Study On The Mechanism Of Dawson Polyoxometalate Inactivating Foodborne Pathogens And Its Antibacterial Membrane

Food safety has become a problem that can not be ignored in today’s society.Food safety accidents caused by a variety of foodborne pathogens can cause serious public health problems.As a new type of food antistaling agent with great development potential,polyoxometalates（POMs）has been paid more and more attention due to its excellent bactericidal activity.However,there are still many deficiencies in the current basic research on the bactericidal mechanism of POMs.It was focused more on its damage to bacterial cell wall structure,cell membrane-related lipids,proteins,and key enzymes（mainly located around the cell membrane that affect the bacterial respiratory chain）.Therefore,in this paper,common foodborne pathogenic bacteria including Escherichia coli O157:H7（E.coli O157:H7）and Staphylococcus aureus（S.aureus）were selected as research objects to explore other possible inactivation mechanisms of the foodborne microorganisms by Dawson Polyoxomethoate（Dawson-POM）,which is of great significance to comprehensively elucidate the biological activity of polyoxomethoate.At the same time,Carr/POM films with significant antibacterial activity were prepared by introducing Dawson-POM with carrageenan as membrane matrix,which provided theoretical basis for the application of polyoxometalates in food preservation.The key findings were summarized as follows:（1）Preparation and structural characterization of Dawson polyoxometalate(K₆[Mo₁₈O₆₂P₂])Dawson-POM(K₆[Mo₁₈O₆₂P₂])was prepared by hydrothermalsynthesis method.The structure of Dawson-POM(K₆[Mo₁₈O₆₂P₂])was verified and characterized by FT-IR and UV-visible absorption spectra.It was proved that the compound had the characteristic peak of Dawson-POM(K₆[Mo₁₈O₆₂P₂]).Through thermal analysis,it was shown that the thermal stability of Dawson-POM was poor,and two obvious weight loss occurred in the range of 30-150℃and 512-800℃,respectively,and the total weight loss rate was 21.91%.（2）Study on the inactivation of foodborne microorganisms by Dawson polyoxomethoate(K₆[Mo₁₈O₆₂P₂])The bacteriostatic effects of Dawson-POM on E.coli O157:H7,S.aureus and L.monocytogenes were studied by Kirby-Bauer disk diffusion method and solid contact test.The results showed that E.coli O157:H7,S.aureus and L.monocytogenes first appeared in obvious inhibition zone when the concentration of Dawson-POM was 4 mg/m L,4 mg/m L and 16mg/m L,respectively.At the same time,the size of the inhibition area was proportional to the concentration of Dawson-POM,which had concentration-dependent manner.In addition,solid contact experiment showed that the medium containing 4 mg/m L Dawson-POM could completely inhibit the growth of E.coli O157:H7,S.aureus and L.monocytogenes with the inhibition rate of 100%.（3）The genomic DNA Degradation of foodborne pathogens by Dawson polyoxometalate(K₆[Mo₁₈O₆₂P₂])The artificial nuclease characteristics of Dawson-POM were studied by fluorescence quenching test,nuclear magnetic resonance（NMR）and p UC19 plasmid cleavage in vitro,and the effect of Dawson-POM on genomic DNA of foodborne pathogens was further explored by agarose gel electrophoresis and TUNEL method.The results showed that Dawson-POM could bind DNA mimics 4-nitrophenylphosphate disodium（NPP）and hydrolyze NPP with phosphatase activity.At the same time,agarose gel electrophoresis experiments showed that Dawson-POM could degrade p UC19 plasmids,and there was a chemical catalytic phenomenon.The rate of DNA degradation at 50℃was much faster than that at 37℃.In addition,agarose gel electrophoresis and TUNEL method further confirmed that Dawson-POM could break the bacterial DNA of E.coli O157:H7and S.aureus,and the fragmentation of genomic DNA of S.aureus by Dawson-POM was stronger than that of E.coli O157:H7 genomic DNA.Therefore,Dawson-POM can exert phosphatase activity on foodborne pathogenic bacteria and cause bacterial genomic DNA breakage,ultimately leading to bacterial death.（4）Response of Dawson polyoxomethoate(K₆[Mo₁₈O₆₂P₂])to DNA breakage in E.coli O157:H7Western Blot protein immunoblotting and Annexin V-FITC detection kits were used to explore the expression of Rec A protein and phosphatidylserine eversion in E.coli O157:H7 after Dawson-POM treatment.The results showed that the expression of Rec A,an important protein for DNA repair,was upregulated after Dawson-POM treated E.coli O157:H7,suggesting that Dawson-POM could disrupt genomic DNA of E.coli O157:H7.In addition,the early apoptosis rate of E.coli O157:H7 was increased to 63.55%after 8 mg/m L Dawson-POM treatment,indicating that Dawson-POM treatment could significantly cause programmed cell death such as phosphatidylserine valgus.Therefore,Dawson-POM treatment of E.coli O157:H7 can significantly up regulate the important protein Rec A of DNA repair and induce phosphatidylserine evagination and other programmed death characteristics of bacterial cells,further suggesting that Dawson-POM can break DNA.（5）Preparation and characterization of Dawson polyoxomethoate(K₆[Mo₁₈O₆₂P₂])antibacterial filmCarrageenan/Polyoxometalate（Carr/POM）films were prepared by flow-casting method,and the effects of Dawson-POM addition on the mechanical properties,thermal stability and microstructure of the films were evaluated.Meanwhile,Kirby-Bauer diffusion experiment,carrier surface disinfection experiment and biodegradation experiment were carried out to investigate the antibacterial performance and biodegradability of the film.The results showed that the prepared Carr/POM film had good bactericidal performance against common foodborne bacteria such as E.coli O157:H7 and S.aureus.In addition,a series of characterization of Carr/POM films also showed that low concentrations of polyoxomethoate（≤4 mg/m L）had little effect on the microstructure of the films,while Carr/POM（@8 mg/m L）showed good antibacterial activity,with a slight decrease in water content and tensile strength.