Purification and characterization of an antioxidative peptide from enzymatic hydrolysate of yellowfin sole (Limanda aspera) frame protein

In order to utilize yellowfin sole ( Limanda aspera) frame protein (YFP), which is normally discarded as industrial waste in the process of fish manufacture, yellowfin sole frame protein hydrolysates (YFPHs) were fractionated using an ultrafiltration (UF) membrane system following hydrolysis with pepsin and mackerel intestines crude enzyme (MICE). The YFPHs were separated into five major types, YFPH-I (30-10 kDa), YFPH-II (10-5 kDa), YFPH-III (5-3 kDa), YFPH-IV (3-1 kDa), and YFPH-V (below 1 kDa) by using UF membranes with molecular weight cut-offs of 30, 10, 5, 3, and 1 kDa, respectively. The antioxidative activity of the YFPHs was investigated and compared with that of a natural antioxidant, alpha-tocopherol, used as a reference. Furthermore, the fraction showing strong antioxidative activity was isolated from the YFPHs using consecutive chromatographic methods on an SP-Sephadex C-25 column, on a Sephadex G-75 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. The molecular mass of the antioxidant was identified as 13 kDa using HPLC on a gel permeation chromatography (GPC) column, and the antioxidative peptide was composed of 10 N-terminal amino acid residues, RPDFDLEPPY.