Preparation Of Gallic Ester And Chitosan Crosslinking Nano-self Assembly And Its Photodynamic Antibacterial Mechanism

Photodynamic inactivation（PDI）,as a new non-thermal sterilization technology,inactivates microbial cells by activating photosensitizers（PSs）to produce reactive oxygen species with specific wavelengths of light.At present,researchers are mainly using exogenous organic small molecule photosensitizers（such as curcumin）to improve the effectiveness of PDI in food applications.Our previous study found that alkyl gallate（such as octyl gallate）not only showed excellent broad-spectrum antibacterial activity,but also showed excellent bactericidal and biofilm removal properties after coupling with light irradiation（such as blue light）,which is expected to be developed into a dual-effect food additive with antibacterial and photodynamic activity.However,the hydrophobic alkyl chains in the molecular structure of alkyl gallate reduce its solubility in aqueous solutions,thus limiting the application of alkyl gallate mediated PDI technology in the food industry.Based on the above findings,a new PDI system based on amphiphilic chitosan derivatives nano-self-assembly was constructed for the first time in this study.That is,through enzymatic reaction,lauryl gallate（GAC12）was covalently cross-linked with CS（GAC12-G-Cs）,and amphiphilic,photosensitive and nano-self-assembly properties were given to the system.Based on the formation of nanoscale aggregate（self-assembly behavior）induced by hydrophilic and hydrophobic action,the purpose of rapid sterilization and removal of biofilm was realized in collaboration with blue light irradiation,and the antibacterial mechanism of the PDI system was revealed from the molecular level.The main research results are as follows:（1）The morphology,structure and particle size of GAC12-g-CS nano-self-assembly were characterized by DLS and TEM.The results show that the GAC12-g-CS nano-self-assembly is a stable dispersed spherical nanoparticle with an average hydrodynamic diameter of 136.5 nm,a polydispersion coefficient of 0.211,a Zeta potential of 34.9 m V,and a critical micellar concentration of 1.709 mg/m L.In addition,the GAC12-g-CS nano self-assembly has significant antioxidant and high efficiency photodynamic antibacterial activity.The DPPH·scavenging efficiency of GAC12-g-CS nano self-assembly（0.8 mg/m L）was 6.8 times that of natural CS.（2）Under synergic blue light conditions,compared with CS and GAC12,GAC12-G-Cs nanoself-assembly showed stronger photodynamic antibacterial activity against E.coli,S.ureus and P.aeruginosa,and it was dependent on irradiation dose.Among them,the PDI effect of P.aeruginosa was the best,killing live cells within 10 min（initial colony number:4×10⁶ CFU/m L,GAC12-g-CS nano self-assembly:0.8 mg/m L,blue light:420 nm 213m W/cm²）.（3）The antibacterial mechanism of GAC12-g-CS nano self-assembly mediated PDI was deeply discussed from two aspects:cell membrane damage,the composition of active species,and interaction with biomolecules.（1）The mechanism of cell membrane damage induced by GAC12-g-CS nano self-assembly was analyzed by propidium iodide uptake analysis,membrane potential analysis,and dissipative quartz crystal microbalance（QCM-D）technique.The results showed that rapid membrane binding occurred mainly through the strong electrostatic interaction between protonated chitosan amino group and polyanion on the bacterial surface,resulting in the collapse of cell membrane potential and serious damage to the integrity of the cell membrane.In addition,under experimental conditions in the study,such as particle size（137 nm and 512 nm）and concentration（0.8 mg/m L and 0.08 mg/m L）of the self-assembly and p H（p H4 and p H6）of the system,the mode of action（membrane binding）of the GAC12-g-CS nano-self-assembly with cell membrane was basically not significantly affected.But to different degrees.More specifically,the membrane binding degree is better when the particle size is small,the concentration is high and the p H is low.（2）The mechanism and composition of ROS were studied by fluorescence quantitative method.GAC12-g-CS nano self-assembly can induce a large number of toxic ROS in the cell,including H₂O₂,·OH,·O₂^-and ¹O₂ generation,and·OH is the main component.It is speculated that the ROS production mechanism may be the direct production of GAC12-g-CS nano self-assembler induced by blue light irradiation or the production of 1O2 by bacterial endogenous porphyrins stimulated by blue light,resulting in the oxidation of phenolic esters and H2O2,which is converted to high activity·OH by photolysis,resulting in bacterial inactivation.（3）The effect of ROS on bacterial DNA genome was studied by DNA gel electrophoresis.The findings affect the function of the DNA genome and thus the expression of bacterial genes.（4）The influence of protein quantification,CLSM visualization,and plate reculture on biofilm was explored.It was found that GAC12-g-CS nano self-assembly mediated PDI could effectively clear mature biofilms,and the biofilm biomass was reduced by 45.5%after only15 min irradiation compared with the control group（GAC12-g-CS nano self-assembly 4mg/m L,blue light:420 nm 213 m W/cm²）.