AOAC Official Method 2002.01 Measurement of alpha.gif (53 bytes)-Amylase Activity in White Wheat Flour, Milled Malt, and Microbial Enzyme Preparations Ceralpha Assay First Action 2002

A. Principle

Test portions are extracted with salt solution or buffer at room temperature or 40°C. Extracts are clarified by centrifugation or filtration and diluted. Aliquots of diluted extract are incubated with the substrate mixture under defined conditions of pH, temperature, and time. The substrate is nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable -glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, -glucosidase, or -amylase. When the substrate is cleaved by endo-acting -amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Results are expressed in Ceralpha units; 1 unit is defined as the amount of enzyme required to release (in the presence of excess -glucosidase) 1 µmol p-nitrophenol per minute at 40°C