Assessment of the Anti- Listeria Effect of Citrus limon Peel Extract In Silico, In Vitro, and in Fermented Cow Milk During Cold Storage

Abstract

In this study, the antimicrobial effect of Citrus limon peel extract against Listeria monocytogenes was analyzed in silico, tested in vitro, and validated in fermented cow milk during cold storage. The in silico analysis revealed that 4,5-di-O-caffeoyquinic acid interacts with L. monocytogenes proteins involved in colonization and intracellular survival. The in vitro experiments demonstrated that the anti-Listeria activity of Citrus limon peel extract is primarily attributed to limonene and phenolic compounds. In fermented milk stored at 4 °C for 7 days, the addition of Citrus limon peel extract resulted in a 2 Log reduction of L. monocytogenes compared to the control. Using the Baranyi and Roberts model, a significant decrease in the maximum growth rate (-0.021 h^-1) and the concentration of L. monocytogenes from 5.95 to 3.67 log CFU/mL was observed in fermented milk supplemented with a 2×MIC level of Citrus limon peel extract during storage at 4 °C. The findings from all three approaches highlighted that the inhibitory effect of Citrus limon peel extract against L. monocytogenes is primarily due to chlorogenic acid derivatives, especially 4,5-di-O-caffeoyquinic acid, and limonene. Beyond its antimicrobial properties, the supplementation of fermented milk with Citrus limon peel extract also enhances the milk antioxidant capacity and total organic acids content.

Keywords: Citrus limon peel extract; Listeria monocytogenes; docking visualization; fermented milk.

Figure 1

Two-dimensional and three-dimensional binding interaction of 4,5-di-O-caffeoyquinic acid toward the active site of (a) PDB: 1XEU, (b) PDB: 2WQV, (c) PDB: 4L3A, (d) PDB: 5KZS, (e) PDB: 6TIF, and (f) PDB: 7WJP.

Figure 2

Effect of LPE at different concentrations (1, 2, and 4×MIC) on Listeria monocytogenes in vitro. LM: Listeria monocytogenes, MIC: minimum inhibitory concentration.

Figure 3

Survival of L. monocytogenes in fermented milk samples with 2×MIC of LPE (25 mg/mL) or without LPE during storage at 4 °C. LPE: lemon peel extract; LM: L. monocytogenes.

Figure 4

Curves of lactic acid bacteria (a) and mesophilic bacteria (b) in fermented milk, inoculated or not inoculated with L. monocytogenes and with or without LPE (2×MIC) during storage at 4 °C. LPE: Citrus limon peel extract; LM: L. monocytogenes.

Figure 5

Changes in pH (a) and acidity (b) of fermented milk samples with or without lemon peel extract (2×MIC) during storage at 4 °C. Different letters indicate significant differences between samples (p < 0.05, Tukey’s test). LPE: lemon peel extract.