Abstract: In order to obtain highly bioactive peptides and to achieve high-valued utilization of aquatic product proteins, this study determined the optimal conditions for the hydrolysis of tuna dark muscle by a mixture of trypsin and alcalase using combination of one-factor-at-a-time method and response surface methodology. The molecular mass of peptides in the prepared hydrolysate was measured by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), and the functions of the dominant peptides were predicted using the CDocker module in Discovery Studio. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging capacity and angiotensin-converting enzyme (ACE) inhibitory activity of the peptides synthetized in vitro were compared with those of the hydrolysate. The optimal processing conditions that provided maximal degree of hydrolysis of 61.01% were determined as 1:1, 1:4, 51 ℃, 2% and 5 h for mass ratio between the two enzymes, solid-to-liquid ratio, hydrolysis temperature, enzyme concentration and time, respectively. Molecular docking showed that the dominant peptides VSSK and EPR could separately bind to Kelch-like ECH-associated protein 1 (Keap1) and ACE to exert antioxidant and hypotensive effects, respectively. Further, in vitro tests showed that the half maximal inhibitory concentration (IC50) values of the hydrolysate, VSSK and EPR were 0.204, 3.050 and 3.368 mg/mL for DPPH radical scavenging activity; and 2.321, 2.914 and 4.857 mg/mL for ACE inhibitory activity, respectively. These data illustrated that the hydrolysate had stronger antioxidant and hypotensive effects. The results of this study provide a theoretical basis for the high-value utilization of tuna dark muscle.

Key words: tuna dark muscle; hydrolysis; polypeptide; molecular docking; in vitro activity