Antibiofilm effect of caffeine against Listeria monocytogenes and Escherichia coli in grape and apple fruit juices

This study evaluated the antibiofilm activity of caffeine against Listeria monocytogenes and Escherichia coli 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 E. coli and 37.5 mM for L. monocytogenes. 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 E. coli and 98.7% for L. monocytogenes, 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.