Determination of Free Amino Acids
This was performed according to the instructions of Hitachi amino acid analyzer (L-8900, Hitachi High-Technologies Corporation, Tokyo, Japan). Briefly, the samples (1 mL) were thoroughly mixed with 1 mL of 10% (w/v) sulfosalicylic acid and kept at 4 °C for 6 h. These were treated by centrifugation (14000g , 10 min) to obtain transparent supernatants, which were filtered through a 0.22 μm membrane before injection (20 μL) for detection. The percentage of total free amino acids in total proteins (0.96%) in pH 7-OB suspension was calculated, which could be used to quantitatively estimate the exopeptidase activity. The percentage of TCA-soluble peptides could be obtained by subtracting percentage of total amino acids from TCA−NSI, which could be used to quantitatively explain the endopeptidase activity.