Study On Quorum Sensing Of Hafnia Alvei By AHLs And Its Spoilage Ability Isolated From Scophthalmus Maximus

Fish is currently the main species of marine aquaculture in China,accounting for about 60%of the total output of aquaculture aquatic products.However,after the catch of aquatic products,a series of deterioration concerning the color,flavor,texture and nutrition ingredients will occur,or even food safety problems will take place with the action of enzyme,microbial and lipid oxidation.Microorganisms are the major reason of spoilage in aquatic products,and the expression of microbial growth and characteristics is controlled by quorum sensing system(QS),and N-acyl-homoserine lactones(AHLs)are the most common signal molecules in the induction system of Gram-negative bacteria.Most studies have focused on the isolation and identification of spoilage bacteria,the prediction of shelf life and analysis the ability of spoilage,little is known about the pathogenesis of microbial pathogenesis,especially spoilage mediated by QS and its pathogenicity.In this study,Hafnia alvei separated from spoilage turbot were used as test strain,and to study the influence of different environmental factors on AHLs secretion and biofilm formation.At the same time,the spoilage ability was studied when symbiosis with Pseudomonas fluorescens through inoculation sterile turbot fillets.This study aimed to clarify the spoilage mechanisms of fish caused by microorganism,to provide theoretical basis for the development of new seafood preservation techniques based on the targeted inhibition of the spoilage bacteria communication underlying the QS systems.1.Selective medium was used to separate and purify bacterial samples from refrigerated turbot stored at 4?,combining with VITEK-2 and 16S rRNA sequencing identification to build the phylogenetic tree.The biosensors CV026 and A136 were used to detect quorum sensing phenomenon of the bacteria,and the production of biofilms at different growth stages was studied,at the same time,the relationship between AHLs and biofilm formation was studied by adding exogenous signal molecules.The result showed that the strain was Hafnia alvei,and named Ha-01.The bacteria could produce AHLs,the amount of biofilm formation was positively correlated with incubation and reached a maximum at 72 h,followed by gradual stabilization.The addition of exogenous standard AHLs could promote the biofilm formation.2.The production of AHLs under different culture conditions(carbon source,NaCl concentration,pH,temperature and incubation time)of strain Ha-01 were examined by CV026 bio-sensors and gas chromatography-mass spectrometry(GC-MS).The results showed the maximum secretion of AHLs was 277.82 g/L at 16 h during the growth stages.When xylose was used as carbon source,the signal molecules production reached the highest,was 271.62?g/L,the different carbon source could influence activity of AHLs,it was:xylose>glucose>fructose>maltose>lactose>sucrose.Low concentration(0.5%-2%)of NaCl could enhance the AHLs activity of Ha-01,while C10-HSL produced only in the salt concentration was 1%,and the secreted of AHLs reached the peak when the concentration of NaCl was 1.5%,224.22 g/L.The ability of Ha-01 producing AHLs was reduced by alkalescent environment,the optimum pH of Ha-01 secreting AHLs was pH 6.0,AHLs secretion was 258.0 ?g/L,C6-HSL and C8-HSL as the main signal molecules.The ability of AHLs secretion of strain Ha-01 was strong at 4? and 37?,206.75?g/Land 237.65 ?g/L,respectively.In conclusion,the AHLs production of Hafnia alvei(Ha-01)was influenced by environmental factors.3.To investigate the process of biofilm formation of strain Ha-01,and to examine the influences of different culture conditions(carbon source,pH,NaCl concentration,adhesive material)on the biofilm formed by Ha-01 and its growth properties by microtiter-plate method,ultrasonic plate method and scanning electron microscopy(SEM),and also assessed the relationship between exogenous AHLs and the biofilm formation.The result indicated that the strains Ha-01 biofilm formation was closely related to incubation time.The biofilm formation ability of Ha-01 varied depending on carbon source,when xylose was used as carbon source,the ability reached the strongest level.The biofilm formation ability of Ha-01 was stronger under neutral culture.At a NaCl concentration was 2%,the biofilm production was the highest.The biofilm formation ability of Ha-01 on different material surfaces was deceased in the order of Al,Zn,and glass.The biofilm formation ability became stronger with the increase of exogenous C6-HSL.In conclusion,culture conditions could affect the biofilm formation of Ha-01,which could be regulated by AHLs.4.The microbial indices,physical and chemical indications(including the total number of colonies,TVB-N value,K value,WHC,Ca2+-ATPase,total sulfhydryl content and change of muscle tissue)of sterile turbot fillets which inoculated strains Hafnia alvei(Ha-01),Pseudomonas fluorescens(PF15),Ha-01 and PF15 mixed bacteria at 4? were measured.The result indicated that the total number of colonies,TVB-N value,K value of fillets which inoculated strains Ha-01,PF15 and mixed bacteria increased faster with the extension of storage time than the control group,and mixed bacteria group rised fastest;the WHC,Ca2+-ATPase,total sulfhydryl content decreased speed of inoculation groups was obviously faster than the control group,the mixed bacteria group decreased fastest;and the muscle tissue of inoculation groups could be clearly observed to produce degradation.The shelf life of turbot sterile fillets which inoculated strains Ha-01 and PF15 during 4? storage were 6-7 days;the control group was 15 days.In conclusion,Ha-01 and PF15 could cause turbot spoilage,when they were co-culture,they can produce symbiotic role during refrigerated storage.