| Quorum sensing is a way to communicate by signaling molecules between bacteria.The spoilage of seafood is mainly caused by the bacterial growth,QS an important regulatory system in microorganism controlled by bacterial communication signals,acylated homoserine lactones(AHLs),has become a hot topic in life science field.However,most studies of QS focused on the virulence factors regulations in pathogenic bacteria.The roles of QS in seafood spoilage and the regulation mechanisms of QS in spoilage bacteria were still poorly elucidated.In view of above reasons,this paper took turbot(Scophthalmus maximus)as the research object,separated and identified spoilage bacterium-Aeromonas sobria in the process of refrigeration,then studied its secretion signal molecule dynamics and environmental factors on the impact of AHLs secretion,at the same time,the influence of AHLs on outside or inside spoilage ability by A.sobria was studied,this study aimed to clarify the spoilage mechanisms of fish caused by microorganism deeply,to provide theoretical basis for the development of new seafood preservation techniques based on the targeted inhibition of the spoilage bacterial communication underlying the QS systems.The main results obtained in this study were as follows:1.The selective medium was used to separate and purify bacterial samples from 4℃refrigerated turbot and get a single colony,combining with VITEK-2 and 16S rRNA sequencing identification to build the phylogenetic tree,at the same time the biosensors CV026 and A136 were used to detect quorum sensing phenomenon of the bacteria.The result showed that the strain was accurately Aeromonas sobria AS7,the bacterium could decompose CV026 to produce purple pigment and decompose A136 to react blue X-gal,this result indicated that strain AS7 could produce both short chain AHLs and long chain or acyl side chain of C-3 carbonyl instead of AHLs.2.The meteorological chromatography-mass spectrometry(GC-MS)was established,and signaling molecules standard qualitative and quantitative analysis by internal standard method bacteria was supplemented to measure concentration of signal molecules and the kinds of content in the crude extract of strain AS7.Results showed that the strain AS7 was detected including at least five AHLs,C4-HSL,C6-HSL,Cg-HSL,C10-HSL and C12-HSL,when using sucrose as carbon source,C8-HSL and C10-HSL were as the main signal molecules,theit concentrations were 144.7 and 515.9μg/L,separatedly.The concentrations of C4-HSL,C6-HSL and C12-HSL were 2.1,5.2 and 3.4 μg/L,respectively.3.GC-MS was used to research quantitatively the effect of environmental conditions(NaCl concentration,temperature,carbon source,pH)and the incubation time on the production of AHLs of strain AS7,the results showed that the types and contents of AHLs were obviously different under different conditions,the total AHLs secretion was at the maximum,reached 514.4 μg/L,C4-HSL and C6-HSL were the main signal molecules at 32h,C10-HSL disappeared after 20hThe total AHLs secretion were at 582.6 μg/L,C8-HSL and C10-HSL were also the main signal molecules at pH=8,,then a significant decrease at pH=9,signal molecules were unstable and easy to open-loop at alkaline environment,and then losed the role of signal molecules;the maximum total AHLs secretion was at 0.7%salt concentration,595.3 μg/L,C8-HSL and C10-HSL as the main signal molecules,when at 4%salt concentration,C4-HSL and C6-HSL signaling molecules disappeared;the capability of strain AS7 secreted AHLs to use the carbon source:sucrose>maltose>glucose>lactose>fructose>xylose,when xylose was as the carbon source,the signal molecules production reached the lowest,221.1 μg/L,at the same time,sucrose,glucose and maltose was as the carbon source,respectively,the signal molecule secretion was at peak,671.4,623.3 and 623.3 μg/L,respectively;C8-HSL and C10-HSL were also the advantage signaling molecules;at low temperature environment(4 and 10℃),the ability of AHLs secretion of strain AS7 was strong,the signal molecule secretion capacity was strong at 28 and 25℃,while the production of signal molecules was fell sharply at 37℃,Cg-HSL and C10-HSL as the main signal molecules,at 28 and 25℃ the amount of AHLs secretion was at peak,as 582.6 and 585.2 μg/L,respectively,at 4 and 10℃,the strain could also secrete AHLs at a higher level,but under the condition of low temperature,did not check out the C4-HSL and C6-HSL.4.The relationship between different treatment(the nutrition of substrate,symbiotic cultivation and adding different kind of AHLs)and the spoilage factors(siderophore,protase and biofilm)were further studied,the results showed that the secretion of siderophore was affected by medium quality,that could secret at natural substrate such as skim milk,while could not produce siderophore at synthetic medium;adding AHLs had a significant influence to the production of siderophore,C8-HSL affected significantly,the production of siderophore presented positive correlation with the increase of adding AHLs(P<0.05),but C6-HSL effect was not significant(P>0.05);the production of siderophore of Pseudomonas fluorescens and Shewanella putrefaciens was obvious promoted when symbiotic cultivation with AS7;adding AHLs could also promote the production of protase and biofilm,and to present the positive correlation(P<0.05),showed the AHLs could regulate the production of extracellular protease,siderophore and biofilm in AS 7.5.The regulation of quorum sensing to the spoilage ability of AS7 was studied by measuring the freshness indicators(pH,TVB-N,water binding capacity,K-value,total bacterial count)of turbot sterile fish blocks inoculated strains AS7 refrigerated at 4℃,contrasted to the samples adding different kinds of AHLs standard signal molecules,the results showed that adding C6-HSL had no significant impact on the spoilage ability of bacteria(P>0.05),and C4-HSL and C8-HSL respectively showed significant correlation in different freshness index,for the TVB-N had remarkable influences(P<0.01),the quorum sensing could regulate the spoilage ability of AS7 by AHLs. |
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