC55 and Chromobacterium violaceum CV026 were used as test bacteria for AHLs production.As a sturgeon SSO,A.veronii LP-11 could produce AHLs detected by both A.tumefaciens KYC55 and C.violaceum CV026.When further analyzed by thin-layer chromatography-biosensor,high-performance liquid chromatography/ion trap mass spectrometry,High-performance liquid chromatography triple quadrupole tandem mass spectrometry and High-performance liquid chromatographyquadrupole time-of-flight mass spectrometry,it was found that A.veronii LP-11 could produce four different AHLs:C6-HSL,3-oxo-C8-HSL,3-OH-C8-HSL and C8-HSL.(3)The changes of phenotypes related to spoilage and proteins of A.veronii LP-11 were analyzed when its QS system was disturbed by quorum quenching(QQ)enzyme.The results showed that the growth of bacteria was not changed,while protease production,motility and adhesion to colon adenocarcinoma cell line HT-29 were reduced.SDS-PAGE analyses showed that intracellular and extracellular proteins bands of A.veronii LP-11 were different markedly.Proteome and qRT-PCR analyses showed that the transport ability for carhonhydrate,amino acid and other nutrition substances was reduced,carbon and nitrogen metabolism was inhibited,protein synthesis ability was reduced and secretion of extracellular enzymes were inhibited in QS disturbed cells,leading to the metabolic activity decrease in QS disturbed cell;the metabolism of tricarboxylic acid cycle was enhanced and ATP output was increased,which would provide energy and intermediate metabolites for metabolisms proceeding in QS disturbed cells,thus it was benefit for maintaining bacteria growth;selective transcription and translation were induced in QS disturbed cell,which would regulate part biological function in QS disturbed bacteria;the cell wall synthesis was enhanced and environmental adaptability was decreased in QS disturbed cells.In conclusion,the growth of A.veronii LP-11 was not regulated by QS,while the biological function of spoilage was regulated by QS by changing the ability of absorption and utilization of nutrient,extracellular enzymes activities and metabolic activity.(4)The effect of QQ enzyme and QQ enzyme combined with nisin on the quality of vacuum packed sturgeon stored at 4℃ were analyzed by determining microbiology,biochemical and sensory changes.The results showed that 6.6 U/mL QQ enzyme could suppress the growth of microflora,including total viable counts,Aeromonas,Psychrotrophs and Enterobacteriaceae during storage.Meanwhile,the increase of TVB-N and biogenic amine levels were significantly delayed in 6.6 U/mL QQ enzyme treated samples compared to the control samples.6.6 U/mL QQ enzyme or 2000 IU/mL nisin extended the sturgeon fillets’ shelf life to 3 days.Compared with QQ enzyme or nisin independently,the effect of delaying sturgeon spoilage was better for the combination of QQ enzyme and nisin,6.6 U/mL QQ enzyme combined with 2000 IU/mL nisin extended the sturgeon fillets’ shelf life to 5 days.