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Alpha-amylase assay and uses thereof

a technology of alpha-amylase and assay, applied in the field of alpha-amylase assay, can solve the problems of reducing the value of flour for baking and food production, sticking or doughy bread, and unsuitable commercial production

Inactive Publication Date: 2006-06-29
VICAM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Provides a more accurate and reliable measurement of α-amylase activity, enabling better prediction of flour performance and value, and allowing for the determination of both endogenous and exogenous α-amylase levels in flour samples.

Problems solved by technology

For example, excess starch breakdown in flour can result in sticky or doughy bread that can't be cut in automated loaf-slicing machinery and is therefore unsuitable for commercial production.
Insufficient α-amylase activity in flour can also reduce the value of a flour for baking and food production.
Insufficient α-amylase activity in flour can result flour that lacks the necessary levels of sugars for proper fermentation and yeast activity in baking.
Although the Falling Number test is currently accepted as the industry standard, it does not measure the actual α-amylase enzyme activity level directly, and it is the activity of the enzyme that affects baked good texture and value.
Alternative methods that directly measure α-amylase enzyme activity have been developed, but are not used to fluorometrically test flour, amylase concentrate, or stock samples, which limits their usefulness.

Method used

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  • Alpha-amylase assay and uses thereof
  • Alpha-amylase assay and uses thereof
  • Alpha-amylase assay and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Introduction

[0073] Wheat or fungal α-amylase activity in flour samples is tested. The method may also be used to measure the activity of other types of microbial α-amylase, such as bacterial α-amylase activity.

Methods

Preparation of a Fluorescent Starch Substrate for Use in Assaying α-amylases

[0074] Starch, for example potato starch or waxy maize starch, is activated by reaction with alkaline cyanogen bromide using the method of Cuatrecasas, P. and Anfinsen, C., Meth. Enzymol. 22:351-378, 1971. Activation is followed by reaction with a fluorescent dye that has a free amino group. The amino group of the fluorescent molecule reacts with the cyanogen-bromide-activated starch according to the following reactions, wherein R=ligand (e.g. fluorescent or enzyme).

[0075] The amount of derivatization of the starch is kept to 1 in 300 to 1 in 1300 D-glucose residues to allow α-amylase to react with the derivatized starch. The labeling ratio can be determined by NMR using standard methods....

example 2

Methods

[0082] The following buffers were used as indicated in the Examples below.

[0083] Reaction Buffer Stock was prepared by dissolving 17.6 ml Acetic acid, 16.0 gm anhydrous sodium acetate, 29.2 gm sodium chloride, and 5.6 gm calcium chloride in de-ionized (DI) water to a final volume of 1 liter. The Reaction Buffer used in the assays was prepared by diluting the Reaction Buffer Stock 1:10 v / v with DI.

[0084] Stop Buffer was prepared by mixing 115.5 ml acetic acid with DI water to a final volume of 1 liter.

[0085] Phosphate Buffer (Reaction Buffer for Cereal Amylase) was prepared by dissolving 1.2651 gm anhydrous monobasic sodium phosphate and 1.562 gm anhydrous dibasic sodium phosphate in DI to a final volume of 1 liter.

[0086] Substrate Tubes used in the reactions were AMYLease™ substrate tubes (Vicam, Watertown, Mass.) containing labeled starch substrate.

Test for Cereal Amylase

Introduction

[0087] The objective of this study was to measure cereal amylase activity.

[0088] Th...

example 3

Test For Fungal Amylase in Wheat Flour and Fungal Concentrates

Introduction

[0090] The objective of this study was to utilize to measure fungal amylase activity in wheat flour and in concentrated fungal amylase preparations (e.g., amylase concentrates). Tests were conducted with two types of commercial fungal amylase preparations. Doh Tone™ (American Ingredients, Inc. Anaheim, Calif.) contains 5.5% by weight neat fungal amylase, and it also contains fungal proteinases, wheat starch and silica. Doh Tone™ is usually dosed at 2 g per 100 lb wheat flour. Doh Tone™ II (American Ingredients, Inc.) contains 2.75% by weight of neat fungal amylase, wheat starch and silica. Doh Tone™ II does not contain proteinases. Doh Tone™ II is usually dosed at 4 g per 100 lb wheat flour.

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PUM

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Abstract

This invention relates to methods and kits for measuring α-amylase activity in samples such as flour, stock, or amylase concentrate.

Description

RELATED APPLICATION [0001] This application is a continuation-in-part of U.S. application Ser. No. 10 / 230,969, filed Aug. 29, 2002, now pending.FIELD OF THE INVENTION [0002] This invention relates to methods and kits for measuring α-amylase activity in grain products such as flour. BACKGROUND OF THE INVENTION [0003] The use of flour produced through the processing of cereal grains such as wheat, rye, and oats is an important feature in nutrition and food production around the world. Grains are grown, harvested, and milled into flours, which are used to make breads, bakery products, pastas, all of which are staples in the diet of many individuals world wide. Grains and grain products are also utilized for brewing and fermentation. [0004] Wheat flour is an important ingredient in home baking and is the foundation for almost every commercially baked product and pasta. Of the grains available for the production of flour, wheat is unique in that it is the only cereal grain with sufficien...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/573C12Q1/40G01N33/10
CPCC12Q1/40G01N33/10
Inventor COHEN, BARB ARIELPOWERS, STEPHEN P.FAN, KESUO
Owner VICAM