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TOTAL STARCH HK
ASSAY PROCEDURE
(AMG / -AMYLASE / HK METHOD)
K-TSHK 02/14
(100 Assays per Kit)

A Modification of:
AOAC Method 996.11
AACC Method 76.13
ICC Standard Method No. 168

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
produced 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).
More recently, thermostable -amylases that are active and stable
at lower pH values have become available.10 Consequently, we
have updated our total starch methodology to incorporate such an
enzyme.11 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), which is resistant to hydrolysis
by amyloglucosidase and -amylase. The method has been further
modified by adjusting the D-glucose determination to a hexokinase/
glucose-6-phosphate dehydrogenase/NADP+ based format.
The Megazyme total starch analysis procedure (K-TSHK) allows the
measurement of total starch in most 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 and samples high in D-glucose and maltodextrins are
washed with aqueous ethanol before analysis. 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 5.0, 100C
maltodextrins
(1) Resistant starch + H2O
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).

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

D-glucose

D-Glucose is phosphorylated by the enzyme hexokinase (HK) and

adenosine-5-triphosphate (ATP) to glucose-6-phosphate (G-6-P) with
the simultaneous formation of adenosine-5-diphosphate (ADP) (1).

(HK)
(4) D-Glucose + ATP

G-6-P + ADP

In the presence of the enzyme glucose-6-phosphate dehydrogenase

(G6P-DH), G-6-P is oxidised by nicotinamide-adenine dinucleotide
phosphate (NADP+) to gluconate-6-phosphate with the formation of
reduced nicotinamide-adenine dinucleotide phosphate (NADPH) (2).

(G6P-DH)
(5) G-6-P + NADP+
gluconate-6-phosphate + NADPH + H
The amount of NADPH formed in this reaction is stoichiometric with
the amount of D-glucose. It is the NADPH which is measured by the
increase in absorbance at 340 nm (Figure 1).

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 0.324 mg of D-glucose (or
0.292 mg starch)/L of sample solution at the maximum sample volume
of 2.00 mL. The detection limit is 0.649 mg D-glucose (or 0.584 mg
starch)/L, which is derived from an absorbance difference of 0.020
with the maximum sample volume of 2.00 mL.
The assay is linear over the range of 4 to 80 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 2.00 mL, this corresponds to a D-glucose concentration
of approx. 0.162 to 0.324 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. 40 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 are available from
Megazyme. The kits contain the full assay method plus:
Bottle 1:
Thermostable -amylase [12 mL, 8300 U/mL

on soluble starch (or 1,600 U/mL on Ceralpha
reagent12 ) at pH 5.0 and 40C].

Stable for > 3 years at 4C.
Bottle 2:
Amyloglucosidase [10 mL, 3300 U/mL on

soluble starch (or 200 U/mL on p-nitrophenyl
-maltoside13)] at pH 4.5 and 40C.

Stable for > 3 years at 4C.
Bottle 3:
Buffer (15 mL, pH 7.6) plus sodium azide (0.02% w/v)

as a preservative.
Stable for > 2 years at 4C.
Bottle 4: NADP+ plus ATP.

Stable for > 5 years at -20C.
Bottle 5:

Hexokinase plus glucose-6-phosphate dehydrogenase

suspension, 2.25 mL.
Stable for > 2 years at 4C.

Bottle 6:

D-Glucose standard solution (5 mL, 1.0 mg/mL).

Stable for > 2 years at 4C.

Bottle 7:

Standardised regular maize starch control. Starch

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

PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:

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.

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.

3. Use the contents of bottle 3 as supplied.

Stable for > 2 years at 4C.

4. Dissolve the contents of bottle 4 in 12 mL of distilled water.

Divide into appropriately sized aliquots and store in

polypropylene tubes at -20C between use and keep cool

during use if possible. Once dissolved, the reagent is stable for

> 2 years at -20C.
5.

Use the contents of bottle 5 as supplied. Before opening for

the first time, shake the bottle to remove any enzyme that
may have settled on the rubber stopper. Subsequently, store
the bottle in an upright position. Swirl the bottle to mix
contents before use. Stable for > 2 years at 4C.

6.

Use the contents of bottle 6 as supplied.

Stable for > 2 years at 4C.

7.

Use the contents of bottle 7 as supplied.

Stable for > 5 years at room temperature.

REAGENTS (NOT SUPPLIED):

1. Reagent 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.

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. Reagent 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. Reagent 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.
5

4. Reagent 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. Reagent 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.
EQUIPMENT (RECOMMENDED):
1. Glass test tubes (round bottomed; 16 x 100 mm).
2. Disposable plastic cuvettes (1 cm light path, 3.0 mL).
3. Micro-pipettors, e.g. Gilson Pipetman (20 L and 100 L).
4. Positive displacement pipettor, e.g. Eppendorf Multipette

- with 5.0 mL Combitip (to dispense 0.1 mL aliquots of
amyloglucosidase solution, imidazole buffer and NADP+/ATP
solution).

- with 25 mL Combitip (to dispense 2.0 mL aliquots of
distilled water).
- with 50 mL Combitip (to dispense 3 mL aliquots of bacterial
-amylase solution).
5.

Bench centrifuge (required speed 3,000 rpm; approx. 2,000 g).

6.

Analytical balance.

7.

Spectrophotometer set at 340 nm.

8.

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

9.

Thermostated water bath set at 40.0C.

10. Boiling water bath with tube rack.

11. Stop clock.
12. Whatman No.1 (9 cm) filter papers.
6

PROCEDURE:
Wavelength:
340 nm
Cuvette:

1 cm light path (glass or plastic)
Temperature:
~ 25C
Final volume:
2.27 mL
Sample solution:
4-80 g of D-glucose per cuvette


(in 0.05 - 2.00 mL sample volume)
Read against air (without a cuvette in the light path) or against water
Pipette into cuvettes
Blank
Sample
distilled water (at ~ 25C)
2.05 mL
2.00 mL
sample
-
0.05 mL
solution 1 (buffer)
0.10 mL
0.10 mL
solution 2 (NADP+/ATP)
0.10 mL
0.10 mL
Mix*, read the absorbances of the solutions (A1) after approx. 3 min
and start the reactions by addition of:
suspension 3 (HK/G-6-PDH)
0.02 mL
0.02 mL
Mix*, read the absorbances of the solutions (A2) at the end of the
reaction (approx. 5 min). If the reaction has not stopped after 5 min,
continue to read the absorbances at 2 min intervals until the
absorbances remain the same over 2 min**.
* for example with a plastic spatula or by gentle inversion after sealing
the cuvette with a cuvette cap or Parafilm.
** if the absorbance continues to increase, this may be due to effects
of colour compounds or enzymes in the sample. These interfering
substances may be removed during sample preparation.
CONTROL:
With each set of determinations, a standard flour or starch sample
should be included.
CALCULATION:
Determine the absorbance difference (A2-A1) for both blank and
sample. Subtract the absorbance difference of the blank from the
absorbance difference of the sample, thereby obtaining AD-glucose.
The value of AD-glucose should as a rule be at least 0.100 absorbance
units to achieve sufficiently accurate results.
The concentration of starch can be calculated as follows:
c

V x MW
x 162
x d x v
180
7

AD-glucose

[g/L]

where:
V
=
MW =

=

=
d
=
v
=
162/180 =

final volume [mL]

molecular weight of D-glucose [g/mol]
extinction coefficient of NADPH at 340 nm
6300 [l x mol-1 x cm-1]
light path [cm]
sample volume [mL]
Adjustment from free D-glucose to anhydro D-glucose
(as occurs in starch).

It follows for starch:

c

2.27 x 180.16
6300 x 1 x 0.05

x 162
180

1.1685 x AD-glucose

AD-glucose

[g/L]
[g/L]

If the sample has been diluted during preparation, the result must be
multiplied by the dilution factor, F.
When analysing solid and semi-solid samples which are weighed out
for sample preparation, the content (g/100 g) is calculated from the
amount weighed as follows:
Content of starch:
=
cstarch [g/L sample solution]
x 100
weightsample [g/L sample solution]

[g/100 g]

NOTE: These calculations can be simplified by using the Megazyme

Mega-CalcTM, downloadable from where the product appears on
the Megazyme website (www.megazyme.com).

SAMPLE PREPARATION:
1. Sample dilution.
The amount of D-glucose present in the cuvette (i.e. in the 0.05 mL of
sample being analysed) should range between 4 and 80 g. The sample
solution must therefore be diluted sufficiently to yield a D-glucose
concentration between 0.02 and 0.4 g/L.

Dilution Table
Estimated concentration
of D-glucose (g/L)

< 0.4
0.4-4.0
4.0-40
> 40

Dilution
with water

Dilution
factor (F)

No dilution required
1 +
9
1 +
99
1 + 999

1
10
100
1000

If the value of AD-glucose is too low (e.g. < 0.100), weigh out more
sample or dilute less strongly. Alternatively, the sample volume to
be pipetted into the cuvette can be increased up to 2.00 mL, making
sure that the sum of the sample and distilled water components in the
reaction is 2.05 mL and using the new sample volume in the equation.
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.

5.

Place the tube in a bath at 50C. Add 0.1 mL of the contents

of bottle 2 (amyloglucosidase, 20 U). Stir the tube on a vortex
mixer and incubate it 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. Typically, no further dilution is required and
a sample volume of 0.05-0.1 mL is satisfactory.

ALTERNATIVELY, at Step 6, if the starch content is unknown

and is thought to be less than 10% w/w, adjust the volume to
10 mL (10 g) with distilled water (taking note of the original weight
of sample plus tube) and then centrifuge the tubes at 3,000 rpm for
10 min.
For samples containing 1-10% w/w starch content, use this solution
directly for analysis. Typically, a sample volume of 0.05-0.1 mL is
satisfactory.
For samples containing 10-100% starch, dilute 1.0 mL of this
solution to 10 mL with distilled water before analysis. Typically, a
sample volume of 0.05-0.1 mL is satisfactory.
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).

10

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 Reagent 5 (200mM

sodium acetate buffer, 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 product to pass a 0.5 mm screen.

2.

Add flour 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 Reagent

3 (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 2).

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 Reagent 2 (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.

Proceed from step 6 of example (a).

11

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.

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.

12

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.

Use the clear, undiluted filtrate for the assay. Typically, no further
dilution is required and a sample volume of 0.1-0.2 mL is
satisfactory.

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. 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.

If the solution is turbid, centrifuge the tube at 2,000 g for 10 min

in a bench centrifuge (or at 12,000 rpm in a Microfuge). Use
the clear filtrate for the assay. Typically, either no dilution, or a
dilution of 10-fold is required and a sample volume of 0.05 - 0.1
mL is satisfactory.

13

h) Determination of enzyme resistant starch.

Absorbance, 340 nm

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).

Blank
Standard

Incubation time, min

Figure 1: Increase in absorbance at 340 nm on incubation of 50 g of
D-glucose with hexokinase and glucose 6-phosphate dehydrogenase in
the presence of NADP+ and ATP.
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.
15
14

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. Crabb, D. W. and Shetty, J. Improving the properties of amylolytic

enzymes by protein engineering. Trends in Glycoscience and
Glycotechnology, 15 (82), 115-126.
11. McCleary, B. V. and Draga, A. (2009). unpublished.
12. McCleary, B. V., Bouhet, F. and Driguez, H. (1991). Measurement of

Amyloglucosidase using P-Nitrophenyl -Maltoside as Substrate.

Biotech. Tech., 5, 255-258.
13. McCleary, B. V., McNally, M., Monaghan, D. and Mugford, D. C. (2002).
Measurement of -Amylase Activity in White Wheat Flour, Milled
Malt, and Microbial Enzyme Preparations using the Ceralpha Assay:
Collaborative Study. J. AOAC Int., 85, 1096-1102.
14. 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.
15. McCleary, B. V. and Monaghan, D. A. (2002). Measurement of resistant
starch. J. AOAC Int., 85, 665-675.

Figure 2. Arrangement of ice-water bath over a magnetic stirrer for

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

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 and D-glucose
was determined with hexokinase/glucose-6-phosphate dehydrogenase.

Sample

Total starch (as is basis)a

AOAC
Method 996.11

Regular maize starch

Wheat flour
High maize starchb
ACS soluble starch
Novelose 240b
Hylon VIIb
Amylose (potato)b
Oat bran
Chicken feed
a The average of duplicate analyses
b high amylose starches.

85.7
68.4
77.8
83.5
66.3
76.0
64.5
34.7
9.3

Current Method
(pH 5, HK)
86.7
69.6
77.3
83.4
66.3
77.5
64.5
34.6
9.4

by two separate analysts.

Table 2. Results of an interlaboratory evaluation of the total

starch assay procedure (AOAC Official Method 996.11;
examples b and modification with DMSO). [Note:
glucose assays were performed with GOPOD reagent]

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

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Table 2. (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.

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17

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IRELAND.
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