Hydrolysis of sample was done by measuring 0.1 g of sample into 5 mL of 6 M HCl and kept at 110 ºC for 24 h
to break the protein into constituent amino acids according to AOAC 994.12. The sample was cooled and filtered
through 0.45 μm Whatman filter paper to remove any remaining particles. The filtrate was injected into the HPLC
and fractionated using gradient composition system (Acetonitrile 60 %, 1.0 mL · min–1 , 37 ºC, column 3.9 mm ×
150 mm). The HPLC analysis separate various individual amino acids (Excitation = 250 nm, emission = 395 nm)
(references). Amino acids react with ninhydrin reagent that detects amino acids. The amount of an amino acid
standard is analyzed at the same time to quantify the amount of amino acid that may be present in the sample.