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

GARCÍA FILLERÍA, S. F.; TIRONI, V. A.; RODRIGUEZ MARIELA

Faro

Congreso; XII CIBIA, Iberoamercan Congress of Food Engineering, Faro, Portugal, 2018.; 2019

Diverse methodologies for the simulated gastrointestinal digestion are used in order to study bioactivity. In our laboratory, we have applied a simplified protocol focused in protein digestion of amaranth protein isolate (I) to release antioxidant peptides. The aim of the present work was to compare this protocol with other ones based on an international consensus (COST-INFOGEST) in order to evaluate the effect of digestion conditions and matrix in the protein hydrolysis and the generation of antioxidant peptides. The in-house protocol was applied to the I digestion obtaining Id1, the standardized method was applied to digest I and amaranth flour (F) obtaining Id3 and Fd3, as well as a modified version using the same enzyme/substrate ratio than in our lab method (Id2 and Fd2). Protein hydrolysis degree (HD, TNBS method) was similar for the three digests from I (about 60%), but lower for F digests (45% for Fd2 and 34% for Fd3). All digests presented comparable protein solubility values (about 70%). Polypeptide composition was analyzed (SDS-PAGE and tricine-SDS-PAGE, gel filtration chromatography FPLC); low molecular mass molecules (< 6.5 kDa) appeared in all cases, and only small differences in non-hydrolyzed and released peptides proportions among the digests were registered. Antioxidant activity was evaluated by the ORAC and HORAC methods. In the case of ORAC, soluble fractions from Id1, Id2, Id3 and Fd3 presented similar activity (IC50 = 0.020-0.024 mg protein/ml, respectively), while activity was greater for Fd2 (IC50 = 0.012 mg/ml). For HORAC assay, only small differences among digests were registered (IC50 = 1.13-1.61 mg protein/ml). All FPLC fractions showed some level of ORAC activity, but those in the 0.2-0.6 kDa range presented the greater potency. Active HORAC fractions were in the range of 0.55-3.5 kDa. Results evidenced that the modification of the digestion conditions (phases, kind and concentration of salts, pH, enzyme activities) on I digestion produced only small differences in the molecular composition of digests but did not have effect neither on HD nor antioxidant activity, while the presence of other food components (F) had incidence on HD.