Green Extraction of High-Quality Protein from Cassava Leaves Using Deep Eutectic Solvents: Nutritional and Functional Comparison with Alkaline Extraction

Abstract

Cassava (Manihot esculenta) leaves, rich in protein content ranging from 20–30% and abundant in essential amino acids (EAAs), represent a valuable and sustainable source of plant-based protein alternatives. Deep eutectic solvents (DESs) represent a promising green alternative to conventional alkaline extraction- isoelectric precipitation (AE-IP) for recovering plant proteins with minimal denaturation. In this study, eight different DESs formations were evaluated for extracting protein from detoxified cassava leaves (DCP), focusing on physical properties, hydrogen bond capacity and their interactions. The protein isolate from DES-based extraction (DCPI-Dn) obtained were characterized nutritionally, structurally, thermal and functionally. Principal component analysis (PCA) integrating all response variables identified DES of mixture of Lactic acid and glycerol (DES-8) as the optimal extractant of DCPI. Extraction with DES-8 produced a protein content of 22.16 ± 0.16 mg/g dm and a recovery yield of 72.77 ± 1.05 % with as modest extraction yield of 16.85 ± 0.41 %. Compared to AE-IP, DES-8 markedly reduced antinutrient levels while preserving nutritional compounds. DCPI D8 revealed enhanced secondary structures, supplemented by favourable free sulfhydryl content and zeta potential that correlated with stable and ordered microstructures. Thermogravimetric analysis demonstrated increased thermal stability in DCPI-D8. Functionally, DCPI-D8 exhibited significant improvements in foaming capacity (243 %) and emulsifying capacity (350 %), with moderate gains in foaming stability (4.2 %) and emulsion stability (47 %) as compared to DCPI-A. Water-holding capacity increased 1.46-fold relative to AE-IP isolates, while oil-holding capacity declined slightly (1.15-fold). Amino acid profiling confirmed that the mild acidity of lactic acid and glycerol’s stabilizing effects maintained higher total (91.24 %) and essential amino acid contents (40.36 %) relative to AE-IP, in which alkaline conditions promoted partial denaturation and exposure of hydrophobic residues. DCPI-D8 also enhanced in vitro digestibility, making DES a promising sustainable alternative for protein extraction with improved nutritional, structural and functional quality.