Listeria monocytogenes is a common foodborne pathogen causing severe listeriosis, with a mortality rate of 20–30%. This pathogen spreads through biofilm formation in the food industry. Antimicrobial peptides are widely used in food preservation to combat harmful pathogens. Thanatin, a 21-amino acid peptide from Podisus maculiventris, combats various pathogens effectively. This study aimed to evaluate the antimicrobial efficacy of thanatin peptide against L. monocytogenes ATCC 19115. The minimum inhibitory concentration (MIC) of thanatin against L. monocytogenes was determined to be 17 μg/mL. Analysis using a scanning electron microscope (SEM) confirmed its antibacterial efficacy. Thanatin treatment inhibited swimming motility, biofilm formation, auto-aggregation, cytotoxicity, hemolysin activity, adhesion, and host cell invasion by L. monocytogenes. Concentrations ranging from 1/16MIC (27%) to MIC (92%) effectively prevented biofilm formation, and existing biofilms experienced significant reduction (9.41%–80.66%) after thanatin treatment. Moreover, the thanatin peptide exhibited significant cytotoxic effects on Caco-2 cancer cells (IC50 = 27.04 μg/mL) and decreased the hemolytic activity of L. monocytogenes (2.66%–7.33%). Additionally, thanatin effectively suppressed the expression of virulence genes in L. monocytogenes, decreasing its pathogenicity. In summary, thanatin peptide exhibits beneficial antibacterial effects, suggesting its potential as a natural bacteriostatic agent for further research and practical implementation.

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