Optimization of enzymatic hydrolysis and evaluation of thermal treatment for improving amino acid and volatile profiles of soy protein hydrolysates

Enzymatic hydrolysis has attracted attention as a safer and more sustainable alternative to conventional acid hydrolysis for producing hydrolyzed soy protein (HSP). However, the effects of protease combination, reaction conditions, and post-hydrolysis thermal treatment on amino acid and volatile compound formation remain limited. This study investigated the effects of five commercial proteases and their combinations on the amino acid and volatile profiles of HSP and optimized hydrolysis conditions using response surface methodology (RSM). Among the enzyme combinations tested, Alcalase + Protana Prime produced the most favorable amino acid profile, characterized by elevated sweet- and umami-related amino acids and relatively low levels of bitter amino acids. Subsequent heat treatment markedly increased pyrazines, furans, aldehydes, and sulfur-containing compounds, resulting in volatile profiles comparable to those of acid-hydrolyzed products. RSM identified optimal conditions of 15.91 h reaction time and 5.85% enzyme concentration, which increased total, sweet, and umami amino acid contents while reducing enzyme usage. Under the optimized conditions, taste activity values of umami and sweet amino acids were approximately twofold higher than those under the high-enzyme-concentration treatment. These findings demonstrate the potential of combining optimized enzymatic hydrolysis and controlled thermal processing as a clean-label strategy for producing flavor-enhanced HSP.