Structural and functional properties of lupin proteins and bioactive peptides

Abstract

This thesis investigated the structural, physicochemical, and techno-functional properties of white lupin (Lupinus albus) and blue lupin (Lupinus angustifolius) proteins across multiple fractionation levels and explored enzymatic strategies to enhance functionality and generate bioactive peptides. Protein isolates were produced by alkaline extraction and isoelectric precipitation, followed by solubility-based fractionation into albumin, globulin, and glutelin classes, with globulins further separated into vicilin (7S) and legumin (11S) sub-fractions. All fractions were characterized for molecular structure, amino acid composition, and techno-functional properties at pH 3–9. Both isolates had similar essential amino acid profiles, with sulfur-containing amino acids, valine, and lysine being limiting, and achieved emulsifying performance comparable to soybean protein isolate at neutral and alkaline pH. Higher α-helix-to-β-sheet ratios at pH 7 and 9 were associated with smaller oil droplet sizes and improved emulsion stability. Fractionation revealed that isolate-level behavior masks the contributions of individual components: white lupin globulin and vicilin were uniquely not limited in sulfur-containing amino acids (107% and 127% amino acid scores), blue lupin vicilin emerged as the most versatile fraction with high emulsion stability (95–100%) and foaming capacity (75–85%), and white lupin legumin exhibited the highest thermal stability and lowest gelation concentration (8%). Transglutaminase-catalyzed cross-linking (10–15 U/g) enhanced emulsifying properties at pH 3 by inducing structural rearrangements, whereas excessive cross-linking (20–25 U/g) compromised functionality. The bioactive potential was further explored by generating arginase-inhibitory peptides. White lupin flavourzyme hydrolysate showed the highest inhibitory activity (~35% at 800 μg/mL). Peptide purification yielded ten sequences, with DMGAVA and VVALKKVGVLKKPGPT showing the highest inhibition. Kinetic analysis demonstrated mixed-type inhibition, with DMGAVA affecting both Km and Vmax (Ki ~266 μM) and VVALKKVGVLKKPGPT exhibiting predominantly competitive behavior (Ki ~589 μM). A complementary in-silico strategy generated 24 candidate peptides; of these, DQR exhibited the highest inhibitory activity (17% at 20 μg/mL) with mixed-type inhibition. Discrepancies between computational predictions and experimental outcomes highlighted the limitations of binding affinity as a sole predictor of inhibitory function. Collectively, this thesis establishes structure-function relationships governing lupin protein functionality and identifies lupin proteins as a promising, underexplored source of bioactive peptides for functional food development.