Caffeine as a natural inhibitor of <i>Salmonella</i> biofilms in fruit juices

Caffeine holds promise for applications in food safety due to its antioxidant and antibacterial properties. Given rising antimicrobial resistance, its natural antimicrobial potential is valuable for controlling foodborne pathogens and reducing reliance on synthetic preservatives. This study aimed to explore caffeine as an alternative to control Salmonella biofilms in fruit juice substrates. Salmonella Enteritidis and S. Typhimurium biofilm were developed in brain heart infusion (BHI) broth (control) and grape and apple juice before and after caffeine application. Biofilm inhibition was quantified by crystal violet staining, exopolysaccharide (EPS) production, and visualization through confocal and scanning electron microscopy. Swimming motility assays assessed caffeine's impact on bacterial motility. Both strains formed biofilms in the tested juices. The minimum inhibitory concentration (MIC) of caffeine was 9.37 mM mL-1 for S. Typhimurium and 18.75 mM mL-1 for S. Enteritidis. Biofilm inhibition was observed for treatments before and after caffeine application, with varying levels depending on the matrix. EPS production and inhibition were higher in biofilms formed in grape and apple juices compared to the control (BHI). Sub-inhibitory concentrations of caffeine reduced motility in both strains. These findings suggest that caffeine may be a promising approach to control Salmonella biofilms in the food industry.