Comparative characterization and nanoencapsulation of seahorse (Hippocampus sp.) protein fractions: Amino acid profile, gastrointestinal release behavior, and antioxidant retention

Seahorses are high-value marine organisms widely used in traditional medicine; however, bioactive peptides from marine invertebrates remain susceptible to proteolytic degradation during gastrointestinal transit, limiting their bioavailability for functional food applications. This study aimed to comparatively characterize three seahorse protein fractions: enzymatic hydrolysate (SPH), pH-shift isolate (SPI), and protein concentrate (SPC) and evaluate their nanoencapsulation performance using chitosan–tripolyphosphate (TPP) ionic gelation. Among the fractionation conditions evaluated, 1.5% Alcalase for SPH (degree of hydrolysis: 77.86%; protein content: 81.76%) and pH 3.0 for SPI (protein content: 73.09%) demonstrated the most favorable balance across fractionation parameters. SPH exhibited the highest total amino acids (63.19 g/100 g), essential amino acids (25.51 g/100 g), antioxidant amino acids (10.53 g/100 g), and taurine precursors (8.51 g/100 g) among all fractions. Nanoencapsulation of SPH (HNE: seahorse protein hydrolysate nanoencapsulate) showed the most favorable physicochemical properties, including particle size (159.73 nm), polydispersity index (PDI: 0.34), zeta potential (+45.70 mV), and encapsulation efficiency (97.16%), outperforming INE and CNE across all parameters. Simulated gastrointestinal studies demonstrated pH-responsive release behavior, with HNE exhibiting the lowest gastric release (18.90%) and the highest intestinal release (95.97%); release kinetics were best described by the Korsmeyer–Peppas (gastric phase, n = 1.09) and Higuchi (intestinal phase) models. Antioxidant activity retention exceeded 94% (DPPH, ABTS, FRAP) following nanoencapsulation of HNE, indicating effective bioactive protection. To the best of our knowledge, this is the first systematic comparative study of Indonesian seahorse protein fractions with parallel nanoencapsulation evaluation, supporting their potential for oral nutraceutical delivery development.