英文名：Total Starch (AA/AMG) Assay Kit
规格：50 assays per kit
The Total Starch (AA/AMG) test kit is used for the measurement and analysis of total starch in cereal flours and food products. This kit now contains an improved α-amylase that allows the amylase incubations to be performed at pH 5.0 (as well as pH 7.0).
Colourimetric method for the determination of Total Starch in
cereal products, feeds, foodstuffs and other materials
(α-amylase, 100°C ± DMSO)
(1) Starch granules + H2O → maltodextrins
(2) Maltodextrins + H2O → D-glucose
(3) D-Glucose + H2O + O2 → D-gluconate + H2O2
(4) 2H2O2 + p-hydroxybenzoic acid + 4-aminoantipyrine →
quinoneimine + 4H2O
Kit size: 100 assays
Method: Spectrophotometric at 510 nm
Total assay time: ~ 90 min
Detection limit: 1-100% of sample weight
Cereal flours, food products and other materials
AOAC (Method 996.11), AACC (Method 76-13.01), ICC (Standard Method
No. 168), and RACI (Standard Method)
- Very competitive price (cost per test)
- All reagents stable for > 12 months after preparation
- Simple format
- Mega-Calc™ software tool is available from our website for hassle-free raw data processing
- Standard included
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.
We feel more comfortable with quadruplicate glucose controls. If the control is incorrect, or questionable, then all the results are in doubt.
Duplicate samples do not have to be measured. We just suggest this for laboratories starting up.
Q4. Does the Regular Maize Starch need to be analysed with pre-treating by DMSO? How do you store this Enclosed Control?
The Regular Maize Starch does not require DMSO pre-treatment. The value should be about 84% with a moisture content of about 12%, the final dry weight value is about 96-97%. Store the sample at room temperature, dry.
If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:
- Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
- 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.
- State the kit lot number being used (this is found on the outside of the kit box).
- State which assay format was used (refer to the relevant page in the kit booklet if necessary).
- State exact details of any modifications to the standard procedure that is provided by Megazyme.
- State the sample type and describe the sample preparation steps if applicable.
Yes. We believe that the DMSO step will solubilise vitrified starch in malt. Make sure that the malt is milled to pass a 0.5 mm screen. You could vary the time of cooking with DMSO to check solubilisation (i.e. 5 minutes, 10 minutes, or even up to 1 hour).
Q7. Is it possible to differentiate between gelatinised and ungelatinised starch in finished products, such as dog food, using the Total Starch Kit?
We think that there is a better chance of success using the Megazyme Starch Damage Kit.
Q8. Is it possible to use the Total Starch Kit to measure starch levels in plasterboard and related products?
There should be no problem in measuring the starch in plasterboard. I suggest that you grind about 100 g in a kitchen blender and then fine mill to pass 0.5 mm screen. Run a standard assay, but adjust volume to 10 mL after alpha-amylase treatment. Keep a close check on the pH. Plasterboard may push the pH value up (pH up to about 8 should be fine). You may be advised to run a DMSO format concurrently just to be sure. When you treat with amyloglucosidase, I would advise that you take 0.2 and 0.4 mL aliquots of digest (to get the colour up), also, be careful about checking the pH.
A 20% fat content could cause a problem for the method. We suggest that the sample be defatted before analysis for starch.
Q10. Do you have any kit or procedures for the determination of extractable starch in corn? This is of particular concern in the corn wet milling. I presume that extractable starch in corn is not the same as total starch?
Our Total Starch Assay Kit could measure starch left in a residue, or starch extracted. No method could measure potential extractable starch, as this will depend on numerous factors, including processing equipment, conditions etc.
Q11. Can I use another buffer instead of the MOPS with your Total Starch Assay Kit? If so, which would be suitable and easily prepared from commonly available laboratory reagents?
You can use phosphate buffer at the same concentration.
Q12. I wish to measure Total Starch in several products. These products contain 10-20% starch + maltodextrins at similar levels. Is it possible to remove the maltodextrins from the sample? Will ethanol work?
Most of the maltodextrins can be removed with 50% ethanol washing. If the starch is not gelatinised, it can be washed with cold water. This will remove all of the soluble maltodextrins, but the starch will spin down. If the starch has been gelatinised, then the best material which can be used for washing is 50% ethanol.
Q13. Could your Total Starch Assay Kit (K-TSTA) be used with success to measure total starch in plant tissues (samples of roots and shoots of maple trees)?
Yes, the Total Starch Kit can be used to measure starch in roots and shoots etc.
Q14. Is the accuracy of the Total Starch test affected by the presence of other inorganic chemicals and ground calcium carbonate in pulp?
We think that calcium carbonate etc. will not cause any problems. However, this of course depends on the amount present and if it changes the pH of the incubation mixture.
Q15. Does the Megazyme Total Starch method work well on all the new chemically modified starches that are now appearing, e.g. highly crosslinked, dextrinised and highly propylene oxide substituted?
The method will work for some chemically modified starches (e.g. crosslinked) however, if the degree of chemical modification is high, there will be an underestimation as the modification will interfere with complete hydrolysis to glucose and subsequent measurement.
You only need to wash samples which you feel may contain glucose and/or maltodextrins, e.g. breakfast cereals. There is little glucose in ground cereals, so it is not necessary to pre-wash these materials.
The enzymes from this kit are stable at room temperature for at least 6 months. At 4˚C, they are stable for several years.
The Starch Damage Kit may be best for this. If the starch is gelatinised and dried before analysis the correct results for gelatinisation will not be obtained.
The Total Starch Kit can accurately measure starch levels as low as 1% w/w.
The absorbance for 100 micrograms of glucose (in 3 mL of GOPOD Reagent) is about 0.97.
Yes, you can reduce the volume of GOPOD to 1 mL and use micro cuvettes. This will increase sensitivity by ~ 3-fold.
DMSO does solubilise resistant starch (crystallise amylose and amylopectin). The only starch material we have had problems in dissolving in DMSO is potato amylose.
Q23. When analysing samples containing sugars, an 80% v/v solution of ethanol is used to solubilise and remove the sugars. About how large are the smallest dextrins that are left in the starch (not solubilised) in this treatment?
We believe that for starch fragments, oligosaccharides of a DP up to 10 would be soluble in 80% alcohol. The degree of solubility of other oligosaccharides would depend on the sugar type and linkage type.
Q24. What is the sensitivity of the Total Starch Method for measurement in liquids containing low levels of starch?
The Total Starch Kit can be used for liquids containing as little as 200 micrograms per mL with some adjustments of conditions, as below:
Mix 0.5 mL of sample with 0.5 mL of 100 mM sodium acetate buffer (pH 4.5). Incubate at 40˚C and add 0.1 mL of Amyloglucosidase and incubate for 30 minutes. Add GOPOD reagent as usual. You will need to run an AMG blank as this enzyme preparation contains a very small amount of glucose.
Q25. AMYLOGLUCOSIDASE. The activity is stated as being 3260 U/mL (Soluble Starch). How was this determined?
The AMG activity was determined with soluble starch as substrate (10 mg/mL) in 0.1 M sodium acetate buffer at pH 4.5 and 40˚C. One Unit is the amount of enzyme required to hydrolyse one micromole of maltose per minute (i.e. to release 2 micromoles of glucose). Glucose release is measured with Glucose Determination Reagent.
Q26. I wish to know if it is possible to perform the assay under acidic conditions? I also need to alter the pH of the MOPS/amylase mixture to pH 3 or 4. Is it known if the amylase supplied with the Total Starch Assay Kit has activity at such a low pH?
We can assure you that the Total Starch Kit will not work if incubations with the thermostable alpha-amylase are performed at pH 3 or 4. This enzyme is inactivated at pH values below 5.0. You may wish to look at a method using just amyloglucosidase which is quite active down to pH 4.0. Check the old AOAC procedure for starch.
Q27. We are currently using your kits for Total Starch, Starch Damage and Sucrose/Glucose/Lactose Assay procedures. How accurate are the volumes contained? Can they be diluted entirely, to avoid wasting the contents while transferring?
When we dispense the enzymes we usually include an extra 5% in each vial, so yes, you can dilute the whole vial. When you do this, please divide into aliquots and store them frozen.
Q28. Which analytical procedure would you recommend for determination of total starch residues in brewing wort and final beer. Megazyme’s procedure – AA/AMG’97, only refers to solid (and not solubilised) starch.
You can use the standard procedure. We would recommend that you treat 2 mL of beer or wort with 8 mL of ethanol, stir and centrifuge (3,000 rpm). Wash the pellet with 10 mL of 80% ethanol. Then dissolve/suspend the pellet in 2 mL of sodium acetate buffer (pH 4.5, 0.1 M) and cook at 100˚C for 10 min. Then add 0.1 mL AMG from the Total Starch Kit and proceed according to the method. You will have to determine the degree of dilution for yourself. Treat 0.1 mL with GOPOD etc.