Comprehensive assessment of hydrolyzed pig hoof keratin (HPHK) as a potential food ingredient: Stability, techno-functional properties, in vitro, and in silico bioactivities

Keratin-rich pig hoof waste remains underutilized due to poor solubility and limited bioavailability. This study aimed to evaluate structural, functional, and bioactive properties of hydrolyzed pig hoof keratin (HPHK) obtained via commercial protease hydrolysis of native keratin (PHK). PHK and HPHK were compared for amino acid profile, molecular weight (SDS-PAGE, SEC), thermal stability (TGA), and structure (FTIR), with further assessment of functionalities and bioactivities. Hydrolysis reduced β-sheet content and molecular weight (~13 → 3.1 kDa), improving solubility and digestibility. HPHK exhibited excellent oil-holding (>4 g/g) and foaming (>200%) capacities but poor emulsification. Antioxidant (IC 50  ~ 1 mg/mL) and ACE-inhibitory (IC 50  ~ 8 mg/mL) activities were confirmed by in vitro and in silico analyses. Acid, heat, and simulated digestion contributed to reduced bioactivity. These findings highlight HPHK's potential as a functional and bioactive ingredient and underscores the need to optimize hydrolysis condition for desired functionalities and bioactivity.