Inhibitory Mechanism Of Plant Essential Oils On Bacterial Quorum Sensing And Essential Oils Technological Design Of Liposome

Aquatic products are an important source of high quality protein for human nutrition and an important variety of common people’s dishes.Their quality directly affects people’s health and safety.However,aquatic products have many kinds of proteases,and often carry a variety of spoilage bacteria,easily spoilage after fishing,so it is of great significance in anti-bacterial preservation.Pseudomonas fluorescens is one of the dominant spoilage bacteria causing spoilage of aquatic products,and Quorum sensing（QS）is the main spoilage mechanism.Blocking the quorum sensing system of Pseudomonas fluorescens with quorum sensing inhibitors can modulate the expression of genes related to spoilage and interfere with many physiological processes,for example,toxin production,biofilm formation,extracellular protease secretion,antibiotic biosynthesis,bioluminescence and so on,inhibit the corruption of aquatic products caused by it.Plant essential oils have special effects in inhibiting quorum sensing,can effectively inhibit the spoilage of aquatic products,and have both the advantages of safety and green,can replace some toxic and harmful chemical preservatives,and reduce the use of antibiotics,the application prospects are very broad.Based on the characteristics and regularity of Pseudomonas fluorescens putrefaction in tilapia,a major aquatic product in Guangxi,the synergistic bacteriostatic effect of Allicin,Terpene-4-ol and Carvone on QS system was studied,the mechanism of antibacterial and fresh-keeping of plant essential oils was revealed from the point of inhibiting quorum-sensing,and the improvement of antibacterial efficiency of essential oils coated by p H-sensitive liposomes was discussed:（1）The combination of EO_ATC（Allicin,Terpene-4-ol and Carvone）,which has synergistic inhibitory effect on Pseudomonas fluorescens QS,was selected from seven essential oils,it was found that the MIC effective concentration of EOATC was 1/8 to 1/10 of that of its single component MIC.EO_ATC could inhibit the QS system of quorum sensing detection bacterium,Bacillus violaceus CV026,and the inhibition rate reached 62.90%after 0.125μl/m L treatment.EO_ATC of sub-MIC concentration had no effect on the normal growth of Pseudomonas fluorescens,but significantly inhibited the formation and movement of biofilm.（2）Gas chromatography-mass spectrometry（GC-MS）was used to analyze the production of signal molecules（AHLs）of Pseudomonas fluorescens.It was found that the two AHLs,C₆-HSL and C₈-HSL,were produced by Pseudomonas fluorescens,EPS and biofilm formation of P.fluorescens were decreased,the secondary and tertiary structure of protein were destroyed,and-helix content of secondary structure was decreased.Molecular docking software（Auto Dock Vina）was used to study the inhibition mechanism of EO_ACT on Pseudomonas fluorescens QS,it was found that allicin,terpene-4-ol and carvone combined with the key amino acids THR145 and ARG104 of Rhl I formed hydrogen bond,and the docking result was better than that of natural ligand C₆-HSL.The amino acid residues TYR 122 and LYS 121 of Rhl R protein could be bound by carvone,which was better than that of natural ligand C₆-HSL.Therefore,EO_ACT can competitively bind Rhl I/R protein with C₆-HSL,thus blocking the QS signal.（3）Two p H-sensitive liposomes containing Oleic acid（OA）and Linoleic acid（LA）were prepared to improve the bacteriostatic efficiency of essential oils.The average particle size of the two liposomes was 104.6 nm and 108.5 nm,theζpotential was-42.8 and-44.3,and the PDI index was 0.209 and 0.218,respectively,the results showed that p H-sensitive liposomes were stable in normal body fluid（p H 7.4）.The release rate of EO_ATC at p H 5.5 is much faster than that at p H 7.4,which provides an idea for the design of p H controlled slow-release anti-bacterial preservative for aquatic products.To sum up,this study provides a theoretical reference for the research and application of plant essential oil bacteriostatic agent,and provides a technical guidance for the antibacterial and fresh-keeping of aquatic products.