Preparation Of Photoactive Hydrophobic Chitosan (GA/OA-g-CS) Film And Photodynamic Eradication Of Bacteria And Biofilms

Food safety is a fundamental issue related to human survival and development and has always been the focus of the masses.According to the World Health Organization（WHO）survey data,foodborne diseases cause about 230,000 deaths yearly,and foodborne pathogens cause 81.4%.As global foodborne pathogens,Escherichia coli（E.coli）and Staphylococcus aureus（S.aureus）are thought to cause diseases such as severe gastroenteritis,hemorrhagic colitis,and hemolytic uremia.It can grow rapidly in nutrient-rich environments,forming biofilms on the surface of food,water,and processing equipment,causing major public health security threats and huge economic losses.Therefore,it is still urgent to develop a safe and efficient new antibacterial strategy to meet the application needs of the food industry.Our previous studies have found that food antioxidants gallic acid and its alkyl esters（such as octyl gallate）have excellent broad-spectrum antibacterial activities.To our great surprise,they are also excellent photosensitivities,showing excellent photodynamic inactivation（PDI）and biofilm clearance properties when coupled to light irradiation.Therefore,inspired by these splendiferous findings,we investigated the potential of grafting gallic acid（GA）and octanoic acid（OA）onto CS to empower CS for ultra-efficient bacterial capture and photodynamic ablation of planktonic bacteria and biofilms with BL irradiation.That is,GA and OA are covalently cross-linked with CS through a green radical induced grafting reaction,and the GA/OA-g-CS has amphiphilic and photosensitive activities.After synergistic blue light irradiation,rapid sterilization and biofilm removal of bacteria were realized,and the antibacterial mechanism of the PDI system was further revealed.The main results are as follows:（1）Through the green radical covalent cross-linking reaction mediated by ascorbic acid/hydrogen peroxide（AA/H₂O₂）,gallic acid（GA）and octanoic acid（OA）were covalently grafted onto natural chitosan（CS）molecules to prepare hydrophobic chitosan with antibacterial and photosensitive activities,namely GA/OA-g-CS.Its structure and physicochemical properties were determined and characterized.The optimal grafting rate of GA in GA/OA-g-CS is 172.13 mg GAE/g（GA equivalents/g）.UV-vis and ¹H NMR spectra showed that both GA and OA were covalently cross-linked with CS.FT-IR analysis further indicated that GA,OA,and CS were covalently cross-linked by amide bond and ester bond,respectively.XRD results show that GA/OA-g-CS is amorphous.DSC atlas and thermal stability analysis showed that the thermal stability of natural CS decreased after GA and OA were introduced into the molecular structure,which further confirmed the covalent cross-linking between GA,OA,and CS.（2）The species and amino content of free radicals in the CS/AA/H₂O₂ reaction system were analyzed qualitatively and quantitatively.In AA/H₂O₂ oxidation reduction system,H₂O₂ and AA redox reaction form the hydroxyl free radical（·OH）and ascorbic acid free radicals(Asc^·-).·OH with CS on the amino group（-NH₂）and hydroxyl（-OH）,formation of CS macromolecular radicals（·CS）.As a nucleophile,·CS reacts with the carboxyl group（-COOH）of GA and OA to form GA/OA-g-CS after covalent cross-linking.（3）The ability of GA/OA-g-CS film to capture bacteria and its bactericidal activity were investigated.The results of laser confocal microscopy（CLSM）showed that the long hydrophobic chain of OA in GA/OA-g-CS molecular structure endows the film with the ability to capture and fix bacterial cells,i.e."bacteria-capturing".At the same time,GA on the graft can give the film effective sterilization activity,that is,"contact-killing".More interestingly,under the synergistic effect of blue light（BL）irradiation,GA/OA-g-CS film（2:1）reduced the initial colony number of 10⁸ Log（CFU/m L）E.coli by 5.2 Log（CFU/m L）within 30 min.The bacteriostatic rate was as high as 99.999%.The initial colony number of10⁷ Log（CFU/m L）of S.aureus decreased by 2.5 Log（CFU/m L）,and the antibacterial rate reached 99.68%.The higher the content of GA in GA/OA-g-CS film,the better the antibacterial effect of PDI.（4）The synergistic sterilization mechanism of GA/OA-g-CS surface contact sterilization and coupled PDI was described from two perspectives of"surface contact-induced cell membrane damage"and"intracellular ROS".DCFH-DA fluorescent probe was used to quantitatively analyze the intracellular ROS levels of E.coli after GA/OA-g-CS and cooperative BL irradiation,and the PI fluorescent probe was used to analyze the damage of GA/OA-g-CS and cooperative BL irradiation on E.coli cell membrane.The results showed that the intracellular ROS level was significantly increased after BL irradiation.Compared with the blank control group,GA/OA-g-CS（2:1）increased the intracellular ROS level by nearly 1000times,and the intracellular ROS level further increased after synergic BL irradiation.The damage to cell membrane integrity caused by the transmembrane insertion of the hydrophobic chain groups of GA/OA-g-CS is the main cause of the E.coli cell death caused by GA/OA-g-CS contact sterilization and its collaboration with BL.（5）Combined with crystal violet staining experiment and CLSM analysis,the inhibitory effect of GA/OA-g-CS film and its cooperative BL irradiation on E.coli biofilm formation and its scavenging activity on mature E.coli biofilm were further investigated.After 30 min of BL irradiation,GA/OA-g-CS（2:1）film co-incubated with E.coli,the maximum biomass reduction rates of E.coli biofilm formation and mature biofilm reached 60%and 69%.According to the observation of CLSM,GA/OA-g-CS（2:1）film and BL irradiation in biofilm samples had the least residual cell amount,the thinnest biofilm thickness,and the most dead bacteria.At the same time,after 30 min irradiation of GA/OA-g-CS（2:1）films in collaboration with BL,the number of viable bacteria in E.coli mature biofilm and its supernatant decreased by 4.36 Log and 3.37 Log（CFU/m L）,respectively.