Antimicrobial Activity of TP4 Recombinant Peptide Alone and in Combination With ZnO NPs on Foodborne Bacteria: Potential as a Milk Preservative

Bacterial resistance, including resistant foodborne pathogenic bacteria, is developing daily, thus becoming a growing public health concern worldwide. Antimicrobial peptides (AMPs) such as tilapia piscidin 4 (TP4) are a class of small peptides widely present in nature that are promising antimicrobial agents that could potentially be considered food preservatives. This study is aimed at investigating the effect of TP4 alone and in combination with zinc oxide nanoparticles (ZnO NPs) on foodborne bacteria in milk. First, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of TP4 and its combination with ZnO NPs (synergistic effect) were measured against four standard foodborne bacteria using the microdilution and checkerboard methods, respectively. According to the results, TP4 showed a high antibacterial effect against four tested strains. Also, TP4 combination with ZnO NPs showed a relative synergistic effect against three tested strains. Then, a time-kill assay was performed to evaluate the antibacterial effect of TP4 over time on selected bacteria. The results showed that within 24 h, a TP4 concentration of ≥1 × MIC prevented the growth of Gram-negative and Gram-positive bacteria. The microbial load of pasteurized and raw milk was also associated with population reduction against the 2 × MIC concentration of TP4. To evaluate the cytotoxicity of TP4, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis was performed on the human embryonic kidney-293 (HEK-293) cell line and the half-maximal inhibitory concentration (IC50) was evaluated as 71.61 μg/mL, indicating the nontoxicity of this peptide. Finally, the results showed that TP4 peptide reduces the microbial load of milk, and due to its safety, it can be used as a food preservative.