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: CCM 4516, CCM 4517, CCM 1961, DBM 4062, CCM 8189, and 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 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 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.