Amino Acid Composition
The determination of total amino acids of the walnut kernel flours (WKF) was performed by high performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies Co., Ltd., Palo Alto, CA, USA, USA) according to the described previously method of Trigui et al [17]. The HPLC system was equipped with two columns (Styragel HMW7 and HMW6; Styragel, Waters, Milford, MA, USA). Two hundred milligrams of samples were hydrolyzed by 15 mL 6 M HCl for 24 h at 120 ºC. Then, the hydrolyzed samples were mixed with 40 mL of 0.2 M citrate buffer (pH 2.2). Norleucine was added as the standard solution with the amount of 1mL. The mixture solution was diluted to 100 mL by citrate buffer. After the samples were filtered by a 0.2 μm nylon filter, which was detected by HPLC. The amino acid contents were expressed as g 100g-1 protein. Amino Acid Score (AAS) of amino acid composition in samples was expressed as following the equation [18].
\(\text{AAS\ }\left(\%\right)=\frac{mg\ of\ amino\ acid\ in\ 1\ g\ of\ test\ protein}{\text{mg\ of\ amino\ acid\ in\ requirement\ pattern}}\times 100\)(1)