Preparation and Identification of the Novel Umami Peptides from Sea Cucumber Viscera Hydrolysate

Sea cucumber viscera by-products are abundant but remain underutilized. Although the development of umami peptides from marine by-products has been well-reported, sea cucumber viscera have received less attention. In this study, an umami-rich hydrolysate was prepared from sea cucumber viscera through synergistic dual-enzyme hydrolysis. Under optimal conditions, the co-hydrolysis using Flavourzyme and aminopeptidase yielded extraction rates of 69.38% for solids, 67.29% for protein, and 66.96% for total sugar, and produced a 1.75-fold higher umami signal intensity (electronic tongue) than the single-enzyme (Flavourzyme) hydrolysate. The target umami fraction was enriched through sensory-guided separation combined with ultrafiltration and ion-exchange chromatography. Thirty-three umami peptides, predominantly derived from actin hydrolysis, were identified in this fraction via peptidomics and virtual screening. Based on docking simulations against the umami receptor T1R1/T1R3, two peptides (DFLDDGPG and SDTGNFGF) with the lowest docking scores were selected. The predictions revealed that two peptides bind to the T1R3 subunit via hydrogen bonds and π-related interactions. The umami-enhancing effect of peptide DFLDDGPG in salty systems was demonstrated by a trained panel (<i>n</i> = 10) across concentration ranges of 0.1-1.0 mg/mL peptide and 0.1-1.0% NaCl, with a positive correlation validated by RSM and ANOVA (<i>p</i> < 0.05). This study identified novel umami peptides from sea cucumber by-products as promising candidates for natural, low-sodium flavor enhancers.