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.

Yes.

From our experience, the level of beta-amylase/pullulanase etc. added is more than adequate to completely hydrolyse any starch likely to be present.  However, if there is a suspicion of incomplete starch hydrolysis, you can check this by running the hydrolysis with the pullulanase/beta-amylase etc. over a range of incubation times and comparing the results.

The method measures inulin and cereal fructans.

Fructan Control Flour (29.6%) 0.496 0.498
Fructose Control 1.133 1.135.

Our reproducibility is usually +/- 5 to 7% of the fructan value.

With all our kits we can obtain approx. 5% c.v. in interlaboratory studies.  In-house these values are closer to 3%.

The fructanase in the kit will completely hydrolyse the fructans in plants.  These are either inulin type or "branched" type (as in oat or wheat stems).  We do not know if the enzymes would act on pure levan.

The value of 54.5 is the actual amount of fructose standard used.  This amount of fructose will give a certain colour in the PAHBAH assay.
Thus; F = factor = [54.5 (micrograms of fructose)] divided by the absorbance value obtained for 54.5 micrograms of fructose in the PAHBAH method.

Yes.  The best enzyme to use for analytical purposes is exo-Inulinase (E-EXOIAN).

Yes, the sugar enzyme can be used to measure sucrose.  The enzyme is an alpha-glucosidase, so it will also hydrolyse maltose if this is present.

Extract solutions are best made and analysed the same day (not stored overnight). But if they are stored overnight in a refrigerator, heat them to 80˚C, and cool before analysing (to ensure all fructan has re-dissolved).

Yes.

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.

Yes , the sucrase does hydrolyse sucrose only and has no action on the other carbohydrates that you mention.  The “sucrase” enzyme is an alpha-glucosidase which we sell as Alpha-Glucosidase (Maltase) E-MALTS.

Fructans are very soluble and will redissolve very readily.  They may precipitate on freezing (at least the polymeric portion) but they will easily re-dissolve at 80˚C.