Formation of pea protein fibrils via novel trypsin-assisted self-assembling process

This study presents a novel approach for forming pea protein fibrils under food-compatible mild pH conditions by combining trypsin hydrolysis with thermal treatment. Pea protein isolates were hydrolyzed by trypsin and then heated at various pH values ranging from harsh to mild, including 2.0, 4.0, 6.0, and 8.0, which were compared to pea protein fibrils formed via the conventional acid-heat process. Fibril formation was characterized using Thioflavin T fluorescence, transmission electron microscopy (TEM), particle size analysis, zeta potential, spectroscopy, and SDS-PAGE. Results indicated that the generated small peptides, primarily 10–20 kDa, served as building blocks for protein fibril assembly. Heating under neutral pH conditions leads to effective fibrillation when appropriate peptide sizes were maintained. In brief, heating at pH 2.0 of the trypsin-generated pea peptides produced short fibrils and their aggregates, whereas heating at pH 8.0 formed large and straight fibrils. Amorphous aggregates and fibril bundles were formed at pH 4.0 and 6.0, respectively, because of the absence of surface charge or reduced electrostatic repulsion. Compared to conventional acid-heat methods, the trypsin-assisted process offers enhanced structural tunability. This work provides mechanistic insight into enzyme-assisted fibrillation and presents a practical strategy for fabricating plant protein fibrils under food-compatible pH conditions, which expands the use of plant proteins in food systems.