2.6. Quantification of Free Amino Acids

An amount of 3 g of sample was ultrasonicated with 20 mL of 0.2 mol/L phosphate buffer solution for 20 min. The mixture was stored at 4 °C for 18 h after vortexing with 20 mL of 5% trichloroacetic acid (TCA) for 1 min. Next, the mixture was centrifuged for 20 min, the supernatant was collected after being filtered through Whatman No. 4 filter paper (Whatman International Co., Ltd., Maidstone, UK), followed by being adjusted to pH 6.0–6.2 with 4 mol/L NaOH. Afterward, the filtrates were transferred into a volumetric flask and diluted to 50 mL with ultrapure water, followed by filtration through a 0.22 μm membrane. Then, 10 μL of sample filtrate and the amino acid standard solution were derivatized as reported by Goh et al. [22].

The separation was performed on a Nova-Pak C18 Column (150 mm × 3.9 mm, 4 μm; Waters, Milford, MA, USA) at 37 °C. The injection volume was 10 μL. The chromatographic conditions were chosen according to a previously reported method [23], using an Alliance 2695 HPLC system with a 2475 Multi λ Fluorescence detector (Waters, Milford, MA, USA). The concentration of amino acids from samples was identified and quantified by comparison with the amino acid mixture (AAS18; Sigma-Aldrich, St. Louis, MO, USA).