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Foodborne disease caused by Listeria monocytogenes is a major global food safety problem. A potential solution is the antimicrobial development of the highly bioactive natural product juglone, yet few studies exist on its antibacterial mechanism against L. monocytogenes. Thus, we aimed to elucidate the antibacterial mechanism of action of juglone against L. monocytogenes by determining the resultant cell morphology, membrane permeability, membrane integrity, and proteome changes. The minimum inhibitory concentration of juglone against L. monocytogenes was 50 μg/mL, and L. monocytogenes treated with juglone had longer lag phases compared to controls. Juglone induced L. monocytogenes cell dysfunction, leakage of potassium ions, and membrane potential hyperpolarization. Confocal laser scanning microscopy and field-emission-gun scanning electron microscope assays revealed clear membrane damage due to juglone treatment. Fourier transform infrared analyses showed that L. monocytogenes responded to juglone by some conformational and compositional changes in the molecular makeup of the cell membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that juglone either destroyed proteins or inhibited proteins synthesis in L. monocytogenes. Therefore, our findings established juglone as a natural antibacterial agent with potential to control foodborne L. monocytogenes infections.

Walnut green husk (WGH) is rich in natural compounds and is valued as a potential source of antioxidant and antimicrobial properties. In this study, the antioxidant and antimicrobial activities of petroleum ether polar fraction, dichloromethane polar fraction, ethyl acetate polar fraction (EAPF), and nbutanol polar fraction from WGH were analyzed. The results showed that EAPF exhibited the highest total flavonoid content (65.74 ± 1.01 mg rutin equivalents [RE]/g dry weight [DW]) and total phenol content (48.73 ± 1.09 mg gallic acid equivalent [GAE]/g DW), with the highest 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical (•OH), and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonate scavenging activity compared with other fractions. EAPF also showed good antibacterial activity against Escherichia coli and Bacillus cereus vegetative cells, with a diameter of inhibition zones of 33.5 and 37.6 mm, respectively, a minimum inhibitory concentration of 31.25 mg/ml and a minimum bactericidal concentration of 62.5 mg/ml, which inhibited the growth of both bacteria.Analysis of the antibacterial mechanism demonstrated that EAPF damaged the integrity of the cell membrane, increased the membrane permeability, and triggered the leakage of intracellular material. In addition, ultrahigh performance liquid chromatography-tandem with mass spectrometry analysis revealed that 8 polyphenols and 14 flavonoids were mainly present in EAPF, such as chlorogenic acid (C 16 H 18 O 9 ), gallic acid (C 7 H 6 O 5 ), vanillic acid (C 8 H 8 O 4 ), ferulic acid (C 10 H 10 O 4 ), epicatechin (C 15 H 14 O 6 ), catechin (C 15 H 14 O 6 ), hesperetin (C 16 H 14 O 6 ), naringenin (C 15 H 12 O 5 ), hyperin (C 21 H 20 O 12 ), luteolin (C1 5 H 10 O 6 ), and so on. Therefore, WGH had the potential to be developed as a natural antioxidant and antibacterial material.K E Y W O R D S antimicrobial mechanism, antioxidant activity, chemical compounds, walnut green husk Meihua Xi and Yujie Hou contributed equally to this work.

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