The Study On The Antibacterial Activity And Mechanism Of Citrus Essential Oils Against Listeria Monocytogenes

Listeria monocytogenes is an important foodborne pathogen that could cause listeriosis and poses potential threats to human health and safety.L.monocytogenes is able to survive in many harsh environments such as low temperature,low p H and high salty,meanwhile,it is able to form biofilm by adhering to the surfaces of food industrial equipment.In this study,fourteen species of citrus essential oils(EOs)from the main cultivated varieties in China were extracted,and their chemical composition were analyzed by GC-MS,the antimicrobial activity of different varieties of EOs were evaluated and those varieties with strong antimicrobial activity were selected,then the relationship between the components of EOs and antimicrobial ability was analyzed by several statistical methods.Through microscopic observation,transcriptomics and proteomics,the antibacterial activity and mechanism of EOs from Citrus Changshan huyou Y.B.Chang(Huyou EO),Citrus.medica var.sarcodactylis Swingle(Fingered citron essential oil,FCEO)and Linalool against L.monocytogenes were explored.The main results are as follows:(1)The chemical composition and antimicrobial activities of fourteen species of citrus EOs from the main cultivated varieties in China were evaluated and compared.A total of more than 200 components were identified,which are mainly composed of terpenes,alcohols,aldehydes,esters,ketones and acids.Antimicrobial ability of fourteen EOs and seven major components were tested against six bacteria and two fungi.The susceptibility of Gram-positive bacteria tested was observed to be greater than Gram-negative bacteria,and the fungi tested were relatively more susceptive than bacteria.Three groups(Strong,Medium and Weak)were divided according to the antimicrobial ability,three characters and tendencies were found out to correlate chemical composition with antimicrobial activity of different citrus EOs.Huyou EO and FCEO exhibited the strongest antimicrobial activity against all tested strains.Linalool showed the strongest antimicrobial activity against all microorganisms except Pseudomonas aeruginosa.(2)Huyou EO exhibited strong antibacterial activity against L.monocytogenes.Firstly,Huyou EO exhibited significantly antimicrobial ability against planktonic L.monocytogenes,both of scanning electron microscopy(SEM)and transmission electron microscopy(TEM)observations confirmed the physical damage,considerable morphological alteration and the leakage of intracellular constituents.Secondly,Huyou EO was able to destroy preformed biofilm through decreasing the biomass and cellular activity.SEM,confocal scanning laser microscopy(CLSM)and COMSTAT analysis revealed the extensive disruption and architectural changes of biofilm.Thirdly,the potential antimicrobial mechanisms were demonstrated by RNA sequencing.A total of2804 genes were differentially expressed with 1709 genes downregulated and 1095 genes upregulated.Enriched GO analysis identified 8 terms related to cellular components(including intracellular part,cyt oplasmic part,cellular component et al.),50 terms for biological processes(including reductive tricarboxylic acid cycle,carbon fixation and organic hydroxy compound transport et al.)and 21 terms for molecular functions(including organic hydroxy compo und transmembrane transporter activity,sulfur compound transmembrane transporter activity and inorganic anion transmembrane transporter activity et al.).(3)FCEO have strong antibacterial activity against L.monocytogenes.Firstly,Obvious alterations of cell morphology were observed by SEM and TEM after exposed to FCEO.Secondly,FCEO exhibited strong anti-biofilm activity on both inhibiting biofilm formation and eradicating preformed biofilm,significantly increased the metabolic active cells in the biofilm,destroyed the intact architecture of biofilm,reduced biofilm biomass,thickness and substratum coverage.Thirdly,from the data of RNA sequencing,a total of 1553 genes were differentially expressed with 794 genes down-regulated and 759 genes up-regulated.GO analysis demonstrated many potential cell responses,including metabolic process,cellular process,single-organism process,cell part,membrane,catalytic activity,binding,and transporter activity.KEGG analysis suggested that L.monocytogenes responded and adapted by(1)increasing motility through the enhancement of flagella rotation;(2)promoting cell tumbles and re-orientating to escape from FCEO;(3)enhancing the uptake of carbohydrates from environment to gain more energy;(4)changin g the uptake of several metallic cations,including iron,zinc,cobalt,and nickel.(4)Linalool showed strong antibacterial activity against L.monocytogenes.Firstly,Linalool exhibited strong anti-listeria activity in the planktonic stage.Seven changi ng stages of cells were classified at microscopic level by SEM and TEM observations.Mesosome-like structures were observed in L.monocytogenes after linalool treatment and its process was explored.Secondly,Linalool also showed significant anti-biofilm activity through both dispersal and killing of cells in the biofilm.Thirdly,from the data of RNA sequencing,a total of 2824 genes were differentially expressed with 1897 genes down-regulated and 927 genes up-regulated.Enriched GO analysis identified several GO terms related to cellular components(including intracellular,cytoplasmic and cell components et al.),biological processes(including metabolic process,single-organism process,single-organism transport et al.),and molecular functions(includin g cation binding and metal ion binding,catalytic activity et al.).Fourthly,comparative proteome analysis was carried out to unravel potential mechanisms of action for linalool.A total of 445 proteins(211 genes down-regulated and 234 genes up-regulated)were differentially expressed.31 GO terms were identified by GO enrichment analysis,which included 12 cellular components(including biological adhesion,biological regulation,carbon utilization et al.),10 biological processes(including cell,cell p art,extracellular region et al.),and 9 molecular functions(including antioxidant activity,binding,catalytic activity et al.)61 KEGG pathways were identified by KEGG enrichment analysis,which included 6 cellular process pathways,6 environmental information processing pathways,3 human disease pathways,40 metabolic pathways,and 2 organic system pathways.