The AHLs Quorum Signal Secretion And The Curcumin Liposome Inhibition Effect Of The Specific Psychrotroph Spoilage Bacteria In Grass Carp

Grass carp(Carpere gramen)is one of the main aquaculture species in China freshwater fish farming industry.Due to its rich protein,soft meat and high moisture content,grass carp has caused a lot of corruption during low temperature transportation and fish processing,resulting in huge economic losses.Although grass carp has a variety of microorganisms involved in the spoilage process,only one or two spoilage bacteria destroy fish spoilage.This spoilage bacteria is called specific spoilage organic(SSO),and research shows that it inhibits specific spoilage bacteria.The ability to spoil can effectively extend the shelf life of grass carp.Several studies at home and abroad have shown that food-specific spoilage bacteria can produce N-acyl-L-homoserine lactones(AHLs)to regulate the growth rate and spoilage ability,so the specific spoilage bacteria that produce AHLs in grass carp are selected And to study its decay and group sensing ability,by inhibiting its group sensing ability to extend the shelf life of grass carp is a new and effective method.In this paper,by analyzing the quality changes of grass carp at different temperatures and the structure of colonies at the spoilage stage,the specific spoilage organic of grass carp were separated and screened,and the specific spoilage bacteria with the ability to produce AHLs were screened using Chromobacterium violaceum(CV026).The HPLC-MS detection method was established to quantify AHLs secreted by specific spoilage organic,preparing curcumin liposomes to act on specific spoilage organic,and observing their inhibitory effect,provide a new idea for the antiseptic research of grass carp.The main research results are as follows:1.To study the quality change of grass carp during storage at three different low temperatures and the identification of cold-tolerant dominant spoilage bacteria.Grass carps are stored at low temperature below 0℃,4℃ and 10℃,the transformation of spoilage progress,freshwater fish pH,colony count(TAC),volatile basic nitrogen(TVB-N)value and sensory evaluation(QIM)score are all Significant increase(p<0.05),among which the rate of decay is 10℃,followed by 4℃,the grass carp stored at 0℃ has the slowest decay;When analyzing the microbial colony structure composition of fresh spot and spoilage end point by dilution coating plate method,the dominant spoilage bacteria of grass carp at spoilage end point were Pseudomonas,Aeromonas and Shewanella.After separating and screening the microorganisms at the end point of 4℃ grass carp spoilage,47 strains were obtained,and the DNA of the strains was extracted for 16S rDNA sequencing,of which 13 were Aeromonas sp.,Accounting for 27.66%;15 were Pseudomonas sp.,Accounting for 31.91%;Shewanella sp.11 strains,accounting for 23.40%;4 strains of Psychrobacter sp.,3 strains of Acinetobacter sp.and 1 strain of Yersinia sp..2.Screened the SSO of grass carp with the ability to produce AHLs among the dominant spoilage organic,and analyzed their AHLs secreted species and content and their ability to decay the sterile grass carp juice.Using biological reporter bacteria CV026 to screen for SSO that can produce purple pigments,two specific spoilage bacteria producing AHLs,A.hydrophila YZ-CY-03 and P.fluorescens YZ-CY-09,were obtained.Through the HPLC-MS detection system,it can quickly and efficiently quantify 4 AHLs(R2>0.99)within 9 min.The secretion amount reached 25.73 μg/L after 48 h of cultivation.P.fluorescens YZ-CY-09 secreted C10-HSL and C12-HSL content reached the highest after 36 h of culture,respectively 1.32 and 1.51μg/L,higher than A.hydrophila YZ-CY-03;Sterile fish juice was inoculated with different SSO.By analyzing the changes of TMA,TVB-N and biogenic amine during the spoilage process,the decay ability of the two SSO producing AHLs was compared.When the fish fillet spoiled until the 10th day,the TMA content of A.hydrophila YZ-CY-03 and P.fluorescens YZ-CY-09 were 1.70 mg/100mL and 1.40 mg/100mL,and the TVB-N content was 36.84 mg/100mL,respectively.And 33.66 mg/100mL,the tryptophan,putrescine,cadaverine,and tyramine in the A.hydrophila YZ-CY-03 group were significantly higher than the P.fluorescens YZ-CY-09 group(p<0.05),reaching 40.59 mg/L,23.49 mg/L,50.04 mg/L and 41.04 mg/L;3.The effect of curcumin liposomes on the secretion of AHLs and biofilms by two SSO was studied.The MIC(Minimum Inhibitory Concentration)of curcumin liposomes against A.hydrophila YZ-CY-03 and P.fluorescens YZ-CY-09 strains was determined to be 640 mg/L by double dilution method.Using CV026 to detect the anti-quorum sensing activity of curcumin liposomes at sub-inhibitory concentration,320 mg/L curcumin liposomes can significantly inhibit the production of purple pigment of CV026;A.hydrophila YZ-CY-03 and P.fluorescens YZ-CY-09 strain growth,curcumin liposomes can eliminate and inhibit A.hydrophila YZ-CY-03 and P.fluorescens YZ-CY-09 biofilms,the highest elimination rate is 40.65%and 51.49%,the highest inhibition rates are 57.01%and 61.54%;Detect the effect of different concentrations of curcumin liposomes on AHLs and find that curcumin liposomes can significantly inhibit the production of A.hydrophila YZ-CY-03 and P.fluorescens YZ-CY-09 C4-HSL,320 mg/L curcumin liposomes inhibit A.hydrophila YZ-CY-03 70.13%at 24 h,P.fluorescens YZ-CY-09 88.41%C4-HSL production,at 48 h inhibit 85.08%,79.13%C4-HSL production.