FULL PAPER
Measurement
Measurement of Starch: Critical Evaluation of Current
Methodology
Barry V. McCleary,* Lucie M. J. Charmier, and Vincent A. McKie
Most commonly used methods for the measurement of starch in food, feeds,
and ingredients employ the combined action of α-amylase and amyloglucosi-
dase to hydrolyze the starch to glucose, followed by glucose determination with
a glucose oxidase/peroxidase reagent. Recently, a number of questions have
been raised concerning possible complications in starch analytical methods. In
this paper, each of these concerns, including starch hydrolysis, isomerization of
maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase,
enzyme purity and hydrolysis of sucrose, and β-glucans have been studied in
detailed. Results obtained for a range of starch containing samples using AOAC
Methods 996.11 and 2014.10 are compared and a new simpler format for starch
measurement is introduced. With this method that employs a thermostable α-
amylase (as distinct from a heat stable α-amylase) which is both stable and
active at 100
C and pH 5.0, 10 samples can be analyzed within 2 h, as
compared to the 6 h required with AOAC Method 2014.10.
1. Introduction
Starch is the major source of energy in the human diet. It is
contained in many staple foods such as cereals, legumes, root
vegetables, and fruits. Widely used foods that contain starch are
bread, pasta, and breakfast cereals.[1] Starch also comprises a
significant portion of many foods and feeds for animals and
finds numerous industrial applications in the native or
chemically modified forms. Starch broadly comprises two
components, amylose which is predominantly an 1,4-α-linked
D-glucan containing very few 1,6-α-linked branch points; and
amylopectin, which has a highly branched structure with a high
proportion of 1,6-α-linked 1,4-α-D-glucan chains. Other poly-
saccharides with very similar structures are glycogen from
animal tissues and phytoglycogen from plant sources and
α-glucans from fungi. These are all hydrolyzed by the digestive
enzymes in the human and animal small intestine and thus
Dr. B. V. McCleary, L. M. J. Charmier, Dr. V. A. McKie
Megazyme
Bray Business Park, Southern Cross RoadBray, County Wicklow
A98YV29, Ireland
E-mail: barry@megazyme.com
© 2018 Megazyme. Published by WILEY-VCH Verlag GmbH & Co.
KGaA, Weinheim. This is an open access article under the terms of the
Creative Commons Attribution-NonCommercial License, which permits
use, distribution and reproduction in any medium, provided the original
work is properly cited and is not used for commercial purposes.
DOI: 10.1002/star.201800146
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behave identically from a nutritional stand-
point. For this reason, the Laboratory
Methods and Services Committee of the
Association of American Feed Control
Officials (AAFCO)[2] with involvement of
industry and researchers developed a
definition for “Dietary Starch,” namely an
alpha-linked-glucose carbohydrate of or
derived from plants, animals, and
microbes from which glucose is released
after gelatinization through the use of
purified α-amylases and amyloglucosidases
(AMG) that are specifically active only on α-
(1–4) and α-(1–6) linkages. Its concentra-
tion in feed is determined by enzymatically
converting the alpha-linked-glucose carbo-
hydrate to glucose and then measuring the
liberated glucose. This definition would
encompass plant starch, glycogen, malto-
dextrins, and maltose/isomaltose.”[3]
Historically, a range of methods have been
employed for the measurement of starch, but
in recent decades, the method of choice involves gelatinization of the
starch at elevated temperatures in the presence of a thermostable
α-amylase to produce a range of linear and branched dextrins, which
are subsequently quantitatively hydrolyzed to glucose with AMG.[4–11]
Released glucose is measured with a glucose oxidase/peroxidase
(GOPOD)reagentor employinghexokinase with glucose 6-phosphate
dehydrogenase in the presence of ATP and NADPþ. The use of heat
stable α-amylase with AMG in the measurement of starch was first
introduced by Baur and Alexander,[4] and more extensively developed
by Batey,[5] Dintiz and Harris,[6] and Karkalas[7] and modified by
several other researchers.[8–11] The method was streamlined by
McCleary et al.[10] and subjected to a combined AOAC International/
AACC International/ICC interlaboratory evaluation. On the basis of a
successful study, the method was adopted as AOAC Method 996.11,
AACC International Method 76–13.01 and ICC Method 168.
A method for measurement of dietary starches in animal
feeds and pet foods was published by Hall in 2009[12] and
successfully subjected to an interlaboratory evaluation under the
auspices of AOAC International (to become AOAC Method
2014.10).[3,13] In this method, the incubation time with a heat
stable α-amylase was increased from 6 min to 1 h and
incubations with both α-amylase and AMG were performed
at pH 5.0, similar to that described in the Megazyme Total Starch
Assay Kit (cat. no. K-TSTA; 2008). The author reported higher
starch values with AOAC method 2014.10 than was obtained
with AOAC Method 996.11, and proposed that this was due to
the formation of maltulose (and thus loss of “starch”) under the
pH/temperature conditions employed with AOAC Method

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996.11 and other methods. The author also reported a non-linear
color response in measurement of glucose with GOPOD
reagent,[14] something that the current authors have never
observed. Hall[3] suggested that all starch methods prior to
AOAC Method 2014.10 are “quasi-empirical.”
On the basis of the observations by Hall,[3,12–14] we decided to
re-look at starch analysis methods with the aim of better
understanding each step in the determination. More specifically,
starch liquefaction with α-amylase, hydrolysis of starch dextrins,
and sucrose by AMG and maltulose production during starch
hydrolysis, and measurement of glucose with GOPOD reagent
have been studied in detail and an improved total starch
analytical procedure has been developed (Figure 1).
2. Experimental Section
2.1. Materials
2.1.1. Chemicals
Bovine serum albumin (cat. no. A-2153), sodium azide (cat. no.
S-8032), maltose (cat. no. M-5895-1Kg), calcium chloride
(CaCl2.2H2O; MW 147.01; cat. no. C-3306-100G) were from
Sigma–Aldrich Ireland Ltd. (Dublin, Ireland). Acetic acid (glacial)
GR and sodium hydroxide were from Merck (Darmstradt,
Germany). Maltotriose (cat. no. O-MAL3), D-Glucose Assay Kits
(cat. nos. K-GLUC and K-GLUHK), and α-Amylase Assay Kit
(Ceralpha1; cat. no. K-CERA) were obtained from Megazyme,
Bray, Ireland). Amyloglucosidase (AMG) (cat. no. E-AMGDF;
3300 U mL1 on soluble starch at pH 4.5 and 40
C) and Bacillus
species thermostable α-amylase (cat. no. E-BSTAA; 2800 U mL1
on Ceralpha1 reagent at pH 5 and 40
C) are from the Megazyme
Total Starch Assay Kit (cat. no. K-TSTA). Unless otherwise
Figure 1. Schematic representation of the rapid total starch (RTS) method.
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stated, AMG (cat. no. E-AMGDF; 3300 U mL1) was used
in all experiments employing AMG. Termamyl 120 L
(Bacillus licheniformis heat stable α-amylase; 1880 U mL1 on
Ceralpha1 reagent at pH 5 and 40
C) was from Novozymes, Novo
Alle, Denmark and Multifect AA 21L (Bacillus licheniformis heat
stable α-amylase; 1870 U mL1 on Ceralpha1 reagent at pH 5.0
and 40
C) was from Genencor International, Rochester, NY, USA.
2.1.2. Samples
Pure starch samples: Corn starch (regular maize starch; cat. no.
S-4126), high amylopectin corn starch (cat. no. S-9679), potato
starch (cat no. S-4251), wheat starch (cat. no. S-5127), rice starch
(cat. no. S-7260), potato amylose (cat. no. A-9262), and ACS
Soluble starch (cat. no. S-21101) were from Sigma–Aldrich
Ireland Ltd. (Dublin, Ireland). Hylon VII1 (Ref. 98GH8401),
Novelose 3301 (Ref. AH17529), and Novelose 2401 (Ref.
96LF10063) were from National Starch and Chemical Company,
Bridgewater, CT, USA (now Ingredion). Fibersol 21 was from
Matsutani Chemical Company, Hyogo, Japan. Fibersym1 was
from MGP ingredients, Atchison, Kansas, USA.
Dry grains, beans, and seeds: Dry grains, beans, and seeds
including cannellini beans, butter beans, organic red kidney
beans, pinto beans, haricot beans, soya beans, mung beans,
chick peas, yellow split peas, black eye beans, lentils verts, blank
baluja lentils, quinoa, spelt grain, roasted buckwheat, long grain
brown rice, oat groats, millet, wheat grain, and linseed were
obtained from Natures Gold, Killincarrig Road, Greystones, Co.
Wicklow, Ireland.
Animal feeds: Connolly’s RED MILLS layee hen pellets were
obtained from Connolly’s RED MILLS, Goresbridge, Co.
Kilkenny, Ireland. Gain soyabean meal, Gain Freedom mix
low starch horse feed, Gain Drive dairy feed complete, Gain
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small dog adult kibble, Gain feeds flaked maize, and Gain
Premium Cuts wet dog food were obtained from Gain Animal
Feeds, Bridge Street, Portlaoise, Ireland. TopSpec Top Chop
alfalfa and TopSpec Ulsakind low starch horse feed were from
TopSpec Equine Ltd, Middle Park Farm, Pickhill, Thirsk, North
Yorkshire, YO7 4JN, UK. Corn silage was obtained as a sample
from a local supplier, County Laois, Ireland.
Processed foods and vegetables: Kellogg corn flakes, Kellogg
Special K1 Original, Kellogg Frosties1, Kellogg All Bran1, and
Shreddies1, and Weetabix1, Roma maceroni (DS 7296), fresh
carrots, sweet potato, swede, button mushrooms, red peppers
(capsicums), red onion, celery, rooster potatoes, cauliflower,
brochelli, mango, and ripe banana were obtained from Tesco
Ireland, Greystones, Co. Wicklow, Ireland. A range of other
beverages and food products as summarized in Table S1–S3,
Supplementary Information, were also obtained from Tesco Ireland.
2.2. Methods
2.2.1. Measurement of Starch Using Modifications to AOAC
Method 996.11
Principle (see Figure 1): Ground or homogenized solid samples
or heated and homogenized liquid samples of starch containing
plant materials, human food, or animal feeds were incubated
with thermostable α-amylase in sodium acetate buffer contain-
ing calcium chloride in a boiling water bath (at 100
C) for
15 min with periodic mixing to gelatinize and hydrolyze the
starch to maltodextrins. After cooling to 50
C over 5 min, AMG
was added which catalyses complete hydrolysis of the maltodex-
trins to glucose within 30 min. The mixture was clarified,
appropriately diluted, and glucose was determined using a
GOPOD reagent that is both sensitive and specific for glucose.
Free glucose in each test sample was determined simultaneously
by running a test portion through the entire procedure but
omitting enzyme addition. For the sake of this analysis, starch
(dietary starch) is defined to include plant starches, microbial
starches/phytoglycogens, animal glycogen and maltodextrins of
degree of polymerization (DP) of two or higher (i.e., the sum of
all linear and branched 1,4/1,6-α-linked glucans and
oligosaccharides).
Apparatus:
a. Grinding mill—Centrifugal, with 12-tooth rotor and 0.5 mm
sieve, or similar device. Alternatively, a cyclone mill can be
used for small test laboratory samples provided the mill has
sufficient air-flow or other cooling to avoid overheating of
samples.
b. Homogenizer/blender to homogenize moist and semi-
moist food or pet food materials—NutriBullet Pro 9001
blender or similar.
c. Magnetic stirrer and stirrer bars.
d. Microfuge—Capable of holding tubes of 2 mL capacity and
of speeds up to 13 000 rpm. High Performance-Wise CF-10,
Product Code: EDHCF-10SN: 04039671231037, Lennox,
Dublin.
e. Microfuge tubes—Eppendorf Safe-Lock microfuge tubes,
2.0 mL. Merck cat. no. T2795-1000EA.
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f. Spectrophotometer set at 505 or 510 nm. Equipment used in
this work were a Shimadzu UV-1800 Spectrophotometer
(cat. no. 206-25400-59) connected to SWA-2 Sample Waste
Unit (cat. no. 206-23820-91) supplied by Mason Technology,
Dublin; a Biochrom Libra S22 UV/Vis Recording Spectro-
photometer (cat. no. 80-2115-20) operated with SWIFT ll
Method Applications Software Version 2.04 and connected
to Julabo CORIOTM C immersion circulator and water bath
supplied by Brennan & Company, Dublin; and a Mega-
QuantTM Wave Spectrophotometer (cat. no. D-MQWAVE-1)
supplied by Megazyme, Dublin.
g. Digestion Tubes—Corning culture tubes (20  125 mm;
Fisher cat. no. 14-933C) with screw caps.
h. Glass test tubes—16  120 mm.
i. Vortex mixer—(e.g., Vortex-Genie1 2 mixer).
j. Brand Bottle-top dispensette S Digital 2.5–25 mL (cat. no.
46003)—capable of accurately dispensing 10.0 and 4.0 mL of
100 mM sodium acetate buffer (pH 5.0) containing 10 mM
CaCl2, 5 mL of 200 mM sodium acetate buffer (pH 5.0)
containing 5 mM CaCl2, 8 mL of 0.6 M sodium acetate
buffer (pH 3.8) containing 5 mM calcium chloride, and of
dispensing 3.0 mL of GOPOD reagent.
k. Micro-pipettor, 200 μL variable—to deliver 100 μL of sample
solution.
l. Positive displacement pipettor—e.g., Eppendorf Handi-
Step1 with 2.5 mL Combitip1 (to dispense 0.1 μL aliquots
of α-amylase and AMG).
m. Water bath with immersion heater—capable of maintaining
temperature of 50  1
C.
n. Boiling water bath—capable of maintaining a temperature
of 95–100
C.
o. Balance—0.1 mg readability, accuracy, and precision.
p. Oven—Mechanical convection, set at 105  2
C.
q. Timer.
r. pH meter.
s. Thermometer—Capable of measuring to 110
C.
t. Filter paper—Whatman No. 1, 9 cm.
u. Freeze drier—VirTis GenesisTM.
v. Moisture content analyzer—MB44, OHAUS, Switzerland
(cat. no. R523).
Reagents:
a. Ethanol (or industrial methylated spirits; IMS) 95% and 80% v/v.
b. Thermostable α-amylase (Megazyme cat. no. E-BSTAA)—
2800 U mL1 on Ceralpha reagent at pH 5.0 and 40
C or
4290 U mL1 at pH 7.0 and 40
C. Stable for >4 years at 4
C.
The thermostable α-amylase must be essentially devoid of
free glucose.
c. Amyloglucosidase (AMG; Megazyme cat. no. E-AMGDF)—
3300 U mL1 on soluble starch (or 200 U mL1 on
p-nitrophenyl β-maltoside) at pH 4.5 and 40
C. Stable for >4
years at 4
C. AMG must be free of enzymes active on β-glucan
and cellulose. AMG must be essentially devoid of free glucose.
d. GOPOD reagent buffer—Potassium phosphate buffer
(0.26 M, pH 7.4), p-hydroxybenzoic acid (0.22 M) and sodium
azide (0.4% w/v).
e. GOPOD reagent enzymes—Glucose oxidase (>12 000 U)
plus peroxidase (>650 U) and 4-aminoantipyrine (80 mg) as a
lyophilized powder.
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f. GOPOD Reagent—Add the GOPOD reagent buffer to
900 mL of demineralized water and dilute to 1 L with
demineralized water. Dissolve the GOPOD reagent enzymes
in the GOPOD buffer and store the solution in a 1 L Duran
bottle. Cover the bottle with aluminium foil to protect the
solution from light and store the reagent at 4
C. Under these
conditions, the reagent is stable for 3 months.
g. D-Glucose standard solution—(1.00 mg mL1) in 0.2 % w/v
benzoic acid. This solution is stable for 5 years at room
temperature.
h. Sodium acetate buffer—100 mM, pH 5.0 plus 5 mM CaCl2.
Add 5.8 mL of glacial acetic acid (1.05 g mL1) 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. Add 0.74 g of calcium
chloride dihydrate and dissolve. Adjust the volume to 1 L and
store the buffer at 4
C. Stable for 3 months at 4
C.
i. Sodium acetate buffer—200 mM, pH 5.0 plus 5 mM CaCl2.
Add 11.6 mL of glacial acetic acid (1.05 g mL1) 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. Add 0.74 g of calcium
chloride dihydrate and dissolve. Adjust the volume to 1 L and
store the buffer at 4
C. Stable for 3 months at 4
C.
j. Sodium acetate buffer—600 mM, pH 3.8 plus 5 mM CaCl2.
Add 34.8 mL of glacial acetic acid (1.05 g mL1) to 800 mL of
distilled water. Adjust the pH to 3.8 by the addition of 2 M (8 g/
100 mL) sodium hydroxide solution. Add 0.74 g of calcium
chloride dihydrate and dissolve. Adjust the volume to 1 L and
store the buffer at 4
C. Stable for 3 months at 4
C.
k. Sodium hydroxide solution (1.7 M)—Add 68 g NaOH to
900 mL of deionized water and dissolve by stirring. Adjust
volume to 1 L. Store in a sealed container. Stable for >4 years
at room temperature.
l. Reagents (b), (c), (d), (e), and (g) are available from
Megazyme in the Total Starch Assay Kit (cat. no. K-TSTA).
Preparation of test samples: Grind ca. 50 g of dry sample in a
grinding mill to pass a 0.5 mm sieve. Transfer all material to a
wide-mouthed plastic jar, seal, and mix well by shaking and
inverting. Freeze-dry high-moisture samples such as wet dog or
cat foods. Grind this material in a NutriBullet Pro 9001
blender. With liquid samples, directly transfer 1.0–5.0 mL with
a HandyStep1 dispenser with a 25 mL tip to a 20  125 screw
cap corning culture tube and proceed to analysis. Weigh 1.0 g
(weighed accurately) of semi-solid materials such as spreads,
yogurts, and mayonnaise-type materials and solid food samples
such as chocolate and peanut butter directly into the analysis
tubes. Add 5 mL of 200 mM sodium acetate buffer, pH 5.0 and
adjust the volume to 10 mL with distilled water using markings
on the analysis tubes. Add 0.1 mL of thermostable α-amylase
and proceed with the analysis as per liquid samples (see section
RTS-liquids method). Homogenize vegetable and fruit samples
in a NutriBullet Pro 9001 blender. Pour the homogenized
slurry onto a pre-weighed aluminium disposable tray and
record the weight of tray plus wet sample. Freeze and freeze-dry
the sample and record the dry weight of the sample and tray.
Calculate wet weight and dry weight of sample and moisture
content of the sample. Analyze samples (100 mg weighed
accurately) using the standard format (see section, RTS
method).
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Enzyme purity: To ensure absence of undesirable enzymatic
activities analyze amorphous cellulose, barley β-glucan, and
sucrose/cellulose controls (10% and 20% sucrose in amor-
phous cellulose) using the standard starch assay procedure (see
section, RTS method).
RTS Method—Measurement of total starch in solid samples:
1. Accurately weigh 100 mg of test sample, in triplicate (one
blank), into Corning culture tubes (20  125 mm; Fisher cat.
no. 14-933C). Record the exact weight. Tap the tube so that
sample falls to the bottom of the tube.
2. To each of the three tubes add 10 mL of 100 mM sodium
acetate buffer, pH 5.0, containing 5 mM CaCl2 using a
Brand Bottle-top dispensette S Digital 2.5–25 mL Cat. No.
4600351. Stir the tubes on a vortex mixer for 5 s.
3. To two of the tubes (sample tubes) add 0.1 mL of undiluted
thermostable α-amylase (Megazyme cat no. E-BSTAA; 280 U
at pH 5.0) using a HandyStep dispenser with 5 mL tip. To
the third tube (sample blank) add 0.1 mL of 100 mM sodium
acetate buffer (pH 5.0).
4. Vortex the tubes for 3 s, cap the tubes loosely and
immediately transfer them to a boiling water bath and
start the timer. After 2 min, tighten the caps and mix the
tube contents vigorously on a vortex mixer. After a further 5
and 10 min, vortex the tube contents again for 5 s and return
the tubes to the boiling water bath. After 15 min (from when
α-amylase was added) remove tubes from the boiling water
bath and mix the contents vigorously for 5 s on a vortex
mixer. Place the tubes in a water bath at 50
C and allow
them to equilibrate to temperature over 5 min.
5. To two of the tubes (the sample tubes), add 0.1 mL of
undiluted AMG (Megazyme cat. no. E-AMGDF; 330 U)
using a HandyStep dispenser with 2.5 mL tip and vortex for
3 s. To the third tube (the sample blank) add 0.1 mL of
100 mM sodium acetate buffer (pH 5.0). Incubate the tubes
at 50
C for 30 min with no further mixing.
6. Remove the tubes from the water bath and allow them to
cool to room temperature over 10 min. Invert the tubes a few
times to ensure condensed water on the inside of the lid is
mixed with liquid in the tube.
7. Carefully transfer 2.0 mL of each solution to a microfuge
tube and centrifuge the tube at 13 000 rpm for 5 min. Using
a Gilson Pipetman dispenser, accurately transfer a 1.0 mL
aliquots of the supernatants to 12  120 mm tube contain-
ing 4 mL of 100 mM sodium acetate buffer (pH 5.0) and
mix the contents on a vortex mixer. (Retain the remaining
9.2 mL of incubation solution and refer to the following
step 10.
8. Accurately transfer duplicate 0.1 mL aliquots of each sample
to the bottoms of 16  120 mm glass test tubes.
9. Add 3.0 mL of GOPOD reagent and incubate the solutions at
50
C for 20 min and measure absorbance at 510 nm.
Concurrently incubate glucose standard solution (0.1 mL,
1.0 mg mL 1) with 3.0 mL of GOPOD reagent in quadrupli-
cate. For reagent blank, incubate 0.1 mL of acetate buffer
(100 mM, pH 5.0) with 3.0 mL of GOPOD reagent in duplicate.
10. Zero the spectrophotometer with the GOPOD reagent blank
(no glucose) and measure all other absorbance values
against this.
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Note: If the GOPOD absorbance values for samples are less
than 0.100, analyze the centrifuged sample solution (see
section, RTS method, steps 6 and 7) without further dilution. If
absorbance values are greater than 1.20, then add 1.0 mL of the
centrifuged sample solution to 10.0 mL of 100 mM sodium
acetate buffer (pH 5.0), mix well and remove 0.1mL aliquots for
analysis using GOPOD reagent. For samples containing less
than 1% (w/w) starch content, increase sample size to 500 mg,
and analyze 0.1 mL of undiluted sample solution.
11. Calculate starch content on a % (w/w) as is basis.
Total starch; % ¼ ΔA  F  102  D  1=1000  100=W  162=180
¼ ΔA  F  D=W  90
where: ΔA ¼ absorbance of sample solution read against reagent
blank, less the absorbance of the sample blank read against the
reagent blank; F ¼ factor co convert absorbance values to μg
glucose (¼ 100 μg glucose divided by the absorbance value
obtained for 100 μg of glucose); 102 ¼ volume correction, i.e.,
0.1 mL taken from 10.2 mL of sample solution; D ¼ further
dilution of sample solution (either undiluted, fivefold or 11-fold);
1/1000 ¼ conversion from μg to mg; 100/W ¼ conversion to
100 mg sample; 162/180 ¼ factor to convert from free glucose, as
determined, to anhydroglucose, as occurs in starch.
Alternatively, calculations can be simplified using a Mega-
zyme Excel based calculator (Supplementary Information).
RTS NaOH method—Measurement of total starch (including
resistant starch) in solid samples: method employing sodium
hydroxide dissolution:
1. Accurately weigh 100 mg of test sample, in triplicate, into
corning culture tubes (20  125 mm; Fisher cat. no. 14-933C).
Record the exact weight. Tap the tube so that sample falls to
the bottom of the tube.
2. Add 0.2 mL of 80% aqueous ethanol and stir the tube on a
vortex mixer to completely wet and disperse the sample.
3. Add 2 mL of ice-cold 1.7 M sodium hydroxide solution (68 g
NaOH/L) using a HandyStep1 dispenser with 25 mL tip and
stir the tube contents on a vortex mixer for 15 s Place the tube
in a rack in an ice water bath for 30 min and during this time,
stir the contents of the tube vigorously on a vortex mixer 2–3
times. Ensure that there are no lumps in the sample slurry.
4. Add 8 mL of 600 mM sodium acetate buffer (pH 3.8)
containing 5 mM calcium chloride using a Brand1 Bottle-
top dispensette1 S (Digital 2.5–25 mL cat. no. 4600351). Stir
the tubes on a vortex mixer. Check that the pH is 5.0.
5. Immediately add 0.1 mL of undiluted thermostable
α-amylase (Megazyme cat. no. E-BSTAA) using a Handy-
Step1 dispenser with a 2.5 mL tip to two of the tubes (sample
tubes). Then add 0.1 mL of undiluted AMG (E-AMGDF;
3300 U mL1) using a HandyStep1 dispenser with a 2.5 mL
tip to the same two tubes. To the third tube (sample blank)
add 0.2 mL of 200 mM sodium acetate buffer (pH 5.0). Cap all
three tubes and vortex the contents for 3 s.
6. Incubate the tubes at 50
C for 30 min.
7. Remove the tubes from the water bath and allow them to cool
to room temperature. Invert the tubes a few times to ensure
condensed water on the inside of the lid is mixed with liquid
in the tube.
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8. Proceed with the assay as described in see section, RTS
method, step 8. Perform calculations as described in section,
RTS method, step 11.
RTS-liquids method—Measurement of total starch in liquid
samples:
1. Accurately transfer 1.0–5.0 mL of test sample, in triplicate, into
corning culture tubes (20  125 mm; Fisher cat. no. 14-933C).
Adjust the volume of each tube to 5 mL by addition of distilled
water.
2. To each tube, add 5 mL of 200 mM sodium acetate buffer, pH
5.0, containing 5 mM CaCl2 using a Brand Bottle-top
dispensette S Digital dispenser (2.5–25 mL cat. no. 4600351).
Stir the tubes on a vortex mixer for 5 s.
3. To two of the tubes, add 0.1 mL of undiluted thermostable α-
amylase (Megazyme cat no. E-BSTAA) using a HandyStep
dispenser with 5 mL tip. To the third tube, add 0.1 mL of
100 mM sodium acetate buffer (pH 5.0). Proceed with the assay
as for solid samples as described in section, RTS method, step 4.
2.2.2. Measurement of Starch Using AOAC Method 996.11
(McCleary et al.)
Supporting Information.
2.2.3. Measurement of Starch Using AOAC Method 2014.10
(Hall)
Supporting Information.
2.2.4. Assay of α-Amylase and AMG and Definitions of
Enzyme Activity
Supporting Information.
2.2.5. Preparation of a D-Glucose Standard Solution
Supporting Information.
2.2.6. Determination of D-Glucose with GOPOD Reagent
Supporting Information.
Preparation and storage of GOPOD reagent: Supporting
Information.
Linearity of glucose-GOPOD standard curve: Supporting
Information.
Stability of GOPOD color complex: Supporting Information.
2.2.7. Gelatinization and hydrolysis of starch by α-amylase
Effect of pH and presence of CaCl2 on the stability of α-amylase at
100
C: Supporting Information.
Stability of maltose under the incubation conditions used for the
hydrolysis of starch by α-amylase: Supporting Information.
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2.2.8. Hydrolysis of Sucrose Under α-amylase Incubation
Conditions
Supporting Information.
2.2.9. Hydrolysis of Starch Dextrins by AMG at 40 and 50
C
Supporting Information.
2.2.10. Purity of AMG
Hydrolysis of cellulose and β-glucan: Supporting Information.
Hydrolysis of sucrose by AMG: Supporting Information.
2.2.11. Accuracy and Reproducibility of Starch Assay Procedures
Supporting Information.
3. Results and Discussion
The quantitative measurement of starch employing hydrolysis
by α-amylase plus AMG, with glucose determination using
GOPOD reagent has several requirements, namely; that glucose
measurement is simple and quantitative; that starch dextrins are
quantitatively hydrolyzed to glucose with no loss through side
reactions, and that starch solubilization and dextrinization is
complete. The method that will be described in detailed here is
outlined in Figure 1. This is a modification of AOAC Method
996.11 incorporating the use of a thermostable α-amylase that is
both active and stable at pH 5.0, meaning that incubations with
both thermostable α-amylase and AMG can be performed at the
same pH. This modification to AOAC Method 996.11 had first
been introduced into the Megazyme Total Starch assay kit
K-TSTA in 2008 and is widely used.
3.1. Preparation of a Glucose Standard Solution
Glucose can be quantitatively measured with either the glucose
oxidase/peroxidase (GOPOD) reagent, or using the hexokinase/
glucose 6-phosphate dehydrogenase (HK/G6PDH) procedure in
the presence of ATP plus NADPþ. The HK/G6PDH procedure
has the advantage of giving an absolute value for measured
glucose on the basis of production of NADPH and employing its
extinction coefficient. This contrasts to the GOPOD procedure
where quantitation requires reference to either a glucose
standard or to a glucose standard curve. Most published
procedures describe the preparation of a glucose standard
solution by adding exhaustively dried crystalline glucose to a
given weight of water. However, we have found that even using
glucose that has been exhaustively dried under vacuum and at
elevated temperatures, the final glucose concentrations is at least
2% lower than that expected based on the weight of the glucose
used. Consequently, we prepared glucose standard solutions by
making a solution of 1.1 mg mL1, determining the exact
concentration of glucose using the HK/G6PDH (measuring
NADPH produced), calculating the concentration of NADPH
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(and thus glucose) from the extinction coefficient of the
NADPH, and then diluting the glucose to the exact concentra-
tion by addition of distilled water by weight (see section 2.2.5).
Benzoic acid was then added to a concentration of 0.2% w/v.
3.2. Linearity of the Glucose Standard Curve with GOPOD
Reagent
The linearity of the glucose standard curve with GOPOD reagent
prepared according to AOAC Method 996.11 (Method 2.2.6.A.a,
Supporting Information) with 4-hydroxybenzoic acid, or alterna-
tively with phenol (Method 2.2.6.A.b, Supporting Information)[7]
and measured in a Shimadzu UV-1800 Spectrophotometer is
shown in Figure 2. Clearly, the standard curve is absolutely linear
with both GOPOD reagents. With GOPOD employing 4-
hydroxybenzoic acid (2.2.6.A.a, Supporting Information), the
standard curve is absolutely linear with an r2 of 1.00 and a
regression equation of glucose (μg) ¼ 94.37  Absorbance.
For the GOPOD reagent employing phenol, the r2 is 0.9994 and
the regression equation is glucose (μg) ¼ 90.65  Absorbance.
In a similar study using a MegaQuantTM Wave Spectrophotom-
eter and GOPOD reagent (2.2.6.A.a, Supporting Information) gave
a regression equation of glucose (μg) ¼ 104.6  Absorbance, and
an r2 value of 0.9994 (Figure S1, Supporting Information) was
obtained.
Clearly, the standard curves are both absolutely linear and also
pass through the origin, which contrasts with the results of Hall
and Keuler.[14] As the reagents were prepared using the same
procedure, we can only assume that the problem of non-linearity
reported by Hall and Keuler[14] must have been associated with
the spectrophotometer employed. In the current study, two
spectrophotometers and one colorimeter were used (see section
Apparatus, f), and linearity was obtained in each case; the
standard curve dissected the origin and r2 was essentially 1.00.
Linearity of the glucose/GOPOD standard curve is essential if
glucose values are to be calculated from a single glucose
concentration rather than by reference to a regression equation.
Use of a regression equation requires that a full standard curve
be prepared for each set of determinations. Clearly, in use of the
GOPOD reagents and the procedure described here, there is no
requirement for a full glucose standard curve, but it is prudent to
Figure 2. Linearity of glucose determination with GOPOD reagents
containing either phenol or p-hydroxybenzoic.
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perform the analysis of the glucose standard in quadruplicate to
allow for potential errors in pipetting.
3.3. Stability of the Glucose/GOPOD Color Complex
The rate of glucose/GOPOD color formation and the stability of
this color in the dark at 40
C was measured using a Biochrom
Libra S22 UV/Vis Recording Spectrophotometer and is shown in
Figure 3. The results of a parallel incubation performed in the
light at 40
C and at 50
C in a conventional water bath as
measured using a Shimadzu UV-1800 Spectrophotometer with
sipper-cell attachment is shown in Figure S2, Supporting
Information. In each case, maximum color formation was
achieved within 20 min. At 40
C, the color complex was stable in
both the light and dark for up to 200 min. At 50
C, there was a
gradual color decrease after 60 min. However, as glucose
standard solutions are incubated concurrently with samples
being analyzed, even the small changes observed at 50
C after
200 min are accounted for. Stability of the color complex
significantly simplifies the assay as there is no need to perform
the incubations and measurements under a strict time regime.
3.4. Solubilization and Hydrolysis of Starch
In initial publications[4,7] on the use of heat stable α-amylase for
hydrolysis of starch in starch determination procedures,
incubation temperatures of 80–85
C were employed in
unbuffered solutions. Under these conditions, B. licheniformis
α-amylase (as in Novozymes 120L) is quite stable. The enzyme
has adequate stability at 100
C when buffered at pH 7.0
(Figure 4) in the presence of Ca2þ. However, at pH 5.0 and in the
absence of Ca2þ, as employed by Aman and Hesselman,[8] the
enzyme is extremely unstable at 100
C, losing all activity within
10 min (Figure 4). In contrast, thermostable α-amylase (Mega-
zyme Cat. No. E-BSTAA, Bacillus sp.) in the presence of Ca2þ, as
used in the current procedure, retains 50% of its activity even
after incubation for 60 min at 100
C and pH 5. In evaluating the
Figure 3. Time course of colour development (and colour stability) on
incubation of glucose (0, 50, and 100 μg/assay) with 3.0 mL of GOPOD
reagent (2.2.6.A.a, Supporting Information) in a recording spectropho-
tometer (in the dark).
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current procedure for measurement of starch in a broad range of
products, an incubation time of just 10 min at 100
C was
required for most samples. However, with some vitreous grain
samples (e.g., milled wheat), an incubation time of 15 min with
α-amylase (E-BSTAA) gave slightly higher starch values (2%
higher); presumably due to the time required to completely
gelatinize the starch in such samples. Consequently, to
standardize the procedure to cover all samples, an incubation
time with thermostable α-amylase of 15 min is recommended.
In the method developed by Hall,[3,13] samples are incubated
at 100
C with heat stable α-amylase (Termamyl 120 L or
Multifect AA 21L) in sodium acetate buffer, pH 5.0 in the
absence of calcium chloride for 60 min. However, under these
conditions, the enzyme is completely inactivated within 10 min
(Figure 4). As this method gives a quantitative measurement of
starch, 10 min of incubation with α-amylase is clearly adequate.
3.5. Stability of Maltose at Higher pH Values
Hall[13] reported that higher values for starch were obtained in
various animal feed samples using AOAC Method 2014.10 than
were obtained with other analytical methods, including AOAC
Method 996.11. The author suggested that this may be due to
partial isomerization of the reducing-end glucose in malto-
oligosaccharides to fructose during the hydrolysis of starch by α-
amylase at 100
C under neutral or slightly alkaline conditions.
Such oligosaccharides are rapidly hydrolyzed by AMG to glucose
and maltulose (4-O-α-D-glucosyl-D-fructose), but the maltulose
is resistant to further hydrolysis by AMG. This in turn leads to an
underestimation of starch. However, the author provided no
experimental evidence that maltulose is actually formed under
the incubation conditions employed in other starch determina-
tion methods. Reference was made to the work of Dias and
Panchal,[15] but in that work, incubations were performed at
much higher temperatures (121–143
C) and at very high starch
dextrin concentration (30 % w/v). In the current work, this
phenomenon was studied under conditions in line with those
used in starch analytical methods. Solutions of maltose
(10 mg mL1) were incubated at pH values from 5.0 to 9.0 at
100
C for various time intervals (2.2.7.B, Supporting
Figure 4. Stability of heat stable α-amylase (Novozymes 120 L) and
thermostable α-amylase (Megazyme cat. no. E-BSTAA) at 100
C in the
presence or absence of calcium chloride at pH 5.0 or 7.0.
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Information). “Loss” of maltose was followed by incubation of
samples with AMG and measurement of glucose using GOPOD
reagent as a measure of remaining maltose. Also, formation of
maltulose was followed employing ion chromatography. The
results of such a study are shown in S.F3; Figure S3, Supporting
Information. Even after incubation for 60 min at 100
C, no loss
of maltose was observed in acetate buffer, pH 5 or MOPS
buffer, pH 7.0; but progressively more maltose was “lost” as
the pH of incubation was increased to pH 9.0. Loss of maltose
and production of maltulose was also followed by analyzing
samples incubated under different conditions by ion chroma-
tography. In Figure 5, ion chromatography patterns for maltose,
maltulose, and various incubation mixtures are shown. Clearly,
on incubation of maltose at pH 7.0 in MOPS buffer at 100
C for
10 min (hydrolysis conditions used in AOAC Method 996.11) no
maltulose was formed, consistent with the results shown in
Figure S3, Supporting Information. In contrast, on incubation of
maltose at pH 8.0 or 9.0 at 100
C for just 20 min, maltulose
formation was observed. These conditions are not used in any
starch analytical methods. On incubation of maltose at pH 5.0
and 100
C, no maltulose was formed even after 60 min of
incubation. In conclusion, maltulose is not formed under the
α-amylase incubation conditions employed in either AOAC
Figure 5. Ion chromatography of maltose and maltulose standards and maltose stored at 100
C and pH 5, 7, 8, or 9 for 10 or 20 min.
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Method 996.11 or 2014.10 or any other published method for
starch measurement.
3.6. Interference of Sucrose in Starch Determination
Plant and food materials often contain substantial quantities of
sucrose, so it is important to understand the stability of this
compound in the presence of α-amylase and AMG under the
conditions used to hydrolyze starch to glucose. Any hydrolysis of
sucrose to fructose and glucose by α-amylase will lead to
overestimation of the starch content of the sample. Hydrolysis of
sucrose (2 mg mL1, 10 mL) by thermostable α-amylase under
the incubation conditions used in the analytical procedure used
to measure starch is shown in Figure S4, Supporting
Information. Sucrose is hydrolyzed, albeit slowly. The rate of
hydrolysis at 100
C, pH 5 both in the presence or the absence of
α-amylase is the same, indicating that the instability is due to the
pH/temperature of incubation, rather than hydrolysis by
α-amylase or a contaminating enzyme activity in the α-amylase
preparation. Under the incubation and dilution conditions
employed, an absorbance of 0.10 equates to a starch value of 1%
w/w. However, this is accounted for in the blank absorbance
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Table 1. Specificity of the RTS method for starch determination:
hydrolysis of cellulose, β-glucan, sucrose, and kestose.
Sample Starch contenta) % (w/w), dwbb)
α-Cellulose 0.03
Barley β-Glucan (Megazyme cat. no. 90803) 0.10c)
Sucrose (10% w/w) in α-cellulose 0.15
Sucrose (20% w/w) in α-cellulose 0.50
Kestose (100 mg) 0.30
a)
All values are the mean of duplicate analyses; b)Dry weight basis; c)The β-glucan
analyzed had a starch content of 0.06%.
value as the hydrolysis is independent of the presence of
α-amylase.
Similar experiments have been performed with the AMG used
in the starch analytical method (namely Aspergillus niger cat. no.
E-AMGDF), an ultra pure A. niger AMG and AMG from Rhizopus
sp. Experimental conditions are described in 2.2.10.B, Support-
ing Information and results are shown in Figure S5, Supporting
Information. Sucrose is hydrolyzed by the pure AMG (E-
AMGDF) and the ultra pure AMG (AMGUP) at the same rate,
indicating that hydrolysis is catalyzed by AMG and not by a
contaminating activity. This is not surprising as AMG inherently
acts on α-linked D-glycosyl residues, such as in maltose,
maltodextrins, and starch.
In the experiment shown here, sucrose concentrations in the
incubation mixture of 1 and 10 mg mL1 was chosen as these
equate to a sucrose concentration in the sample of 10% and
100% (w/w). Most food and plant samples contain less than 10%
sucrose. Under these conditions 0.5 μg of glucose is present in
0.1 mL of reaction mixture after incubation for 30 min with
AMG; which equates to a “starch” value of just 0.05% (w/w),
which is insignificant. At higher sucrose concentrations, the
amount of glucose released is higher. However, even at a sucrose
concentration of 10 mg mL1 (i.e., a sample of 100% (w/w)
sucrose), the glucose released after 30 min (3.8 μg/0.1 mL)
equates to a starch value of just 0.4% (w/w), which is within the
experimental accuracy of the method.
In a separate set of experiments, sucrose/cellulose control
samples with a sucrose content of 10% (w/w) and 20% (w/w)
were analyzed in the standard starch analytical procedure (see
section, RTS method) and results obtained are shown in Table 1.
The determined level of glucose released equated to a “starch”
value of just 0.15% with the 10% (w/w) sucrose containing
sample and 0.50% with the 20% (w/w) sucrose containing
sample. These values are within the experimental accuracy of the
method and thus can be ignored. Commercially available food or
feed samples are unlikely to contain more than 20% (w/w)
sucrose content.
3.7. Hydrolysis of Maltodextrins by AMG
Linear and branched maltodextrin are rapidly hydrolyzed to
glucose by AMG under the standard incubation conditions.
Hydrolysis at 40 and 50
C is shown in Figure S6, Supporting
Information. For complete hydrolysis at 40
C, an incubation

Table 2. Comparison of starch determination methods for the measurement of total starch in a range of animal feeds and pet foods.

Total starch,a) % (w/w) on an “as is”b) basis

Sample details AOAC AOAC Modified AOAC 996.11 Relative bias b (%) (Modified AOAC Relative bias b (%) (Modified AOAC 996.11
996.11 2014.10 (RTS method) 996.11 vs. AOAC 996.11) vs. AOAC 2014.10)

Connolys Red Mills Layee 33.0 32.9 34.2 3.8 4.1

Hen Pellets
Gain Soyabean Meal 7.7 7.7 7.8 1.3 1.3
TopSpec Top Chop Alfalfa 0.7 0.8 0.7 0.7 15.8
TopSpec Ulsakind low 12.1 12.7 12.9 6.2 1.2
starch horse feed
Gain Freedom mix low 9.0 9.2 9.2 2.2 0.0
starch horse feed
Gain drive Dairy feed 17.2 17.1 17.4 1.5 1.8
complete
Gain small dog adult kibble 39.4 40.8 39.3 0.1 3.6
Corn Silage No. 160807 26.3 28.7 26.7 1.5 6.8
Gain feeds flaked maize 66.2 66.7 67.3 1.7 0.8
Gain Premium cuts wet dog 0.5 0.7 0.7 42.1 4.3
food
Regular maize Starch Lot 86.1 85.6 85.9 0.2 0.4
140206
Mean 5.4 1.9

a)
All values are the mean of duplicate analyses; b)Results presented in this table are on an “as is” basis. Moisture content has not been accounted for.

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time of 30 min is required while at 50
C the reaction is complete
within 20 min.
3.8. Analysis of Samples
Starch values determined for a range of animal feed samples
using AOAC Method 996.11, AOAC Method 2014.10, and the
new method (AOAC 996.11 modified method; RTS Method) are
shown in Table 2. The accuracy of the modified AOAC 996.11
method was assessed by calculating the relative bias b (%) for
each sample result using results from the AOAC Method 996.11
or AOAC Method 2014.10 as the reference value in the
calculation as per the following equation:
x
x ref
bð%Þ ¼  100
ref
x Method 2014.10.
where x ¼ Mean result from the modified AOAC 996.11 method.
ref ¼ Mean result from the reference method (AOAC Method
x 996.11 (using acetate buffer) was assessed using 11 milled
996.11 or AOAC Method 2014.10).
For all samples, the determined starch values obtained with
the three methods were very similar. The mean relative bias for
results obtained using the modified AOAC 996.11 method was
5.4% relative to AOAC Method 996.11 results, and 1.9%
relative to AOAC Method 2014.10 results.
The highest level of variation was observed for the alfalfa and
wet dog food samples and was due to inaccuracies associated
with measuring the very low starch contents and thus low
absorbance values obtained. Apart from these two samples all
other relative bias values (n ¼ 18) were less than 7% and of
these only four values were above 4%. With the single highest
relative bias value removed as an outlier from each calculation
the mean relative bias for results obtained using the modified
AOAC 996.11 method was 1.7% relative to AOAC Method
996.11 results and 0.5% relative to AOAC Method 2014.10
results. These results clearly indicate that the modified AOAC
996.11 method generates extremely accurate results across a
range of samples relative to AOAC Method 996.11 and AOAC
The repeatability (intra-assay precision) and intermediate
precision (inter-assay precision) of the modified AOAC Method
samples. For each sample, duplicate extractions were processed
and applied to the assay on each day across four separate days.
The repeatability and intermediate precision across this data set

Table 3. Repeatability study.

Total Starch, % (w/w), dwb,a) Meanb)  2 SD, (CVc)%)

Sample Day 1 Day 2 Day 3 Day 4 Interday mean, 2 SD, (CV,c) %)

Hen pellets 37  0.5 36.1  0.6 38  0.9 38.7  0 37.4  2.1

0.65 0.82 1.17 0.00 2.80
Soyabean meal 7.5  0.3 7.5  0.1 7.9  0.7 7.7  0.5 7.6  0.5
1.79 0.38 4.41 2.95 3.01
Alfalfa 0.3  0 0.3  0 0.3  0.1 0.3  0.1 0.3  0
6.73 2.32 8.32 11.22 7.39
Ulsakind horse feed 14.3  0.4 13.7  0.6 13  1.2 13.9  0.8 13.7  1.2
1.39 2.16 4.74 2.75 4.33
Freedom horse feed 9  1.2 8.6  0.2 9.2  0.3 8.7  1.2 8.9  0.8
6.93 1.32 1.46 6.72 4.65
Dairy feed complete 19.3  0 19.3  1.3 19.4  0.9 19.8  1.5 19.5  0.9
0.11 3.40 2.44 3.68 2.38
Dog adult kibble 40.6  0.6 40.7  0.4 41.4  0.8 41.1  0.7 40.9  0.8
0.73 0.52 0.97 0.86 1.04
Corn silage No. 16087 27.9  0.5 29.4  0.2 28.7  1.2 28.4  0.5 28.6  1.3
0.94 0.41 2.10 0.82 2.25
Flaked maize 74.9  2 75.6  1.8 76.8  2.8 76.9  1.7 76.1  2.4
1.34 1.19 1.83 1.13 1.58
Wet dog food 0.4  0.1 0.4  0.1 0.5  0 0.4  0 0.4  0.1
12.12 11.16 0.00 1.79 12.28
Regular maize starch 97.9  4.4 99.1  1.6 99.5  1.9 99.6  0.8 99.1  2.4
2.27 0.83 0.94 0.43 1.22

Total starch content of animal feeds and pet foods using the RTS method.
a)
All results are presented as total starch on a dry weight basis; b)On each day samples of each material were analyzed in duplicate; c)CV, coefficient of variation.

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(excluding samples with less than 1% starch) was extremely high
with less than 7% for repeatability and less than 5% for
intermediate precision (Table 3). This level of repeatability and
precision indicates that the modified AOAC Method 996.11
(using acetate buffer) is reliable and repeatable, and therefore
suited to the application of measuring total starch in food and
feed samples.
The current rapid total starch (RTS) procedure has been
applied to a range of starch samples, and grain, legumes, and
seed samples and compared to a procedure involving sodium
hydroxide extraction and the results are shown in Table 4
and 5. For grain and seed samples the values obtained by the
RTS method (see section, RTS method) and the NaOH-RTS
method (see section, RTS NaOH method) are very similar,
demonstrating that for most samples, starch gelatinization
and hydrolysis in buffer at elevated temperature is quantita-
tive. In contrast, the starch content of high amylose starches
and potato amylose is underestimated by the RTS method.
This underestimation of starch in these types of samples is
well known, thus for quantitative starch measurement, a
chaotrophic agent such as NaOH, KOH, or dimethyl
sulphoxide is routinely used to facilitate dissolution and
subsequent hydrolysis of the amylose in these samples. The
phosphate cross-linked wheat starch, Fibersym1 is resistant
to hydrolysis by α-amylase and AMG. The phosphate cross-
linking restricts hydration in boiling water and even in
solutions of NaOH or KOH, and thus restrict access by the
enzymes.
Starch contents of a range of breakfast cereals, pasta, and
bread are shown in Table S1, Supporting Information, of
various vegetables in Table S2, Supporting Information, and
of a range of liquid and wet food products are shown in
Table S3, Supporting Information. Samples were prepared
and extracted as described in section ‘Preparation of test
samples’. Starch contents of vegetable and fruit samples
varied from 0% to 67% (w/w) on a dry weight basis of the
various samples, and this is equivalent to 0–15.3% (w/w) on
an as is basis. Of the samples tested, celery had no starch
content, and rooster potatoes were the highest at 15.3% (w/w)
on an as is basis. The starch (including maltodextrins) content
of beer is as high as 25.4% (w/v) with cider as low as 1.2% (w/
v). Values for a range of other liquid and semi liquid samples
are given in Table S3, Supporting Information,. All values are
an average of duplicate determinations. A range of dairy
products were analyzed and shown to contain essentially no
starch content. These samples included Tesco goat cheese,
Philadelphia original soft cheese, The Laughing cow original
cheese, Tesco natural cottage cheese, Tesco Ricotta cheese,
Tesco Mascarpone cheese, Garlic Gold spread, and Stork
Baking and Cooking spread.

Table 4. A comparison of methods for the measurement of total starch in “pure” starch samples.

Starch contenta) RTS Starch contenta)

method % w/w NaOH-RTS method %
w/w
Starch type and lot number Moisture content % w/w as is dwbb) as is dwbb)

Corn Starch; High AMP Sigma S-9679 10.9 88.38 99.2 88.54 99.4
Potato Starch—Sigma S-4251 12.3 83.15 94.8 82.81 94.4
Rice Starch—Sigma S-7260 11.6 85.33 96.5 87.28 98.7
Wheat Starch (native)—Sigma S-512 12.9 87.05 99.9 88.78 101.9
Hi Maize 1043—Batch 02161 10.0 60.08 66.8 87.23 96.9
Corn Starch—Sigma S-4126 11.4 85.95 97.0 87.86 99.2
ACS Soluble Starch—Lot 21101 12.8 86.34 99.0 85.57 98.1
Novelose 240— Native High amylose 11.3 64.37 72.5 87.01 98.1
Hi Maize (HAMS)—Batch CO-343 11.3 74.46 83.94 85.87 96.80
Novelose 330—Native High Amylose 9.8 58.21 63.8 88.69 97.2
Hylon VII)—Lot 50904 10.7 73.45 82.25 86.51 96.9
Amylose (Potato)—Sigma A-9262 10.5 63.78 71.2 88.90 99.3
Fibersol  2  Lot 112181A (Matsutani) 7.5 8.52 9.21 9.06 9.79
Purified Actistar (Cerestar) 7.0 88.36 95.0 90.83 97.7
Regular Maize Starch—Lot 140801 13.7 85.97 99.6 87.17 101.0
Fiberite RW (pre-gelatinated phosphate crosslinked starch) 10.5 68.54 76.6 55.72 62.3
Fibersym RW (not pre-gelatinated phosphate crosslinked starch) 9.9 69.85 77.5 67.59 75.0
Green Banana—Lot 9.1 63.00 69.3 63.35 69.7
High amylose maize starch—Lot 60107 11.2 59.87 67.4 85.61 96.4
Chemically modified Starch—Lot: 21101b 11.6 83.33 94.3 84.30 95.4

a)
All values are the mean of duplicate analyses; b)Dry weight basis.

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Table 5. Starch contents of a range of grains, legumes, and seeds.

Starch contenta)RTS Starch contenta)

method % w/w RTS-NaOH method % w/w
Starch type and lot number Moisture content % w/w as is dwbb) as is dwbb)

Cannellini beans (natures gold) 12.07 34.2 38.9 34.9 39.7

Butter beans (natures gold) 11.22 31.7 35.6 33.2 37.3
Red kidney beans (natures gold) 12.02 31.3 35.6 32.4 36.8
Pinto beans (natures gold) 12.09 32.2 36.6 35.3 40.1
Haricot beans (natures gold) 13.40 33.4 38.6 34.5 39.8
Soya beans (natures gold) 8.70 0.9 1.0 0.7 0.8
Mung beans (natures gold) 11.80 38.9 44.1 40.8 46.3
Chick peas (natures gold) 10.27 39.7 44.2 41.9 46.7
Yellow split peas (natures gold) 12.36 44.7 51.0 46.6 53.2
Black eye beans (natures gold) 11.46 35.2 39.8 38.0 42.9
Lentils verts (natures gold) 10.64 33.0 36.9 33.3 37.2
Black baluga lentils (natures gold) 12.12 36.2 41.1 36.3 41.2
Quinoa (natures gold)c) 12.38 57.2 65.3 57.1 65.2
Spelt grain (natures gold) 12.67 59.8 68.5 59.2 67.8
Roasted buckwheat (natures gold) 4.94 63.9 71.7 70.6 74.3
c)
Long grain brown rice (natures gold) 11.75 72.3 81.9 64.8 73.4
Oat groats (natures gold) 10.82 61.6 69.1 60.2 67.5
Millet (natures gold) 11.28 63.9 71.1 61.5 69.3
Wheat grain (natures gold) 11.24 57.1 64.3 57.2 64.4
Brown linseed (flaxseed) 4.38 1.0 1.0 0.4 0.4
Regular maize starch (control) 13.7 87.0 100.8 87.9 101.9

A comparison of the RTS method with the RTS-NaOH method.

a)
All values are the mean of duplicate analyses; b)Dry weight basis; c) the lower value obtained for this sample with the RTS-NaOH procedure than for the RTS method is
thought to be due to grittiness of the sample, leading to incomplete solubilisation in NaOH.

The method described here, with the minor modifications, as 4. Conclusion

detailed above, has been applied successfully to all of the samples
In this paper, three methods for the measurement of starch in animal
we have evaluated, and is accurate and quantitative over the
feeds were evaluated and compared. One of these methods, the RTS
starch content range of 0.1–100% (w/w). To obtain accurate
method (see section RTS method) for rapid starch measurement, was
values for the starch content of samples containing high levels of
also applied to a much wider range of samples. For animal feeds,
fat e.g., chocolate or sausage meat), the sample should first be
similar starch values were obtained for all samples with each method.
extracted with solvent according to AOAC Method 991.42.
The RTS method gave accurate measurement of starch in all samples
except those that contain high amylose starches or chemically
modified starches. For such samples, treatment with a chaotropic
Table 6. A comparison of method performance statistics for AOAC agent is required to dissolve the amylose. In the RTS-NaOH
Method 996.11 and 2014.10 for the analysis of starch in food and feed (see section RTS NaOH method) method, NaOH is employed as the
samples. chaotropic agent. Accurate starch values were obtained for samples
containing as little as 0.1% (w/w) starch content by analyzing the
AOAC Number of Number of RSDr, RSDR, HorRat Outlier
method samples laboratories % % results, %
undiluted incubation solution. For samples with starch contents
greater than 10% (w/w), the incubation solution was diluted
996.11 16 (8  2) 32 2.1– 2.9– 2.0 2.7 appropriately in 100 mM sodium acetate buffer (pH 5.0); fivefold
3.9 5.7 for samples with 10–40% (w/w) starch content, or 11-fold for samples
2014.10 20 (10  2) 13 1.2– 3.9– 2.7 11.4 with 10–100% starch content. For samples with less than 1% (w/w)
5.0 11.2 starch, the sample size was increased to 500 mg.
Potential problems in the use of starch analytical methods have
RSDr, repeatability relative standard deviation; RSDR, reproducibility relative been highlighted by Hall.[13,14] One such problem is the possible
standard deviation; HorRat, Horwitz ratio, an indication of the precision of the
isomerization of the reducing-end glucose of malto-oligosacchar-
method. Outlier results (Cochran and Grubbs), the percentage of results received
that were not included in the statistical evaluation of the method. ides to fructose (and thus underestimation of starch) during the

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hydrolysis of starch by α-amylase at 100
C. This phenomenon has
been reported at temperatures greater than 100
C and at pH values
above 8, but it does not occur under the incubation conditions
employed in AOAC Method 996.11 (pH 7.0, 100
C, 6 min)
(Figure 5; Figure S3, Supporting Information). A second concern
highlighted by Hall[13,14] relates to the purity of the AMG used to
hydrolyze maltodextrins. As AMG hydrolyses α-linked D-glucose, it
is not unexpected that there might be some action of this enzyme
on sucrose (O-α-D-glucopyranosyl-(1 ! 2)-β-D-fructofuranoside.
The purified AMG used in this work, and that employed by
Hall[13,14] (i.e., Megazyme cat. no. E-AMGDF), has a slight, but
insignificant action on sucrose. As the same degree of hydrolysis of
sucrose was found for an ultra-high purity AMG prepared in this
laboratory, it can be concluded that sucrose hydrolysis is due to the
inherent action of the AMG, rather than to a separate
contaminating enzyme in the AMG preparation. In samples
containing up to 10% (w/w) sucrose, the released glucose
calculates as a “starch” content of approx. 0.15% (w/w). Analysis
of pure sucrose under the RTS-method for starch content gave a
“starch” content of 0.5%, a value well within the experimental
accuracy of the method. Lack of linearity of the glucose/GOPOD
standard curve was a further concern highlighted by Hall.[13,14] The
glucose/GOPOD standard curves typically obtained in our
laboratory are shown in Figure 2. The curves are dead linear
and dissect the origin. Accurate starch determinations can be
performed across the GOPOD range of 0.05–1.20 absorbance units
with a single glucose standard (100 μg/assay). As the value for the
glucose standard, in many ways, is the most important value in the
determinations, this analysis should be performed in quadrupli-
cate with a standard glucose solution of 1 mg mL1 (100 μg per
assay). If desired, a glucose standard curve can be performed with
each set of determinations, but simply is not necessary.
The method performance data for AOAC Methods 996.11 and
2014.10 are shown in Table 6. For the evaluation of method 996.11,
16 samples (eight blind duplicates) were analyzed by 32
laboratories. With AOAC method 2014.10, 20 samples (ten blind
duplicates) were analyzed by 13 laboratories. The ranges in the
relative standard deviations for repeatability (RSDr) and relative
standard deviations for reproducibility (RSDR) for both methods
were very similar as was the HorRat ratio. However, with AOAC
Method 996.11 the percentage outliers (Cochran and Grubbs) was
2.7%, while that for AOAC Method 2014.10 was much more
significant at 11.4%. This demonstrates that in the hands of a wide
range of users, AOAC Method 996.11 is considerably more robust
than AOAC Method 2014.10. Both of these methods as well as the
newly introduced modification of AOAC Method 996.11 (the RTS
method) give statistically similar analytical results over a wide
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range of samples. A major advantage of the RTS method is that it is
simple, rapid and versatile. Ten samples can be analyzed within
2 h, as compared to 6 h with AOAC Method 2014.10.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgments
Some initial experimental work associated with this study was performed
by Ms. Ruth Ivory, Senior Analyst, Megazyme.
Conflict of Interest
The authors of this paper are all employees of Megazyme.
Keywords
animal feed, cereal grains, dietary starch, foods, thermostable α-amylase,
total starch
Received: May 13, 2018
Revised: June 13, 2018
Published online: July 3, 2018
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