FOOD COMPOSITION AND ADDITIVES

Measurement of Total Starch in Cereal Products by

Amyloglucosidase
-Amylase Method: Collaborative Study
MCCLEARY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 3, 1997
BARRY V. MCCLEARY
Megazyme International Ireland Ltd., Bray Business Park, Bray, County Wicklow, Ireland
TREVOR S. GIBSON
NSW Agriculture, Biological and Chemical Research Institute, PMB 10, Rydalmere, New South Wales, 2116, Australia
DAVID C. MUGFORD
Bread Research Institute of Australia, Inc., PO Box 7, North Ryde, New South Wales, 2113, Australia
Collaborators: O. Lukow; D.S. Jackson; E. Rabe; N. Patel; P.C. Williams; J. Gelroth; M.E. Camire; R.N. Chibbar; M. Ingelin;
C. Niemann; L.A. Grant; D.M. Peterson; H. Corke; P. Sanders; J. Muir; M. Choct; J. Schmidt; R. Walker; A.B. Blakeney; S.
Logue; A. Tarr; T. Gibson; I. Batey; J. Dynes; T. Miklaszewicz; J. Panozzo; B. McCleary; B.W. Li; P. Hofer; E. Arndt; M.
Thomas; R. Depalo

An American Association of Cereal Chemists/AOAC collaborative study was conducted to

evaluate the accuracy and reliability of an enzyme
assay kit procedure for measurement of total
starch in a range of cereal grains and products.
The flour sample is incubated at 95C with thermostable -amylase to catalyze the hydrolysis of
starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze
the dextrins to glucose. Glucose is measured with
glucose oxidaseperoxidase reagent. Thirty-two
collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included
chicken feed pellets, white bread, green peas, highamylose maize starch, white wheat flour, wheat
starch, oat bran, and spaghetti. All samples were
analyzed by the standard procedure as detailed
above; 4 samples (high-amylose maize starch and
wheat starch) were also analyzed by a method that
requires the samples to be cooked first in dimethyl
sulfoxide (DMSO). Relative standard deviations for
%, and
repeatability (RSDr) ranged from 2.1 to 3.9%
relative standard deviations for reproducibility
%. The RSDR value for
(RSDR) ranged from 2.9 to 5.7%
high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%
%; the value

Submitted for publication April 12, 1996.

The recommendation was approved by the Methods Committee on
Commodity Foods and Commodity Products, and was adopted by the
Official Methods Board of the Association. See Official Methods Board
Actions (1996) J. AOAC Int. 79, 76A, and Official Methods Board
Actions (1996) The Referee, June issue.

was reduced to 2.9%

% when the DMSO procedure
was used, and the determined starch values increased from 86.9 to 97.2%
%. The amyloglucosidase
-amylase method for measurement of total starch
in cereal products has been adopted first action by
AOAC INTERNATIONAL.

any procedures for analysis of total starch do not give

quantitative measurements for high-amylose starches
and many processed cereal products (1). This problem was addressed by the development of an enzymic procedure in which starch is dispersed in dimethyl sulfoxide
(DMSO) and then quantitatively hydrolyzed to glucose by sequential treatment with thermostable -amylase, pullulanase
-amylase, and amyloglucosidase (glucoamylase; 1). Glucose
is measured colorimetrically with a glucose oxidaseperoxidase (GOPOD) reagent. This procedure is quantitative for a
wide range of modified starches and cereal products. An interlaboratory evaluation of precision demonstrated its high repeatability and reproducibility (2), and it received first-approval
status from the American Association of Cereal Chemists
(AACC Method 7612). However, many users of this assay
report that the method is too complicated, requiring too many
manipulations.
In another starch assay procedure, starch is hydrolyzed by
sequential treatment with thermostable -amylase and amyloglucosidase (38). Widespread use of this approach has been
limited by the prohibitive cost of the high-purity enzymes required, especially amyloglucosidase free of contaminating activities of cellulase and catalase. Cellulase contamination contributes to false high starch values because of cellulose
hydrolysis, and catalase reduces the stability of the chromogen
formed in glucose assay methods based on the use of GOPOD
reagent. A cost-effective amyloglucosidase of the necessary pu-

rity is now available commercially and was used in evaluating

a new rapid starch assay procedure.
Collaborative Study
Sixteen homogenous test samples were provided as 8 blind
duplicates to 32 collaborators, who were asked to become familiar with both the standard (non-DMSO) and DMSO procedures of the method by repeated analysis of the reference sample. All samples were assayed by the standard procedure, and
samples of high-amylose maize starch and wheat starch were
also assayed by the DMSO procedure. Collaborators assayed each test sample once only and reported the analyses
on an air-dry basis. Results were adjusted for moisture content
prior to statistical analysis.
The results were analyzed as described by McCleary et al.
(2), according to AOAC guidelines (9). Cochrans extreme
variance test for repeatability and Grubbs test for reproducibility (both P < 0.01) were used. Outliers identified by these tests
were omitted from analysis of variance. Within (sr) and between (sR) laboratory standard deviations were determined
from analyses of variance of results for each sample. We also
calculated repeatability (r) and reproducibility values (R) as 2.8
sr and 2.8 sR, respectively, and relative standard deviations
(RSDr and RSDR) from sr and sR were calculated as percentages
of the mean values. Horwitz ratios (HORRAT; 10) were calculated as follows:
RSDR (determined)
RSDR (predicted)
where RSDR (predicted) = 21 0.5 log C and C is starch concentration as a decimal fraction (1% = 0.01; 13, 14). HORRAT
values normalize the RSDR values with respect to concentration.
996.11 Starch (Total) in Cereal Products,
Amyloglucosidase
-Amylase Method
First Action 1996
(Applicable for determination of total starch in cereal
products.)
Caution: See Appendix B, safety notes on ethanol. Glucose
oxidaseperoxidaseaminoantipyrine buffer mixture, 3-(Nmorpholino)propanesulfonic acid (MOPS), and acetate buffers
contain sodium azide. Avoid contact with skin and eyes. In case
of contact, immediately flush contact surfaces with plenty of
water. Disposal of these reagents into sinks with copper or lead
plumbing should be followed immediately with large quantities of water to prevent potential explosive hazards. Dimethyl
sulfoxide is a skin irritant and should be used with caution. Dispose of waste solvents according to applicable environmental
rules and regulations.
Method Performance:
See Table 996.11 for method performance data.

A. Principle
Samples are hydrated and starch is hydrolyzed to maltodextrins with thermostable -amylase at 95100C. Temperature
and pH are adjusted, and maltosaccharides are quantitatively
hydrolyzed to glucose with highly purified amyloglucosidase.
Glucose is determined with high-purity glucose oxidaseperoxidase reagent. Samples containing high-amylose starches or
resistant starch are pretreated with dimethyl sulfoxide (at 95C)
before treatment with -amylase. Starch content is calculated
and reported as percentage of sample on as is basis.

B. Apparatus
(a) Grinding mill.Centrifugal, equipped with 12-tooth
rotor and 0.5 mm sieve, or similar device. Alternatively, cyclone mill can be used for small samples.
(b) Bench centrifuge.Capable of holding 16 120 mm
glass test tubes, with rating of ca 1000 g.
(c) Water bath.Capable of maintaining 50 0.1C.
(d) Boiling water bath.Capable of boiling (e.g., appropriate deep-fat fryer filled with H2O) at 95100C.
(e) Vortex mixer.
(f) pH meter.
(g) Stop-clock timer (digital).
(h) Top-loading balance.
(i) Analytical balance.
(j) Laboratory oven.With forced-convection; capable of
maintaining 103 1C; used for determining dry weight of
sample.
(k) Spectrophotometer. Capable of operating at 510 nm.
(l) Pipets.Capable of delivering 100 and 200 L; with
disposable tips. Alternatively, motorized hand-held dispenser
can be used.
(m) Positive-displacement pipetter.Equipped with
5.0 mL tips; capable of accurately delivering 100 and 200 L;
and 50 mL tips, capable of delivering 3.0 mL.
(n) Dispenser.500 mL capacity set to deliver 3.0 mL.
Used for glucose oxidaseperoxidaseaminoantipyrine buffer
mixture.
(o) Glass test tubes.16 120 mm, 17 mL capacity, suitable for centrifugation at ca 1000 g; and 18 150 mm.
(p) Test tube racks.48 place, capable of holding 16
120 mm and 18 150 mm tubes.
(q) Thermometer.Capable of reading 103 1C.
(r) Filter paper.Fast, ashless.

C. Reagents
(a) 3-(N-Morpholino)propanesulfonic acid (MOPS) buffer.pH 7.0. Contains 50 mM MOPS, 5 mM calcium chloride,
and 0.02% sodium azide. In 1 L volumetric flask dissolve
11.55 g MOPS in 900 mL H2O and adjust pH to 7.0 with 1M
HCl (ca 17 mL). Add 0.74 g calcium chloride dihydrate and
0.2 g sodium azide. Dilute to volume with H2O. Buffer is stable
at room temperature.
(b) Thermostable
-amylase
solution.10 mL;
3000 U/mL. Dilute 1 mL -amylase solution (in 50% glycerol)
to 30 mL with MOPS buffer, (a). Thermostable -amylase so-

lution is stable up to 3 years when stored frozen. (Note: One

unit [U] of -amylase activity is amount of enzyme required to
release 1 mol p-nitrophenol from end-blocked p-nitrophenyl maltoheptaoside in presence of saturating levels of
-glucosidase and amyloglucosidase [i.e., Ceralpha -amylase
assay reagent] at 40C and pH 6.0; 11) Thermostable -amylase solution should be free of detectable levels of free glucose.
(c) Amyloglucosidase solution.10 mL; 200 U/mL. Use
directly without dilution. Solution is viscous; for dispensing
use positive-displacement dispenser. Amyloglucosidase solution is stable up to 3 years when stored at 4C. (Note: One unit
[U] of enzyme activity is amount of enzyme required to release
1 mol p-nitrophenol from p-nitrophenyl -maltoside in the
presence of saturating levels of -glucosidase [i.e., amyloglucosidase assay reagent] at 40C and pH 4.5; 12) Amyloglucosidase solution should be free of detectable levels of free glucose.
(d) Glucose oxidaseperoxidaseaminoantipyrine buffer
mixture.Mixture of glucose oxidase, >12000 U/L; peroxidase, >650 U/L; and 4-aminoantipyrine, 0.4 mM.
Prepare buffer concentrate by dissolving 13.6 g potassium
dihydrogen orthophosphate, 4.2 g sodium hydroxide, and 3.0 g
4-hydroxybenzoic acid in 96 mL distilled H2O. Adjust to
pH 7.4 with either 2M HCl or 2M NaOH. Dilute solution to
100 mL, add 0.4 g sodium azide and mix until dissolved. Buffer concentrate is stable up to 3 years when stored at 4C.
To prepare glucose oxidaseperoxidaseaminoantipyrine
buffer mixture, dilute 50 mL buffer concentrate to 1.0 L. Use
part of diluted buffer to dissolve entire content of vial containing freeze-dried glucose oxidaseperoxidase mixture. Transfer
contents of vial to 1 L volumetric flask containing diluted buffer. Reagent is stable 23 months when stored at 4C and 2
3 years when stored at 20C. Color formed with glucose is stable several hours. Note: Glucose oxidase must not be
contaminated with - and/or -glucosidase, and chromogen
color complex must be stable at least 60 min.
Check color formation and stability of glucose oxidaseperoxidaseaminoantipyrine buffer mixture by incubating (in duplicate) 3.0 mL glucose oxidaseperoxidaseaminoantipyrine
buffer mixture with certified glucose standard (100 g dried
crystalline glucose in 0.2 mL 0.2% sodium benzoate solution).
After 15, 20, 30, and 60 min incubation, read absorbance, A, of
solution at 510 nm. Maximum color formation should be
achieved within 20 min, and color should be stable at least
60 min at 50C.
(e) Aqueous ethanol.About 80% (v/v). Dilute 80 mL
95% ethanol (laboratory grade) to 95 mL with H2O.
(f) Sodium acetate buffer.(1) 200 mM, pH 4.5.Pipet
11.8 mL glacial acetic acid (1.05 g/mL) to 900 mL H2O. Adjust
pH to 4.5 with 1M NaOH solution (ca 60 mL is required). Add
0.2 g sodium azide and dilute to 1 L with H2O. (Caution: Sodium azide should not be added until pH is adjusted. Acidification of sodium azide releases poisonous gas.) Buffer is stable
12 months when stored at room temperature. (2) 50 mM,
pH 4.5.Dilute an aliquot of 200 mM acetate buffer 4-fold
with H2O.
(g) Dimethyl sulfoxide (DMSO) solution.Laboratory
grade.

(h) Glucose standard stock solution.1 mg/mL. Before

preparing solution, dry powdered crystalline glucose (purity
>97%) 16 h at 60C under vacuum.
(i) Corn starch.Containing known content of starch (e.g.,
ca 98% dry weight).
Items (a), (b), (c), (h), and (i) are supplied in Total Starch
Assay kit available from Megazyme International Ireland Ltd.,
Bray Business Park, Bray, County Wicklow, Ireland.

D. Preparation of Test Samples, Standards, and

Reagent Blank
(a) Test sample.Grind ca 50 g sample in grinding mill to
pass 0.5 mm sieve. Transfer all material into wide-mouthed
plastic jar and mix well by shaking and inversion.
(b) D-Glucose standard working solutions.50 and
100 g. Add 50 and 100 L D-glucose standard stock solution,
C(h), to separate test tubes, and adjust volume in each tube to
100 L with H2O. Prepare solutions immediately before use.
(c) Reagent blank.Transfer 0.1 mL H2O into test tubes
and proceed with total starch determination by standard assay
procedure starting from step E(a)(7).

E. Determination of Total Starch

(a) Standard procedure (AA/AMG).Run D-glucose
working standard solutions (in quadruplicate), reagent blank
(in duplicate), and corn starch with each set of test samples. Use
reagent blank to zero spectrophotometer.
(1) Accurately weigh 90100 mg ground test sample directly into glass test tube. Tap tube gently on laboratory bench
to ensure that all particles of sample drop to bottom of tube.
Note: When analyzing cereal products containing high levels of glucose (processed cereal products such as breakfast cereals and all samples of unknown or uncertain composition, for
example samples containing free glucose or maltodextrins),
preextract 90100 mg ground test sample 2 with 10 mL 80%
aqueous ethanol, C(e), at ca 80C at 10 min/extraction. Centrifuge slurry at 1000 g and discard supernatant. Use sediment
for analysis.
(2) Add 0.2 mL 80% aqueous ethanol to tube and stir on
Vortex mixer to ensure sample is wet. Add 3.0 mL thermostable
-amylase, C(b), and mix contents of tube on Vortex mixer to
ensure complete dispersion of sample.
(3) Immediately place tube in boiling water bath. Incubate
2 min, remove from water bath, and mix vigorously on Vortex
mixer. Return tube to boiling water bath for additional 3 min
and then mix contents vigorously on Vortex mixer. Note: Part
of sample will adhere to side of test tube; however, this will not
affect analysis because tube contents are treated with enzyme
in this step.
(4) Place tubes in water bath set at 50C and allow to equilibrate 5 min. Add 4.0 mL 200 mM sodium acetate buffer,
C(f)(1), and 0.1 mL amyloglucosidase solution, C(c), and vigorously mix contents on Vortex mixer. Cap tube with marble
and incubate 30 min at 50C.
(5) Quantitatively transfer entire contents of test tube to
100 mL volumetric flask. Use water wash bottle to rinse tube
contents thoroughly. Dilute to 100 mL with H2O. (Note: If sam-

ple contains <10% starch, adjust volume to 10.0 mL instead of

100 mL. Make appropriate adjustments to calculations.) Thoroughly mix contents of flask. Centrifuge portion of suspension
10 min at 1000 g. Alternatively, filter portion through filter
paper.
(6) Carefully and accurately transfer 0.1 mL portion of
each supernatant (or filtrate) to bottoms of separate test tubes;
use 2 tubes/supernatant (or filtrate).
(7) Add 3.0 mL glucose oxidaseperoxidaseaminoantipyrine buffer mixture, C(d), to each tube (reaction solutions
from test sample and corn starch, reagent blank, and D-glucose
standard working solutions), and incubate 20 min at 50C.
(8) Measure and record absorbance, A, of each sample at
510 nm against reagent blank. Average A values for each sample and use in calculations, G.
(b) Modified procedure (DMSO/AA/AMG).For samples
containing enzyme-resistant starch.
(1) Accurately weigh 90100 mg ground test sample directly into glass test tube. Tap tube gently on laboratory bench
to ensure all particles of sample drop to bottom of tube. (Note:
When analyzing cereal products containing high levels of glucose, preextract 90100 mg ground test sample 2 with 10 mL
aqueous ethanol, C(e), at ca 80C at 10 min/extraction. Centrifuge slurry at 1000 g and discard supernatant. Use sediment
for analysis.)
(2) Add 0.2 mL 80% aqueous ethanol to tube and stir on
Vortex mixer to ensure that sample is wet.
(3) Immediately add 2 mL DMSO solution, C(g), and stir
tube on Vortex mixer. Place tube in vigorously boiling water
bath and remove after 5 min. Add 3.0 mL thermostable -amylase solution, C(b), and mix contents of tube on Vortex mixer
to ensure complete dispersion of sample.
(4) Immediately proceed according to standard procedure
(AA/AMG), starting from step E(a)(3).

F. Determination of Enzyme-Resistant Starch

(Optional)
(Note: This part of method was not validated in collaborative study and, therefore, was not included in method performance statistical analysis.)
Level of enzyme-resistant starch in samples depends on nature of original starch (e.g., high amylose) and on processing
conditions. This may vary from 0.1 to 30% total starch in sample. Determine level of enzyme-resistant starch as follows:
(1) Analyze samples according to standard procedure,
E(a)(1)(4).
(2) Centrifuge reaction solution 10 min at 1000 g and
carefully pour supernatant into 100 mL volumetric flask.
(3) Resuspend pellet in 5 mL 50 mM sodium acetate buffer, C(f)(2), by vigorous stirring on Vortex mixer. Add 5 mL
50 mM sodium acetate buffer, mix, and then centrifuge 10 min
at 1000 g.
(4) Combine supernatant with that from step (2) and dilute
to volume. Analyze for starch by the standard procedure, starting with step E(a)(6).

(5) To pellet from (3), add 2 mL DMSO and analyze for

starch by modified procedure E(b), starting from step E(b)(3)
[proceed to procedure E(a) as stated].
(6) At step E(a)(5), adjust volume to 10 mL (instead of
100 mL).
(7) Proceed with standard procedure, E(a), starting from
step E(a)(6). Make appropriate adjustments to calculations.

G. Calculations
Calculate total starch content (%, on as is basis) in test sample as follows:
Total starch, % = A F 1000
=A

100 162
1

1000 W 180

F
90
W

where A = absorbance of reaction solutions read against reagent

blank; F = factor to convert absorbance values to g glucose =
100 g glucose/absorbance value for 100 g glucose; 1000 =
volume correction, i.e., 0.1 mL taken from 100 mL; 1/1000 =
conversion from g to mg; 100/W = conversion to 100 mg sample; 162/180 = factor to convert from free glucose, as determined, to anhydroglucose, as occurs in starch.

Negative Controls
The negative controls used to demonstrate assay specificity
were -cellulose, barley -glucan, and wheat arabinoxylan.
These were assayed by the standard assay procedure E(a) with
incubation with -amylase and amyloglucosidase. In another
control, the incubation contained buffers and enzymes only
(i.e., no sample was added). Portions from each of these controls were incubated with glucose oxidaseperoxidase buffer
mixture, and the absorbance was read against the reagent blank
(glucose oxidaseperoxidase buffer plus distilled water) D(c).
For all control solutions, the color formed was negligible, that
is,<0.001 absorbance unit.
Ref.: J. AOAC Int. 80, 571579(1997)
Results and Discussion
The precision for both assay procedures [E(a) and E(b)] was
calculated according to AOAC guidelines (9). Total starch values
determined by each collaborator by the non-DMSO, (E(a), and
DMSO, E(b), procedures as a percentage on a dry-weight basis,
are shown in Tables 1 and 2. Means, repeatability values (r), repeatability relative standard deviations (RSDr), reproducibility
values (R), reproducibility relative standard deviations (RSDR),
HORRAT values, and ranges for test results on each sample also
are given. Results requested but not supplied by the collaborator
are identified by a dash. The Cochran test identified as outliers the
results for the following samples: high-amylose maize starch (collaborator 12, non-DMSO procedure, and collaborator 19, DMSO
procedure), white wheat flour (collaborator 32), oat bran (collaborator 5), spaghetti (collaborator 19), and wheat starch (collaborator 5, DMSO procedure). The Grubbs test identified as outliers
the results of collaborator 18 for green peas.

RSDr values ranged from 2.1% to 3.9%, and RSDR values

ranged from 2.9% to 5.7%. The RSDR value was 5.7% for highamylose maize starch analyzed by the non-DMSO procedure.
The value was reduced to 2.9% when the recommended
DMSO procedure (for these types of samples) was used. HORRAT values ranged from 1.4 to 2.8, with the lowest and highest
values derived from analyses of high-amylose maize starch by
the DMSO and non-DMSO procedures, respectively. The differences in the performance of the 2 assay procedures for
analysis of high-amylose maize starch emphasizes the need for
DMSO pretreatment for these sample types. They also highlight some problems experienced in analysis of samples containing high levels of resistant starch.
Although use of thermostable -amylase followed by amyloglucosidase in measurement of starch has been reported (3
8), to date no reports exist of collaborative studies to establish
the precision of this method. Results of this current study show
that this method (both the non-DMSO and the DMSO procedures) demonstrates a greater precision (RSDr range, 2.1
3.9%; RSDR range, 2.95.7%) than that determined for AACC
Method 7612 (2), for which RSDr and RSDR values are 1.5
7.3% and 4.111.3%, respectively (Table 3). The better precision of the current method is particularly noticeable for the spaghetti, oat bran, and chicken feed samples. For these samples,
the older procedure gives RSDR values of 9.411.3% whereas
the current procedure gives RSDR values of 4.65.0%. HORRAT values with the current method are also lower than reported for AACC Method 7612 (1) and approach the theoretical acceptable value of 2 (13, 14). The lower precision
parameters for the modified procedure reported here probably
reflect the simplification of the procedure, to the benefit of analysts less familiar with the assay. Precisions are lower than
those reported for a precision study on both a polarimetric and
an alkaline dispersion/enzymic procedure on purified starches
(Analytical Working Party of the Starch Experts Group 1987;
15). The low sr and RSDr values reported elsewhere for analytical procedures to measure total starch in food and cereal products (3, 4, 6, 7) were derived from analyses by a single laboratory, which was the laboratory responsible for developing the
respective procedure.
The availability of a cost-effective amyloglucosidase free of
cellulase and catalase activities and simplification of an enzymic test procedure previously described by several authors
have markedly improved the convenience and precision of
quantitative starch analysis in cereal products.
Recommendation
On the basis of the study results, it is recommended that
the amyloglucosidase-amylase method for measurement
of total starch in cereal products be adopted first action.
Acknowledgments
We thank the following collaborators:
Odean Lukow, Agriculture Canada Research Station, Winnipeg, Manitoba, Canada

David S. Jackson, Department of Food Science and Technology, University of Nebraska, Lincoln, NE
E. Rabe, Federal Centre for Cereal, Potato and Lipid Research, Detmold, Germany
Naresh Patel, Campden and Chorleywood Food Research
Association, Chipping Campden, United Kingdom
Philip C. Williams, Canadian Grain Commission, Grain Research Laboratory, Winnipeg, Manitoba, Canada
Janette Gelroth, American Institute of Baking, Manhattan, KS
Mary Ellen Camire, University of Maine, ME
Ravindra N. Chibbar, National Research Council, Saskatoon, Canada
Mark Ingelin, Kansas State University, Department of Grain
Science, Manhattan, KS
Claudia Niemann, Technische Universitt Berlin, Institut
fr Lebensmitteltechnologie, Berlin, Germany
Linda A. Grant, U.S. Department of Agriculture, Biosciences Research Laboratory, Agricultural Research Services,
Fargo, ND
David M. Peterson, U.S. Department of Agriculture-ARS,
Cereal Crops Research Unit, Madison, WI
Harold Corke, University of Hong Kong, Department of
Botany, Hong Kong
Peter Sanders, NIKO, Groningen, The Netherlands
Jane Muir, Deakin University, School of Nutrition and Public
Health, Victoria, Australia
Mingan Choct, CSIRO Division of Human Nutrition,
OHalloran Hill, Australia
Jenny Schmidt, GFW Ingredients Limited, Tamworth, Australia
Richard Walker, Bread Research Institute of Australia, Inc.,
North Ryde, Australia
A.B. Blakeney, NSW Agriculture, Yanco Agriculture Institute,
Yanco, Australia
Sue Logue, The University of Adelaide, Waite Campus,
Glen Osmond, Australia
Allen Tarr, Western Australian Department of Agriculture,
South Perth, Australia
Ian Batey, CSIRO Grain Quality Research Laboratory,
North Ryde, Australia
John Dynes, Defiance Milling Company, Toowoomba, Australia
Teresa Miklaszewicz, Bunge Albury Mills, Albury, Australia
Joe Panozzo, Victorian Institute for Dryland Agriculture,
Horsham, Australia
Betty W. Li, Agricultural Research Service, U.S. Department
of Agriculture, Beltsville, MD
Petrea Hofer, Plant and Soil Science Department, Montana
State University, Bozeman, MT
Elizabeth Arndt, Con Agra, Omaha, NE
Michelle Thomas, Weston Cereals Laboratories, Australia
Rochelle Depalo, Arnotts Biscuits Ltd., Homebush, Australia
References
(1) McCleary, B.V., Solah, V., & Gibson, T.S. (1994) J. Cereal
Sci. 20, 5158
(2) McCleary, B.V., Gibson, T.S., Solah, V., & Mugford, D.C.
(1994) Cereal Chem. 71, 501505

(3) Batey, I.L. (1982) Starch 34, 125128

(4) Aman, P., & Hesselman, K. (1984) Swedish J. Agric. Res. 14,
135139
(5) Blakeney, A.B., & Matheson, N.K. (1984) Starch 36, 265269
(6) Karkalis, J. (1985) J. Sci. Food Agric. 36, 97106
(7) Holm, J., Bjorck, I., Ostrowska, S., Drews, A., & Asp, N.-G.
(1986) Starch 38, 224226
(8) Henry, R.J., Blakeney, A.B., & Lance, R.C.M. (1990) Starch
42, 468470
(9) Guidelines For Collaborative Study Procedure To Validate
Characteristics Of A Method Of Analysis in Official Methods of Analysis (1990) 15th Ed., AOAC, Arlington, VA

(10) Peeler, J.T., Horwitz, W., & Albert, R. (1989) J. Assoc. Off.
Anal. Chem. 72, 784806
(11) McCleary, B.V., & Sheehan, H. (1987) J. Cereal Sci. 6,
237251
(12) McCleary, B.V., Bouhet, F., & Driguez, H. (1991) Biotechnol. Tech. 5, 255258
(13) Horwitz, W., Albert, R., Deutsch, M.J., & Thompson, J.N.
(1990) J. Assoc. Off. Anal. Chem. 73, 661680
(14) Horwitz, W., Albert, R., Deutsch, M.J., & Thompson, J.N.
(1992) J. Assoc. Off. Anal. Chem. 75, 227239
(15) Anon. (1987) Starch 39, 414416

Table 996.11. Method performance for determination of total starch in processed cereal products by amyloglucosidase
-amylase method
Sample
Chicken feed pellets
White bread
Green pea
High-amylose maize starch
White wheat flour
Wheat starch
Oat bran
Spaghetti
High-amylose maize starch (DMSO method)
Wheat starch (DMSO method)
a
b
c

Calculated on as is basis.
r = 2.8 sr.
R = 2.8 sR.

Mean total starch,a %

Moisture, %

No. of labs

a a
sr

44.9
60.9
38.5
74.8
68.0
85.3
38.5
67.5
84.2
84.7

11.4
10.6
12.4
13.4
12.8
12.2
18.8
11.8
13.4
12.2

32
32
31
26
31
26
31
31
31
31

1.4
1.6
1.3
2.2
2.0
2.8
1.5
2.6
1.8
2.6

sRa

RSDr, %

RSDR, %

bb

c c

2.1
3.0
1.9
3.6
2.9
3.3
1.9
3.2
2.4
3.9

3.1
2.6
3.4
2.9
2.9
3.3
3.9
3.9
2.1
3.1

4.7
4.9
4.9
4.8
4.3
3.9
4.9
4.7
2.9
4.6

3.9
4.5
3.6
6.2
5.6
7.8
4.2
7.3
5.0
7.3

5.9
8.4
5.3
10.11
8.1
9.2
5.3
9.0
6.7
10.91

Table 1. Results for determination of total starch in processed cereal products and plant materials (part 1)
Chicken feed pellets

White bread

Laboratory No.

11
12
13
14
15
16
17
18
19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Moisture, %
d
Number of labs
e
Outliers
Average, %
f
sr
g
RSDr
h
r
i
sR
j
RSDR
k
R
l
HORRAT
Range

49.75
54.94
50.54
52
53.69
46.25
48.39
50.54
51.66
48.05
51.89
45.23
56.51
49.97
54.26
49.07
49.75
49.86
55.39
49.75
51.10
51.21
49.97
52.57
49.63
51.78
50.54
51.21
53.69
49.52
52.23
54.37

49.86
53.81
47.60
50.65
49.29
45.35
50.31
49.63
52.23
47.60
49.07
45.46
51.10
51.89
50.31
49.63
48.84
49.97
55.27
50.65
49.75
48.73
52.23
50.54
49.63
54.03
50.87
51.10
50.08
51.10
51.78
49.75

66.59
70.51
69.28
71.52
63.46
62.67
67.15
68.38
68.49
67.49
64.91
60.44
77.11
70.84
78.79
64.35
65.71
69.17
71.29
67.61
67.71
72.64
68.38
70.96
66.37
69.95
67.49
67.15
62.56
70.51
68.94
73.87

66.03
70.73
65.36
71.52
62.67
63.01
64.02
68.72
69.05
67.15
67.15
63.46
69.28
70.06
70.96
62.90
67.82
69.39
69.50
67.71
66.03
69.17
68.38
70.51
66.59
72.86
67.82
67.38
64.47
67.26
67.82
71.85

a
b
c
d
e
f
g
h
i
j
k
l

11.35
32
0
50.7
1.6
3.1
4.4
2.4
4.6
6.6
2.1
45.455.3

10.65
32
0
68.1
1.8
2.7
5.2
3.4
5
9.5
2.4
62.074.9

Green pea
C

42.69
43.26
46.00
46.00
42.35
42.58
44.98
44.75
40.41
40.75
36.64
42.12
44.86
43.84
44.29
44.52
45.09
43.72
42.58
43.95
44.18
43.38
40.87
44.29
42.24
44.06
45.21
46.00
43.49
40.87
44.18
41.89
43.04
43.72
c
c
26.48c
23.52c
46.92
47.95
44.63
43.38
43.38
42.47
45.55
46.58
48.63
45.55
44.29
44.86
43.95
42.58
46.23
45.78
44.29
44.41
43.95
43.15
49.89
43.84
43.49
39.61
43.26
44.41
44.75
47.03
12.4
31
c c
1
44
1.5
3.4
4.2
2.1
4.8
6
2.1
39.447.4

= result not supplied.

Cochran outlier.
Grubb outlier
Number of labs = number of laboratories included in calculations.
Outliers = number of outlier laboratories, not included in calculations.
sr = repeatability standard deviation.
RSDr = repeatability relative standard deviation.
r = repeatability value (2.8 sr).
sR = reproducibility standard deviation.
RSDR = reproducibility relative standard deviation.
R = reproducibility value (2.8 sR).
HORRAT = Horwitz ratio, an indication of the precision of the method.

Maize starch
D

81.02
81.25
76.98
80.55
a a

78.94
79.86
90.36
87.94

88.98
88.40
80.78
83.21

b
b
87.25b
72.48b
86.90
90.83
90.83
88.86
89.90
86.56
83.67
86.67
86.79
85.05
88.52
90.48

85.29
87.71
83.44
81.13
96.83
86.67
86.90
88.75
89.32
89.21
83.21
83.90
90.71
91.06
88.52
88.98
85.98
86.56
75.59
85.29
88.29
84.25
86.21
89.32
88.63
90.36
13.35
26
b b
1
86.9
2.5
2.9
6.9
4.9
5.7
13.8
2.8
78.7100.7

White wheat flour

G

76.95
76.72
81.31
79.47
76.83
80.16
82.91
78.10
77.52
72.36
69.50
73.74
76.26
75.92
77.75
77.41
79.24
81.77
76.61
76.49
78.78
75.23
74.43
69.84
83.37
88.19
79.70
80.50
80.85
75.34
72.82
74.54
75.92
77.87
79.47
79.47
80.96
75.46
77.64
76.49
75.92
73.39
87.16
78.21
78.67
79.24
80.16
78.78
76.83
77.18
81.88
79.13
78.33
77.52
78.21
78.33
79.36
78.67
76.61
76.72
79.82
79.01
c
b
83.37c
69.15b
12.8
31
b b
1
78
2.2
2.9
6.3
3.3
4.2
9.2
2
71.685.8

Table 2. Results for determination of total starch in processed cereal products and plant materials (part 2)
Wheat starch
Laboratory No.
11
12
13
14
15
16
17
18
19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Moisture, %
c
Number of labs
d
Outliers
Average, %
e
sr
f
RSDr
g
r
h
sR
i
RSDR
j
R
k
HORRAT
Range
a
b
c
d
e
f
g
h
i
j
k

95.84
95.84
101.991
100.741
a a

93.45
93.11
96.52
99.03

95.50
100.061
95.38
95.95

89.23
102.451
99.49
110.541
101.991
100.511
98.80
91.51
87.98
95.61
97.21
94.25
96.07
95.84

96.87
96.30
98.80
95.38
92.99
99.60
99.15
96.41
97.89
98.12
96.18
95.95
99.49
99.94
97.44
96.75
96.52
96.98
95.84
96.75
92.31
91.85
101.541
100.971
102.791
97.66
12.25
26
0
97.2
3.2
3.3
9.0
3.7
3.8
10.4
1.9
191.8105.0

Oat bran

Spaghetti

40.79
43.09
37.17
44.63
b
44.85b
45.72
42.11
44.19
42.11
42.65
40.68
39.91
42.76
43.31
42.87
39.91
40.90
44.63
45.07
41.56
46.38
42.11
43.20
43.97
41.34
46.60
44.74
43.09
41.23
41.01
43.75
39.36

41.23
43.97
40.24
44.74
b
23.68b
47.92
40.02
41.78
42.65
40.90
41.45
38.49
42.21
42.43
37.50
39.69
41.67
43.20
42.32
42.76
40.46
45.18
41.67
42.54
40.46
43.75
40.90
41.78
42.11
39.36
40.90
40.57

8.8
31
b b
1
42.2
1.6
3.8
4.5
2.1
5.0
6.0
2.2
38.746.8

Maize starchDMSO
N

76.87
75.62
81.41
78.57
77.78
75.28
80.73
80.61
78.57
67.46
71.88
74.38
79.48
76.64
78.46
78.23
81.63
81.41
76.30
77.10
69.61
79.93
74.38
72.45
76.76
63.49
79.02
75.62
72.79
73.02
71.20
73.70
76.30
77.89
73.24
73.36
b
b
83.45b
17.69b
77.44
77.44
76.08
73.81
77.78
85.71
77.10
78.46
78.80
78.12
76.98
77.10
80.39
79.82
77.21
78.12
77.21
77.55
77.21
76.42
70.86
71.32
80.84
77.55
80.39
76.87
11.8
31
b b
1
76.6
3.0
3.9
8.4
3.7
4.8
10.3
2.3
70.181.8

= result not supplied.

Cochran outlier
Number of labs = number of laboratories included in calculations.
Outliers = number of outlier laboratories.
sr = repeatability standard deviation.
RSDr = repeatability relative standard deviation.
r = repeatability value (2.8 sr).
sR = reproducibility standard deviation.
RSDR = reproducibility relative standard deviation.
R = reproducibility value (2.8 sR).
HORRAT = Horwitz ratio, an indication of the precision of the method.

94.86
93.59
98.33
99.48
101.671
99.60
101.671 102.141
96.94
89.44
100.061 101.441
96.02
100.291
97.40
97.63
99.37
97.63
96.13
95.21
89.32
93.94
94.40
102.371
98.67
100.081
97.17
97.29
100.981
99.02
93.25
95.44
98.56
99.37
97.17
97.86
b
10.39b b103.29b
100.751
98.56
94.06
95.44
100.521
94.40
97.17
97.52
96.36
98.79
95.79
96.48
97.29
97.52
94.75
94.86
94.40
94.86
95.44
94.86
97.63
97.40
94.75
96.71
99.02
95.33
13.35
31
b b
1
97.2
2.0
2.1
5.7
2.8
2.9
7.8
1.4
191.6101.9

Wheat starchDMSO
H

94.47
94.25
99.03
97.55
97.44
96.18
89.23
100.401
b
b
2.96b
78.52b
104.841 103.251
97.55
93.79
96.18
96.98
98.46
92.42
94.36
95.61
86.72
85.36
89.12
95.84
97.66
98.12
98.80
99.03
84.33
94.36
94.70
94.93
100.171 103.251
98.12
98.12
101.421 105.981
100.971
99.26
94.59
96.41
103.131
95.61
96.30
95.04
97.44
98.69
92.99
92.42
99.60
99.03
96.07
95.61
94.59
94.70
99.49
97.55
94.25
91.85
98.12
88.66
105.071
99.37
12.25
31
b b
1
96.5
3.0
3.1
8.4
4.4
4.6
12.4
2.3
186.0104

Table 3. Comparison of results obtained from interlaboratory evaluation of analytical procedures for the
measurement of total starch contents of a range of samples
Sample
White wheat flour

Method
a a

A
B
A
B
A
B
A
B
A
B
A
B
A
B
c c
C

b b

White bread
Chicken feed
Oat bran
Green peas
Spaghetti
High amylose maize starch

a
b
c

Starch content, %
(dry wt)

No. of
outliers

RSDr

RSDR

80.0
78.0
69.8
68.1
47.5
50.7
45.6
42.2
44.2
44.0
75.1
76.6
98.2
86.3
97.2

1
1
2
0
0
0
0
1
0
1
1
1
1
1
1

2.6
2.9
1.5
2.7
5.7
3.1
7.3
3.8
3.6
3.4
6.2
3.9
1.5
2.9
2.1

5.4
4.2
4.1
5
10.91
4.6
9.4
5
5.7
4.8
11.31
4.8
4.9
5.7
2.9

Method using DMSO, -amylase, pullulanase-amylase, and amyloglucosidase (McCleary et al.).

Method using -amylase and amyloglucosidase (no DMSO) (McCleary et al.)
Method using -amylase and amyloglucosidase with DMSO.