4.3. Free Amine Groups Analysis by TNBS Test

In order to determine free amine groups released during the enzymatic hydrolysis of the amaranth proteins, the 2,4,6-Trinitrobenzenesulfonic acid (TNBS) test was performed, with some modifications, according to the protocol described by Sashidhar et al. [59]. Briefly, 5% TNBS solution (Sigma-Aldrich, (St. Loui, MO, USA) was diluted in 0.21 M phosphate buffer (pH 8.2) to a final concentration of 1% (v/v). Two milliliters of the prepared substrate was added to 2 mL of phosphate buffer (0.21 M; pH 8.2), and 0.25 mL of the test sample. The mixture was incubated during 1 h at 50 °C and the reaction was stopped by the addition of 2 mL of 0.1 N HCl. The absorbance was read at 340 nm. Results were plotted against a calibration curve prepared by different glycine concentrations (0, 0.05. 0.1, 0.15, 0.2, and 0.25 mg/mL) using the following equation (R = 0.9972):

Results are expressed as milligrams of free amines per liter (mg/L NH:⁻).