Comparative binding and functional behavior of three benzaldehyde-based food flavorants with pepsin: a combined...

Vanillin (Van), ethylvanillin (Eva), and veratraldehyde (Ver) are three widely used food flavorants that belong to the benzaldehyde family of compounds. In this study, their interaction mechanisms with pepsin (PEP) was systematically investigated using computational simulations and multi-spectroscopic techniques, along with enzymatic activity and antioxidant capacity assays. Molecular docking revealed that all three ligands occupied the catalytic cleft near Asp32 and Asp215 with comparable binding energies, although Eva exhibited a slightly more favorable interaction. Molecular dynamics simulations further uncovered distinct differences in the dynamic stability of the ligand-PEP complexes. Spectroscopic and thermodynamic analyses indicated that Van binding was mainly driven by electrostatic interactions, whereas the binding of Eva and Ver was governed by hydrogen bonds and van der Waals forces. All three ligands formed stable complexes with PEP via static quenching, with moderate binding constants ranging from 10⁴ to 10⁵ M^-1. Synchronous and three-dimensional fluorescence confirmed that complex formation significantly increased the polarity of the Tyr and Trp residue microenvironments. CD and FT-IR spectra revealed ligand-induced conformational rearrangements of PEP, with the β-sheet content increasing by 12.8% upon Van binding. Enzymatic assays showed concentration-dependent inhibition of PEP by Van and Eva, reaching 28.79% and 28.13%, respectively, whereas Ver exhibited negligible inhibition. Complex formation diminished the antioxidant activities of the flavorants, reducing DPPH and ABTS radical-scavenging capacities by up to 11.5% and 7.0%, respectively, with Eva showing the greatest reduction. These results demonstrate that free phenolic hydroxyl groups are critical for PEP inhibition by the three food flavorants, whereas alkoxy substituents exert minimal effects. Overall, this study provides molecular-level insights into the structure-dependent interactions between these three benzaldehyde compounds and PEP during gastric digestion, although the conclusions are limited by the small compound set and the use of a single enzyme.