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

## TL;DR

This study examines how pepsin and trypsin affect the stability and antibiofilm properties of nanoemulsions made with pea protein and carvacrol at different pH levels.

## Contribution

The novel contribution is the pH-dependent analysis of how proteolytic enzymes influence nanoemulsion stability and antibiofilm activity.

## Key findings

- Trypsin improved nanoemulsion stability and antibiofilm performance at neutral and alkaline pH.
- Pepsin caused destabilization under acidic conditions due to strong electrostatic interactions.
- Molecular docking explained enzyme-protein interactions affecting emulsion behavior.

## Abstract

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.

•Pepsin and trypsin were incorporated into pea protein-stabilized nanoemulsions.•Nanoemulsion stability was strongly governed by pH and enzyme type.•Trypsin improved interfacial stability and enhanced antibiofilm performance.•Pepsin induced emulsion destabilization under acidic conditions.•Molecular docking clarified enzyme–protein interactions driving emulsion behavior.

Pepsin and trypsin were incorporated into pea protein-stabilized nanoemulsions.

Nanoemulsion stability was strongly governed by pH and enzyme type.

Trypsin improved interfacial stability and enhanced antibiofilm performance.

Pepsin induced emulsion destabilization under acidic conditions.

Molecular docking clarified enzyme–protein interactions driving emulsion behavior.