*3.6. Measurement of Degree of N-Acylation*

The measurement of the degree of N-acylation was prepared, as described by Habeeb [39]. Briefly, to the protein suspensions, 1 cm<sup>3</sup> of 4% NaHCO<sup>3</sup> solution and 1 cm<sup>3</sup> of 0.1% trinitrobenzenesulfonic acid (TNBS) solution were added. After heating at 60 ◦C for 2 h, the samples were cooled down to room temperature. Next, 1 cm<sup>3</sup> of 10% SDS and 0.5 cm<sup>3</sup> of 0.1 N HCl were added. The absorbance of the solutions was read at 335 nm in a Rayleigh UV-2601 PC spectrophotometer (Beijing, China) against a reagent blank. The absorbance of the control protein concentrate was set equal to 100% free amino groups, and the extent of acetylation of the modified samples was calculated based on the decrease in absorbance because fewer amino groups were able to react with the TNBS reagent.
