Phytase activity was determined by measuring the amount of liberated inorganic phosphate. The reaction mixture consisted
of 0.9 ml of acetate buffer (0.2 M, pH 5.5) containing 1 mM phytate, and 0.1 ml of the enzyme solution. After incubation
for 30 min at 37 C, the reaction was stopped by the addition of 1 ml of 10% trichloroacetic acid. The aliquot was subsequently analyzed for inorganic phosphate as described earlier (Fiske and Subbarow 1925; Singh and Satyanarayana 2006).