Formation and characterization of plant-based amyloid fibrils from hemp seed protein

Amyloid fibrils from plant-based food protein sources bear a large unexploited potential for applications in food and other biomaterials due to their techno-functional features. However, their low solubility and highly complex, inhomogeneous protein composition often hamper fibrillization. The objective of this study was to evaluate the feasibility of amyloid fibril production from hemp seed protein, known as a sustainable and low-allergenic protein source. Hemp protein concentrate (HPC), primarily constituted of the 11 S globulin edestin, with 89.0% protein solubility (0.25% w/w HPC, pH 2) was extracted using gentle micellization. Fibrillization of HPC (2% w/w, pH 2, 90 °C, 300 rpm) was monitored over 5 h by ThT fluorescence, exhibiting a steep increase in fluorescence signal after a lag phase of 180 min. SDS-PAGE analysis indicated progressive polypeptide hydrolysis upon heating and the formation of large proteinaceous aggregates after 160 min. Conformational changes towards increased β-sheet content were demonstrated by CD and FTIR. The morphology of the formed fibrillar aggregates was characterized by TEM and AFM. While essentially linear, branching effects of the fibrils became visible and kept increasing with incubation time. After a relatively short incubation time of 4 h, fibrils had an average height of 7.8 nm, a contour length of 1.8 μm, and a persistence length of ∼2.7 μm. These results suggest, that under the chosen conditions for protein extraction and incubation, HPC forms relatively flexible amyloid fibrils with a high aspect ratio and tendency to form branches. By revealing the potential of hemp seed proteins for amyloid fibril formation, these results contribute to expand the understanding of plant protein fibrillization.