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α-Amylase SD Assay Kit

0:03  Introduction
1:03  Principle
1:57  Reagent Preparation
3:42  Enzyme Extraction
5:47  Procedure
8:18  Calculation

α-Amylase SD Assay Kit
alpha-Amylase SD Assay Kit High Sensitivity Method K-AMYLSD
   
Product code: K-AMYLSD
€217.00

160 / 320 assays (manual) / 640 assays (auto-analyser)

Prices exclude VAT

Available for shipping

Content: 160 / 320 assays (manual) / 640 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: α-Amylase
Assay Format: Spectrophotometer, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 400,
405  
Signal Response: Increase
Limit of Detection: 0.05 U/mL
Reproducibility (%): ~ 3%
Total Assay Time: ~ 20 min
Application examples: Sprout damaged wheat grain and food products such as jam sauces conserves and ice cream.
Method recognition: AACC Method 22-02.01, AOAC Method 2002.01 and ICC Standard No. 303

The Amylase SD Method is a highly sensitive colourimetric method for the determination of α-amylase in sprout damaged wheat grain (also known as pre-harvest sprouting or weather damaged wheat grain) and “late maturity α-amylase” wheat grain. Can also be used for measuring α-amylase in confectionery, soft drinks, brewing and fermentation, jams, sauces, conserves, ice cream and baby food.

Advantages
  • Extremely high sensitivity - 2.4-fold increase over Ceralpha (K-CERA) 
  • Very cost effective 
  • All reagents stable for > 2 years after preparation 
  • Very specific 
  • Simple format 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included 
  • Suitable for Manual and auto-analyser formats
Validation of Methods
Publications
Megazyme publication
Investigation into the use of the amylase SD assay of milled wheat extracts as a predictor of baked bread quality.

Mangan, D., Szafranska, A., McKie, V. & McCleary, B. V. (2016). Journal of Cereal Science, 70, 240-246.

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.

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Megazyme publication

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

Cornaggia, C., Ivory, R., Mangan, D. & McCleary, B. (2016). Journal of the Science of Food and Agriculture, 96(2), 404-412.

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 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. 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.

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Megazyme publication
A rapid, automated method for measuring α-amylase in pre-harvest sprouted (sprout damaged) wheat.

McKie, V. A. & McCleary, B. V. (2015). Journal of Cereal Science, 64, 70-75.

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.

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Publication

Evaluation of Commercial &alpha;‐Amylase ELISA Test Kits for Wheat.

Kiszonas, A. M. & Morris, C. F.  <i>Cereal Chemistry</i>, In Press.

<b>Background and objectives:</b> There are few methods in wheat to directly measure &alpha;-amylase, which is the result of pre-harvest sprouting (PHS) and late-maturity α-amylase (LMA). Both of these conditions are associated with reduced wheat and flour quality. Those methods that do exist for measuring &alpha;-amylase in wheat are based on enzyme activity. The objective of this study was to evaluate commercially available enzyme linked immunosorbent assay (ELISA) kits on sound grain, sprouted grain, and grain that had both PHS and LMA. <b>Findings:</b> Three kits were evaluated; human AMY1 and AMY2, and a “plant α-amylase” kit. The wheat samples were also assayed for &alpha;-amylase activity using the Megazyme <i>SD</i> &alpha;-amylase method. All three ELISA kits functioned very well based on calibration curves (<i>R<sup>2</sup></i> > 0.99). Of the three, the AMY1 and plant &alpha;-amylase kits appeared to show no useful detection of wheat &alpha;-amylase. The AMY2 kit, however, detected a 2.7 fold increase in &alpha;-amylase protein in the PHS and PHS/LMA grain lots, similar to a 2.7 fold difference in activity. <b>Conclusions:</b> It appears that the particular human AMY2A ELISA kit, and perhaps others, can detect &alpha;-amylase in wheat and may be a useful alternative technology to enzyme kits. These kits may also be useful in predicting PHS and LMA potential damage. <b>Significance and novelty:</b> Thus far, &alpha;-amylase studies have focused on enzyme kits, whereas this study examined three commercially available ELISA kits to measure &alpha;-amylase content in sound, sprouted, and PHS/LMA grain. Both PHS and LMA are highly detrimental to grain and result in low prices paid to growers.

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Safety Information
Symbol : GHS07
Signal Word : Warning
Hazard Statements : H315, H319, H335
Precautionary Statements : P261, P264, P271, P280, P302+P352, P304+P340
Safety Data Sheet
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