Protease-assisted microencapsulation of carvacrol in pea protein systems for enhanced and durable antibiofilm activity

Abstract

Developing dry antimicrobial delivery systems that combine bioactive protection with biofilm-matrix disruption remains a major challenge. Building on a previously established protease-modulated pea protein isolate (PPI)-carvacrol nanoemulsion system, this study developed protease-assisted microcapsules designed to preserve carvacrol and promote the removal of preformed biofilms. Carvacrol-loaded nanoemulsions were prepared at pH 3.5, 7.0, and 10.0 and supplemented with pepsin or trypsin before spray-drying or freeze-drying with maltodextrin. Protease-specific interfacial modification markedly affected emulsion stability, powder structure, and encapsulation performance. At pH 7.0 and 10.0, trypsin reduced droplet size from 284.60 to 232.52 nm and from 149.13 to 140.38 nm, respectively. The resulting spray-dried microcapsules exhibited high encapsulation efficiency (>96%) and low surface carvacrol contents (0.95–1.24 mg/g). In contrast, pepsin caused pronounced destabilization under acidic conditions, particularly after freeze-drying, yielding porous powders with an encapsulation efficiency of 48.18% and a surface carvacrol content of 69.51 mg/g. Trypsin-assisted microcapsules achieved 90–99% removal of preformed Listeria innocua biofilm biomass within 1 h, whereas enzyme-free and pepsin-containing formulations generally remained below 40%. Microscopic observations confirmed extensive disruption and detachment of the biofilm structure. After one year at 4 °C, trypsin-loaded formulations retained high biofilm biomass removal activity (85–99% after 2 h). These findings demonstrate that protease-assisted microencapsulation can couple interfacial regulation during particle formation with enzyme-mediated biofilm-matrix disruption after rehydration, providing a sustainable carvacrol delivery platform with durable antibiofilm functionality.