α-Amylase SD Assay Kit (High Sensitivity Method)

The quality of wheat for baking is critically dependent on the level of α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1), which can be present as “late maturity α-amylase” (LMA), or due to pre-harvest sprouting due to high rainfall and humidity at the time of harvesting. The most commonly used method to measure α-amylase in wheat grain is the Hagberg Falling Number method, but values are also influenced by rheological properties of starch in the grain. In this study we describe a simple, rapid, automated method (Amylase SD) for measurement of α-amylase in pre-harvest sprouted (sprout damaged) wheat grain. The method (Amylase SD) measures the release of p-nitrophenol from 4,6-O-ethylidene-α-4-nitrophenyl-maltoheptaoside by α-amylase in the presence of α-glucosidase. The absorbance of p-nitrophenolate measured at 405 nm in a ChemWell^®-T auto-analyser is directly related to the level of α-amylase activity present in the milled wheat grain extract. The Amylase SD method generated < 6%CV and correlation to the Falling Number method was represented by an inflection point at ∼160 s. The precision, sensitivity and speed of this method provides an ideal alternative to the Falling Number method for measurement of α-amylase (sprout damage) in wheat grain in wheat breeding programmes or at grain receival points.

α-Amylase content in milled wheat is a key quality parameter in the baking industry. This metric is usually determined using the Hagberg Falling Number (FN) method, the results of which can be used to segment grains into a range of quality categories. The authors describe herein an alternative to the traditional FN assay, namely the Amylase SD assay, as a predictor of baked bread quality. 144 Wheat grain samples with known FN values were analysed using the Amylase SD assay and a simple model for the conversion of Amylase SD units into “predicted FN” (PFN) values has been described. To test the usefulness of this approach, 11 separate wheat grain samples were analysed using the FN method, the Amylase SD assay, and also for gluten content. These grain samples were then milled to produce flour which was re-analysed using the Amylase SD assay and then formulated into dough that was subjected to Mixolab analysis, before being baked to produce pan bread. Correlations of baked bread volume and common Mixolab parameters to both FN and PFN values have been investigated for each sample. The Amylase SD assay has been shown to be a potentially promising alternative to the FN method for use as a baked bread quality predictor.

Background 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 lowα-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. Results 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. Conclusion 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.