www.megazyme.

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TOTALSTARCH
ASSAYPROCEDURE
(AMYLOGLUCOSIDASE/
-AMYLASE METHOD)
K-TSTA 09/14
(100 Assays per Kit)

AOAC Method 996.11

AACC Method 76-13.01
(and improvements)

Megazyme International Ireland 2014

INTRODUCTION:
Starch determination methods are broadly grouped into acid
hydrolysis or enzymic procedures.1 Acid hydrolysis procedures
can only be applied to pure starch samples and thus have limited
application. Enzymic procedures vary in pre-treatment steps,2 starch
gelatinisation, liquefaction and dextrinisation, hydrolysis of dextrins to
glucose and glucose measurement.3 AACC Method 76-114 specifies
starch gelatinisation under aqueous conditions in an autoclave,
followed by starch conversion to glucose with amyloglucosidase and
glucose measurement. AACC Method 76-11 underestimates starch
content in a range of samples and materials, including high amylose
maize starches and many processed cereal products. Most methods in
use today incorporate treatment with thermostable -amylase either
during or immediately following the starch gelatinisation step.5,6 For
samples which are difficult to gelatinise (such as high amylose maize
starch) solvents such as sodium hydroxide or dimethyl sulphoxide
(DMSO)7,8 have been employed. In a procedure to ensure complete
solubilisation of starch in dietary fibre determination, Englyst and
Cummings (1988)7 included treatment with the starch debranching
enzyme, pullulanase.
To satisfy the need for an extremely simple, yet quantitative and
reliable, procedure for the measurement of total starch Megazyme
produces, and offers, a total starch assay kit based on the use of
thermostable -amylase and amyloglucosidase (McCleary et al.9). This
method has been adopted by AOAC (Official Method 996.11) and
AACC (Method 76.13.01).
More recently, thermostable -amylases that are active and stable
at lower pH values have become available. Consequently, we have
updated our total starch methodology to incorporate such an
enzyme.10 The major advantage of this improvement is to allow both
the thermostable -amylase and amyloglucosidase incubation steps
to be performed at the same pH (pH 5.0) which, in turn, simplifies
the assay and minimises the possibility of production of maltulose
(4--glucopyranosyl-D-fructose)11, which is resistant to hydrolysis by
amyloglucosidase and -amylase.
The Megazyme total starch analysis procedure allows the
measurement of total starch in a wide range of food, feed, plant and
cereal products (natural or processed). For most samples (e.g. wheat
flour), starch is completely solubilised on incubating the sample at
approx. 100C in the presence of thermostable -amylase. Samples

containing high levels of resistant starch (e.g. high amylose maize

starch), require pre-dissolution in cold 2 M KOH or hot DMSO. For
samples containing soluble starch or maltodextrins, cooking with
thermostable -amylase is not required.
PRINCIPLE:
Thermostable -amylase hydrolyses starch into soluble branched and
unbranched maltodextrins (1).

-amylase, pH 7.0 or 5.0, 100C
(1) Starch + H2O
maltodextrins
Where necessary, resistant starch in the sample is pre-dissolved
by stirring the sample with 2 M KOH at approx. 4C, followed by
neutralisation with sodium acetate buffer and hydrolysis with
-amylase (2). Alternatively, dissolution in DMSO at 100C is
effective.

KOH then neutralisation + -amylase
(2) Resistant starch + H2O
maltodextrins
Amyloglucosidase (AMG) quantitatively hydrolyses maltodextrins to
D-glucose (3).
AMG
(3) Maltodextrins

D-glucose

D-Glucose is oxidised to D-gluconate with the release of one mole of

hydrogen peroxide (H2O2) which is quantitatively measured in a
colourimetric reaction employing peroxidase and the production of a
quinoneimine dye.
(glucose oxidase)

(4) D-Glucose + O2 + H2O

D-gluconate + H2O2

(5) 2 H2O2 + p-hydroxybenzoic acid + 4-aminoantipyrine

(peroxidase)

quinoneimine dye + 4 H2O

Samples containing high levels of D-glucose and maltodextrins are

washed with aqueous ethanol (80% v/v) before analysis.
Analysis of a single sample can be performed within 70 min. Twenty
samples can be analysed within 2 h.

SPECIFICITY, SENSITIVITY, LINEARITY AND

PRECISION:
The assay is specific for -glucans (including starch, glycogen,
phytoglycogen and non-resistant maltodextrins).
The smallest differentiating absorbance for the assay is 0.010
absorbance units. This corresponds to 1.0 mg of D-glucose (or
0.9 mg starch)/L of sample solution at the maximum sample volume
of 1.00 mL. The detection limit is 2.0 mg D-glucose (or 1.8 mg
starch)/L, which is derived from an absorbance difference of 0.020
with the maximum sample volume of 1.00 mL.
The assay is linear over the range of 5 to 100 g of D-glucose per
assay. In duplicate determinations using one sample solution, an
absorbance difference of 0.005 to 0.010 may occur. With a sample
volume of 1.00 mL, this corresponds to a D-glucose concentration of
approx. 0.5 to 1.0 mg/L of sample solution. If the sample is diluted
during sample preparation, the result is multiplied by the dilution
factor, F. If, in sample preparation, the sample is weighed, e.g. 10 g/L,
a difference of 0.02 to 0.05 g/100 g can be expected.
INTERFERENCE:
If the conversion of D-glucose has been completed within the time
specified in the assay (approx. 5 min), it can be generally concluded
that no interference has occurred. However, this can be further
checked by adding D-glucose (approx. 50 g in 0.1 mL) to the
cuvette on completion of the reaction. A significant increase in the
absorbance should be observed.
Interfering substances in the sample being analysed can be identified
by including an internal standard. Quantitative recovery of this
standard would be expected. Losses in sample handling and
extraction are identified by performing recovery experiments, i.e. by
adding D-glucose to the sample in the initial extraction steps.
SAFETY:
The general safety measures that apply to all chemical substances
should be adhered to.
For more information regarding the safe usage and handling of this
product please refer to the associated SDS that is available from the
Megazyme website.

KITS:
Kits suitable for performing 100 determinations of total starch are
available from Megazyme. The kits contain the full assay method plus:
Bottle 1:

Thermostable -amylase (10 mL, 3,000 U/mL

on Ceralpha reagent* at pH 6.5 and 40C or 1600
U/mL on Ceralpha reagent at pH 5.0 and 40C).
Stable for > 4 years at 4C.

Bottle 2:

Amyloglucosidase (10 mL, 3300 U/mL on soluble

starch or 200 U/mL on p-nitrophenyl -maltoside*)
at pH 4.5 and 40C.
Stable for > 3 years at 4C.

*Full assay procedure is available at www.megazyme.com - Product

Codes: K-CERA (bottle 1) and R-AMGR3 (bottle 2).
Bottle 3:
GOPOD Reagent Buffer. Buffer (50 mL,
pH 7.4), p-hydroxybenzoic acid and sodium azide

(0.095% w/v).
Stable for > 4 years at 4C.
Bottle 4:
GOPOD Reagent Enzymes. Glucose

oxidase plus peroxidase and 4-aminoantipyrine.
Freeze-dried powder.
Stable for > 5 years at -20C.
Bottle 5:
D-Glucose standard solution (5 mL, 1.0 mg/mL) in

0.2% (w/v) benzoic acid.
Stable for > 5 years at room temperature.
Bottle 6:

Standardised regular maize starch control. Starch

content shown on vial label.
Stable for > 5 years at room temperature.

PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:

Solution 1.

Dilute 1.0 mL of the contents of bottle 1 to 30 mL

with Reagent 1 (100 mM sodium acetate buffer,
pH 5.0; not supplied). Store the diluted enzyme
frozen between use. Divide into appropriately sized
aliquots and store in polypropylene tubes at -20C
between use and keep cool during use if possible.
Stable for > 3 years at -20C.

NOTE:
If the sample is to be analysed according to AOAC Official Method
996.11 (example b), the enzyme is diluted in MOPS buffer (50 mM,
pH 7.0; Reagent 4)
4

Solution 2.
Use the contents of bottle 2 as supplied. This

solution is viscous and thus should be dispensed

with a positive displacement dispenser, e.g.
Eppendorf Multipette with 5.0 mL Combitip (to

dispense 0.1 mL aliquots).

Stable for > 3 years at 4C.
Solution 3.

Dilute the contents of bottle 3 (GOPOD Reagent

Buffer) to 1 L with distilled water. Use immediately.

NOTE:
1. If GOPOD Reagent Buffer is stored at -20C, it will form salt
crystals that must be completely dissolved when this buffer is
diluted to 1 L with distilled water.
2. This concentrated buffer contains 0.095% (w/v) sodium azide.
This is a poisonous chemical and should be treated accordingly.
Solution 4.
Dissolve the contents of bottle 4 in 20 mL of

solution 3 and quantitatively transfer this to the

bottle containing the remainder of solution 3.

Cover this bottle with aluminium foil to protect the

enclosed reagent from light. This is Glucose
Determination Reagent (GOPOD Reagent).
Stable for ~ 3 months at 2-5C or > 12 months at
- 20C.

If this reagent is to be stored in the frozen state,

preferably it should be divided into aliquots. Do not
freeze/thaw more than once.

When the reagent is freshly prepared it may be light

yellow or light pink in colour. It will develop a
stronger pink colour over 2-3 months at 4C. The
absorbance of this solution should be less than 0.05
when read against distilled water.

Solutions
5 & 6.

Use the contents of bottles 5 and 6 as supplied.

Stable for > 5 years at room temperature.

REAGENTS (NOT SUPPLIED):

1. Sodium acetate buffer (100 mM, pH 5.0) plus calcium
chloride (5 mM).

Add 5.8 mL of glacial acetic acid (1.05 g/mL) to 900 mL of

distilled water. Adjust the pH to 5.0 by the addition of 1 M
(4 g/100 mL) sodium hydroxide solution (approx. 30 mL is
required).
Stable for approx. 2 months at 4C.
5

Add 0.74 g of calcium chloride dihydrate and dissolve.

Adjust the volume to 1 litre and store the buffer at 4C.
Stable for > 6 months at 4C.

The stability of this buffer can be increased by adding

sodium azide (0.2 g of sodium azide/L buffer).
Stable for approx. 2 years at room temperature.

NOTE: Sodium azide should not be added until the pH is adjusted.

Acidification of sodium azide releases a poisonous gas.
2. Sodium acetate buffer (1.2 M, pH 3.8).

Add 69.6 mL of glacial acetic acid (1.05 g/mL) to 800 mL of

distilled water and adjust to pH 3.8 using 4 M sodium hydroxide.
Adjust the volume to 1 litre with distilled water.
Stable for 12 months at room temperature.

3. Potassium hydroxide solution (2 M).

Add 112.2 g KOH to 900 mL of deionised water and dissolve by

stirring. Adjust volume to 1 litre. Store in a sealed container.
Stable for > 2 years at room temperature.

4. MOPS buffer (50 mM, pH 7.0) plus calcium chloride (5 mM)

and sodium azide (0.02% w/v). Optional: Only required if
samples are analysed according to example (b).

Dissolve 11.55 g of MOPS (sodium salt, Sigma cat. no. M-9381)

in 900 mL of distilled water and adjust the pH to pH 7.0 by the
addition of 1 M (10% v/v) HCl (approx. 17mL is required).
Add 0.74 g of calcium chloride dihydrate and 0.2 g of sodium
azide and dissolve. Adjust the volume to 1 L.
Stable for 6 months at 4C.

5. Sodium acetate buffer (200 mM, pH 4.5) plus sodium azide

(0.02% w/v). Optional: Only required if samples are analysed
according to example (b).

Add 11.6 mL of glacial acetic acid (1.05 g/mL) to 900 mL of

distilled water. Adjust the pH to 4.5 by the addition of 1 M
(4 g/100 mL) sodium hydroxide solution (approx. 60 mL is
required). Add 0.2 g of sodium azide and dissolve. Adjust the
volume to 1 L.
Stable for 6 months at 4C.
NOTE: Sodium azide should not be added until the pH is adjusted.
Acidification of sodium azide releases a poisonous gas.
6

EQUIPMENT (RECOMMENDED):
1.

Glass test tubes (round bottomed; 16 x 120 mm or

18 x 150 mm).

2. Micro-pipettors, 100 L (e.g. Gilson Pipetman or Rainin

EDP-2 motorised dispenser).
3.

Positive displacement pipettor, e.g. Eppendorf Multipette

- with 5.0 mL Combitip (to dispense 0.1 mL aliquots of

amyloglucosidase solution).

- with 50 mL Combitip (to dispense 3 mL aliquots of bacterial

-amylase solution).

4.

Bench centrifuge (required speed 3,000 rpm; approx. 1,800 g).

5.

Analytical balance.

6.

Spectrophotometer set at 510 nm.

7.

Vortex mixer (e.g. IKA Yellowline Test Tube Shaker TTS2).

8.

Thermostated water bath set at 50C.

9.

Boiling water bath with tube rack.

10. Stop clock.

CONTROLS AND PRECAUTIONS:
1.

The time of incubation with GOPOD reagent is not critical,

but should be at least 20 min. The colour formed should be
measured within 60 min.

2.

With each set of determinations, reagent blanks and glucose

controls (100 g, quadruplicate) should be included.

a) The reagent blank consists of 0.1 mL distilled water + 3.0 mL

GOPOD Reagent.

b) The glucose control consists of 0.1 mL glucose standard

solution (100g/0.1 mL) + 3.0 mL GOPOD Reagent. The
Factor F (pages 13 and 14) is calculated by dividing the
amount of D-glucose analysed (100 g) by the absorbance
obtained for this amount of D-glucose in the standard assay
(e.g. 100/1.038 = 96.339). The absorbance value will
vary.

3.

With each set of determinations, a standard flour or starch

sample should be included.

4.

With each new batch of GOPOD Reagent, the time for maximum
colour formation with 100 g of glucose standard should be
checked. This is usually approximately 15 min.
7

SAMPLE BLANKS:
Sample blanks can be determined using the Standard Assay Procedure
[example (a)] with the modifications that in Step 4, three (3) mL of
distilled water is used and in Step 5, amyloglucosidase is replaced by
water. Alternatively, the need to perform sample blank analysis can be
avoided by pre-extraction of samples with aqueous ethanol (80% v/v)
[see example (e)].
SAMPLE PREPARATION EXAMPLES:
(a) Determination of starch in cereal and food products not
containing resistant starch, D-glucose and/or maltodextrins
(Recommended Procedure; all incubations at pH 5.0).
1.

Mill cereal, plant or food product to pass a 0.5 mm screen.

2.

Add milled sample (~ 100 mg; weighed accurately) to a glass test

tube (16 x 120 mm). Tap the tube to ensure that all of the sample
drops to the bottom of the tube.

3.

Add 0.2 mL of aqueous ethanol (80% v/v) to wet the sample and aid
dispersion. Stir the tube on a vortex mixer.

4.

Immediately add 3 mL of thermostable -amylase (contents of bottle

1 diluted 1:30 in Reagent 1; 100 mM sodium acetate buffer, pH 5.0).
Incubate the tube in a boiling water bath for 6min. (Stir the tube
vigorously after 2, 4 and 6 min).

NOTE: In this step it is essential that the tube be stirred vigorously

to ensure complete homogeneity of the slurry (removal of lumps).
Also, stirring after 2 min prevents the possibility of some of the
sample expelling from the top of the tube when the alcohol is
evaporating.
If polypropylene tubes are used, increase the incubation time to
12 min, with stirring after 4, 8 and 12 min.
5.

Place the tube in a bath at 50C; add 0.1 mL of the contents of

bottle 2 (amyloglucosidase, 330U on starch). Stir the tube on a
vortex mixer and incubate at 50C for 30 min.

6.

Transfer the entire contents of the test tube to a 100 mL

volumetric flask (with a funnel to assist transfer). Use a wash
bottle to rinse the tube contents thoroughly. Adjust to volume
with distilled water. Mix thoroughly. Centrifuge an aliquot of
this solution at 3,000 rpm for 10 min. Use the clear, undiluted
filtrate for the assay.
8

ALTERNATIVELY, at Step 6, adjust the volume to 10 mL with

distilled water and then centrifuge the tubes at 3,000 rpm for 10
min. For samples containing 1-10% starch content, this solution
is used directly in Step 7. For samples containing 10-100% starch,
an aliquot (1.0 mL) is diluted to 10 mL with distilled water before
proceeding to Step 7.
7.

Transfer duplicate aliquots (0.1 mL) of the diluted solution to the

bottom of glass test tubes (16 x 100 mm).

8.

Add 3.0 mL of GOPOD Reagent to each tube (including the

D-glucose controls and reagent blanks), and incubate the tubes at
50C for 20 min.

9.

D-Glucose controls consist of 0.1 mL of D-glucose standard

solution (1mg/mL) and 3.0 mL of GOPOD Reagent. Reagent
Blank solutions consist of 0.1 mL of water and 3.0 mL of GOPOD
Reagent.

10. Read the absorbance for each sample, and the D-glucose control at
510 nm against the reagent blank.
(b) Determination of starch in cereal and food products not
containing resistant starch, D-glucose and/or maltodextrins
(AOAC Official Method 996.11).
1.

Mill cereal, plant or food product to pass a 0.5 mm screen.

2.

Add milled sample (~ 100 mg; weighed accurately) to a glass test

tube (16 x 120 mm). Tap the tube to ensure that all of the sample
drops to the bottom of the tube.

3.

Add 0.2 mL of aqueous ethanol (80% v/v) to wet the sample and aid
dispersion. Stir the tube on a vortex mixer.

4.

Immediately add 3 mL of thermostable -amylase (contents of bottle

1 diluted 1:30 in Reagent 4; 50 mM MOPS buffer, pH 7.0). Incubate
the tube in a boiling water bath for 6min. (Stir the tube vigorously
after 2, 4 and 6 min).

NOTE: In this step it is essential that the tube be stirred

vigorously to ensure complete homogeneity of the slurry (removal
of lumps). Also, stirring after 2 min prevents the possibility of some
of the sample expelling from the top of the tube when the alcohol is
evaporating.
If polypropylene tubes are used, increase the incubation time to
12 min, with stirring after 4, 8 and 12 min.
9

5.

Place the tube in a bath at 50C; add sodium acetate buffer

(4mL, 200mM, pH 4.5), followed by amyloglucosidase (0.1mL,
20U). Stir the tube on a vortex mixer and incubate at 50C for
30 min.

6.

Proceed according to Step 6 of example (a).

(c) Determination of total starch content of samples

containing resistant starch, but no D-glucose and/or
maltodextrins (KOH Format - Recommended).
1.

Mill cereal, plant or food product to pass a 0.5 mm screen.

2.

Add milled sample (~ 100 mg, weighed accurately) to a glass

tube (16 x 120 mm).

3.

Wet with 0.2 mL of aqueous ethanol (80% v/v) to aid dispersion,

and stir the tube on a vortex mixer.

4.

Add a magnetic stirrer bar (5 x 15 mm) and 2 mL of 2 M

KOH to each tube and re-suspend the pellets (and dissolve the
RS) by stirring for approx. 20 min in an ice/water bath over a
magnetic stirrer (Figure 1).

NOTE:
1. Do not mix on a vortex mixer as this may cause the starch to
emulsify.
2. Ensure that the tube contents are vigorously stirring as the
KOH solution is added. This will avoid the formation of a lump
of starch material that will then be difficult to dissolve.
5.

Add 8 mL of 1.2 M sodium acetate buffer (pH 3.8) to each

tube with stirring on the magnetic stirrer. Immediately add

0.1 mL of thermostable -amylase (bottle 1) and 0.1 mL of AMG
(bottle 2), mix well and place the tubes in a water bath at 50C.

6.

Incubate the tubes for 30 min with intermittent mixing on a

vortex mixer.

7.

For samples containing > 10% total starch content;

quantitatively transfer the contents of the tube to a 100 mL
volumetric flask (using a water wash bottle). Use an external
magnet to retain the stirrer bar in the tube while washing the
solution from the tube with a water wash bottle. Adjust to
100 mL with distilled water and mix well. Centrifuge an aliquot of
the solution at 1,800 g for 10 min.

8.

For samples containing < 10% total starch content; directly

centrifuge the tubes at 1,800 g for 10 min (no dilution). For
10

such samples, the final volume in the tube is approx. 10.4 mL

(however, this volume will vary particularly if wet samples are
analysed, and appropriate allowance for volume should be made
in the calculations).

9.

Proceed from Step 7 of example (a).

(d) Determination of total starch content of samples

containing resistant starch, but no D-glucose and/or
maltodextrins (DMSO Format - AOAC Official Method
996.11).
1.

Mill cereal, plant or food product to pass a 0.5 mm screen.

2.

Add milled sample (~ 100 mg, weighed accurately) to a glass

tube (16 x 120 mm).

3.

Wet with 0.2 mL of aqueous ethanol (80% v/v) to aid dispersion,

and stir the tube on a vortex mixer.

4.

Immediately add 2 mL of dimethyl sulphoxide (DMSO) and

stir the tube on a vortex mixer. Place the tube in a vigorously
boiling water bath and remove after 5 min.

5.

Proceed from Step 4 of examples (a) or (b).

(e) Determination of starch in samples which also contain

D-glucose and/or maltodextrins.
1.

Mill cereal, plant or food product to pass a 0.5 mm screen.

2.

Add milled sample (~ 100 mg, weighed accurately) to a glass

centrifuge tube (16 x 120 mm; 17 mL capacity).

3.

Add 5.0 mL of aqueous ethanol (80% v/v), and incubate the tube
at 80-85C for 5 min. Mix the contents on a vortex stirrer and
add another 5 mL of 80% v/v aqueous ethanol.

4.

Centrifuge the tube for 10 min at 1,800 g (approx. 3,000 rpm)

on a bench centrifuge. Discard the supernatant.

5.

Resuspend the pellet in 10 mL of 80% v/v aqueous ethanol and

stir on a vortex mixer. Centrifuge as above and carefully pour
off the supernatant.

6.

Proceed from Step 4 of example (a) or (b).

Alternatively:

Proceed from Step 4 of example (c) if the sample contains

resistant starch.

11

(f) Determination of starch in samples in which the starch is

present in a soluble form and D-glucose and maltodextrins
are not present.
1.

Filter an aliquot of the sample solution through Whatman No.

1 filter paper (or Whatman GF/A Glass fibre filter paper if
necessary). Use the clear filtrate for the assay.

2.

Add 10 mL of this filtrate to a glass tube. Add 2 mL of Reagent

1 (100 mM acetate buffer, pH 5.0) plus 0.1 mL of AMG (bottle
2) diluted 10-fold in Reagent 1 (i.e. 33 U of AMG on starch) and
incubate in a water bath at 50C for 30 min. Adjust volume to
20 mL (or 20 g) with distilled water.

3.

Transfer duplicate aliquots (0.1 mL) of the diluted solution to

the bottom of glass test tubes (16 x 100 mm).

4.

Add 3.0 mL of GOPOD Reagent to each tube (including

D-glucose controls and reagent blanks), and incubate the tubes
at 50C for 20 min.

5.

D-Glucose controls consist of 0.1 mL of D-glucose standard

solution (1mg/mL) and 3.0 mL of GOPOD Reagent. Reagent
Blank solutions consist of 0.1 mL of water and 3.0 mL of
GOPOD Reagent.

6.

Read the absorbance for each sample, and the D-glucose control
at 510 nm against the reagent blank.

(g) Determination of starch in samples in which the starch

is present in a soluble form and D-glucose and maltodextrins
are present.
1.

Filter an aliquot of the sample solution through Whatman No.

1 filter paper (or Whatman GF/A Glass fibre filter paper if
necessary). Use the clear filtrate for the assay.

2.

Add 2 mL of the solution to be analysed to a 17 mL glass test

tube. To this, add 8 mL of 95% v/v ethanol and mix vigorously
on a vortex mixer. Allow to stand at room temperature for 30
min and centrifuge at 1,800 g for 10 min.

3.

Decant the supernatant solution and redissolve the starch

containing pellet in 1 mL of water. If necessary, heat the tube
and contents in a boiling water bath to aid redissolution. Adjust
the volume to 3.9 mL (3.9 g) with Reagent 1 (100 mM acetate
buffer, pH 5.0), taking account of the original weight of the tube.

4.

If necessary, repeat the ethanol precipitation and centrifugation

steps (e.g. for samples containing high levels of free D-glucose
and/or maltodextrins). Decant the supernatant solution and
redissolve the starch containing pellet in 1 mL of water.
12

If necessary, heat the tube and contents at 100C to aid

redissolution. Adjust the volume to 3.9 mL (3.9 g) with water,
taking account of the original weight of the tube.

5.

Add 0.1 mL of AMG (bottle 2) diluted 50-fold in Reagent 1 (i.e.

6.6 U of AMG on starch) and incubate in a water bath at 50C
for 30 min.

6.

Transfer duplicate aliquots (0.1 mL) of the diluted solution to

the bottom of glass test tubes (16 x 100 mm).

7.

Add 3.0 mL of GOPOD Reagent to each tube (including

D-glucose controls and reagent blanks), and incubate the tubes
at 50C for 20 min.

8.

D-Glucose controls consist of 0.1 mL of D-glucose standard

solution (1mg/mL) and 3.0 mL of GOPOD Reagent. Reagent
Blank solutions consist of 0.1 mL of water and 3.0 mL of
GOPOD Reagent.

9.

Read the absorbance for each sample, and the D-glucose control
at 510 nm against the reagent blank.

(h) Determination of enzyme resistant starch.

This can be accurately measured using the Resistant Starch assay kit
(K-RSTAR) supplied by Megazyme. Results obtained closely
simulate those obtained under in vivo conditions. Details of this
method can be obtained from the Megazyme website (www.
megazyme.com; K-RSTAR). This method has been successfully
subjected to interlaboratory evaluation (37 labs, 16 samples) under
the auspices of AOAC INTERNATIONAL (AOAC Official Method
2002.02).12, 13 and AACC International (Recommended Method
32-40.01).
CALCULATIONS (Solid samples):
Starch, % = A x F x FV x 1
x 100 x 162

0.1 1000
W
180
= A x F x FV x 0.9
W

where:
A =

absorbance (reaction) read against the reagent blank.

100 (g of D-glucose)
F =
(conversion from

absorbance for 100 g of glucose
absorbance to g)
13

FV
= final volume (i.e. equals 100 mL or 10 mL in examples

(a) and (b); 100 mL or 10.4 mL in example (c); 100 mL or
10.0 mL in example (d); and 100 mL or 10 mL in example
(e).
0.1

= volume of sample analysed.

1
1000 = conversion from g to mg.
100
W

= factor to express starch as a percentage of flour weight.

W = the weight in milligrams (as is basis) of the flour

analysed.
162 = adjustment from free D-glucose to anhydro D-glucose
180

(as occurs in starch).
Starch % w/w (dry wt. basis):
100
= Starch % w/w (as is) x
100 - moisture content (% w/w)
CALCULATIONS (Liquid samples; mg/100 mL):
Starch

= A x F x 100 x 1
x
0.1
1000

= A x F x D x 1.8

162 x 2 x D
180

where:
A = absorbance (reaction) read against the reagent blank.
100 (g of D-glucose)
F
=
(conversion from

absorbance for 100 g of glucose
absorbance to g)
100

= conversion to 100 mL sample volume.

0.1

= volume of sample analysed.

1
1000 = conversion from g to mg.
162 = adjustment from free D-glucose to anhydro D-glucose
180
(as occurs in starch).
2

= dilution of the sample solution on incubation with AMG.

= further dilution of the incubation mixture (if required).

14

NOTE: These calculations can be simplified by using the Megazyme

Mega-CalcTM, for solid or liquid samples, downloadable from where
the product appears on the Megazyme website (www.megazyme.com).
REFERENCES:
1.

Anon, (1987). Measurement of the starch content of commercial

starches. Starch, 39, 414-416.

2.

Karkalis, J. (1985). An improved enzymic method for the determination

of native and modified starch. J.Sci. Fd. Agric., 36, 1019-1027.

3.

Knudson, K. E. B. (1997). Carbohydrate and lignin contents of plant

materials used in animal feeding. Animal. Fd. Sci. Tech., 67, 319-338.

4.

American Association of Cereal Chemists: Approved Methods of the

AACC. Method 76-11, approved October 1976.

5.

Theander, O. and Aman, P. (1979). Studies on dietary fibres. 1.Analysis

and chemical characterisation of water-soluble and water-insoluble
dietary fibres. Swedish J. Agric. Res., 9, 97-106.

6.

Batey, I. L. (1982). Starch analysis using thermostable alpha-amylase.

Starch, 34, 125-128.

7.

Englyst, H. N. and Cummings, J. H. (1988). Improved method for

measurement of dietary fibre as non-starch polysaccharides in plant
foods. J. AOAC Int., 71, 808-814.

8.

McCleary, B. V., Solah, V. and Gibson, T. S. (1994). Quantitative

measurement of total starch in cereal flours and products. J. Cereal
Science, 20, 51-58.

9.

McCleary, B. V., Gibson, T. S. and Mugford, D. C. (1997). Measurement

of total starch in cereal products by amyloglucosidase - -amylase
method: Collaborative study. J.AOAC Int., 80, 571-579.

10. McCleary, B. V. and Draga, A. (2009). unpublished.

11. Crabb, D. W. and Shetty, J. Improving the properties of amylolytic
enzymes by protein engineering. Trends in Glycoscience and
Glycotechnology, 15 (82), 115-126.
12. McCleary, B. V., McNally, M. and Rossiter, P. (2002). Measurement
of resistant starch by enzymic digestion in starch samples and selected
plant materials: Collaborative Study. J. AOAC Int., 85, 1103-1111.
13. McCleary, B. V. and Monaghan, D. A. (2002). Measurement of resistant
starch. J. AOAC Int., 85, 665-675.

ACKNOWLEDGEMENT:
We acknowledge valuable discussions with Dr. MaryBeth Hall
(Research Animal Scientist, USDA - Agricultural Research Service,
Madison, WI) during the current updates of the total starch assay
procedure.
15

Table 1. Comparison of total starch values determined with AOAC

Method 996.11 and the current method in which -amylase and
amyloglucosidase incubations were performed at pH 5.
Total starch (as is basis)a
Sample

AOAC
Method 996.11

Regular maize starch

Wheat flour
High amylose maize starchb
ACS soluble starch
Chemically modified starch
Oat bran

85.0
69.1
76.8
83.5
81.7
37.2

Current Method
(pH 5)
84.8
68.7
77.2
83.4
82.7
37.0

a The average of duplicate analyses by two separate analysts.

b high amylose starch (total starch value is underestimated).

Table 2. Comparison of total starch values determined with AOAC

Method 996.11 (DMSO modification) and the current modification
in which starch is dissolved in 2 N KOH, pH adjusted and -amylase
and amyloglucosidase incubations performed at pH 5.
Total starch (as is basis)a

Sample

AOAC
Method 996.11

Regular maize starch

High amylose maize starchb
Potato amyloseb
Novelose 240c
Hylon VIIb
a The average of duplicate analyses
b native high amylose starch.
c retrograded high amylose starch.

16

84.3
83.2
86.1
84.5
85.1

Current Method
(KOH / pH 5)
85.2
83.4
86.7
84.7
84.9

by two separate analysts.

Table 3. Results of an interlaboratory evaluation of the total starch assay

procedure (AOAC Official Method 996.11; examples b and
modification with DMSO).

Chicken
White
Green
High
White
Sample Feed Bread Pea Amylose Wheat

Pellets
Maize Starch*
Flour
Moisture %
11.4 10.7 12.4 13.4 12.8
No. of labs.
32
32
31
25
31
Outliers 0 0 1G 1C 1C
Average % 50.7 68.1 44.0 86.3 78.0
Sr
1.6 1.8 1.5 2.5 2.2
RSDr 3.1 2.7 3.4 2.9 2.9
r
4.4 5.2 4.2 7.0 6.3
SR
2.4 3.4 2.1 4.1 3.3
RSDR 4.6 5.0 4.8 4.8 4.2
R
6.6 9.5 6.0 11.6 9.2
HORRAT 2.1 2.4 2.1 2.8 2.0
Range
45.4-55.3 62.0-74.9 39.4-47.4 78.7-96.8 71.6-85.8

Table 3. (cont.)

Wheat
Oat
Spaghetti High Amylose
Wheat
Sample
Starch*
Bran
Maize Starch
Starch

DMSO
DMSO

procedure
procedure
Moisture %
12.3
8.8
11.8
13.4
12.3
No. of labs.
26
31
31
31
31
Outliers 0 1C 1C 1C 1C
Average %
97.2
42.2
76.6
97.2
96.5
Sr
3.2 1.6 3.0 2.0 3.0
RSDr
3.3 3.8 3.9 2.1 3.1
r
9.0 4.5 8.4 5.7 8.4
SR
3.7 2.1 3.7 2.8 4.4
RSDR
3.8 5.0 4.8 2.9 4.6
R
10.4 6.0 10.3 7.8 12.4
HORRAT 1.9 2.2 2.3 1.4 2.3
Range
91.8-105.0 38.7-46.8 70.1-81.8 91.6-101.9 86.0-104.0
Number of Labs. = Number of laboratories included in calculations
Outliers
= Number of outlier laboratories, not included in calculations
(C=Cochran, G=Grubbs outlier)
Sr
= Repeatability standard deviation
RSDr
= Repeatability relative standard deviation
r
= Repeatability value (2.8 x Sr)
SR
= Reproducibility standard deviation
RSDR
= Reproducibility relative standard deviation
R
= Reproducibility value (2.8 x Sr)
HORRAT
= Horwitz ratio, an indication of the precision of the method
*
= With these samples, only 26 sets of results were supplied due to a
misinterpretation of instructions

In this table the statistical evaluation of results from an interlaboratory

evaluation of the methods is shown. Thirty-two laboratories (worldwide)
were involved and sixteen samples (eight blind duplicates) were analysed.
17

Figure 1. Arrangement of ice-water bath over a magnetic stirrer for

treatment of samples with 2 M KOH and dissolution of RS.

NOTES:

18

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