Enzyme-assisted extraction of navy bean protein from whole and dehulled flour: Effects on extractability, structure, and techno-functional properties

The growing demand for sustainable plant proteins calls for alternative extraction methods that preserve protein quality. Navy beans, though rich in hypoallergenic, gluten-free protein, remain underutilized. Conventional extraction methods often cause protein denaturation and aggregation, thereby limiting functional properties. This study evaluated enzyme-assisted (E) and homogenization-enzyme-assisted (HE) extraction of whole (W) and dehulled (D) navy bean flours (protease, pH 10, 0.5 %, 1.5 h), compared with aqueous extraction (Aq). Both extraction method and flour type significantly influenced extractability, structural, and techno-functional properties. HE-D exhibited the highest protein purity (68.99 %), while E-D and HE showed the highest recovery. Compared to Aq-W, solubility improved by 4.5 % in E-W. Enzyme-based extracts showed higher EAI than aqueous controls, with no separation among enzyme-treated groups; ESI varied depending on extraction method and sample matrix, with HE-D yielding the highest values. WAC increased by 32 % in HE-W, whereas OAC declined across enzyme-treated samples. LGC increased from 12 % to 18 % in enzyme-treated flour. Free sulfhydryl content increased in E and HE, whereas foaming ability and surface hydrophobicity decreased. SEM revealed disrupted aggregates and porous structures, while FTIR showed reduced β-sheet and α-helix with increased random coil and β-turn. XRD provided qualitative context on short-range order, and DSC revealed lower denaturation temperatures and enthalpies. Fluorescence and UV-vis spectroscopy indicated partial unfolding in enzyme-treated samples. TNBS primary-amine index (h) supported interpretation of structure-function trends. These findings support enzyme-based extraction as a viable green approach to enhance the structural and functional quality of navy bean proteins for food applications.