Comparing media dependent antimicrobial activity of random peptide mixtures in vitro and in mouse model

Antimicrobial efficacy is strongly influenced by growth medium, yet this dependence is often not accounted for in standard in vitro testing. This consideration is particularly relevant for cultured meat production, where bacterial control must be achieved in complex cell culture media without antibiotics due to tight regulations. Random Peptide Mixtures (RPMs) are potential non-antibiotic alternatives to traditional antibiotics; however, their selectivity in cell culture media remains challenging. In this study, we examined how altering the hydrophobic to cationic residue ratio of RPMs influences antimicrobial activity and mammalian cell viability under conditions relevant to cultured meat production. A series of 20-mer RPMs with varying Leucine:Lysine (L:K) ratios was synthesized and evaluated against Gram-negative and Gram-positive model bacteria in both LB and cell culture media (DMEM). Lower L:K ratios significantly reduced cytotoxicity toward bovine mesenchymal stem cells (MSCs) without compromising antimicrobial activity in DMEM. Notably, activity against S. aureus increased dramatically, with MIC values below 1 µg/mL. Moreover, selected RPMs also demonstrated strong antimicrobial activity in vivo, where their efficacy more closely resembled the activity observed in DMEM than in the traditional LB medium, highlighting the importance of media composition for the discovery and evaluation of new antimicrobial agents. Overall, these findings demonstrate the critical impact of hydrophobic to cationic residues ratio and media composition on AMPs activity and showcase new RPM as promising non-antibiotic antimicrobial agents for cultured meat production.