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.