Measurement of α-amino nitrogen content by 2,4,6-trinitrobenzenesulfonic acid (TNBS)

The TNBS method was modified from that described by Adler-Nissen (1979) as follows. Whey protein hydrolysate (25 μL), 0.2 mL of phosphate buffer (0.2 M, pH 8.2), and 0.2 mL of 0.1% TNBS were mixed, and a color reaction was carried out for 1 h in a dark water bath at 50℃. To quench the reaction, 0.4 mL of 0.1 M HCl was added and the mixture was allowed to stand at 25℃ for 20 min. Distilled water (1.6 mL) was then added and the absorbance was measured at 340 nm using a spectrophotometer. The α-amino acid content of each sample was quantified using the standard curve of L-leucine.