1. Total amino acids analysis
The total amino acids of peptides were determined according to the method described by Dong et al. (2008) with a slight modification. Amino acid compositions were determined after hydrolysis at 110 °C for 24 h with 6 MHCl prior to the derivatization with phenyl isothiocyanate. Alkaline hydrolysis was also done to determine tryptophan. Thereafter, the solution was ultrafiltered through a Millipore filter having a pore diameter of 0.45 μm(Millipore, Bedford,MA). Twentymicroliters of each sample was analyzed using a fully automated amino acid analyzer Hitachi L-8900A (Hitachi Ltd., Japan).
2. Free amino acids analysis
Samples for free amino acids analysis were prepared according to the procedures described by Aro et al. (2010).