NOTE e -Xtra*

Colorimetric Method for Rapidly Predicting Rice Amylose Content

Amogh A. Ambardekar,1 Terry J. Siebenmorgen,1,2 and Tanya Pereira1

Cereal Chem. 88(6):560–563

Amylose content (AC) of head rice (milled kernels that are
three-fourths of their original length) is considered an important
predictor of cooking and processing performance (Juliano and
Hicks 1996). Several methods have been studied to standardize
AC measurement of rice flour, including iodometric (Juliano et
al 1968; Juliano 1971; Sowbhagya and Bhattacharya 1971), am-
perometric (Williams et al 1970), near-infrared reflectance (NIR)
spectroscopy (Delwiche et al 1995, 1996), chromatographic
(Grant et al 2002), and thermal methods (Moorthy et al 2006;
Stawski 2008).
The most popular method to quantify AC is the iodometric
method, which is based on the blue helical inclusion complex
formed because of the affinity of iodine with amylose chains
(Williams et al 1958). Juliano (1971) modified the original iodo-
metric method developed by Williams et al (1958); rice starch
was defatted in order to avoid interference by lipid-amylose com-
plexes and developed standard curves for AC with potato amy-
lose. Absorbance was measured at 620 nm. Fitzgerald et al (2009)
further modified the AC method by measuring the absorbance at
720 nm under the assumption that most of the light at that wave-
length was absorbed by amylose molecules. A standard curve
was prepared with rice flours of predetermined AC. In an attempt
to optimize the colorimetric test, Williams et al (1970) compared
it with an amperometric test that measured electric current pro-
duced by the reaction of potassium iodide with amylose from
pure rice starch and rice flours that had been dissolved in a
potassium hydroxide solution to determine the equivalence point
of iodine. Although the results of this method showed a high
degree of accuracy and precision, the procedure was cumber-
some and costly to perform.
Whereas iodometric and amperometric methods have been
shown to be time consuming, NIR and thermal measurements are
considered rapid and reasonably accurate. However, these meth-
ods require expensive equipment that may not be readily available
in many processing facilities.
The majority of the studies discussed previously used wet-chem-
istry methods and involved lengthy sample-preparation techniques,
including grinding rice kernels to flour, starch isolation, and defat-
ting of starch for determination of AC. However, a recent study by
Avaro et al (2009) described a rapid classification procedure with a
color chart for estimating amylose levels in milled rice-flour sam-
ples after staining. Seven cultivars, ranging from 0 to 34% amylose,
were grouped into five categories of increasing AC. An amylose-
iodine solution was prepared according to the method of Juliano
(1971) and applied to each cultivar within the respective groups.
The resulting color formation was from the reaction of iodine with
the amylose of the rice flours. The amylose-containing solutions
were described as reddish yellow, grayish olive, grayish green,
grayish greenish blue, and grayish blue, corresponding to very low,
low, intermediate, high, and very high AC levels, respectively.
Color strips were developed to match the color of each solution.
Although rapid and cost effective, prediction was limited to a broad
range of AC among cultivars, from very low to very high. The
method also failed to quantify color formation of the amylose-
iodine complex through a standard color scale. Tuano et al (2009)
stained cultivars containing low, intermediate, and high AC with
iodine solution and differentiated the milled-rice color stains by
assigning scores on a scale of 1–4, 1 being low and 4 being high
AC. The color scoring designed by this method is subjective and
covers a broad range of AC within each score.
The goal of this study was to develop a rapid colorimetric
method to objectively predict AC in head rice based on instrumental
color measurement of the amylose-iodine complex in rice cultivars.
A Commission Internationale de l’Eclairage (CIE) L* and b* color
scale was used to correlate L* and b* values with the concentra-
tions of amylose in waxy, short-grain, medium-grain, and long-
grain rice, as measured with the iodometric method. The purpose of
developing this method is to provide quick estimates of AC in head
rice for rice producers, processors, and breeders through cost-effec-
tive methods that require minimal resources.
MATERIALS AND METHODS
Standard Curve
A standard curve for AC was generated with ground rice flour
samples of predetermined AC ranging from 0 to 25% (Fig. 1)

* The e-Xtra logo stands for “electronic extra” and indicates that Figure 2 appears
in color online.
1 Post-doctoralassociate, professor, and graduate assistant, respectively. Depart-
ment of Food Science, University of Arkansas, Fayetteville, AR 72704.
2 Corresponding author. Phone: 479-575-2841. Fax: 479-575-6936. E-mail:

tsiebenm@uark.edu
Fig. 1. Standard curve for amylose content based on rice flours of known
values (International Rice Research Institute). Each data point represents
http://dx.doi.org/10.1094 / CCHEM-04-11-0052 a mean absorbance and standard deviation resulting from the replication
© 2011 AACC International, Inc. of four absorbance values for each standard.

560 CEREAL CHEMISTRY

obtained from the International Network for Quality Rice labora- cultivars were washed with deionized water and stained with 27
tory at the University of Arkansas Department of Food Science, mL of iodine solution (0.5 g of iodine and 2.5 g of potassium
which previously procured the samples from the International Rice iodide in 1 L of deionized water) for durations of 1, 2, 5, 10, 15,
Research Institute (IRRI), Los Baños, Philippines. These samples, and 20 min. Note: it is important that, prior to color measurement,
henceforth referred to as standards, were refrigerated upon arrival residual staining solution needs to be removed completely from
and equilibrated at 25°C for 2 hr before standard-curve preparation. the stained rice sample to avoid variation in color quantification
AC determination was performed according to the modified method resulting from the staining solution. Removal was done by plac-
of Juliano (1971). Duplicate 100-mg (± 0.5 mg) samples of each ing the stained head rice in a strainer with a 2-mm pore size and
standard were steeped for 12 hr in 9 mL of 1N NaOH solution to manually pressing the rice with a spatula to remove the staining
allow for starch-granule breakdown and release of amylose. Each solution completely.
solution was brought to a final volume of 100 mL with deionized Color of the stained head rice was measured according to the
water and mixed for 1 min with a vortex mixer. A 0.5-mL aliquot of CIE L* and b* color scales with a colorimeter (Colorflex, Hunter
amylose solution was pipetted into a test tube and mixed with 0.1 Association Laboratory, Reston, VA). The colorimeter was cali-
mL of 1N acetic acid and 0.2 mL of iodine solution (0.2 g of iodine brated daily with black and white tiles supplied by the manufac-
and 2.0 g of potassium iodide in 100 mL of aqueous solution) and turer. Immediately after the iodine solution was drained, stained
then adjusted to a final volume of 10 mL with deionized water. The head rice samples were placed in a clear plastic sample cup and
solution was mixed for 1 min with a vortex mixer and allowed to covered with a black plastic cup prior to color measurement in
stand for 20 min. The absorbance of the solution was measured in order to avoid interference from ambient light. After the first color
duplicate for each sample with a spectrophotometer (DU 520, reading was taken, the sample was rotated approximately 180°
Beckman, Fullerton, CA) at 720 nm and plotted against the known and a second reading was performed. The procedure was repeated
percentage of AC. The procedure was repeated for each of the stan- four times to yield eight readings. The average of eight readings
dards, and absorbance was plotted against AC. was reported for each of the 9-g head rice subsamples of a culti-
var. The L* and b* values, representing kernel lightness and blue
Test Sample Analysis intensity, respectively, were measured within 1 min of straining.
One waxy, three short-grain, 10 medium-grain, and 10 long- For both samples, L* values and b* values decreased up to 10
grain rice samples (Table I) were obtained from the University of min, after which no further differences were observed. Thus, a 10-
Arkansas Rice Research and Extension Center near Stuttgart and min staining duration was used for the primary study.
the Rice Experiment Station of the California Co-operative Rice Refrigerated head rice samples were equilibrated for 4 hr at
Research Foundation during 2009 and 2010. Rough rice samples 25°C before staining. Iodine staining and color measurements
were cleaned with a dockage tester (Carter-Day, Seedburo, IL). were performed on all ten 9-g head rice subsamples of each of the
Cleaned samples were conditioned with a temperature and rela- four milling replicates of a cultivar, as described in the staining-
tive humidity controller (AA5582, Parameter Generation and duration optimization procedure.
Control, Black Mountain, NC) at 26°C and 53% rh to achieve an
equilibrium moisture content of 12.5% as predicted by the Statistics
Chung-Pfost equation (ASABE 2009). Moisture content was veri- The L* and b* values of each test sample were plotted
fied by drying 15-g samples in an oven at 130°C for 24 hr. against the sample’s measured AC. The prediction models for
Following conditioning, four 150-g replicate samples of rough
rice from each cultivar were dehulled in a laboratory sheller TABLE I
(THU, Satake, Tokyo) with a clearance of 0.048 cm (0.019 in.) Cultivars, Harvest Seasons, and Percent Amylose Content
between the rollers. The resultant brown rice samples were milled (mean ± SD, n = 4) of Rice Used in the Study
for 45 sec with a laboratory mill (McGill No. 2, RAPSCO, Brook- Cultivar Harvest Season Amylose Contenta (%)
shire, TX) that had a 1.5-kg weight on the lever arm, situated 15
cm from the milling chamber. Head rice was then separated from Waxy cultivar
CM-101 2010 0.1 ± 0.0
broken kernels with a sizing device (Seedburo Equipment, Chi- Short-grain cultivars
cago, IL). The surface lipids of head rice were stripped off by CA-201-1b 2010 5.0 ± 0.1
swirling the rice in a 70% ethanol solution for 3 min and further CA-201-2b 2010 6.4 ± 0.5
drying the rice. Koshihikari 2010 8.2 ± 0.6
Furthermore, head rice samples of each of the four milling Medium-grain cultivars
replicates were divided into 11 subsamples (ten 9-g subsample Jupiter-1b 2009 10.1 ± 0.2
STG07M 2009 11.4 ± 0.3
sets for color measurements and one 5-g subsample set for a wet- STG05AC 2009 12.2 ± 0.3
chemistry test for AC determination). A 5-g subsample of head Arborio 2010 12.9 ± 0.4
rice from each milled sample was ground into flour with a cy- Neptune 2009 12.7 ± 0.2
clone mill with a 0.5-mm sieve (Udy, Fort Collins, CO). Ground Jupiter-2b 2009 13.1 ± 0.2
flour samples and remaining head rice were stored separately in M207 2009 13.1 ± 0.2
plastic ziplock bags at 4°C. Each flour sample was equilibrated at M204 2009 14.7 ± 0.4
Medark 2009 15.3 ± 0.4
25°C for 2 hr prior to AC analysis. AC of duplicate 100-mg ali- Bengal 2009 15.7 ± 0.8
quots of flour samples was determined according to the method Long-grain cultivars
described for the standards. The absorbance of the solution was L-206 2010 18.0 ± 0.2
measured in duplicate for each aliquot with the spectrophotometer XL756 2010 18.7 ± 0.3
at 720 nm. AC of each sample was estimated by the equation gen- CLXL729 2010 20.2 ± 0.3
erated from the standard curve. XP752 2010 20.4 ± 0.2
XL745 2010 20.4 ± 0.2
XP751 2010 22.1 ± 0.0
Color Measurements Wells 2010 22.7 ± 0.3
Head rice samples allocated for color tests were soaked in L-205 2010 23.3 ± 0.6
iodine solution for a sufficient duration to induce a stable form of CL181 2010 23.3 ± 0.3
the blue helical inclusion complex between amylose and iodine. Cheniere 2010 26.1 ± 0.4
In a preliminary study to establish this duration for staining head a Determined using the modified method of Juliano (1971).
rice, 9-g samples of medium-grain Jupiter and long-grain Wells b Cultivars followed by -1 and -2 were procured from different locations.

Vol. 88, No. 6, 2011 561

Fig. 2. Color intensity of iodine-stained head rice samples having the indicated percent amylose content (AC).
Fig. 3. Relationship of L* and b* values with the amylose content of test
cultivars representing lightness and blue color indices, respectively. Slopes
of regression models (L* and b* versus amylose content) are averages
of 40 subsamples (n = 40).
AC that employ L* and b* values were developed through lin-
ear regression (JMP, version 8.0, SAS Institute, Cary, NC). The
repeatability of color measurements of each milling replicate
and precision of each measurement were evaluated by compar-
ing the slopes of regression lines of milling replicates and
measurement repetitions with analysis of covariance at a sig-
nificance level of α = 0.05.
562 CEREAL CHEMISTRY
RESULTS AND DISCUSSION
Figure 1 illustrates the linear relationship between absorb-
ance and known amylose values of the standard flours. AC
values estimated from this equation for each of the cultivars
tested in this study are shown in Table I. The AC of waxy rice
was measured as 0.09%. The AC of short-, medium-, and long-
grain cultivars were 5.0–8.2, 10.1–15.7, and 18.0–26.1%, re-
spectively. Visual differences in the color of iodine-stained
head rice as a function of AC are shown in Figure 2. The waxy
rice with 0.09% AC showed a yellowish color after iodine
staining. The intensity of the purple color increased with in-
crease in AC.
Regression lines of L* and b* values versus AC are shown
in Figure 3. R2 values of 0.96 and 0.97, respectively, indicated
a strong relationship between hue and blue-color intensity of
iodine-stained head rice and corresponding sample AC. The
mean L* and b* values for the waxy cultivar were 69.7 and
12.5, respectively. The L*-value ranges for short-, medium-,
and long-grain cultivars were 59–55, 53–48, and 48–34,
respectively. Similarly, b*-value ranges for short-, medium-,
and long-grain cultivars were 8–4, 3–0.7, and –4 to –6, respec-
tively. The root mean square error (RMSE) for the L* predic-
tion model was 1.75, and RMSE was 0.89 for the b* model.
Precision and repeatability of the experiments were found to
be acceptable because no statistical differences were observed
in the mean slopes of both regression models across measure-
ment repetitions and milling replicates, respectively. In order
to achieve low standard deviations that arise from chalky ker-
nels, practitioners are encouraged to implement multiple sam-
ple replications during the iodine-staining process.
CONCLUSIONS
Results of this study indicated that AC of an individual sam-
ple can be predicted from CIE L* and b* values of stained head
rice kernels. The findings indicate strong potential for the
development of a rapid and cost-effective method for predicting
AC in head rice samples. The linearity, coefficients of deter-
mination, and RMSE of the regression models correlating color
values with AC can be further strengthened by incorporating
very high AC (>26%) test cultivars and standards.
ACKNOWLEDGMENTS
The authors would like to thank the Kellogg Company for initiating the
project of rapid AC estimation in medium- and long-grain cultivars and
Dr. Ya-Jane Wang for providing the IRRI rice flour standards for
standard-curve development. The authors also appreciate the support of
Dr. Kent McKenzie for provision of California-grown short-, medium-,
and long-grain cultivars for this study.
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