pH-dependent effects of pepsin and trypsin on the stability and antibiofilm functionality of pea protein-stabilized carvacrol nanoemulsions.

This study investigated how proteolytic enzymes influence the physicochemical stability and bioactivity of pea protein isolate (PPI)-stabilized nanoemulsions encapsulating carvacrol at pH 3.5, 7.0, and 10.0. Pepsin or trypsin (0.1 wt%) was incorporated into pre-formed nanoemulsions (1 wt% PPI, 5 wt% carvacrol) to evaluate pH-dependent effects on structure and function. Under neutral and alkaline conditions, nanoemulsions showed submicron droplets (≈180-254 nm), high ζ-potentials (-26.5 to -41.9 mV), and excellent stability (4 weeks, CI < 5 %). Trypsin introduction enhanced emulsion stability through moderate interfacial hydrolysis, while pepsin caused destabilization under acidic conditions (≈1.5 μm, PDI ≈ 1.0). Carvacrol determined antibacterial activity (MIC = 312.5 μg/mL), whereas antibiofilm performance was pH- and enzyme-dependent: trypsin markedly enhanced biofilm eradication at pH 7.0-10.0, and pepsin had limited effect. Molecular docking revealed strong electrostatic pepsin-protein interactions causing destabilization, while trypsin bound non-catalytically, reinforcing interfacial cohesion.