Summary Ultrasound was incorporated to processing of fish protein hydrolysate to facilitate homogenate pretreatment and enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein. Their effects on Flavourzyme hydrolysis and biological activities of the tilapia hydrolysate were examined. The ultrasound‐assisted hydrolysis caused reduction in degree of hydrolysis ranging from 23% to 35% relative to that of the conventional process. The 70 W ultrasound‐assisted hydrolysis process increased DPPH radical‐scavenging activity and reducing power of tilapia hydrolysate prepared from the non‐pretreatment homogenate by 33% and 45%, respectively. All hydrolysates have no cytotoxicity on RAW264.7 cell lines at the maximum concentration of 20 mg protein mL−1. The 70 W ultrasound pretreatment at 30 and 45 min combined with conventional hydrolysis is the suitable condition for producing tilapia hydrolysate with nitric oxide inhibitory and antioxidative activities on RAW264.7 cell lines, respectively. As a result, ultrasound could be applied to enzymatic protein hydrolysis either as pretreatment or during the hydrolysis.

Nitric oxide (NO)-inhibitory and antioxidative activities of tilapia hydrolysates were prepared using ultrasound pretreatment at 70 W for 30 and 45 min, respectively, followed by Flavourzyme hydrolysis for 1 h. Both hydrolysates were fractionated using size exclusion chromatography on Sephadex G-25 column and purified by RP-HPLC. The amino acid sequence of the most potent and purified fractions was determined using LC/MS/MS. The antioxidant peptide (KAFAVIDQDKSGFIEEDELKLFLQNFSAGARAGDSDG DGKIGVDEFAALVK, MW: 6334.49 KDa) and NO-inhibitory peptide (AFAVIDQDKSGFIEEDELK LFLQNFSAGARAGDSDGDGKIGVDEFAALVK, MW: 6309.49 Da) produced no cytotoxicity in RAW264.7 macrophage cell lines at the concentration of 100 mg mL À1 . The purified peptides at the concentration 100 lg mL À1 possessed antioxidative and NO-inhibitory activities 83.0 AE 1.1% and 40.9 AE 0.2%, respectively, which were about 100 times those of their counterpart crude hydrolysates.