Antimicrobial and Antifungal Activities of Proline-Based 2,5-Diketopiperazines Occurring in Food and Beverages and Their Synergism with Lactic Acid

2,5-Diketopiperazines (2,5-DKPs), naturally occurring in food and beverages, have demonstrated notable antimicrobial properties. However, their synergism with other secondary metabolites in real food matrices remains largely unexplored. In our study, a group of microbially produced 2,5-DKPs, including proline-based dilactams (Pro-DKPs), was synthesized and evaluated for their efficacy against common foodborne pathogens: Staphylococcus aureus CCM 4516, Escherichia coli CCM 4517, Pseudomonas aeruginosa CCM 1961, Penicillium chrysogenum DBM 4062, Aspergillus niger CCM 8189, and Fusarium culmorum DMF 0109. We also investigated the impact of the polarity of 2,5-DKPs on their antimicrobial effect. Among the four synthesized 2,5-DKPs, cyclo(l-Leu-l-Pro) (Pro-DKP-1) inhibited the growth of F. culmorum DMF 0109 by up to 83%, as determined using the poisoned plate method. Further experiments investigated the synergistic effects of Pro-DKPs in combination with lactic acid at food-relevant concentrations. The addition of lactic acid considerably enhanced the antimicrobial activity of all three Pro-DKPs, with inhibitions reaching up to 99% against F. culmorum DMF 0109. Our findings suggest that employing commercial starter cultures capable of producing 2,5-DKPs, including Pro-DKPs, may offer a promising strategy for extending the shelf life of food products and beverages.