There is a growing research interest on products with antimicrobial activity. Antimicrobial polymers are one of the most surefire procedures to combat microbes. In the present study, the ability of Βeta-casein- one of the milk major self assembly proteins with high polymeric film production capability—as a fusion partner of Ib-AMP4 antimicrobial peptide was investigated. Also, the antimicrobial activities of Βeta-casein- IbAMP4 fusion protein antimicrobial against common food pathogens were assessed. The pET21a-BCN-Ib-AMP 4 construct was transformed to Escherichia coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg mL−1 fusion protein by ultrafiltration. 5 μg mL−1 H2O2 was applied for accelerating the formation of two necessary disulfide bonds. Antimicrobial assays were performed against E. coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus and Candida albicans. Results of antimicrobial tests confirmed the efficiency of BCN-IbAMP4 against all tested microorganisms. Overall, the combination of thymol plus BCN-IbAMP4 increased their antimicrobial activities. MIC, MBC, MFC, FICI and FBCI values showed strong synergistic activity between the two examined compounds. Time kill and growth kinetic studies indicated significant reduction of cell viability during first period of exposure to BCN-IbAMP4 and thymol combination.
