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.