*2.4. Amino Acid Profile*

An RP-HPLC apparatus (Young Lin Acme 9000, YL Instruments, Anyang, Korea) equipped with a reverse-phase column (150 mm × 4.6 mm × 5 μm; RP-C18 ODS-A, Barcelona, Spain), a fluorescence detector (LC305; Lab Alliance, State College, PA, USA), and a mobile phase of acetate buffer (50 mM at pH 3.4, with a flow rate of 1.3 mL/min) were used to determine the amino acids in *Spirulina* protein hydrolysates. For this purpose, the hydrolysate was intensively treated with HCl (6 M) at 110 ◦C for 24 h. The digested sample was derivatized with orthophthaldehyde and injected to the HPLC column. The amount of amino acids in hydrolysates was expressed as mg/100 g protein. The biological value (*BV*) and amino acid score (*AAS*) of hydrolysates, as nutritional parameters, were determined using the following equations [16]:

$$AAS = \frac{\% \text{ Essertial amino acids in sample}}{\% \text{ Essenential amino acids recommended by FAO}} \tag{2}$$

$$BV\left(\%\right) = 10^{2.15} \times Ly^{0.41} \times \left(Phc + Tyr\right)^{0.6} \times \left(Met + Cys\right)^{0.77} \times Thr^{0.24} \times Tr p^{0.21} \tag{3}$$

where each amino acid symbol is expressed as % amino acid in sample/% amino acid FAO pattern.
