Antibacterial Mechanism And Application Of 3-carene Against Escherichia Coli And Pseudomonas Aeruginosa

3-carene is the main volatile component of plant essential oils such as pepper oil.which has antibacterial effect and broad prospects in the field of food preservation.This study investigated and preliminarily discussed the antibacterial mechanism of 3-carene on Escherichia coli and Pseudomonas aeruginosa.The effects of 3-carene on the antimicrobial activity,morphological structure,nucleic acid,respiratory metabolism of the tested bacteria were studied to gradually reveal the antibacterial mechanism in combination with metabolomics.At the same time.3-carene was applied to cooked meat products for preservation experiments,which provided solid theoretical basis for the development of 3-carene as a new food preservative.The experimental results were as follows:(1)The antibacterial activity of 3-carene on Escherichia coli.Salmonella typhimurium,Pseudomonas aeruginosa and Listeria monocytogenes was determined by plate gradient dilution method.It was found that 3-carene had antibacterial effects,among the bacteria,it had better antibacterial effects against Escherichia coli and Pseudomonas aeruginosa.Thus,they were selected as tested bacteria.Growth curve indicated that 3-carene could effectively inhibit the growth of tested bacteria.(2)The cell morphology of the tested bacteria was reflected by scanning electron microscopy,the cell structure was studied by measuring the electrical conductivity,protein leakage,membrane potential and AKP content.The results showed that after 3-carene treatment,the normal morphology of Escherichia coli and Pseudomonas aeruginosa was damaged and changed.In terms of structure,the cell membrane was damaged,the membrane permeability was increased,and the membrane integrity was damaged,causing leakage of intracellular electrolytes and macromolecular substances such as protein.In addition.3-carene led to a decrease in the membrane potential of the tested bacteria and depolarization of the cell membrane,resulting in abnormal cellular metabolic activity.(3)The nucleic acid content of tested bacteria was determined by the staining method.The results showed that the nucleic acid content of Escherichia coli and Pseudomonas aeruginosa decreased after 3-carene treatment.(4)The enzyme activities of the test bacteria in the TCA cycle were determined by SDH and MDH assay kits,the organic acid content was determined by HPLC.the metabolic activity and respiratory chain dehydrogenase were measured.The results showed that after 3-carene treatment,the intracellular protein content of Escherichia coli and Pseudomonas aeruginosa decreased continuously,and the activities of SDH and MDH were inhibited,which affected TCA cycle.Pyruvate acid and acetic acid of the tested bacteria leaked and accumulated.For citric acid.Escherichia coli showed little change.Pseudomonas aeruginosa accumulated in the later period,the metabolism of bacterial organic acid was affected.In addition,the metabolic activity and respiratory chain dehydrogenase of the tested bacteria decreased significantly,the respiratory chain was damaged and the respiratory metabolic process was destroyed.(5)Through metabolomics studies,differential metabolites in Pseudomonas aeruginosa before and after 3-carene treatment were identified and screened,and metabolic pathways were analyzed.The results showed that there were 69 differential metabolites,including amino acids,terpenoids.glycerol phosphate amines,quaternary ammonium salts,organic acids,etc.Among them.24 metabolites were up-regulated and 45 metabolites were down-regulated.Differential metabolites participated in 30 metabolic pathways,and 16 metabolic pathways were selected,mainly involving amino acid metabolism and synthesis.TCA cycle,purine metabolism and pyrimidine metabolism.(6)The application of 3-carene in preservation of cooked meat products was studied by measuring the pH value and the total plate counts.The results showed that 3-carene could slow down the increase of pH value and total plate counts,and had antiseptic effect on cooked meat products.In summary.3-carene had antibacterial effect,and the antibacterial mechanism against Escherichia coli and Pseudomonas aeruginosa was:firstly.3-carene could act on the cell membrane of tested bacteria,change and destroy the normal morphology,increase the permeability of cell membrane,destroy the integrity of cell membrane,cause leakage of intracellular substances,lead to depolarization of cell membrane and abnormal metabolism of cells.Secondly.3-carene reduced nucleic acid content,inhibited nucleic acid synthesis and influenced related metabolic processes.3-carene inhibited the respiratory metabolism of the tested bacteria.TCA cycle was inhibited,respiratory chain was destroyed,and organic acid metabolism was also affected.3-carene could play an antimicrobial role by affecting 69 different metabolites and 16 related metabolic pathways in Pseudomonas aeruginosa.Eventually,the bacteria went to death.Meanwhile.3-carene could affect the growth of bacteria in cooked meat products,slow down the process of spoilage and deterioration,and play a role in preservation of cooked meat products.