Defatted soybean meal, a by-product of soybean oil extraction, was pretreated by ultrasonic and hydrolyzed with neutrase. The DSMH pretreated at 400 W of ultrasonic power, identified to possess the strongest antioxidant activity, was fractionated according to molecular weight into three fractions of DSMH-I (>10 kDa), DSMH-II (5–10 kDa), and DSMH-III (<5 kDa) using ultrafiltration. The fraction, DSMH-III (<5 kDa), exhibited the highest antioxidative activity and was further purified using ion-exchange chromatography. The DSMH-III was separated into five fractions (A, B, C, D, and E). Fraction C with molecular weight of 2434 Da exhibited the strongest free radical scavenging, which was evidenced by the electron spin resonance of 1,1-diphenyl-2-pycryl hydrazyl and hydroxyl radicals. Fraction C was subjected to reverse-phase high performance liquid chromatography and the sequences of the highest activity peptide were determined by liquid chromatography tandem mass spectrometry. The strongest antioxidant activity peptide had the amino acid sequence of Glu–Glu–Gln–Glu–Trp–Pro–Arg–Lys–Glu–Glu–Lys. In conclusion, ultrasonic treatment and ultrafiltration could enhanced antioxidant activity of DSMH.
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