2.4. Protein Hydrolysis and Determination of Peptide Content

Proteins in extracts were hydrolyzed through gastrointestinal digestion or with Alcalase enzyme. Digestions were carried out in a Thermomixer (Eppendorf, Hamburg, Germany). Simulated gastrointestinal digestion consisted of a first digestion with pepsin followed by digestion with pancreatin enzymatic mixture. Pepsin digestion was performed by mixing the extract with the enzyme at a 1:35 enzyme:substrate ratio and adjusting the pH to 2.0 with 1 M HCl. The mixture was incubated at 37 °C for 1 h. Next, pancreatin digestion was performed by mixing the extract with the enzyme at a 1:25 enzyme:subtrate ratio and adjusting the pH to 7.0–8.0 with 1 M NaOH. This digestion step was also carried out at 37 °C for 2 h.

Digestion with Alcalase enzyme was carried out by mixing 0.15 UA/g protein, adjusting the pH of extracts to 6.5–8.5, and incubating the mixture for 4 h at 50 °C and 750 rpm. In all cases, reactions were stopped by increasing the temperature to 100 °C for 10 min and centrifuging at 7000× g for 10 min. Supernatants, containing peptides, were collected.

The peptide content in hydrolysates was determined by the OPA assay [16]. The OPA reagent (9 mL) was prepared by mixing 4.5 mL of 100 mM sodium tetraborate, 1.8 mL of 5% SDS (w/v), 18 μL of β-ME, 180 μL of 40 mg/mL OPA in MeOH, and 2.5 mL of water. OPA reagent (100 μL) was mixed with 2.5 μL of sample in 96-well plates and left to stand for 8 min at RT. Afterwards, the absorbance was measured at 340 nm. A calibration curve was prepared using GSH (0–2 mg/mL) as peptide standard, and the peptide concentration of extracts was determined by interpolation in that calibration curve.