Amylase activity was measured with 1.0% soluble starch in 0.1 M sodium acetate buffer (pH 5.0) as a
substrate. One mL of crude enzyme extract and 1 mL of soluble starch were mixed and incubated at 30°C for
10 min. After incubation, one mL of 3, 5-dinitrosalicylic acid was added, reacted in a boiling water bath for
15 min, cooled to room temperature and added with 9 mL of deionized water. The amylase activity was
analyzed spectrophotometrically at 540 nm. One unit of enzyme activity was defined as the amount of
enzyme that released 1 mM of reducing sugar as maltose per minute under the assay condition [11]. The
enzyme activity was reported per gram of dry koji used in the initial extraction. All the samples were analyzed
in triplicate