Research On Quorum Sensing AHLs Of Pseudomonas Fluorescens With Aeromonas Hydrophila In Co-culture System

Aquatic products are rich in nutrients and proteins.If they are not effectively treated,they will undergo a series of changes in color,flavor,texture and nutrients.And they even cause food safety problems with the action of enzymes,microorganisms and fat oxidation.Microbial action is the main reason that causes spoilage of aquatic products,and the growth as well as characterization of microorganisms are regulated by quorum sensing(QS).N-acyl-homoserine lactones(AHLs)are typical signal molecules in the gram-negative QS system.Due to the complexity of microbial species in foods,the interactions between multi-bacterial species are closely related to quorum sensing.However,little research has been conducted on quorum sensing in the two strains coexisting system.In view of above reasons,The spoilage turbot-derived Pseudomonas aeruginosa and Aeromonas hydrophila were studiedin this paper.To investigate the effects of Aeromonas hydrophila on the growth of Pseudomonas fluorescens,the secretion of AHLs and the spoilage effect under different conditions were tested.At the same time,the spoilage ability of Pseudomonas fluorescens and Aeromonas hydrophila was investigated by inoculation on sterile turbot.This study aimed to reveal the pathogenic mechanism of microorganisms induced by the microorganism,which provided the theoretical basis for the preservation of aquatic products based on the control of spoilage bacteria.The main conclusions were shown as follows:1.The meteorological chromatography-mass spectrometry(GC-MS)was established to measure qualitatively the kinds of signaling molecules included in the crude extract of strain Pseudomonas fluorescens(PF-04)and Aeromonas hydrophila(AH-11).Results showed that the signaling molecules of C4-HSL and C8-HSL were detected in the strain PF-04,and the signaling molecules of C4-HSL,C6-HSL and C8-HSL could be detected the strain AH-11.Under co-culture conditions,Aeromonas hydrophila AH-11 could promote the secretion of Pseudomonas fluorescens PF-04 signaling molecules;under pure culture conditions.The ability of P.fluorescens PF-04 to use carbon sources to secrete AHLs was:sucrose>maltose>lactose>glucose>fructose>xylose;The ability of A.hydrophila AH-11 to use carbon sources to secrete AHLs was:maltose>glucose>sucrose>fructose>lactose>xylose.The ability of PF-04 co-culture with AH-11 to use carbon sources to secrete AHLs were:maltose>sucrose>glucose>fructose>lactose>xylose.Tryptone was the best nitrogen source for pure culture and co-culture of strains PF-04 and AH-11.Urea was not the ideal nitrogen source for both strains to grow and secrete AHLs.Under pure culture conditions,the ability of strain PF-04 to utilize nitrogen sources was:tryptone>yeast extract powder>ammonium chloride>beef extract>urea;the ability of strain AH-11 to utilize nitrogen source was:tryptone>ammonium chloride>yeast extract>beef extract>urea.Under co-culture conditions,the ability of strain AH-11 to utilize nitrogen source was the same as pure culture conditions.The availability of AHLs could be affected by pH.AHLs secretion of strain PF-04 reached the maximum at pH 7.0,and AHLs secretion of strain AH-11 reached the maximum at pH 8.0.The strains were more favorable for the secretion of AHLs under the condition of neutral acidification.2.The biofilm formation was studied by crystal violet staining and observed by scanning electron microscopy.Aeromonas hydrophila AH-11 cells and AHLs could promote the formation of Pseudomonas fluorescens PF-04 biofilm,and the secretion of AH-11 signaling molecules could obviously promote the biofilm formation of PF-04.The results of further verification using standard foreign signal molecules were the same.Aeromonas hydrophila AH-11 enhanced the clustering,migration and rubbing ability of Pseudomonas fluorescens PF-04 by detecting the motility of the strains.Further showed that there was a close relationship between motility and bacterial biofilm.3.The production of extracellular protease was studied by defatted milk plate method.The production of extra-ferritin was assayed by CASAD method.The strain was inoculated into myofibrillar protein and sarcoplasmic protein extract,SDS-PAGE method was used to explore the different microbial system of muscle protein degradation.The results showed that both Pseudomonas fluorescens PF-04 and Aeromonas hydrophila AH-11 could secrete extracellular proteases,and the ability of Pseudomonas fluorescens PF-04 to secrete extracellular proteolytic enzymes was slightly stronger than that of Aeromonas hydrophila AH-11.The strain AH-11 also promoted the secretion of strain PF-04 extracellular proteolytic enzyme when co-cultured.The addition of exogenous signal molecule standard C4-HSL increased the Pseudomonas fluorescens proteolytic circle in a concentration-dependent manner.The addition of AHLs affected the production of Pseudomonas fluorescens PF-04,and the effect of C4-HSL was significant.With the addition of AHLs,there was a positive correlation between the addition of AHLs.However,the addition of C8-HSL had no significant effect on the secretion of magnetophils.When co-cultured,Aeromonas hydrophila AH-11 could significantly promote the production of siderophore by P.fluorescens PF-04.Both Pseudomonas fluorescens and Aeromonas hydrophila suspension could degrade myofibrillar protein to a certain extent,and the signal molecules could accelerate the degradation of myofibrillar protein.Different bacterial suspensions mainly promote muscle 180 kDa-25kDa fibroblast degradation.Pseudomonas fluorescens and Aeromonas hydrophila could degrade sarcoplasmic protein,in particular,they could promote sarcoplasmic protein degradation between 63kDa-35kDa.However,there was no significant difference in the ability to degrade sarcoplasmic protein between different bacterial suspensions.4.Pseudomonas fluorescens PF-04 and Aeromonas hydrophila AH-11,as well as the two mixed bacteria were inoculated into the turbot fish,the deterierated potential of co-culture system of two strains was studied by detecting the changes of quality index during storage,including the total viable counts,TVB-N,pH,TBA,K value,water holding capacity,Ca2+-ATPase activity and total sulfhydryl content.The results showed that total viable counts,TVB-N,TBA and K value in each bacteria-inoculation group were significantly higher than those of the control group when the storage time was prolonged at 4℃.The rate of increase of inoculation mixed bacteria group was higher than inoculated single bacteria group.The water-holding capacity,Ca2+-ATPase activity and the total sulfhydryl content of all the inoculation groups also decreased faster than that of the control group,of which the mixed inoculation group decreased most rapidly.The indexes of the combination groups showed that both Pseudomonas fluorescens PF-04 and Aeromonas hydrophila AH-11 could cause spoilage of turbot,and the speed of spoilage could be accelerated by mixing the two strains.