Cascade Catalytic Effect Of Enzyme-carrying Metal Polyphenols And Detection And Killing Of Foodborne Pathogens

Foodborne microbial contamination is one of the major causes of illness and death for tens of millions of people each year.Escherichia coli,Staphylococcus aureus and other popular foodborne pathogens accounted for the highest proportion of foodborne microbial pollution.The existing detection technology has high requirements for time and technical personnel operation ability,which cannot meet the current demand for rapid food detection.In addition,the abuse of antibiotics leads to the emergence of some drug-resistant foodborne bacteria,which puts forward higher requirements for the killing technology of foodborne pathogens.In recent years,nanomases have become a hot material in the field of antibacterial due to their excellent catalytic ability and stable structure.In this paper,a novel GGFzyme nanozyme with cascade catalytic ability（glucose oxidase-peroxidase）was designed to catalyze the generation of reactive oxygen species from substrate glucose.Reactive oxygen species enable rapid detection of glucose concentration and foodborne pathogen activity in real samples by oxidizing 3,3’,5,5’-tetramethylbenzidine（TMB）.At the same time,the catalytic production of a large number of reactive oxygen species made GGFzyme nanozyme exhibit excellent killing ability of drug-resistant Staphylococcus aureus（MRSA）.The nano-enzyme can effectively improve the detection and killing efficiency of foodborne pathogens.The main research contents of this paper are as follows:1.Synthesis and cascade catalytic activity of GGFzyme nanozymeGGFzyme nanozyme was prepared by hydrothermal method.Scanning electron microscopy（SEM）and transmission electron microscopy（TEM）were used to observe the ellipsoidal morphology and uniform distribution of the materials.The particle size of the materials was determined to be 20～50 nm by nano-laser particle size analyzer.Fourier transform infrared spectrometer（FTIR）,X-ray photoelectron spectroscopy（XPS）and energy dispersive analysis（SEM-EDS）were used to determine the distribution and valence bond of each element in the synthetic material,which also confirmed the successful synthesis of the material.When hydrogen peroxide（H₂O₂）was not added in the system,the GGFzyme nanozyme could generate free radicals to oxidize the chromogenic substrate TMB using glucose as substrate,indicating that GGFzyme nanozyme had the catalytic ability of glucose oxidase and peroxidase.The optimal reaction temperature of GGFzyme nanozyme was 37°C,p H was 4,and reaction time was 20 min.The synthesis method of GGFzyme nanozyme is simple and does not require complex instruments.It has high detection accuracy in a wide range of temperature and p H,good catalytic activity and stable structure.2.Detection of foodborne bacteria based on GGFzyme nanozyme cascade catalysisUsing GGFzyme nanozyme cascade catalysis as a method,a rapid detection method for foodborne pathogenic bacteria activity was established by utilizing the consumption of glucose in the metabolic process of foodborne pathogenic bacteria.The test results showed that there was a good linear relationship（R²=0.993）between glucose and absorbance value when the glucose concentration in the system was between 1～500μM,and the lowest detection limit（LOD）was 0.43μM.In this chapter,it is verified that GGFzyme nanozyme has good specificity to substrate,which enables the quantitative detection of glucose in systems with unknown concentration by absorbance value.In the actual sample test,apple juice was taken as the test object.After the pretreatment of apple juice,the absorbance value of apple juice was determined and the content of glucose in apple juice was calculated.Based on the principle that the growth and reproduction of foodborne pathogenic bacteria will consume glucose,the absorbance changes after contamination of foodborne pathogenic bacteria were detected.In this experiment,E.coli,S.aureus,S.typhimurium,L.monocytogenes and E.sakazakii were detected at concentrations of 10⁷ CFU m L^-1.3.Efficient killing of MRSA by cascade catalysis based on GGFzyme nanozymeThe glucose-triggered GGFzyme nanozyme cascade catalyzed free radicals as bactericidal components to carry out green and efficient sterilization of MRSA.The bactericidal ability of GGFzyme nanozyme against MRSA was determined by plate coating method firstly,the results showed that the bactericidal rate of the material was up to 99.4%against 10⁷ CFU m L^-1 bacteria concentration within 30 min.4’,6-diaminyl-2-phenylindole（DAPI）,propidium iodide（PI）fluorescence imaging results also showed that MRSA would die heavily under the combined action of glucose and GGFzyme nanozyme.Scanning electron microscopy was used to explore the bactericidal mechanism of GGFzyme nanozyme.The results showed that the free radicals generated by GGFzyme nanozyme catalysis destroyed the cell membrane of drug-resistant food-borne pathogens,resulting in severe cell membrane damage and cell contents flowing out.By electron spin resonance（ESR）,hydroxyl radical and superoxide anion were identified as the types of free radicals generated.It was confirmed that free radicals could destroy the membrane structure of food-borne pathogens and affect the metabolic process of bacteria,thus killing food-borne pathogens.Using MRSA infected wounds of hyperglycemic mice as a model,it was confirmed that the GGFzzyme nanozyme can also use glucose to kill pathogens in complex biological systems.At the same time,combined with MTT cell toxicity test in vitro and mouse organ section,the GGFzyme nanozyme has good biological safety,which lays a solid foundation for further research on killing foodborne pathogens in the field of food.