Simulated gastrointestinal digestion of amaranth flour and protein isolate: Comparison of methodologies and release of antioxidant peptides

The effect of both the simulated gastrointestinal digestion conditions and the matrix over protein hydrolysis and antioxidant peptides generation was evaluated by comparing an in-house method with COST INFOGEST-based SGD protocols. The in-house protocol was used to digest amaranth protein isolate I (Id1), while the standardized method and a modified version (similar enzyme/substrate ratio than in our lab) were used to digest I and amaranth flour F (Id3 and Fd3, Id2 and Fd2). Protein hydrolysis degree (TNBS method) was similar for the three I digested (about 60%), but lower for F digested (45 and 34% for Fd2 and Fd3, respectively). The five digested obtained presented comparable protein solubility and only small differences in the polypeptide/peptide composition (SDS-PAGE, tricine-SDS-PAGE, gel filtration FPLC), similar antioxidant activity by the ORAC assay (IC50 values between 0.023 and 0.034 mg.mL(-1)) and some mild differences by the HORAC assay (IC50 values between 1.13 and 1.30 mg.mL(-1) for Id1, Fd2, and Fd3; 1.50 mg.mL(-1) for Id2; 1.61 mg.mL(-1) for Id3). All the FPLC fractions presented high ORAC activity, while only fractions between 0.43 and 3.5 kDa showed HORAC activity (due to peptide concentration). Differences in activity and potency among fractions were registered, especially for F digested. The modification of digestion conditions produced only small differences in the molecular composition but did not affect the proteolysis degree and the antioxidant activity in the case of digested from protein isolate. The presence of other components and changes in the digestion method had an impact on proteolysis, composition and antioxidant activity of flour digested.