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J
OURNAL OF
AOAC I
NTERNATIONAL
V
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. 98, N
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. 2, 2015
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for application in the assay for dietary starch. The enzyme
preparation used must be validated within laboratory to
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the dietary starch assay and using a free glucose value of zero
in calculations. Analyses with candidate enzyme should give
values of [mean ± standard deviation (SD)] glucose: 90 ± 2%,
starch: 100±2%, and sucrose: 0.7 ± 0.3% on a dry matter basis.
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with the enzymatic portion of the dietary starch assay. Recovery
of these substrates should be less than 0.5% on a dry matter
basis (20). Use AOAC approved methods for determination
of dry matters of the samples. Enzyme preparations must not
contain appreciable concentrations of glucose (<0.5%), or
background absorbance readings will interfere with test sample
measurements.
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—Dilute concentrated
amyloglucosidase with 100 mM sodium acetate buffer,
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give 1 mL of solution per test portion with 2 to 5 mL excess.
Add 1/3 of needed buffer to an appropriately sized graduated
cylinder. Pipet concentrated amyloglucosidase into buffer,
rinsing tip by taking up and expelling buffer in the graduated
cylinder. Bring to desired volume with additional buffer. Cap
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The concentrated amyloglucosidase used should not contain
greater than 0.5% glucose, and should have a pH optimum of
4.0 and pH stability between 4.0–5.5 (example of concentrated
amyloglucosidase:
Product
E-AMGDF,
Megazyme
International Ireland, Ltd., Bray, Co. Wicklow, Ireland; origin:
$VSHUJLOOXV QLJHU
, or equivalent).
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the amount of enzyme required to release 1 μmole glucose/min
at pH 4.5 and 40°C; 21).
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1 μmole
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pH 4.5 and 40°C; 22).
The enzyme used must be validated within laboratory to
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.—Weigh 2.0 g benzoic
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2
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been made volumetric by weighing or transferring 1 L water
into the vessel and then etching the meniscus line for the known
volume.
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.—Prepare by dissolving 9.1 g
1D
2
HPO
4
(dibasic, anhydrous) and 5.0 g KH
2
PO
4
in ca 300 mL
H
2
2 LQ D / YROXPHWULF ÀDVN 8VH +
2
O to rinse chemicals into
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(ACS grade) and 0.15 g 4-aminoantipyrine. Use H
2
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Add glucose oxidase (7000 U) and peroxidase (7000 U), rinse
HQ]\PHV LQWR ÀDVN ZLWK +
2
O, and swirl gently to dissolve
without causing excessive foaming. Bring to 1 L volume with
H
2
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B
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s
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amber bottle at ca 4°C. Reagent life: 1 month. Before use in
test sample determinations, determine a standard curve for the
reagent using a 5-point standard curve using
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according to
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accurately determine glucose concentrations of glucose
standard solutions and give values equivalent to the values
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enzymatic hydrolysis portion of the dietary starch procedure and
using a free glucose value of zero in calculations. The glucose
values of the working standard solutions should be predicted
±6 μg glucose/mL. On a dry matter basis, the control sample
glucose should give a dietary starch value (mean ± SD) of 90 ±
2%, corn starch at 100 ± 2%, and sucrose 0.7 ± 0.3%.
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—0, 250, 500,
750, and 1000 μg/mL. Determine the dry matter of powdered
crystalline glucose (purity >99.5%) by an AOAC-approved
method. Weigh approximately 62.5, 125, 187.5, and 250 mg
portions of glucose and record weight to 0.0001 g. Rinse each
portion of glucose from weigh paper into a separate 250 mL
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swirl to dissolve. Bring each standard to 250 mL volume with
0.2% benzoic acid solution,
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glucose standard solutions. The 0.2% benzoic acid solution,
C
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d
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of glucose by dry matter percentage and percentage purity as
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divide by 250 mL to calculate actual glucose concentrations of
the solutions. Prepare solutions at least one day before use to
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solutions may be stored at room temperature for 6 months.
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.—Powdered crystalline
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starch sample, crude protein as nitrogen content × 6.25 and ash
should be determined to determine the nonprotein organic matter
content of the sample. For use in recovery calculations, actual
starch content of the corn starch control sample is estimated as
100%minus ash% and minus crude protein%, all on a dry matter
basis. Analyze 100 mg of each sample with each batch of test
samples. Glucose will allow evaluation of quantitative recovery,
and starch will allow evaluation of quantitative recovery and
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.—The method as described relies
on accurate volumetric additions in order to use the sum of
volumes to describe test solution volume. Accuracy of volume
additions can be evaluated before the assay by the following
procedure: Using 1–2 L distilled water at ambient temperature,
determine the g/mL density of the water by recording the weight
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