Isolation and identification of antioxidant peptides derived from whey protein enzymatic hydrolysate by consecutive chromatography and Q-TOF MS

To isolate and identify antioxidant peptides from enzymatically hydrolysed whey protein, whey protein isolate was hydrolysed by different protease (trypsin, pepsin, alcalase 2.4L, promatex, flavourzyme, protease N). The hydrolysate generated by alcalase 2.4L had the highest antioxidant activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radicals and in a linoleic acid peroxidation system induced by Fe2+. The IC50 values of DPPH and superoxide radical scavenging activities of the hydrolysate decreased significantly (6.89 and 38.88%, respectively) after treatment with macroporous adsorption resin. Seven different peptides showing strong antioxidant activities were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography and high-performance liquid chromatography. The molecular mass and amino acids sequences of the purified peptides were determined using a Quadrupole time-of-flight mass spectrometer (Q-TOF MS). One of the antioxidative peptides, Trp-Tyr-Ser-Leu, displayed the highest DPPH radical scavenging activity (IC50 = 273.63 mu M) and superoxide radical scavenging activity (IC50 = 558.42 mu M). These results suggest that hydrolysates from whey proteins are good potential source of natural antioxidants.