FOOD COMPOSITION AND ADDITIVES Measurement of a-Amylase Activity in White Wheat Flour, Milled Malt, and Microbial Enzyme Preparations, Using the Ceralpha Assay: Collaborative Study

This study was conducted to evaluate the method performance of a rapid procedure for the measure-ment of a-amylase activity in flours and microbial enzyme preparations. Samples were milled (if nec-essary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing a-amylase) were incubated with sub-strate mixture under defined conditions of pH, tem-perature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of ex-cess quantities of thermostable a-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, a-glucosidase, and b-amylase. When the substrate is cleaved by endo-acting a-amylase, the nitrophenyl oligosaccharide is im-mediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of a-glucosidase present in the sub-strate mixture. The reaction is terminated, and the phenolate color developed by the addition of an al-kaline solution is measured at 400 nm. Amylase ac-tivity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 mmol p-nitrophenyl (in the presence of excess quantities of a-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories ana-lyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal a-amylase had been added, milled malt, and fungal and bacterial enzyme prepara-tions. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility rela-tive standard deviations ranged from 5.0 to 16.7%