Abstract
Amyloid fibrils (AFs) are highly ordered nanofibers composed of proteins rich in β-sheet structures. In this study, the impact of heating conditions relevant in food processing on AF formation of wheat gluten (WG) was investigated. Unheated and heated WG samples were treated with proteinase K and trypsin to solubilize the nonfibrillated protein, while protein fibrils were extracted with 0.05 M sodium phosphate buffer (pH 7.0) from the undissolved fraction obtained by the same enzymatic treatment. Conditions (i.e., heating at 78° for 22 h) resembling those in slow cooking induced the formation of straight fibrils (ca. 700 nm in length), whereas boiling WG for at least 15 min resulted in longer straight fibrils (ca. 1-2 μm in length). The latter showed the typical green birefringence of AFs when stained with Congo red. Their X-ray fiber diffraction patterns showed the typical reflection (4.7 Å) for inter-β-strand spacing. These results combined with those of Fourier transform infrared and thioflavin T spectroscopy measurements validated the identification of β-rich amyloid-like fibrils (ALFs) in dispersions of boiled WG. Boiling for at least 15 min converted approximately 0.1-0.5% of WG proteins into ALFs, suggesting that they can be present in heat-treated WG-containing food products and that food-relevant heating conditions have the potential to induce protein fibrillation.