Thermal pretreatment-assisted dual-enzyme hydrolysis of steamed skipjack tuna dark muscle for safety-controlled bioactive hydrolysate production

Steamed skipjack tuna dark muscle (STDM), a protein-rich by-product of tuna canning, is underutilized because of microbial contamination and histamine-related safety concerns. This study developed a safety-controlled hydrolysate through heat pretreatment and sequential Alcalase (AL)-Flavourzyme (FL) hydrolysis. An AL:FL ratio of 4:1 (w/w), 20 mg total enzyme, and sequential addition yielded 56.38% hydrolysis after 5 h. Heat treatment reduced total aerobic bacteria, coliforms, and Escherichia coli below detection limits and was associated with lower relative histamine-forming bacteria-associated DNA signals. It limited further histamine and biogenic amine accumulation during room-temperature exposure without removing preformed amines. Hydrolysis increased total and free amino acids approximately 2.5- and 5.6-fold, respectively, in the resulting hydrolysates. Hydrolysates exhibited over 93% in vitro ABTS radical scavenging activity and 92.42-92.76% in vitro ACE inhibitory activity, alongside altered taste and aroma profiles. These findings support potential use of STDM hydrolysates as protein-based food ingredients for food applications.