Antibiofilm effect of caffeine against <i>Listeria monocytogenes</i> and <i>Escherichia coli</i> in grape and apple fruit juices

This study evaluated the antibiofilm activity of caffeine against <i>Listeria monocytogenes</i> and <i>Escherichia coli</i> within grape and apple juice matrices. Caffeine, a purine alkaloid recognized for its antimicrobial properties, was tested at the minimum inhibitory concentration (MIC) against planktonic and sessile cells. MIC values were established at 9.37 mM for <i>E. coli</i> and 37.5 mM for <i>L. monocytogenes</i>. Biofilm formation was evaluated through crystal violet staining, both when bacterial cells were exposed to caffeine during the initial stages of biofilm development (co-treatment) and after the biofilm had been established (post-treatment). The extracellular polymeric substances (EPS) produced within the biofilms were quantitatively measured. Bacterial viability was assessed by time-kill kinetics assays and further visualized by scanning electron microscopy (SEM). Caffeine treatment significantly inhibited biofilm formation by 98.1% for <i>E. coli</i> and 98.7% for <i>L. monocytogenes</i>, and by 98.8 and 99.0%, in co-treatment and post-treatment, respectively. No consistent correlation was observed between EPS quantity and biofilm inhibition. SEM analysis confirmed caffeine-induced structural disruption of the biofilm matrix and damage to bacterial cell integrity. This investigation introduces a novel application of caffeine as an anti-biofilm agent in unpreserved fruit juice systems and demonstrates, for the first time, its efficacy against foodborne pathogens.