The flour must be stored at room temperature.  Storage in a refrigerator, or particularly in a freezer, will affect the nature of the water in the sample and can lead to starch retrogradation and thus lowered starch damage values.

The procedure was developed for wheat flour.  You will have to define your own parameters for pasta.  We would suggest that the pasta is milled to pass a 0.5 mm screen and then you run the standard assay for flour.  You will need to dilute with a larger volume of sulphuric acid to get values on scale.

If the fungal alpha-amylase is not properly suspended before taking aliquot for dilution, there will be problems.  It is essential that the vial of fungal alpha-amylase is mixed (hand swirl) before an aliquot of the solution is removed.  The enzyme is a colloidal suspension.

We do not have a specific method for starch gelatinisation measurement, however our starch damage method works on this type of principle.  The damaged starch granules hydrate and swell and are susceptible to hydrolysis by fungal alpha-amylase.

All kit components are stable for 2-3 years if stored as supplied at 2-8˚C.  Diluted enzymes should be frozen in polypropylene tubes.  In this form they are stable for 2-3 years.  Enzymes can be freeze/thawed 2-3 times with no loss of activity.

The standard deviation within a run is 3% and from day to day is 3-5%.

With higher levels, one of two things could be limiting:
1) The level of fungal alpha-amylase or
2) The Glucose Determination Reagent.  
Could we suggest the following: first terminate the fungal alpha-amylase reaction with 5 mL of acid as per method; then dilute an aliquot four fold in 0.2% sulphuric acid; then analyse this and allow for dilution in calculation.  If the absorbance is above 1.20, the solution, after alpha-amylase treatment should be diluted 2-fold and the AMG step repeated.  If a dilution of much more than 2 fold is required, then you should use less starch in the initial step (say 50 mg).

The absorbance value for the glucose standard should be 1.10 to 1.20.  The enclosed starch control will give a value of about 0.90.

We would expect the value obtained to vary by no more than 0.2 for the sample supplied.  We have noticed that over a period of years, the measured level of starch damage actually falls slightly (e.g. by 0.2% for a flour with 6.2% starch damage, over a period of 4 years).  I believe that this is possibly due to water movement in the sample.

The flour supplied is a standard bread making flour from a commercial mill.  We assume the particle size is less than 0.5 mm.  It is hard to give the exact size.  The finer the material is milled the higher the starch damage. 

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme ([email protected]). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
3. State the kit lot number being used (this is found on the outside of the kit box).
4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
6. State the sample type and describe the sample preparation steps if applicable.

You may need to reduce the sample size to 50 mg and dilute up to 5 fold, i.e. 1 part filtrate plus 4 parts water.  The colours obtained for the samples should be less than that for the glucose control.

The Starch Damage method can be used to measure the degree of starch damage in any material, so it is applicable to all samples.  The only likely complication occurs when the degree of damage is more than 10%.  In this case, the solution after addition of sulphuric acid must be further diluted before treatment with amyloglucosidase.