Novel assay procedures for the measurement of α-amylase in weather-damaged wheat.

Abstract

The measurement of α-amylase (EC 3.2.1.1) in sprout-damaged grains is a crucial analysis yet a problematic one owing to the typically low α-amylase levels in ground wheat samples. A number of standardised methods such as the Falling Number method and the Ceralpha method exist which are routinely used for the assay of α-amylase. These methods, however, are either highly substrate-dependent or lack the required sensitivity to assess sprout damage. Novel colorimetric and fluorometric reagents have been prepared (Amylase HR, Amylase SD, BzCNPG7 reagent and BzMUG7 reagent) for the direct and specific assay of α-amylase activity in sprout-damaged wheat. Assays employing these reagents have been developed and optimised to include a decolourisation step using activated charcoal. When used in a convenient assay format, Amylase SD--containing EtNPG7 (II) as the colorimetric substrate and α-glucosidase as the ancillary enzyme--was found to be an excellent reagent for the assessment of sprout damage in wheat with incubation times as short as 5 min. The assay using Amylase SD is completely specific for α-amylase. The use of the Amylase SD assay represents a sensitive and valid alternative to the traditionally used Falling Number values for the assessment of sprout damage in wheat samples.