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AACC International Approved Methods - AACC Method 22-02.01. Measurement of alpha-Amylase in Plant and Microbial Materials Using the Ceralpha Method

AACC International Approved Methods

Enzymes

AACCI Method 22-02.01
Measurement of alpha-Amylase in Plant and Microbial Materials Using the Ceralpha Method

VIEW METHOD

Objective
Microbial alpha-amylases find widespread application in the modification of starch in cereal products and in cereal processing. The level of endogenous alpha-amylase in cereal grains and products significantly affects the industrial exploitation of these commodities. In breadmaking, the level of alpha-amylase must be sufficient to produce saccharides that can be absorbed and utilized by yeast but not so high as to cause excessive starch dextrinization, which can lead to sticky crumb and problems in processing. In the brewing industry, the level of malt alpha-amylase is a key quality parameter. alpha-Amylase also finds application as a silage additive, to assist in the degradation of starch and thus to provide fermentable sugars for bacterial growth. The procedure described here (the Ceralpha method) can be used to measure cereal flour, malt, and fungal and bacterial alpha-amylases. Samples are extracted with appropriate buffer and, following filtration or centrifugation (if required), are suitably diluted. Aliquots of diluted extract are incubated with substrate solution under defined conditions of temperature, time, and pH, and the reaction is stopped with an alkaline solution that also develops the color.

The assay is absolutely specific for alpha-amylase. The substrate mixture contains the defined oligosaccharide “nonreducing end-blocked p-nitrophenyl malto­heptaoside” (BPNPG7) in the presence of excess levels of a thermostable alpha-glucosidase (which has no action on the native substrate due to the presence of the “blocking group”). On hydrolysis of the oligosaccharide by endo-acting alpha-amylase, the excess quantities of alpha-glucosidase in the mixture give instantaneous and quantitative hydrolysis of the p-nitrophenyl maltosaccharide fragment to glucose and free p-nitrophenol. The absorbance at 400 nm is measured, and this is a direct measure of the level of alpha-amylase in the sample analyzed. The assay can be used over the pH range 5.2-7.0 and at temperatures of up to 60°. The optimal pH for cereal and fungal alpha-amylases is 5.4 and for bacterial alpha-amylase is 6.5.

A spreadsheet calculator accompanies this method.