Characterization by in vitro and in silico studies of bioactive peptides obtained from a digestive simulation of α-lactalbumin hydrolysates

NARDO AGUSTINA E.; PAULINO, MARGOT; MEDRANO ALEJANDRA; FERNÁNDEZ ADRIANA; AÑÓN M. CRISTINA; BAÉZ JESSICA; BONIFACINO CIRO; TIRONI A. VALERIA

Campinas, Sao pablo

Congreso; 1st Ibero-American Congress on Bioactive Peptides; 2019

Institute of Food Technology (Ital), Campinas, Brazil

The objective of this work was the in vitro and in sílico bioactivity study of identified peptides from fractions obtained after gastrointestinal digestion simulation of α lactalbumin hydrolysates with Alcalase enzyme. The hydrolysates (degree of hydrolysis: 27,4±0,5%) were purified and separated by RP-HPLC. 52 fractions were obtained, 13 of those were selected for protein concentration and antioxidant capacity (ABTS and ORAC-FL) analysis. Peptide identification of the most active fractions was performed by separating in a nano-HPLC (reverse phase) associated with a quadruple-orbitrap hybrid mass spectrometer (Q-Exactive Plus, Thermo). Peptide sequences were assigned by comparison with databases using the Mascot program (Matrix Science). Thus, three sequences corresponding to α-lactalbumin were identified: IWCKDDQNPH (1255.38 Da), DKFLDDDLTDDIM (1555.68 Da), KFLDDDLTDDIM (1440.60 Da), which were synthesized to evaluate its in vitro activity. The IC50 values of antioxidant capacity were 0.016, 0.18 and 0.19 mg/mL (ORAC FL) for these peptides, respectively, and 0.45mg/mL (ABTS) for IWCKDDQNPH. The other peptides did not present any activity according to ABTS.Considering the relevance of multifunctional peptides, the angiotensin converting enzyme (ACE) inhibitory activity was also studied. Only IWCKDDQNPH was active in the evaluated concentration range with an IC50 value of 3.91 mg/ml. To study the structure-activity relationship (SAR) of peptides, their structure was modeled in aqueous phase using molecular dynamics simulations of 5 ns (with NaCl as counter ion, AMBER12 force field and periodic boundary conditions) with MOE.2015 (Chemcomp Ltda.). The trajectories obtained and final structures were analyzed, evaluating the surface exposed to the solvent, distribution of surface charges and presence of hydrophobic zones, as well as the exposure, mobility and change of amino acids associated with the antioxidant capacity such as Cys, Thp and His. It was possible to identify 3 active sequences, being the decapeptide IWCKDDQNPH the one with the highest antioxidant and antihypertensive activity.