Antimicrobial Susceptibility and Antibacterial Mechanism of Limonene against Listeria monocytogenes

Abstract

Limonene is a monoterpenoid compound, which is founded in a lot of plants' essential oils with good antibacterial activity against food-borne pathogens, but it has an ambiguous antimicrobial susceptibility and mechanism against Listeria monocytogenes (L. monocytogenes). In this study, the antimicrobial susceptibility of Limonene to L. monocytogenes was studied, and some new sights regarding its antibacterial mechanism were further explored. Scanning electron MICroscopy (SEM) verified that limonene caused the destruction of the cell integrity and wall structure of L. monocytogenes. The increase in conductivity and the leakage of intracellular biomacromolecules (nucleic acids and proteins) confirmed that limonene had an obvious effect on cell membrane permeability. The results of Propidium Iodide (PI) fluorescence staining were consistent with the results of the conductivity measurements. This indicated that limonene treatment caused damage to the L. monocytogenes cell membrane. Furthermore, the decrease in ATP content, ATPase (Na + K + -ATPase, Ca 2+ -ATPase) activity and respiratory chain complex activity indicated that limonene could hinder ATP synthesis by inhibiting the activity of the respiratory complex and ATPase. Finally, differential expression of proteins in the respiratory chain confirmed that limonene affected respiration and energy metabolism by inhibiting the function of the respiratory chain complex.Molecules 2020, 25, 33 2 of 15 terpenes in nature and is widely found in the volatile oils of various plants (black pepper, lemon and orange, etc.) [9]. Limonene has broad application prospects in antibacterial and food preservation due to its broad-spectrum bactericidal activity, safety, and low toxicity [10]. Moreover, D-limonene can significantly inhibit gram-negative and gram-positive bacteria as well as fungal activity [11]. In addition, many researchers have confirmed that D-limonene can effectively inhibit the growth of spoilage bacteria, such as Aspergillus niger, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli [12,13]. Limonene, which is the main ingredient of lemon essential oil, was found to have antimicrobial activities against L. monocytogenes in minced beef meat [14]. However, few studies have investigated the antibacterial mechanism of limonene.The aims of this study were to determine the antibacterial susceptibility and its antibacterial mechanism of limonene against L. monocytogenes. The minimum inhibitory concentration (MIC) was used to evaluate the anti-L. monocytogenes susceptibility of limonene. The growth curves of the bacterial were determined for evaluating the effect of limonene on the growth and reproduction of L. monocytogenes. The mechanism of action of limonene was explored by analysing its influence on the cell morphology, membrane permeability and changes in the protein, nucleic acid, ATP, ATPase (Na + K + -ATPase, Ca 2+ -ATPase), respiratory chain complex I~V, and differential protein expression of the respiratory chain complex of L. monocytogenes. Resu...

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