The free amino acid was measured using method of Kim et al. (13) with modification.
A fixed quantity of free amino acid for analysis was obtained by adding ethanol (30 mL) to 10 mL samples then leaving the mixture for 12 h at room temperature.
The protein precipitation was removed and centrifuged after separating the upper level liquid for 15 min, and heated it in a double boiler.
Citrate buffer 10 mL was then added (pH 2.2) before dilution using a filtrate with a 0.45-μm membrane filter as the amino acid autoanalyzer (L-8800; Hitachi Co., Toyko, Japan).