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

In this study, the antimicrobial effect of <i>Citrus limon</i> peel extract against <i>Listeria monocytogenes</i> 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 <i>L. monocytogenes</i> proteins involved in colonization and intracellular survival. The in vitro experiments demonstrated that the anti-<i>Listeria</i> activity of <i>Citrus limon</i> peel extract is primarily attributed to limonene and phenolic compounds. In fermented milk stored at 4 °C for 7 days, the addition of <i>Citrus limon</i> peel extract resulted in a 2 Log reduction of <i>L. monocytogenes</i> 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 <i>L. monocytogenes</i> from 5.95 to 3.67 log CFU/mL was observed in fermented milk supplemented with a 2×MIC level of <i>Citrus limon</i> peel extract during storage at 4 °C. The findings from all three approaches highlighted that the inhibitory effect of <i>Citrus limon</i> peel extract against <i>L. monocytogenes</i> 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 <i>Citrus limon</i> peel extract also enhances the milk antioxidant capacity and total organic acids content.