Effects Of Dual-functional And High-efficient Photosensitizer OG On Antibacterial Activity、Mechanism And Biofilm Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus（V.parahaemolyticus）is a foodborne pathogen widely distributed in coastal areas.V.parahemolyticus is the leading cause of gastroenteritis in people who eat improperly processed seafood.V.parahaemolyticus tends to form biofilms in food processing equipment,resulting in better drug resistance.Therefore,there is an urgent need in the food field to develop an efficient method to kill V.parahaemolyticus and eradicate its biofilm.In recent years,photodynamic inactivation（PDI）technology is a non-thermal processing technology gradually emerging.PDI is a photochemical reaction occurring in the presence of light,photosensitizer and dissolved oxygen,which can kill food-borne pathogens efficiently and quickly.Our research group has been engaged in the research on the inhibitory activity and mechanism of phenolic acid derivatives on various foodborne harmful bacteria for many years.On this basis,this study confirmed for the first time that OG is a food additive with both bacterioinhibitory and photosensitive properties,and developed an efficient killing and elimination technology for V.parahemolyticus and its biofilm,namely,PDI technology combining OG with 420 nm blue light（BL）.At the same time,this study further clarified the antibacterial mechanism of OG-mediated PDI technology for V.parahemolyticus.In addition,Octyl gallate（OG）is a food additive approved by the U.S.Food and Drug Administration（FDA）,the European Union（EU）and the World Health Organization（WHO）,which provided an important theoretical basis for the development of green efficient antibacterial agents and photosensitizers in the future.Firstly,the antibacterial activity of OG-mediated PDI technique against V.parahaemolyticus was investigated.By determining the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of GACs with different alkyl chain lengths against V.parahaemolyticus,The MIC and MBC values of OCtyl gallate（OG）against V.parahaemolyticus were 0.05 m M and 0.1 m M,respectively,indicating that OG had the best antibacterial activity.Therefore,choosing OG as a photosensitizer,we continued to investigate the bacteriostatic activity of OG combined with BL against V.parahaemolyticus.The results showed that when the synergy of 0.2 m M OG and BL was 15 min（light dose was191.7 J/cm²）,The colony count of V.parahaemolyticus decreased to 0 Log CFU/m L,indicating that OG-mediated PDI technique can effectively kill V.parahaemolyticus.Secondly,this study continued to investigate the bacteriostasis mechanism of OG-mediated PDI technique against V.parahaemolyticus.The fluorescence intensity of cells treated with OG-mediated PDI was the highest（536.5±56.0）by the absorption of 2-aminopethyl diphenylborate（DPBA）,indicating that OG had a better affinity with bacteria than gallic acid（GA）.Intracellular uptake of OG by bacteria is a key factor in the death of V.parahaemolyticus.Next,reactive oxygen species（ROS）production in V.parahaemolyticus cells was assessed using 2’,7’-dichlorofluorescein diacetate（DCFH-DA）probe and laser confocal focus（CLSM）.The results showed that the fluorescence intensity of ROS in V.parahaemolyticus cells after OG-mediated PDI treatment was the highest（1005.27）and the fluorescence quantity in confocal images was the highest,indicating that ROS production played an important role in the bactericidal mechanism.In addition,ROS species were identified by adding ROS scavengers,and it was found that after adding CAT,DMSO and TEMPOL scavengers,the number of colonies increased to 2.2 Log CFU/m L,4.7 Log CFU/m L and 3.2 Log CFU/m L,respectively.In addition,hydroxyphenyl fluorescein（HPF）probe and flow cytometry were used to verify the results.It was found that the fluorescence intensity of·OH was the highest（～100000）and the number of cells was the highest after OG-mediated PDI treatment.All these experimental phenomena indicated that the types of ROS generated are mainly·O^2-,·OH and H₂O₂,among which·OH played a major role in sterilization.In this study,the QCM-D experiment was continued to investigate the main reasons for the better affinity between OG and cell membrane compared with GA.It was found that when GA acted on simulated membrane,the f₁₁ and D₁₁ values almost remained unchanged,while when OG acted on simulated membrane,the f₁₁ value continued to decrease and the D₁₁ value changed little.This phenomenon showed that GA hardly interacted with the bacterial model membrane,and OG could continuously enter the lipid bilayer by transmembrane insertion.After that,through propidium iodide（PI）uptake experiment,it was found that the fluorescence intensity of PI in bacterial cells after OG-mediated PDI treatment was the highest（5901.6）,which indicated that the transmembrane insertion of OG and the production of ROS would cause certain damage to the integrity of cell membrane.According to DPBA and PI uptake rate of the experimental result showed that when low concentration OG in cell membranes,under the irradiation of BL cell membrane injury still happened,this phenomenon indicated that not only intracellular ROS production and OG transmembrane insertion could destroy the cell membrane,but also extracellular ROS may be produced when OG was inserted on the cell membrane,thus affecting the structure of cell membrane.SEM experiments showed that after OG and BL treatment,the shape of bacteria became irregular,the surface of bacteria became rough,and even broken bacteria appeared,which further intuitively proved that OG-mediated PDI would cause significant damage to cell membrane.Then,through polyacrylamide gel electrophoresis experimental study,it was found that after OG-mediated PDI treatment,the color of V.parahaemolyticus membrane protein bands became darker,and even new protein bands appeared,which indicated that OD-mediated PDI technology could lead to degradation and aggregation of bacterial membrane protein.Fourier transform infrared spectroscopy（FTIR）was used to further analyze the membrane protein,and it was found that the secondary structure of the membrane protein was changed after OG and BL treatment,indicating that the chemical interaction between OG and membrane protein would occur under the irradiation of BL.In addition,through DNA gel electrophoresis,it was found that the DNA bands of V.parahaemolyticus disappeared after the co-treatment of OG and BL,indicating that the OG-mediated PDI technology could damage the integrity of the DNA gene of V.parahaemolyticus.It was also found by molecular butt technology that both OG and its oxidized product quinone could interact with DNA through hydrogen bonds.The lowest binding energy of OG to DNA was-7.26 kcal M^-1,which contained one conformation,and the lowest binding energy of quinone to DNA was-7.53 kcal M^-1,which contained 20conformations.The experimental results showed that both OG and quinone had important effects on the structure of DNA.Compared with OG,the conformation of quinone combined with DNA was more stable,and the interaction between quinone and DNA was stronger.The effects of OG-mediated PDI on the formation and maturation of biofilms of V.parahaemolyticus were further studied.By crystal violet staining of the biofilm and OD₆₀₀measurement,48 hours was the best time for the growth of V.parahaemolyticus biofilm(OD₆₀₀=1.5633).After OG-mediated PDI treatment,the biomass of the forming biofilm and the mature biofilm was significantly reduced,which was reduced by 55.2%and 84%after 30min and 60 min of 0.2 m M OG and 106.5 m W/cm² BL treatment,respectively.In addition,CLSM was used to observe the morphology of biofilms.The results showed that after OG-mediated PDI treatment,both the forming and mature biofilms showed incomplete structure,uneven surface shape,and obvious dead bacteria appeared in the biofilm.All these experimental phenomena indicated that OG-mediated PDI technology could not only effectively prevent the formation of V.parahaemolyticus biofilm,but also can remove the mature biofilm.In addition,this study continued to investigate the activity of bacteria in the biofilm after OG-mediated PDI treatment,and found that the number of live bacteria in the biofilm decreased by 2.54 Log CFU/m L.This experimental phenomenon indicated that OG-mediated PDI technology also had certain bacteriostatic effect on bacteria in the biofilm.Finally,chitosan solution containing OG was prepared and applied to the surface of salmon.It was found that there was no significant change in the number of bacteria on the surface of salmon at 12 days,which was about 3.62 Log CFU/m L.In addition,the salmon still retains its original flavor and color at 15 days,indicating that OG can effectively inhibit the growth of microorganisms on the surface of salmon slices,and has the potential of application for storage and preservation of salmon slices.