Simple determination of amylase activity using blue starch as substrate

II. Application to the control of koji making and sake bottling process

Abstract

Blue starch is a colored insoluble starch covalently labelled with a colored substance, Cibachron Blau F 3 G-A. The enzymatic hydrolysis of such insoluble starch yields soluble starch split products carrying the colored marker. The blue starch is commercially available.
1) A rapid and simple determination of a-amylase activity of rice-koji was established. Enzymes in 10g rice-koji was extracted with 100ml of O.5% NaCl solution containing 5ml of O.1 M acetate buffer of pH 5.0, homogenizing it for 4 min. In a test tube, 1.5 ml of the enzyme solution and 4.5 ml water were taken and prewarmed at 30°C. After addition of O.1 g blue starch preparation, the reaction mixture was stirred well and then stood still for 15 min at 30°C. Reaction was stopped by addition of 1N NaOH solution (0.5ml) followed by stirring. Blue color of the filtrate was measured at 620 nm.
2) Activities measured by the blue starch method were well correlated with α-amylase and the overall saccharogenic activities measured by the ordinary method; coefficient (γ) was 0.937 for α-amylase and 0.967 for the overall saccharogenic activity (sample number =35).
3) Sake was added with either heat labile or heat stable α-amylase and then pasteurized. The sake added with the heat labile a-amylase lost its activity by heating at 55°C for 1.0 min or above 60°C. Amylase activity, however, was still alived by heating at 60°C for 2.0 min in the sake added with the stable α-amylase; It was lost by heating at 65°C for 1.0 min. Therefore, it will be possible to control the pasteurization of the bottled sake checking the a-amylase actvity by the blue-starch method.