*3.5. Measurement of Degree of N-Acylation*

The measurement of degree of N-acylation was prepared, as described by Habeeb [65]. The procedure involved the addition of 1 cm<sup>3</sup> of 4% NaHCO<sup>3</sup> solution and 1 cm<sup>3</sup> of 0.1% TNBS solution to protein suspensions. The samples were heated in a water bath at 60 ◦C for 2 h. Then, they 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 to protein solutions. The absorbance of 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.
