Preparation And Bactericidal Mechanism Of Polyphenol Anchor Based Fungicides

Foodborne pathogens are one of the main reasons for frequent occurrence of food poisoning incidents,which seriously threaten human health.Rational design and preparation of nano fungicides is one of the effective strategies for killing foodborne pathogens.However,the performance of traditional single type of nano fungicides is limited,it is difficult to detect bacterial infection in time,and it is unable to achieve precise targeting and controllable sterilization.Therefore,it is of great significance to develop new,safe and efficient killing technology for foodborne pathogens to ensure food biosafety.The responsive composite based on natural polyphenol compounds is a kind of green,natural and mild fungicide;Compared with traditional fungicides,polyphenol based responsive fungicides have the function of timely detection of infection and activation of sterilization on demand.Bactericidal process can avoid bacterial resistance and prevent the formation of bacterial biofilm and other characteristics.Based on this,this paper designed and synthesized a series of polyphenol responsive biological fungicides,and studied their related bactericidal mechanism,and evaluated their practical application value.The main contents and results of this paper are as follows:1.Preparation and bactericidal mechanism of near-infrared triggered polyphenol anchor based nano fungicidesPrecise targeting and controlled sterilization of fungicides are crucial to food safety.This study proposes a demand-activated sterilization strategy.Polyethylene glycol modified tungsten trioxide nanoflower(PEG-WO_3-x)was prepared by a one-pot solvothermal method.Combined with ultrasound-assisted methods,EL was successfully loaded onto the surface of PEG-WO_3-xNFs through hydrogen bond interaction using the phenol hydroxyl group（-OH）in eugenol（EL）and the ether bond（-O-）of PEG in PEG-WO_3-xNFs to form PEG-WO_3-x@EL NFs.Its morphology,structure and chemical composition were characterized by SEM,TEM,XRD,FT-IR and XPS.Then,the agar plate counting method was used to evaluate its lethal effect on Salmonella and methicillin-resistant Staphylococcus aureus.The elimination mechanism of bacterial biofilm was investigated by combining fluorescence staining and crystal violet staining.The results showed that PEG-WO_3-x@EL NFs nanofungicides have photothermal properties and near-infrared（NIR）mediated EL release properties,confirming that near-infrared as a“trigger switch”can achieve efficient elimination of on-demand release of active substances（polyphenols）assisted by photothermal effect enhanced bacterial biofilms in the application process.2.Preparation of near-infrared triggered polyphenol anchored hydrogel,in situ identification of drug-resistant bacteria and study on its bactericidal mechanismIt is of great significance for food safety to improve the efficacy of powder fungicide and broaden its practical application field.In this study,a strategy of transforming powdered fungicides into non-powdered fungicides gels and functionalizing them was proposed to build a one-stop platform for identification and efficient killing of superbacteria triggered by near infrared.Lignosulfonate（LS）,as an anionic surfactant,can combine with cationic guar gum（CG）to form hydrogel through electrostatic interaction.Then,Ag₂O NPs are diffused and fused inward to form LS/CG/Ag₂O nanocomposite hydrogel.Its structure and morphology were characterized by SEM and FT-IR,and its mechanical properties were tested by rheometer and universal testing machine.The bactericidal mechanism of hydrogel was studied by agar plate counting,bacteria fixation observation,electron spin resonance and infrared thermography.The results showed that hydrogel could catalyze the formation of oxidized TMB（ox TMB）from 3,3’,5,5’-tetramethylbenzidine（TMB）,and differentiate Staphylococcus aureus and methicillin-resistant Staphylococcus aureus by using the different inhibition ability of superbacteria to TMB and hydrogel.Finally,it was confirmed that the near infrared driven ox TMB photothermal effect can effectively kill Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.It has reference significance for further development of fungicides to identify superbacteria.3.Preparation of near-infrared triggered polyphenol anchored hydrogel,in situ identification of drug-resistant bacteria and bactericidal mechanismTimely detection and prevention of bacterial infection is particularly important for food safety.In this study,copper divalent and catechol（CuCL）were used as coordination assembled nanosheets and bromothymol blue（BTB）were embedded together in the network of red algal natural polysaccharide carrageenin to build a Carr/BTB/CuCL hydrogel platform for pH sensitive diagnosis and photothermal effect to kill bacteria.The structure and morphology were characterized by SEM and FT-IR,and the rheological properties of gel were measured by rheometer.The bactericidal activity and related mechanisms of hydrogels were studied by agar plate counting,bacteria fixation observation and infrared thermography.The results showed that compared with Carr hydrogel alone,CuCL nanoflakes made Carr/BTB/CuCL hydrogel have stronger mechanical properties,hydrophobicity and thermal stability.BTB endowed Carr/BTB/CuCL hydrogels with pH response,enabling visual real-time tracking of bacterial infections.Meanwhile,Carr/BTB/CuCL hydrogel produced local thermal effect under near infrared light,killing E.coli and methicillin-resistant Staphylococcus aureus in time.This provides new insights into the development and implementation of“perception-therapy”systems for bacteria,which are critical for food safety.4.Preparation of pH and fluorescent double response polyphenol anchor based hydrogel and its remote monitoring and bactericidal effect on pathogenic bacteriaThe realization of remote intelligent real-time monitoring of bacterial infection and timely sterilization status is of great significance for food safety monitoring and food protection.In this study,catechol was further used as a stabilizer to peel WS₂nanosheet into high-quality nanosheet dispersion.A simple one-pot synthesis technique was used to encapsulate CL/WS₂nanoenzyme and bicolorimetric substance（Bromothymol blue-fluorescein thiocyanate）in a hydrogel matrix with polyethyl alcohol（PVA）,dextran（Dex）and borax.Its structure and morphology were characterized by SEM and FT-IR,and the mechanical properties were tested by rheometer and universal testing machine.The bactericidal mechanism of hydrogel was studied by agar plate counting,bacteria fixation observation,infrared thermography and free radical scavenging test.The results showed that the phenol hydroxy group could effectively improve the uniform dispersion and stability of CL/WS₂nanoenzyme in the hydrogel,and enhance the adhesion of the hydrogel.The adhesion strength of the hydrogel was 8.3±0.6 k Pa,which was 1.66 times higher than that of the commercial adhesive.Based on the pH response characteristics of the hydrogel colorimetric fluorescence probe,the visual color and fluorescence signals generated by changes in the bacterial microenvironment can be recorded by smart phones,which can be converted into on-site pH signals for remote intelligent real-time monitoring of bacterial infection.The hydrogel has high photothermal effect and antioxidant function of endogenous enzyme like activity,and can kill infected bacteria in a timely,broad-spectrum and efficient manner.5.Safety evaluation and application in food of polyphenol anchored responsive bactericideThe safety characteristics of the use of nano fungicides are essential for food safety.In this study,the biosafety of prophase fungicides was evaluated in vitro and in vivo,and the biosafety of prophase fungicides was evaluated by erythrocyte hemolysis test,in vitro cytotoxicity test and in vivo acute toxicity test.The results showed that there was no significant difference in cytotoxicity and cell survival rate under normal dose conditions,and the hemolysis rate was extremely low,which was lower than the allowable value（5%）.At the same time,it has no adverse effects on the body weight and main organs of mice,indicating that the nano bactericide developed in this study has certain safety.Food application research found that it can achieve awareness of bacterial infection and kill bacteria in time,which has a good effect in tomato preservation.