Multiscale structural and functional characterisation of phenolic-reduced rapeseed protein isolates: Effects of phenolic removal and drying methods

Rapeseed proteins remain underutilised compared with soy and pea proteins despite their high protein content and balanced amino acid composition, largely because phenolic compounds impair solubility, structural integrity, and techno-functional performance. This study investigated how phenolic reduction and drying method collectively influence the structural organisation and hydrocolloid-related functionality of rapeseed protein isolates from brown and yellow varieties. Proteins were extracted following ethanol-assisted phenolic reduction, alkaline extraction (pH 10), and isoelectric precipitation (pH 4.5), followed by freeze drying or spray drying. Phenolic contents were markedly reduced from ∼68 to 93 mg GAE g −1 in rapeseed meals to ∼0.55–7.64 mg GAE g −1 in the final isolates, while nitrogen solubility index increased from ∼34 to 37% to ∼80–89%, indicating enhanced hydration and dispersibility after disruption of protein–phenolic interactions. SDS-PAGE and SEC analyses confirmed preservation of cruciferin- and napin-like proteins, whereas FTIR and CD analyses showed that freeze drying preserved β-sheet-rich structures and conformational stability, while spray drying promoted conformational rearrangement and aggregation. Nano-DSC further demonstrated higher denaturation enthalpies and greater thermal reversibility in freeze-dried isolates. These structural modifications strongly influenced functionality. Freeze-dried proteins exhibited higher dispersibility (∼28–39%), water-holding capacity (∼3.2 g/g), emulsifying stability (∼62–76 min), and stable gel formation at 10% protein concentration, whereas spray-dried isolates showed reduced rehydration and failed to form self-supporting gels even at 15% concentration. Overall, the results establish clear structure–process–function relationships linking phenolic reduction and drying-induced structural modification with hydrocolloid functionality in rapeseed protein systems.