# Formation and Characterization of Bifunctional Nanoparticles Fabricated from Insoluble Rice Peptide Aggregate: Effect of Enzymes

## TL;DR

This study shows how different enzymes affect rice peptide nanoparticles, with Alcalase producing the most stable and antioxidant-rich particles for emulsions.

## Contribution

The study reveals that enzyme selection critically determines the functional properties of rice peptide nanoparticles for emulsion stabilization.

## Key findings

- RPNs-alc had the smallest particle size, highest hydrophobic amino acid content, and strongest antioxidant activity.
- RPNs-alc and RPNs-typ stabilized HIPEs with solid-like behavior and regular microstructure.
- RPNs-pro resulted in unstable HIPEs with droplet aggregation and flocculation.

## Abstract

This study systematically investigates the effects of enzyme type (Alcalase, Trypsin, Protamex) on the properties of rice peptide nanoparticles (RPNs) and their efficacy in stabilizing high internal phase emulsions (HIPEs). RPNs prepared with Alcalase (RPNs-alc) exhibited the smallest particle size (≈379.6 nm), a uniform unimodal distribution, the highest content of hydrophobic amino acid, and the strongest DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity (57.32%). In contrast, RPNs from Protamex (RPNs-pro) showed larger, heterogeneous particles with a bimodal distribution and lower antioxidant capacity. Interfacial characterization revealed that RPNs-alc had a superior three-phase contact angle, indicating enhanced interfacial activity. Structural stability analysis confirmed that hydrophobic interactions and hydrogen bonds are the primary forces maintaining all RPNs. Consequently, HIPEs stabilized by RPNs-alc and RPNs-typ displayed solid-like behavior and a regular network microstructure, leading to exceptional physical stability. Conversely, RPNs-pro led to unstable HIPEs with non-uniform droplets and interfacial aggregation, promoting droplet flocculation. These findings demonstrate that enzyme selection critically determines the functional properties of RPNs, with Alcalase-derived RPNs being the most effective bifunctional particles, offering a viable pathway for valorizing proteolytic by-products in fabricating stable, antioxidant-rich Pickering emulsions.