Study On Antibacterial Mechanism Of Basil Essential Oil Against Listeria Monocytogenes And Application Of Its Nanofibers In Pork Preservation

LM is a zoonotic pathogen with a wide distribution and strong stress resistance,refrigerated foods,ready-to-eat foods,dairy products and meat products are easier to be contaminated by LM,thus causing serious clinical symptoms such as septicaemia,meningitis or uterine infection to humans after eating LM-contaminated foods.In view of the potential harm to human health caused by traditional chemical preservative,a highly effective,safe and natural antimicrobial substance is urgently needed to reduce food safety risks caused by LM.For this reason,the natural and safe BEO was selected in this study.Firstly,we evaluated the inhibitory effect of BEO against LM,and revealed the inhibitory mechanism of BEO on LM from membrane barrier,intracellular genetic materials,virulence genes,metabolites levels.Furthermore,in order to improve the chemical stability of BEO,a BCL was prepared by using stearamine as the cationic regulator to encapsulate BEO.Finally,using the electrospinning technology to prepare antibacterial nanofibers as packaging materials by applying BCL as the antibacterial agent and phospholipid as substrate materials.The nanofibers were applied to the packaging of pork to study the changes in the physicochemical properties,microbial population and sensory qualities of pork during storage,so as to evaluate the packing properties of nanofibers in foods.The research work and main conclusions of this paper were shown as follows:(1)BEO treatment impacted the membrane barrier function of LM.Firstly,by measuring the changes of fatty acid,phase transition temperature,ANS fluorescence and Raman spectrum of membrane phospholipid,it was concluded that BEO treatment increased the content of unsaturated fatty acid and decreased the phase transition temperature of membrane phospholipid,thus improving the fluidity of LM membrane.Furthermore,the changes of permeability indexs including propidium iodide fluorescence,electrical conductivity,?-galactosidase and protein contents indicated that the action target of BEO on LM was cytomembrane.FTIR and ¹H NMR experiments indicated that BEO affected the functional properties of LM cytomembrane by acting on the fatty acyl group of the membrane phospholipid.Finally,by measuring the expression of LM redox related genes,ROS fluorescence and ESR,it was found that BEO stimulated the oxidative stress response of LM cells,leading a large amount of alkyl peroxide radicals,alkoxy radicals,alkyl radicals and superoxide radicals generated and accumulated on the cell membrane of LM.(2)BEO treatment interfered with the intracellular genetic materials and metabolites of LM.Through the changes of genomic DNA,plasmid DNA and topoisomerase activity of LM,it was concluded that BEO treatment can impact the transcription and replication process of intracellular DNA,as well as destroyed the superhelical structure of LM plasmid.The binding constant of linalool(the main component of BEO)with DNA calculated by the Stern-Wolmer equation was 0.6×10⁴M^-1,indicating the groove binding of BEO with LM DNA.Subsequently,by detecting the expression levels of LM-related virulence genes(plc A,plc B,hly,act A and mpl)and hemolytic properties,it was found that BEO reduced the hemolytic activity of LM by interfered with the production and secretion of hemolysin.Finally,through ¹H NMR metabolomics technology,six different metabolites were found,which were identified to be associated with following metabolic pathways:glyoxylic acid and decarboxylation metabolism,glutathione metabolism,glycine and serine and threonine metabolism,glycolysis and glycogenogenesis.Glycolysis was the main way of glycolysis and energy acquisition in microbial cells.BEO affected the glycolysis pathway of LM by interfering with the activities of glucokinase,6-phosphofructokinase and pyruvate kinase.(3)BCL was prepared by a thin film hydration method using stearamine as the cationic regulator to improve the stability of BEO.The prepared BCL had a particle size of 165.83 nm,with PDI and zeta potential at 0.2724 and 30.22 m V respectively,the encapsulation efficiency was 49.55%.QCM,FTIR,AFM and UV spectra confirmed that BEO was successfully embedded in BCL,and BCL exhibited a relatively stable spherical structure with the slow-release effect.Due to the negative charge of LM surface,BCL was easier to combine with LM surface through electrostatic adsorption,showing a higher antibacterial effect.In addition,the dual channel confocal laser microscope test also confirmed the improved inhibitory effect of BCL against LM.(4)Using electrospinning technology to prepare PSB NFs as packaging materials by applying PEO/SL as the base materials and BCL as the bacteriostatic agent,to alleviate the shortcoming of easy deposition and uneven distribution in BCL application.The changes of rheological properties,electrical conductivity,SEM,physical and mechanical properties confirmed that the ratio of PEO:SL in the spinning solution at5:1 was optimal for making nanofibers.The prepared PSB NFs exhibited a typical porous fiber structure with an average diameter of 164 nm,showing the best physical and mechanical properties.Furthermore,FTIR,TG,DSC and AFM tests demonstrated the PSB NFs showed stable chemical and thermodynamic properties with improved surface roughness.Finally,the inhibition zone and release rate tests confirmed the antibacterial effect of the prepared PSB NFs against LM.Since LM can secrete phospholipase,BEO in PSB NFs showed stimulus response release to LM.(5)The effects of PSB NFs on the preservation properties of pork were studied.Firstly,by measuring rheological properties,low field NMR,malonaldehyde,volatile base nitrogen and carbonyl contents,it was found that PSB NFs can maintain the viscoelastic properties and WHC of pork by controlling the oxidation of fat and proteins in pork samples.In addition,the changes of color and texture properties of pork samples indicated that PSB NFs can maintain the sensory properties and texture properties of pork samples by controlling the reproduction of spoilage microorganisms and LM in pork samples,which laid a theoretical foundation for the application of PSB NFs in food industries.