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Rice AACC International Method 61-03.01

Page 1 of 4

Amylose Content of Milled Rice

First approval October 15, 1997; Reapproval November 3, 1999

Objective
To determine the apparent amylose content of milled rice, an indicator of
cooking and processing qualities, by colorimetric determination at 620 nm of
the greenish-blue starch-iodine complex developed at pH 4.5–4.8 in acetate
buffer. See Notes 1 and 2. This rapid screening method is applicable to all well-
milled raw, parboiled, and precooked rices. Corrections for differences in
moisture content are needed for precooked rices, rice noodles, etc. Brown rice
must be milled or defatted.
Apparatus
1. Grinder, cyclone type or Wig-L-Bug amalgamator, capable of reducing
milled rice to a flour that passes through a 0.5-mm screen.
2. Colorimeter or spectrometer with matching cells capable of measuring
absorbance at 620 nm.
3. If defatting samples, extraction apparatus such as Soxhlet or Goldfisch.
4. 100-ml volumetric flasks, pipettes, etc.
Reagents
1. Amylose, potato; about 16% iodine affinity, dry basis. Equilibrate to labor-
atory environment. See Note 3.
2. Amylopectin. Prepare defatted waxy rice flour, 1–2% amylose.
Commercial amylopectin has at least 5% amylose. Equilibrate to laboratory
environment. See Note 3.
3. Methanol, 85% (toxic).
4. Ethanol, 95%.
5. NaOH, 1.0N.
6. NaOH, 0.09N.
7. Acetic acid, 1.0N.
8. Iodine solution, 0.2% I2 and 2.0% KI in distilled water.
Procedure
Preparation of rice samples
1. In addition to test samples, prepare a set of check samples of several rices
that represent all amylose levels to be encountered in test samples. Check sam-
ples should be prepared as both defatted and undefatted, if test samples are not
defatted. Grind at least 20 grains in cyclone mill with a 0.5-mm screen or grind
10 grains in a small ball mill such as a dental amalgamator (e.g., Wig-L-Bug
Amalgamator) for 40 sec. Ensure that the ball-milled sample is reduced to a fine,
uniform powder. Allow 2 days for moisture content equilibration. See Note 3.
2. Optional. See Note 4. Defat flour by refluxing with 85% methanol (toxic) or
95% ethanol for 16 hr in a Soxhlet extractor or for 4 hr in a Goldfisch extractor

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Rice AACC International Method 61-03.01
Page 2 of 4

Amylose Content of Milled Rice (continued)

at the rate of 6 drops/sec. Spread defatted sample in a thin layer. Allow 2 days to
evaporate residual alcohol and to equilibrate moisture content.

Dispersion of rice samples

3. Weigh duplicate 100 mg of rice flour (undefatted or defatted) and transfer
quantitatively to 100-ml volumetric flasks. See Note 5.
4. Add 1 ml of 95% ethanol, carefully washing down any sample adhering to
side of tube. This wets the sample. For best dispersion, vortex tubes thoroughly.
5. Add 9 ml 1N NaOH to each sample. To disperse starch, keep samples at
room temperature for 15–24 hr without shaking. Alternatively, let tubes sit at
room temperature for 10 min; then heat 10 min in a boiling water bath and cool
to room temperature. After heating in the water bath, allow samples to stand at
room temperature for at least 2 hr (can be overnight) before continuing with the
procedure.
6. After dispersion of starch, make solutions to 100-ml volume with distilled
water and vortex vigorously.

Solutions for standard curve

7. Prepare solutions (1 g/liter) of potato amylose and of waxy rice flour
(amylopectin) by the same procedure as in steps 3–6 above. Note that more
waxy rice flour solution is required. Use as stock mixtures for working
solutions. See Note 6.
8. Determine quantities needed and prepare working solutions of
amylose/amylopectin mixtures for the standard curve as in the following table.

Volume ratio of stock solutions

(ml/100 ml)
Amylose in
Milled Rice Amylose (1 Amylopectin 0.09N
(% dry basis) mg/ml) (1 mg/ml) NaOH
0 0 7.0 3
10 1.0 6.0 3
20 2.0 5.0 3
25 2.5 4.5 3
30 3.0 4.0 3

Iodine color measurement for standard curve

9. Pipette 5 ml of amylose or amylopectin solutions and check sample solu-
tions into 100-ml volumetric flasks, each containing about 50 ml distilled water.
For a blank, prepare a flask using 5.0 ml 0.09N NaOH.
10. Add 1.0 ml 1N acetic acid and mix.
11. Add 2 ml iodine solution.
12. Make up to 100 ml volume with distilled water, mix, and let stand 20 min.

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Rice AACC International Method 61-03.01
Page 3 of 4

Amylose Content of Milled Rice (continued)

13. Read color absorbance at 620 nm, using the blank to zero the
spectrometer.
14. Plot absorbance at 620 nm against amylose concentration of working solu-
tions for a standard curve. Using absorbance values obtained on the defatted
check samples, read their apparent amylose content to the nearest first decimal
place from the standard curve. Values obtained for the defatted checks can then
be assigned to the undefatted check samples. See Notes 4c and 5.

Iodine color measurement for test samples

15. Pipette 5-ml aliquots of alkaline dispersions of undefatted test and check
samples in 100-ml volumetric flasks, each containing about 50 ml of distilled
water. For blank, use 5.0 ml 0.09N NaOH.
16. Repeat steps 10–13 as for the standard curve.
17. Plot absorbance at 620 nm of undefatted checks against assigned amylose
content. Read amylose values of the test samples to the nearest first decimal
place from the standard curve obtained with the undefatted check-samples.

Notes
1. The green color is due to excess iodine, stable at acidic pH. The term
“apparent amylose” content was proposed for colorimetric iodine-blue color
first determined by Williams et al (Ref. 10) and simplified by the use of acetate
buffer (Ref. 3). The higher iodine-complexing of high-amylose rice starch may
be due to iodine complexing long-chain branches of amylopectin, rather than to
more amylose. See Ref. 9. Such amylopectin with high iodine affinity likely
contributes to cooked rice hardness, as does amylose.
2. Measured apparent amylose content will vary depending upon the
amylose and amylopectin sources used as standards. The potato amylose
standard is only about 80% pure (16% iodine affinity) See Ref. 1. Amylose
standards are now available nearly free of amylopectin. Using an amylose-
amylopectin standard and assuming 90% starch in milled rice (dry weight
basis) have resulted in values as low as 24% for some “high-amylose” (>25%)
samples, due to overcorrection for the amylopectin interference. Reduction of
starch content of the standard mixture to 70% from 90% by reduction of
amylopectin produced values similar to those obtained by the method of
Williams et al (Ref 10). To obtain more accurate results in research studies,
use test samples and check rices defatted with refluxing 95% ethanol rather
than undefatted rices.
3. Because all materials (amylose, waxy flour, and test and check samples) are
conditioned in the same environment to the same equilibrium moisture content,
no correction for moisture content is necessary. Results are usually expressed on
a milled-rice dry weight basis rather than on a starch basis.

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Rice AACC International Method 61-03.01
Page 4 of 4

Amylose Content of Milled Rice (continued)

4. a. If samples are not defatted, rice should be well milled (10% by weight
removal of bran and polish), and degree of milling must be kept constant to
minimize lipid interference with amylose determination.
b. If rice flour samples are not defatted, amylose values will be low due to
interaction of native starch lipids with some of the amylose. Alternative correc-
tions include increasing the amylose content obtained on an undefatted sample
by 2% or by the difference between a similar determination on a standard rice
flour sample and its known amylose content, which has been previously deter-
mined on its defatted starch (see Ref. 6).
c. Defatting of well-milled rice increases apparent amylose content by
approximately 2%. Undefatted milled rice run with an amylose/defatted waxy rice
flour standard curve may be corrected by adding 2% to amylose values. Methanol
(85%) is a more effective defatting agent than 95% ethanol, but it is toxic.
5. Samples can be run in other quantities, such as 60 mg in 60 ml of liquid or 45
mg in 45 ml. For large numbers of samples, this facilitates the dispersion process.
Preferably, the amylose/waxy rice flour standard and defatted milled rice
check standards are run together to determine the values for the milled rice
check samples. The undefatted milled rice counterparts of these same check
samples are then used for the standard curve for undefatted milled rice samples
for routine analyses, provided the degree of milling is comparable.
6. Only 90 mg of amylose or waxy rice starch is used in the stock solutions
based on the mean starch content of milled rice of 90% dry basis. Amylose
content (% dry basis) is usually expressed on a milled-rice basis rather than on a
starch basis.

References
1. IRRI. 1988. International Rice Research Institute Annual Report for 1987. International Rice
Research Institute, Manila, Philippines, p. 43.
2. ISO. 1987. Rice-Determination of Amylose Content, 1st ed. ISO 6647. International Organiza-
tion for Standardization. Geneva, Switzerland.
3. Juliano, B. O. 1971. A simplified assay for milled-rice amylose. Cereal Sci. Today 16:334.
4. Juliano, B. O. 1979. Amylose content in rice—A review. Chemical aspects of rice grain quality.
Proc. workshop. International Rice Research Institute, Los Banos, Philippines.
5. Juliano, B. O. 1985. Criteria and tests for rice grain qualities. Page 443 in: Rice: Chemistry and
Technology. B. O. Juliano, ed. Am. Assoc. Cereal Chem., St. Paul, MN.
6. Juliano, B. O., Perez, C. M., Blakeney, A. B., Castillo, T., Kongseree, N., Laignelet, B., Lapis, E.
T., Murty, V. V. S., Paule, C. M., and Webb, B. D. 1981. International cooperative testing on the
amylose content of milled rice. Starch/Starke 33:157.
7. Paule, C. M., Gomez, K. A., Juliano, B. O., and Coffman, W. R. 1979. Variability in amylose
content of rice. Riso 28:15.
8. Perez, C. M., and Juliano, B. O. 1978. Modification of the simplified amylose test for milled
rice. Starch/Starke 30:424.
9. Takeda, Y., Hizukuri, S., and Juliano, B. O. 1987. Structures of rice amylopectin with low and
high affinities for iodine. Carbohydr. Res. 168:79.
10. Williams, V. R., Wu, W. T., Tsai, H. Y., and Bates, H. G. 1958. Varietal differences in amylose
content of rice starch. J. Agric. Food Chem. 8:47.

Amylose content calculator for samples measured by Method 61-03

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