Abstract
Five deep eutectic solvents were used to extract T. grandis nut protein, with water as comparison. ChCl:Urea was the most effective, preserving integrity and functionality of extracted proteins. Cold ethanol purification yielded highest protein recovery rate 6.89 % ± 0.12 %. Time, temperature, and solid-liquid ratio, were optimized using response surface methodology and a Box-Behnken design. Extracted proteins were characterized via SDS-PAGE to assess digestibility and functional properties. Proteins extracted by DES were further analyzed via LC-MS/MS. De novo sequencing identified 600 peptides with 235 proteins in DES extract, compared to 349 peptides and 143 proteins in the water extract. Furthermore, 87 functional oligopeptides in DES, compared to 41 in water. DES extract contain 63 antioxidant peptides, with three distinct peptides GGE, CME, and YIY. CME peptide had highest antioxidant activity. These findings highlighted DES for extracting smaller functional proteins and peptides from T. grandis, demonstrating potential in food industry applications for protein extraction.