Multiscale analysis of enzyme combinations and hydrolysis methods: Effects on bitterness, structure, functional...

To address bitterness in soybean meal hydrolysates, this study compared simultaneous and sequential hydrolysis using endopeptidases (Alcalase, Protamex, Papain, and Trypsin) with Flavourzyme, focusing on taste, structure, functional properties, and peptide profiles. Hydrolysis progressively degraded β-conglycinin and glycinin, enhanced peptides <5 kDa accumulation, and yielded DH values of 14.73 ± 0.63%-39.30 ± 1.69%. Bitterness initially increased and then decreased, with sequential hydrolysis producing lower bitterness. Structural changes included chromophore exposure, altered hydrogen bonding, increased β-turn content, reduced particle size, and decreased zeta potential, whereas enzyme specificity affected free sulfhydryl content, surface hydrophobicity, emulsifying properties, and antioxidant activity. Although nutritional improvements were limited, emulsifying and antioxidant activities were enhanced. Protamex-Flavourzyme provided the most favorable balance of debittering and functional improvement. Cleavage patterns and potential bitter peptide profiles indicated that Flavourzyme reduced N-terminal Val/Ile/Leu enrichment in Protamex-generated peptides, partly explaining its debittering effect and guiding functional soybean meal hydrolysate design.