Sustainable protein and amyloid fibrils production from tofu wastewater

We have successfully valorised soy whey, a byproduct widely generated by tofu production, by developing a sustainable method for protein recovery and enhancing protein functionality through self-assembly into amyloid fibrils. Using 2-Methyltetrahydrofuran (2-methyloxolane) for defatting, a biomass-derived green solvent, soy proteins were extracted and upscaled from tofu wastewater with a recovery yield of ∼58%, achieving a ∼75% purity of soy protein concentrates. To improve the solubility of the recovered proteins, primarily consisting of glycinin, we applied heat-induced acidic hydrolysis and then produced soy amyloid fibrils. Favourable conditions for fibrillization were identified by varying protein concentration (2–4%), temperature (85–90°C), and exposure time (2–24 h), focusing on the mesoscopic behaviour of soy amyloid fibrils. Higher temperature accelerated the hydrolysis of soy proteins (2% w/w, pH 2), but prevented the yield of smaller peptides. Peptides with a molecular weight of ∼2 kDa were observed at 85°C, which promoted fibrillization. The soy amyloid fibrils produced from tofu wastewater displayed a rich β-sheet secondary structure, as confirmed through CD and FTIR spectroscopy. TEM and AFM revealed that the recovered soy proteins self-assembled into two distinct populations of amyloid fibrils: straight and semiflexible amyloids formed during the growth phase of fibrillization; and shorter, worm-like amyloids induced by prolonged fibrillization. Notably, the introduction of 2-MeTHF increased the rigidity of the amyloid fibrils by 2–3 times. The gelling, foaming, and emulsifying properties of the waste-derived soy proteins were improved by fibrillization. This sustainable up-scale of tofu wastewater may contribute to developing biocompatible and eco-friendly materials for various nanotechnological applications. • Soy proteins (∼80% purity) were recovered from tofu wastewater • Protein extraction with 2-MeTHF provided ∼58% recovery yield • Higher protein concentration inhibited the growth phase of fibrillization • Lower temperature promoted the release of low molecular weight peptides • Defatting solvent determined the persistence length of soy amyloid fibrils • Hydrogel rheological properties were promoted by flexible soy amyloids