Studies On The Antibacterial Activity And Subcellular Mechanism Of Hexyl Ferulate Against Listeria Monocytogenes

Ferulic acid(FA)is a kind of renewable phenolic aromatic compound,which is widely found in Angelica sinensis,Ferula fukanensis K.M.Shen,cohosh and other Chinese herbs.It has a broad biological as well as pharmacological activities as a result of the phenobroad lic hydroxyl and carbon-carbon double bond groups in the molecules.FA can be used in various aspects such as anti-thrombus,anti-aging,anti-tumor,improving human immunity etc.However,it is difficult for it to penetrate the liposoluble bimolecular layer in biofilm due to its short alkyl group and poor fat-solubility,which limits its application in complex food system.Therefore,in order to improve its lipophilicity and broaden its application in food systems,particularly that are rich in oils and fats,the current study aims to modify FA by attachment of fatty alcohol via enzymatic and chemical methods.Firstly,hexyl ferulate(FAC6)was prepared by both chemical method(Knoevenagel)and biological method(lipase catalytic method).The product was purified by thin-layer chromatography and silica gel column chromatography.The structure of FAC6 was subsequently characterized and identified by NMR,MS and IR.Secondly,the antimicrobial activity of FAC6 against Listeria monocytogenes was studied.The MIC and MBC of FAC6 against L.monocytogenes were 0.1 mM and 0.4 mM respectively.The time-killing kinetic curve and growth curve showed that the inhibitory effects of FAC6 on L.monocytogenes was in a concentration-and time-dependent manner.Meanwhile,the autolytic activity of L.monocytogenes increased as a function of time.Next,the subcellular mechanism of FAC6 on L.monocytogenes was further discussed through the influences of FAC6 on the cell membrane,membrane protein of L.monocytogenes,and the interaction between FAC6 and DAN.The results of PI uptake and leakage of intracellular macromolecular proteins and nucleic acids suggested that FAC6 significantly changed the integrity and permeability of L.monocytogenes cell membrane and the Zeta-potential of cell membrane surface was also found to be hyperpolarization.Moreover,scanning electron microscopy(SEM)showed the disappearance of the cell wall of L.monocytogenes,the formation of holes on the cell membrane as well as the cell cytolysis after FAC6 pretreatment.Transmission electron microscopy(TEM)observation showed that the surface of the cells exposed to FAC6 was ruptured and the cytoplasm of the cells was lost.All these results suggest that the cell membrane is one of the sites of FAC6 acting on L.monocytogenes.In terms of the effect of FA6 on the composition of membrane protein,after 6 hours of treatment of FAC6 on extracted membrane protein of L.monocytogenes,it was indicated in SDS-PAGE that the protein bands became either lighter,deeper,partially disappeared,or even forming new bands.In addition,fluorescence spectra showed that FAC6 quenched the phenylalanine(Phe),tryptophan(Trp),tyrosine(Tyr)residues on the surface of the membrane,accompanied by red shift,which indicated the alteration of microenvironment and the conformation of membrane proteins.The results of the interaction between FAC6 and genomic DNA(using UV-Vis absorption spectroscopy,fluorescence spectroscopy,melting point and molecular docking)showed that FAC6 and DNA interact through groove binding.Finally,the effect of FAC6 on the total number of Sturgeon caviar during storage was studied.The results showed that FAC6 could significantly inhibit the growth of L.monocytogenes.