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United States Department of Agriculture

Agricultural Research Service

Research Project: Postharvest Quality and Processing of Sugarcane and Sweet Sorghum for Sugar and Ethanol Production

Location: Commodity Utilization Research

Title: General Subject 2. Report to ICUMSA on the determination of carry-over alpha-amylase activity in white and refined sugars by a spectrophotometric method

Author
item Eggleston, Gillian

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: September 24, 2012
Publication Date: September 1, 2013
Citation: Eggleston, G. 2013. General Subject 2. Report to ICUMSA on the determination of carry-over alpha-amylase activity in white and refined sugars by a spectrophotometric method. In: Proceedings of the International Commission for Uniform Methods in Sugar Analysis, September 1, 2013, Cambridge, UK. p. 88-100.

Interpretive Summary: In recent years, there has been increased concern over carry-over or residual activity of high temperature (HT) and very high temperature (VHT) stable amylases (enzymes that break down starch) in white, refined sugars from refineries to various food manufacturing industries and other end-users. There was an urgent need for an industrial method to measure carry-over amylase in crystalline sugar products. An international report is given on the development of a method using Phadebas® blue starch, which is highly specific for alpha-amylase activity. The conditions of the method are described as well as method validation results.

Technical Abstract: A report is given on a new industrial method for the determination of carry-over alpha-amylase activity in raw and refined sugars, as well as a recommendation. In recent years, there has been increased concern over carry-over activity of mostly high temperature (HT) and very high temperature (VHT) stable amylases in white, refined sugars from refineries to various food manufacturing industries and other end-users. HT and VHT stable amylases were developed for much larger markets than the sugar industry with harsher processing conditions. There was an urgent need for an industrial method to measure carry-over amylase in crystalline sugar products. A selective method has been developed using Phadebas® blue starch, which is highly specific for alpha-amylase activity, has few reagents, and only uses existing equipment at the factory and refinery. Raw or refined cane sugars are diluted 60:40 w/v in calcium chloride-Tris buffer (pH 6.0) and mixed with a Phadebas® tablet and incubated at 37 degrees C for 45 min. The absorbance of the solution after gravity filtration is measured directly on a spectrophotometer at 620 nm. The presence of Ca2+ ions dramatically enhanced the amylase activity. Prolonged incubation past the optimum time of 45 min only reduced the sensitivity. The most accurate results were obtained when a standard curve was generated from standards that included a 60 Brix white beet sucrose solution. The mean activity range detected for refined sugars known to have detrimentally affected the texture of starch puddings was 101 to 145 COU/L (Carry Over Units/Liter) and 272 to 552 COU/L for raw sugars. The lower limit of detection for the industrial method was approximately 25 (COU/L). Precision generally improved with increased activities. The best data and performance statistics were observed for a raw sugar of mean activity of 552.3 COU/L and RSD value of 8.4%. The method is written up in ICUMSA format and attached at the end of the report.

Last Modified: 4/17/2014