Peptidomic evidence for pH-stat flavourzyme-mediated debittering of Alcalase-derived rice protein hydrolysates

Flavourzyme-assisted two-stage hydrolysis is a recognized debittering strategy for protein hydrolysates, yet peptidomic-level evidence for the underlying mechanism-particularly the role of pH control mode-remains limited. This study compared pH-stat and free-fall pH Flavourzyme treatments (3 and 4 h) of an Alcalase-derived rice protein hydrolysate (A6; degree of hydrolysis by the o-phthalaldehyde method [DH-OPA] 24.69%) using nanoLC-ESI-Q-TOF de novo sequencing to characterize peptide-profile changes associated with bitterness reduction. Formal sensory evaluation (15 trained panelists, paired comparison with Bonferroni correction for 10 comparisons) of the ultrafiltered (<2500 Da) fractions placed A6-s3 (pH-stat, 3 h) at the least-bitter end of the ranking, although it did not differ significantly from A6-s4 after multiplicity correction. The apparent ranking differed from that observed in the preliminary evaluation of unfiltered hydrolysates, which used a smaller panel and a different sensory design. De novo sequencing identified 963-1226 peptides per hydrolysate. A6-s3 exhibited the lowest mean Q value (1430 cal mol^-1; Kruskal-Wallis, p < 10-22) and the lowest predicted bitter peptide proportion (49.6%). A6 and A6-s3 shared only one exact PEAKS-assigned sequence among >1900 combined detections, indicating extensive differences in the detectable peptide profiles. Database-assisted annotation against the Oryza sativa proteome showed a markedly lower assignment rate for A6-s3 (0.2%) than for A6 (52.8%, predominantly glutelins); this was treated as a descriptive analytical observation because I/L ambiguity, de novo sequencing uncertainty, stochastic data-dependent acquisition (DDA) sampling, and database coverage may contribute. A6-s3 also showed the lowest C-terminal strongly hydrophobic residue frequency (62.3%; I, L, F, W, Y, P) and the highest N-terminal amino acid diversity (Shannon entropy, 3.91 bits). These findings indicate that maintaining pH during Flavourzyme treatment was associated with a more favorable bitterness-related peptide profile. The results are compatible with established terminal processing accompanied by broader changes in the detectable peptide population, but they do not establish or quantify a distinct contribution from internal cleavage.