Antibacterial Activity And Mechanism Of Monocaprin Against Escherichia Coli And Staphylococcus Aureus

With the increasing globalization of the food supply,food spoilage and foodborne diseases caused by foodborne pathogens have gradually become serious public health and food safety problems.To prevent great economic losses caused by foodborne pathogens,many chemical preservatives,such as potassium sorbate and sodium benzoate,have been widely used in the food industry.However,the continuous use of chemical preservatives may lead to the emergence of drug-resistant strains,which brings great potential safety risks.Therefore,it is of great significance to develop natural preservatives with high efficiency,safety and environmental protection.Monocaprin is an emulsifier with excellent antibacterial properties and is natural found in coconut oil,palm kernel oil and camphor seed kernel oil.It has the potential to be a multifunctional food additive.Our previous studies found that monocaprin has good antibacterial activity against a variety of foodborne pathogens,but its mechanism of action has not been clarified.In this study,potassium sorbate and sodium benzoate were used as positive controls to investigate the antibacterial activity of monocaprin against common foodborne pathogens.In vitro antibacterial experiments,proteomics combined with gene knockout technology,transcriptomic and untargeted metabolomic integrated analysis were used to comprehensively investigate the antibacterial mechanism of monocaprin against Escherichia coli and Staphylococcus aureus from the cell level,protein,transcriptional and metabolism levels.In addition,apple juice,carrot juice and fresh chicken breast meat were used as food models to evaluate the preservation effect of monocaprin in food during storage,and to provide a theoretical and application basis for the development of monocaprin as a green and safe food preservative.The main findings are as follows:(1)The antibacterial activity of monocaprin against Gram-negative bacteria was evaluated by measuring the minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC)and the effect of p H on MIC.The results showed that the MIC and MBC of monocaprin against E.coli,Vibrio parahaemolyticus and Shigella dysenteriae were 1.25,0.625 and 0.625 m M,respectively,which were significantly lower than those of potassium sorbate and sodium benzoate.Under different p H conditions,the antibacterial effect of monocaprin remained stable.In addition,monocaprin showed poor inhibitory effect against Salmonella paratyphi-β,Salmonella Typhimurium,Pseudomonas aeruginosa and Proteus vulgaris,with MIC values greater than 10 m M.Bactericidal kinetics analysis showed that 1.25 m M monocaprin rapidly inactivated E.coli within 15 min.The results of membrane integrity assay,permeability of inner and outer membrane,proton motive force and simulation of phospholipid bilayer interactions in vitro indicated that monocaprin rapidly penetrated the outer membrane of E.coli,then destroyed the phospholipid bilayer structure of the inner membrane,as well as dissipated the proton motive force,resulting in increases of the permeability of the outer and inner membranes.Scanning electron microscopy and transmission electron microscopy observations further confirmed that monocaprin rapidly damaged the inner membrane of E.coli,causing leakage of intracellular substances.(2)To improve the antibacterial broad-spectrum of monocaprin against Gramnegative bacteria,the micro-checkerboard dilution method was used to investigate the synergistic effect of monocaprin and plant essential oil against E.coli O157:H7 and S.Typhimurium.The results showed that the combination of monocaprin and carvacrol had synergistic antibacterial effect on both pathogens,and the fractional inhibitory concentration index(FICI)was less than 0.375.The effects of monocaprin and carvacrol on cell membrane integrity,membrane potential and ROS accumulation were detected by a variety of fluorescent probes,and the changes of inner and outer membrane permeability and cell morphology were analyzed.The results showed that carvacrol could rapidly increase the outer membrane permeability of E.coli O157:H7and S.Typhimurium,allowing monocaprin to enter the cell of both pathogens,and damage the plasma membrane,and monocaprin increased the ROS accumulation effect of carvacrol,resulting in cell death.(3)The i TRAQ proteomic was used to analyze the effect of monocaprin on protein expression in E.coli.A total of 500 differentially expressed proteins were screened,among which 235 proteins were significantly up-regulated and 265 proteins were down-regulated.Bioinformatics analysis of differentially expressed proteins showed that monocaprin could inhibit the transport and metabolism of amino acids of E.coli,promote protein synthesis,leading to excessive consumption of intracellular amino acids.It could also inhibit the carbohydrate transport,promote pyruvate oxidation,inhibit glycolysis and TCA cycle,interfere with electron transport,leading to the disorder of carbohydrate metabolism,block ATP synthesis,and ultimately cause a limitation of energy supply.Meanwhile,monocaprin could inhibit the stress response of E.coli and reduce its stress resistance.In addition,the expression of several outer membrane proteins,including Omp A,Omp C,Omp F,Omp W and Omp X,was significantly changed after treatment with monocaprin.Omp X was found to play a key role in regulating the entry of monocaprin into E.coli by constructing knockout strains and growth inhibition rate assays.The interaction between monocaprin and E.coli outer membrane phospholipase A was investigated by gene knockout,ultraviolet absorption spectroscopy and molecular docking experiments.The results showed that monocaprin bound to outer membrane phospholipase A through non-polar interaction,activating its phospholipase activity and hydrolyzing outer membrane phospholipids,resulting in the compensatory transport of phospholipids to the outer membrane,causing the consumption and contraction of the inner membrane,and "membrane wall separation" phenomenon.(4)The antibacterial effect of monocaprin against Gram-positive bacteria,including S.aureus,Bacillus subtilis and Bacillus cereus was investigated by measuring MIC and MBC,effects of p H on MIC,growth curve and time-kill curve.The results showed that monocaprin had good antibacterial activity against these bacteria,and its MIC and MBC were 0.625 m M,0.312 m M,and 0.625 m M,respectively,which were lower than those of potassium sorbate and sodium benzoate.When p H increased from3 to 9,the MIC values of monocaprin against three bacteria remained unchanged.The disturbance effect of monocaprin on bacterial membrane was evaluated by measuring the changes of cell morphology,cell membrane integrity,cell composition changes,cell membrane fatty acid composition changes,cell membrane protein content changes,and expression changes of genes related to fatty acid synthesis.Our results showed that the content of fatty acids,the ratio of SCFAs/BCFAs and the distribution of membrane proteins of S.aureus,B.subtilis and B.cereus were significantly changed after treatment with monocaprin,leading to a significant decrease in membrane fluidity.All three bacteria need to synthesize more phospholipids to reduce cell membrane damage caused by monocaprin.(5)The effects of monocaprin on m RNA and metabolite levels in S.aureus were investigated by integrated transcriptome and untargeted metabolome analysis.The results showed that 255 genes and 95 metabolites(45 in positive ion mode and 50 in negative ion mode)were significantly altered after treatment with monocaprin(0.312 m M).Correlation analysis of transcriptomic and metabolomic results revealed that these differentially expressed genes and metabolites were mainly involved in amino acid metabolism,glycolysis,pyruvate metabolism,TCA cycle,pyrimidine metabolism and other metabolic pathways.It was found that monocaprin caused increased cell membrane permeability in S.aureus,leading to the leakage of intracellular solutes such as amino acids,N-acetylated amino acids,oligopeptides and amines,and an imbalance in intracellular osmotic pressure.Monocaprin inhibited amino acid metabolism,TCA cycle and electron transport chain of S.aureus,blocked aerobic respiration and reduced virulence factor activity,leading to its growth retardation.Sublethally damaged S.aureus required increased specific protein synthesis and pyrimidine metabolism,enhanced glycolysis,activation of anaerobic respiration,and promotion of biofilm formation for survival after monocaprin stress.(6)Apple juice,carrot juice and fresh chicken breast meat were used as simulation systems to evaluate the preservation effect of monocaprin in food during storage by colony counting,physical and chemical analysis,sensory evaluation assays.The results showed that adding 0.625 m M moncaprin effectively reduced the viable counts of E.coli and S.aureus in apple and carrot juices without adverse effects on their physicochemical and sensory properties.Marinating chicken breast with 1.25-2.5 m M monocaprin for 20 min could delay bacterial proliferation,lipid oxidation and TVB-N value increase,inhibit the deterioration of sensory quality and prolong the storage time of chicken breast meat.