Identification of novel antibacterial peptides isolated from a commercially available casein hydrolysate by autofocusing technique.

Autofocusing, as a simple and safe technique, was used to fractionate casein hydrolysate based on the amphoteric nature of its peptides. The antibacterial activity of casein hydrolysate and its autofocusing fractions (A1-10) was examined against Escherichia coli and Bacillus subtilis. The basic fraction A9 exhibited the highest activity with minimum inhibitory concentration (MIC) of 150 μg/mL, whereas casein hydrolysate showed MIC values ranging from 2000 to 8000 μg/mL. The antibacterial peptides in A9 were purified by using a series of size exclusion and reversed phase chromatographies. Three peptides exhibited the most potent antibacterial activity with MIC values ranging from 12.5 to 100 μg/mL. These peptides were generated from α(s2)-casein, α(s1)-casein, and κ-casein and identified as K165 KISQRYQKFALPQYLKTVYQHQK188, I6KHQGLPQEV15, and T136EAVESTVATL146, respectively. Therefore, the results revealed that casein hydrolysate had potent antibacterial peptides that could be isolated by autofocusing technique.