Total Starch Assay Procedure (Megazyme Amyloglucosidase/: Objective

Starch AACC International Method 76-13.
01
Page 1 of 6
Total Starch Assay Procedure

(Megazyme Amyloglucosidase/α-Amylase Method)
First approval November 8, 1995; Reapproval November 3, 1999
Objective
This method describes determination of total starch in cereal products using a
kit sold by Megazyme International Ireland Ltd. (Bray, Ireland). For samples
that do not contain high levels of resistant starch (e.g., wheat flour), complete
solubilization of starch is achieved by cooking the samples in the presence of
thermostable α-amylase followed by amyloglucosidase hydrolysis to glucose.
Samples that contain high levels of resistant starch (e.g., high-amylose maize
starch) are completely solubilized by pretreatment with dimethyl sulfoxide at
100°C, followed by thermostable α-amylase treatment. The maltodextrins are
hydrolyzed to glucose with glucoamylase, and the glucose produced is
measured using glucose oxidase/peroxidase reagent. Samples containing high
levels of glucose or maltodextrins are washed with aqueous ethanol before
analysis. Analysis of a single sample can be completed within 70 min. Twenty
samples can be completed within 2 hr.
Apparatus
1. Laboratory mill capable of reducing sample to pass 0.6-mm screen.
2. Glass test tubes, round-bottomed, 16 × 120 mm or 18 × 150 mm.
3. Micropipetters, 100-µl,.
4. Positive displacement pipetter
a. with 50-ml tip (to dispense 3-ml aliquots of bacterial α-amylase
solution)
b. with 5.0-ml tip (to dispense 0.1-ml aliquots of amyloglucosidase
solution).
5. Bench centrifuge, required speed 3000 rpm.
6. Filter paper, Whatman no. 1, or equivalent.
7. Analytical balance.
8. Spectrophotometer set at 510 nm.
9. Vortex mixer.
10. Thermostatted water bath set at 50.0º.
11. Boiling water bath with tube rack.
12. Stop clock.
Reagents
Supplied with kit
1. Thermostable α-amylase, 10 ml, 3000 U/ml; stabilized solution. Dilute
aliquot (1.0 ml) to 30 ml with 50 mM MOPS buffer, pH 7.0 (reagent 7). Store
frozen between uses.
http://dx.doi.org/10.1094/AACCIntMethod-76-13.01
Starch AACC International Method 76-13.01
Page 2 of 6
Total Starch Assay Procedure (Megazyme

Amyloglucosidase/α-Amylase Method) (continued)
2. Amyloglucosidase, 10 ml, 200 U/ml; stabilized solution. Use directly with-

out dilution. Dispense with positive displacement dispenser. Store concentrate at
4ºC.
3. Glucose determination reagent (GOPOD), for 1 liter. Reagent concentra-
tions after dissolution in buffer:
a. Glucose oxidase, >2000 U/liter
b. Peroxidase, >650 U/liter
c. 4-Aminoantipyrine, 0.4 mM
See Note 1.
4. Glucose reagent buffer (concentrate), 50 ml. Dilute entire contents to 1 liter
with distilled water and use to dissolve GOPOD reagent. Divide GOPOD
reagent into aliquots of desired volume for storage. See Note 2.
5. Glucose standard solution (100 µg/0.1 ml in 0.2% benzoic acid).
6. Regular maize starch (starch content ~98% dry weight).
Not supplied with kit

7. 3-[N-morpholino]propanesulfonic acid (MOPS) buffer (50 mM, pH 7.0)
plus calcium chloride (5 mM) and sodium azide (0.02%).
a. Add 11.55 g MOPS to 900 ml distilled water. Adjust solution to pH 7.0
by addition of 1M (10%) HCl. Approximately 17 ml is required.
b. Add 0.74 g calcium chloride dihydrate and 0.2 g sodium azide and dis-
solve. Adjust volume to 1 liter, and store at room temperature. Caution:
Do not add sodium azide until pH is adjusted. Acidification of sodium
azide releases a poisonous gas. See Note 3.
8. Sodium acetate buffer, 200 mM (pH 4.5) plus sodium azide, 0.02%.
a. Add 11.8 ml glacial acetic acid (1.05 g/ml) to 900 ml of distilled water.
Adjust solution to pH 4.5 by addition of 4 g/100 ml 1M sodium hydrox-
ide solution. Approximately 60 ml is required.
b. Add 0.2 g sodium azide and adjust volume to 1 liter. Store at room tem-
perature. See Note 3.
9. Dimethyl sulfoxide (DMSO), laboratory grade. See Note 4.
10. Ethanol, aqueous, 80% (v/v).
Procedure
Preparation of standards and blanks
1. With each set of determinations, reagent blanks and glucose standards (100
μg quadruplicate) should be included.
a. Reagent blank consists of 0.1 ml distilled water + 3.0 ml GOPOD
reagent (reagent 3).
Page 3 of 6

b. Glucose standard consists of 0.1 ml glucose standard (reagent 5, 100

μg/0.1 ml) + 3.0 ml GOPOD reagent.
2. With each set of determinations, a standard flour or starch sample should
be included.
3. Sample blanks can be determined using the standard assay method with the
modifications that in step 4, 3 ml of distilled water is used and in step 5, amylo-
glucosidase is replaced by water. Alternatively, the need to perform sample
blank analysis can be avoided by preextraction of samples with aqueous ethanol
(see below).
Pretreatment of samples containing glucose and maltosaccharides

1. Add sample (∼100 mg; weighed accurately) to glass centrifuge tube (16 ×
120 mm; 17 ml capacity). Weigh and record weight of tube plus sample.
2. Add 5.0 ml of aqueous ethanol (80% v/v), and incubate tube at 80–85°C
for 5 min. Mix contents on vortex stirrer and add another 5 ml of 80% aqueous
ethanol.
3. Centrifuge the tube for 10 min at 1000 × g (about 3,000 rpm) on bench
centrifuge. Discard supernatant.
4. Resuspend pellet in 10 ml 80% aqueous ethanol, and stir on vortex mixer.
Centrifuge as above, and carefully pour off supernatant.
5. Proceed from step 4 of standard assay method, or from step 4 of modified
assay method if DMSO treatment is required.
Standard assay method (Method A)

1. Mill cereal product to pass 0.5-mm screen.
2. Add sample (∼100 mg; weighed accurately) to glass tube (16 × 120 mm).
Tap tube to ensure that all sample falls to bottom of tube. Weigh and record
weight of tube plus sample.
3. Wet with 0.2 ml aqueous ethanol (80% v/v) to aid dispersion, and stir tube
on vortex mixer.
4. Immediately add 3 ml thermostable α-amylase (300 U) in 50 mM MOPS
buffer (pH 7.0) (reagent 1), and vigorously stir on vortex mixer. Incubate tube
in boiling water bath for 5 min.
5. Place tube in bath at 50°; add 4 ml acetate buffer (200 mM, pH 4.5)
(reagent 8), followed by 0.1 ml amyloglucosidase (20 U) (reagent 2). Stir tube
on vortex mixer and incubate at 50° for 30 min.
6. Remove tube from water bath and dry with tissue.
7. Weigh tube and contents and adjust total weight to 10.0 g plus weight
recorded in step 2 (i.e., weight of tube plus sample). (Note that 1.0 ml of water
Page 4 of 6

TABLE I
Starch Determination: Reproducibility of Assay Methods A and B
Moisture Starch Content, %

Content (dry weight)
Sample (%) Method A Method B
Waxy maize starch 13.6 99.1 ± 1.8 99.3 ± 2.0
High-amylose maize starch 11.9 84.4 ± 2.1 94.1 ± 1.8
Wheat flour 12.0 76.1 ± 2.2 75.8 ± 1.9
weighs 1.0 g and contribution to weight from buffer salts is negligible.) See
Notes 5 and 6.
8. Centrifuge the tube at 3000 rpm for 10 min, or filter contents through filter
paper.
9. Remove aliquots (1.0 ml) of supernatant and add to 9.0 ml of distilled
water (i.e., 10-fold dilution); mix thoroughly.
10. Transfer aliquots (0.1 ml) of diluted solution to bottom of glass test tubes
(16 × 100 mm) in duplicate.
11. Add 3.0 ml of GOPOD reagent (reagent 3) to each tube (including
glucose standards and reagent blanks), and incubate tubes at 50° for 20 min. See
Note 7.
12. Read absorbance at 510 nm for each sample, and read glucose standards
against reagent blank.
13. See Table I for information on reproducibility.
Modified assay method (Method B, for samples containing resistant starch)

1. Mill cereal product to pass 0.5-mm screen.
2. Add sample (∼100 mg, weighed accurately) to glass tube (16 × 120 mm).
Weigh and record weight of tube plus sample.
3. Wet with 0.2 ml aqueous ethanol (80% v/v) to aid dispersion, and stir tube
on vortex mixer.
4. Immediately add 2 ml DMSO and stir tube on vortex mixer. Place tube in
vigorously boiling water bath, and remove after 5 min.
5. Proceed according to standard assay method from step 4.
6. See Table I for information on reproducibility.
Calculations
1 100 162
Starch, % = ΔE × F × 100 × 10 × × ×
1,000 W 180
F
= ΔE × × 90
W
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where: ΔE = absorbance (reaction) read against the reagent blank, F = 100 (μg
of glucose) divided by absorbance for 100 μg of glucose (conversion from
absorbance to μg), 100 = volume correction (0.1 ml taken from 10 ml), 10 =
dilution factor, 1/1000 = conversion from μg to mg, 100/W = factor to express
“starch” as a percentage of flour weight, W = weight in mg (“as is” basis) of
flour analyzed, and 162/180 = adjustment from free glucose to anhydro glu-
cose (as occurs in starch).
Conversion to dry weight basis

100
Starch, % (dry weight basis) = starch, % (as - is) ×
100 − moisture content, %
Notes
1. With each new batch of GOPOD reagent, check the time for maximum
color formation with 100 μg of glucose standard. This is usually about 15 min.
2. The stability of the GOPOD reagent is 2–3 months at 4º and 12 months at
–20º.
3. Sodium azide is a toxic chemical and should be treated accordingly. It is
added to buffers solely as a preservative. It can be deleted from buffer recipes,
but buffers should then be stored at 4°.
4. DMSO is a skin irritant and thus should be used with caution.
5. Since 2 ml of DMSO weighs 2.2 g, when using the modified method, the
sample weight should be adjusted to 10.2 g (i.e., 10.0 ml).
6. Alternatively, at step 6, transfer the entire contents of the test tube to a 100-
ml volumetric flask and adjust to volume with distilled water. Mix thoroughly.
Centrifuge (3000 rpm, 10 min) an aliquot of this solution, and proceed from step
10 of the method.
7. The time of incubation with GOPOD reagent is not critical but should be at
least 20 min. The color formed should be measured within 60 min.
8. This approach gives relatively large errors for samples containing low lev-
els of resistant starch because it involves subtraction of one high absorbance
value from another.
References
1. Anonymous 1987. Measurement of the starch content of commercial starches. Starch/Staerke
39:414-416.
2. Batey, I. L. 1982. Starch analysis using thermostable alpha-amylase. Starch/Staerke 34:125-128.
3. Englyst, H. N., and Cummings, J. H. 1988. Improved method for measurement of dietary fiber as
non-starch polysaccharides in plant foods. J. Assoc. Off. Anal. Chem. 71:808-814.
4. Karkalis, J. 1985. An improved enzymic method for the determination of native and modified
starch. J. Sci. Food Agric. 36:1019-1027.
Page 6 of 6

5. McCleary, B. V., Gibson, T. S., and Mugford, D. C. 1997. Measurement of total starch in cereal
products by amyloglucosidase/alpha-amylase method: Collaborative study. J. AOAC Int. 80:571-
579.
6. McCleary, B. V., Gibson, T. S., and Mugford, D. C. 1997. Collaborative evaluation of a simplified
assay for total starch in cereal products (AACC Method 76-13). Cereal Foods World 42:476-480.
7. Theander, O., and Åman, P. 1979. Studies on dietary fibers. I. Analysis and chemical characteri-
zation of water-soluble and water-insoluble dietary fibers. Swed. J. Agric. Res. 9:97-106.

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Starch AACC International Method 76-13.

Total Starch Assay Procedure

Total Starch Assay Procedure (Megazyme

2. Amyloglucosidase, 10 ml, 200 U/ml; stabilized solution. Use directly with-

Not supplied with kit

Total Starch Assay Procedure (Megazyme

b. Glucose standard consists of 0.1 ml glucose standard (reagent 5, 100

Pretreatment of samples containing glucose and maltosaccharides

Standard assay method (Method A)

Total Starch Assay Procedure (Megazyme

Moisture Starch Content, %

Modified assay method (Method B, for samples containing resistant starch)

Total Starch Assay Procedure (Megazyme

Conversion to dry weight basis

Total Starch Assay Procedure (Megazyme

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