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Title: The need for and development of a method to measure carry-over amylase activity in raw and refined sugars

item Eggleston, Gillian
item Gober, Jessica
item St Cyr, Eldwin

Submitted to: Sugar Industry Technologists Meeting
Publication Type: Proceedings
Publication Acceptance Date: 5/20/2013
Publication Date: 9/1/2013
Citation: Eggleston, G., Gober, J., St Cyr, E. 2013. The need for and development of a method to measure carry-over amylase activity in raw and refined sugars. In: Proceedings of the Sugar Industry Technologists. 72(1050):80-99.

Interpretive Summary: The activities or strengths of commercial amylases used at sugarcane factories and refineries could not be directly compared because there was no uniform method used by vendors to measure the activity. A report is given on a new industrial method for the determination of the activity of commercial amylase (an enzyme that is used to break down and control starch in sugarcane factories and sometimes refineries). The method uses Phadebas® blue starch, which is highly specific for amylase activity. The reactions conditions were chosen to simulate industrial conditions, and single laboratory validation results are described.

Technical Abstract: In recent years, there has been increased world-wide concern over carry-over activity of mostly high temperature (HT) and very high temperature (VHT) stable amylases in refined sugars to various food and end-user industries. 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 raw and refined sugars. A 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 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 calcium ions dramatically enhanced the sensitivity, whereas prolonged incubation past 45 min only reduced sensitivity. The most accurate results were obtained when a standard curve was generated from standards that included a 60 Brix pure sucrose solution. When carry-over activity was detected in raw sugars they tended to be higher than in refined sugars because less processing time is available to deactivate the enzyme. 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 for raw sugars 272 to 552 COU/L. The lower limit of detection for the method was approximately 25 (COU/L). Precision generally improved with increased activities. Relative standard deviations (RSDs) for refined sugars ranged from ~12 to 20%.