Gel Filtration Chromatography-Guided Sequential Hydrolysis of Sthenoteuthisoualaniensis Protein: Peptide Distribution and Functional Properties

The compact muscle architecture and poor aqueous dispersibility of Sthenoteuthis oualaniensis protein limit its use as a food ingredient. This study developed a gel filtration chromatography (GFC)-guided papain–alcalase sequential hydrolysis strategy using target-window peak area (Atarget) as a peptide-distribution response. Box–Behnken optimization identified an enzyme dosage of 1110 U/g, a papain/alcalase mass ratio of 3:5, and 8 h hydrolysis, yielding an Atarget of 0.0648 ± 0.0012 a.u. O-phthaldialdehyde (OPA)-derived degree of hydrolysis (DH) values were 10.72 ± 0.08%, 35.32 ± 0.12%, and 25.03 ± 0.37% for SPH-Pap, SPH-Alc, and SPH-opt, respectively, showing that the highest Atarget did not coincide with the highest DH. The essential-to-total amino acid ratio remained 38.18–39.20%, while lysine and methionine changed modestly. SPH-opt maintained high solubility across a broad pH range and exhibited a peptide profile distinct from those of the single-enzyme hydrolysates. Radical-scavenging capacity was assessed only post-optimization. At 5 mg/mL, SPH-opt showed DPPH and ABTS scavenging rates of 23.75% and 22.84%, respectively; without external standards, these values support only relative within-study comparisons. Overall, Atarget and DH provided complementary information on peptide distribution and bond cleavage, and no causal relationship between Atarget and radical-scavenging capacity was established.