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Field test for amylose class for rice quality classification

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PHILIPP
Field AGRIC
Test for AmyloseSCIENTIST
Type of Milled Rice ISSN
Arvin 0031-7454
Paul P. Tuaño et al.
Vol. 92 No. 2, 228-232
June 2009

Research Note

Field Test for Apparent Amylose Type for Milled Rice Quality
Classification
Arvin Paul P. Tuaño, Leslie T. Roferos and Bienvenido O. Juliano*

Funded by the Philippine Rice Research Institute Central Experiment Station, Science City of Muñoz, Nueva Ecija 3119,
Philippines
Philippine Rice Research Institute Los Baños, University of the Philippines Los Baños Campus, College, Los Baños,
Laguna 4031, Philippines
*Author for correspondence; e-mail: bienvenidojuliano@yahoo.com.ph; Tel.: 63-49-536-3631 to 33; Fax: 63-49-536-3631

A field test for apparent amylose content (AC) type is a promising tool for use by rice quality inspectors,
institutions and restaurants that purchase milled rice in bulk to classify rice quality into three clusters
based on AC and gelatinization temperature (GT) combination. The field test for AC type based on the
iodine staining method of Kongseree et al. (2002) was adapted to Philippine rice to identify low-AC,
intermediate-AC and high-AC milled rice. Milled rice was surface-defatted with 70% 2-propanol or
ethanol and stained with 0.01% iodine in 0.1% potassium iodide in distilled water. The degree of stain
was reduced by oxidants in tap water and 70% alcohol, pH of tap water, degree of milling and freshly
milled rice. A companion GT test based on the alkali test of Little et al. (1958) was also tested on raw
milled rice to distinguish low GT from intermediate-high GT within 15–20 min. Since the rapid method
was inferior to the standard 23-h soak in 1.7% KOH, the latter method is recommended.

Key Words: alkali spreading value, amylose content, field test, gelatinization temperature, iodine stain, rice cluster
type
Abbreviations: AC – apparent amylose content, GT – gelatinization temperature, KI – potassium iodide, KOH –
potassium hydroxide, NSIC Rc – National Seed Industry Council rice, PhilRice – Philippine Rice Research Insti-
tute, PSB Rc – Philippine Seed Board rice, UPLB – University of the Philippines Los Baños

INTRODUCTION and traditional varieties. Some milled rices labeled as

More than 80 PSB/NSIC Rc varieties of varied grain
quality have already been released to farmers. Surveys
in 2001 and 2003 (Mangaoang et al. 2002; Roferos et
al. 2004 unpublished data) revealed an increase of in-
termediate apparent amylose content (AC) varieties in
the market compared with earlier surveys (Unnevehr
et al. 1985; Juliano et al. 1989) with trend moving to-
ward intermediate-low AC type varieties. This trend
indicates that the breeding objective for 18–22% AC
(from 20–25%) has already reached the market and
the consumers. Amylose, a major component of rice
starch, plays a major role in determining the quality of
rice (Juliano 2007). It is routinely measured by the colo-
rimetric method using iodine-amylose blue complex
(Juliano 1971; Juliano et al. 1981). However, mislabeling
is still very much in evidence since milled rices in the
market are only labeled with the names of good quality
IR64 or Sinandomeng still turned out to be hard and
fluffy when cooked (Roferos et al. 2004 unpublished
data). The inflow of many PSB/NSIC Rc varieties has
aggravated the situation. Millers and traders need a guide
as to which varieties to combine during milling to ob-
tain the eating quality desired. Consumers, on the other
hand, must be assured of the quality of the rice they
buy, while rice farmers should have a clear bargaining
power on the price of their produce.
Rice classification based on eating and cooking prop-
erties will surely benefit the rice stakeholders. The rice
classification (clustering) developed in 2003–2004 based
on texture of cooked rice predicted by starch AC-gela-
tinization temperature (GT) combination (Roferos et al.
2006) is a good benchmark. This will guide millers and
traders as to which varieties to combine during pro-
cessing and assure consumers of the eating quality of
the product they pay for. In the classification system,

228 The Philippine Agricultural Scientist Vol. 92 No. 2 (June 2009)

Field Test for Amylose Type of Milled Rice
Table 1. Milled rice quality classification based on starch
amylose content (AC)-gelatinization temperature (GT)
combination (Roferos et al. 2006).
Grain Quality Cluster AC GT
Score a Score b
Soft texture (cluster 1) L L
I IH
Intermediate/medium texture (cluster 2) I L
H IH
Hard texture (clusters 3 and 4) H L
aL = low AC, I = intermediate AC, H = high AC
bL = low GT, IH = intermediate-high GT
cluster 1 is the soft-textured cooked rice type (low AC-
low GT, intermediate AC-intermediate GT); cluster 2 is
the intermediate/medium-textured type (intermediate
AC-low GT, high AC-intermediate GT); and clusters 3
and 4, the hard-textured varieties (high AC-low GT)
(Table 1). However, if proposed for adoption in the
market, an accompanying field test is needed to ensure
the implementation of the classification. Final GT is the
temperature when > 90% of the starch granules gelati-
nize in hot water. It is measured in the Philippine rice
breeding program based on the alkali spreading value
of Little et al. (1958).
A field test for AC type was developed by Kongseree
et al. (2002) to quantify the admixture of higher-AC
milled rice in Thai jasmine or Khao Dawk Mali 105
milled rice with low AC. In this test, milled rice (3 g) is
defatted for 45 s with 15 mL 70% 2-propanol (rubbing
alcohol), rinsed with demineralized water for 30 s and
stained for 45 s with 15 mL 0.005N sodium acetate (pH
8) containing 0.01% iodine in 0.1% potassium iodide
(KI) before rinsing again with water. Low AC rice stains
pink-purple color and intermediate-high AC grain stains
blue or dark color. The higher AC rice was separated
from the low AC milled rice, both were weighed and
the percent contamination was calculated. A maximum
value of 20% higher AC rice in Khao Dawk Mali 105
was allowed. Defatting of surface fat was necessary
because of the higher content of lipids at the milled rice
surface (Juliano 2007).
The objective of this study was to adapt the stain-
ing method of Kongseree et al. (2002) to Philippine rice
to distinguish among low-AC, intermediate-AC and high-
AC milled rices. The effect of various factors on the
stain was studied.
A companion field test for GT was likewise tried,
based on the alkali test of Little et al. (1958), to comple-
ment the AC test. The GT test used higher potassium
hydroxide (KOH) concentration than 1.7% (0.30 N) to
be able to differentiate low-GT from intermediate-high-
The Philippine Agricultural Scientist Vol. 92 No. 2 (June 2009)
Arvin Paul P. Tuaño et al.
GT types within 20 min. In the alkaliviscogram of rice
starch, low GT rice starch gelatinizes instantly at 0.29–
0.36 N KOH and intermediate-high GT rice starch gela-
tinizes at 0.40–0.49 N KOH (Suzuki and Juliano 1975).
A higher KOH concentration is required to gelatinize
whole-grain rice.
MATERIALS AND METHODS
Materials
Samples used included milled rice from the 2005 dry
season crop at PhilRice Los Baños of PSB Rc varieties
already analyzed for AC (Juliano 1971; Juliano et al.
1981) and alkali spreading value (Little et al. 1958) (Table
2). Rough rices were dehulled with a Satake THU-35A
dehuller and 100 g brown rice was milled for 1 min
with a McGill miller no. 2. Part of the milled rice was
passed through a Udy Cyclone mill with 60-mesh sieve
for AC analysis. Some 2007 wet season samples from
PhilRice Maligaya were also tested. A Kett Pearlest mill
was also used to mill small (5 g) samples of brown rice
for 1 min.
Apparent Amylose Test
Stock solutions consisted of 0.2% iodine in 2.0% KI in
distilled water stored in thick amber-colored bottle (good
for up to 3 mo); 70% by vol. ethanol (made from dena-
tured 95% ethanol) or 2-propanol (commercial rubbing
alcohol). Diluted (0.01%) iodine staining solution was
prepared shortly before use by mixing 1.5 mL stock
iodine solution with 30 mL of distilled water.
Milled rice (3 g) in 150-mL beaker was surface
defatted for 5 min with 10 mL 70% ethanol or 2-pro-
panol with 5–10 sec initial swirling, decanted and dried
completely (air-drying or wiping the grains with paper
towel). Then, the defatted rice was stained for 5 min
with 10 mL diluted (0.01%) iodine staining solution,
with 5–10 sec initial swirling, decanted and air-dried
for 10 min before scoring. The stained milled rice was
scored as follows: Wx = waxy — no stain; L = low AC
— slight stain to very light purplish stain; I = intermedi-
ate AC — light purplish blue with pinkish tinge to purple
blue; and H = high AC — dark purple blue to dark/
intense blue (Fig. 1). The stain was stable even after 3
d. The linear correlation coefficient between AC values
and AC type in the field test was calculated by assign-
ing Wx = 1, Low = 2, Intermediate = 3 and High = 4.
Gelatinization Temperature Test
Samples with homogeneous alkali spreading values of
7.0 (IR60) and 5.0 (PSB Rc2) were selected. Ten
undefatted half grains of IR60 and PSB Rc2 were
229
Field Test for Amylose Type of Milled Rice Arvin Paul P. Tuaño et al.

Table 2. Correspondence of field test for AC typea by two analysts with laboratory test values for AC, together with
reported AC type of the variety when released. 2005 dry season samples.

Variety ACb Field Test AC Variety AC b Field Test AC

Name (%) Name (%)
1 2 1 2

PSB Rc1 23I I I PSB Rc62 29I H H

PSB Rc2 27H H H PSB Rc64 22I I I
PSB Rc3 19H L* L* PSB Rc66 25H I I
PSB Rc4 19I I I PSB Rc68 28H H H
PSB Rc5 22H I I PSB Rc70 25IH I I
PSB Rc6 26H H H PSB Rc78 22I I I
PSB Rc7 25I I I PSB Rc80 21I I I
PSB Rc8 25H H H PSB Rc82 20I I I
PSB Rc10 24H I I PSB Rc84 25I H H
PSB Rc12 22I I I PSB Rc86 20I I I
PSB Rc14 22I H* H* PSB Rc88 20I I I
PSB Rc16 29H H H PSB Rc90 25IH H H
PSB Rc18 21I I I PSB Rc92 24IH I I
PSB Rc22 22I I I PSB Rc96 25H I I
PSB Rc24 23H I I PSB Rc98 21H I I
PSB Rc28 19LI I I PSB Rc100 26I H H
PSB Rc30 21I I I PSB Rc102 26H H H
PSB Rc32 20I I I PSB Rc106 24IH I I
PSB Rc34 21I I I IR8 25H H H
PSB Rc36 28H H H IR29 4Wx Wx Wx
PSB Rc38 27H H H IR42 25H H H
PSB Rc42 20I I I IR60 27H H H
PSB Rc44 26H H H IR64 23I I I
PSB Rc46 26H I* I* IR2071-137-5 11L L L
PSB Rc48 25H H H Sinandomeng 15L L L
PSB Rc50 23IH I I NSIC Rc13c 1Wx Wx Wx
PSB Rc52 18IL I I NSIC Rc19c 0Wx Wx Wx
PSB Rc54 21I I I Maligaya Sp9c 1Wx Wx Wx
PSB Rc56 20H I I Maligaya Sp12c 3Wx Wx Wx
PSB Rc58 24I I I Maligaya Sp15c 2Wx Wx Wx
PSB Rc60 25H H H

Accuracy (%) 95 95
Repeatability (%) 100

aAmylose type: Wx = waxy (0–4%); L = low (< 18%), I = intermediate (18–25%) and H = high (> 25%). * denotes different from current
AC type and AC type when released.
bLetters after AC data refer to AC type assigned when the variety was released.
cWaxy samples from PhilRice Maligaya 2007 wet season crop. Sp = Special.

soaked in 8 mL of 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 M
KOH for 20 min and scored for reaction: some reaction
— low GT and no reaction –– intermediate-high GT. In
the standard alkali digestibility test of Little et al. (1958),
six grains of raw milled rice were soaked in 10 mL of
1.7% KOH for 23 h and scored for alkali spreading
value 1–7 according to Little et al. (1958).
Duplicate three surface-defatted milled rice of the
56 2005 dry season crop were cut in half and soaked
for 20 min in 10 mL of 0.5 M KOH and classified into
low and intermediate-high GT by two technicians.
RESULTS AND DISCUSSION
Apparent Amylose Test
Reproducible results were obtained using distilled wa-
ter as solvent for the iodine solution. Chlorinated tap
water and scented rubbing alcohol should be avoided
because of possible reaction with iodine. The 70% etha-
nol is preferably prepared from denatured 95% ethanol
to ensure the absence of oxidants. Drying the defatted
rice completely by airdrying or blotting with paper towel
before iodine staining gave better and faster results than

230 The Philippine Agricultural Scientist Vol. 92 No. 2 (June 2009)

Field Test for Amylose Type of Milled Rice
Waxy Low-AC
High-AC Intermediate-AC
Fig 1. Field test for the four apparent amylose content
(AC) types.
when rinsed with distilled water. Likewise, the rinsing
of stained rice with distilled water may be omitted with-
out effect on the stain. Chalky portion of grains hardly
stained: thus, only translucent grains were scored. Waxy
grains did not stain also.
Freshly-milled rice from the McGill mill had poor
staining right after milling but gave good results after 1
wk storage (Table 3). By contrast, milled rice from the
Kett Pearlest mill tended to be undermilled and required
at least 2 mo storage to give satisfactory results (not
shown). Overmilling for 3 min in both the McGill miller
Table 3. Comparison of scores for AC type of check samples a milled for 1 min
using the McGill Mill No. 2 and aged at room temperature prior to testing and
freshly milled for 2 or 3 min.
Milling/Storage Conditions
High AC
Milled for 1 min (fresh, unstored)
Stored for 1 wk at room temperature
Stored for 2 wk at room temperature
Stored for 1 mo at room temperature
Stored for 2 mo at room temperature
Overmilled for 2 min (fresh)
Overmilled for 3 min (fresh)
aStandard check samples used were: IR60 = High AC; IR64 = Intermediate AC; Low AC =
Sinandomeng
b
AC type scores: Wx = Waxy; VL = Very Low; L = low; I = Intermediate; HI = high-intermediate;
H = high
The Philippine Agricultural Scientist Vol. 92 No. 2 (June 2009)
Arvin Paul P. Tuaño et al.
No. 2 and the Kett Pearlest mill reduced the fat content
of overmilled rice and did not require aging for the freshly
milled rice to stain properly (Table 3). Presumably, tri-
glycerides are less extractable by 70% alcohol than free
fatty acids and the residual lipids in the surface-defat-
ted milled rice interfere with the iodine staining.
Gelatinization Temperature Test
KOH concentration of 0.6–0.9 M was better than 0.5
M and 0.4 M KOH, where the low GT sample IR60 did
not react. The intermediate GT sample PSB Rc2 showed
some reaction in 0.6–0.9 M KOH, thus, 0.55 M KOH
was used for the final test. But the differentiation be-
tween IR60 and PSB Rc2 was still not reproducible.
Hence, the standard alkali spreading value of Little et al.
(1958), based on the reaction of soaked milled rice in
1.7% KOH for 23 h, is recommended for the field test
on raw milled rice, although it requires a longer time.
Operator time is, however, short.
GT of surface-defatted milled rice of the 2005 dry
season crop showed 98% accuracy for the two techni-
cians and 100% reproducibility (PhilRice unpublished
data). Only PSB Rc18 was misclassified as low GT,
instead of intermediate GT, among 56 rices. However,
the defatted grains were soft and tended to crumble
when cut.
Validation and Application of the Field Method for
Apparent Amylose Type
The AC field test takes 30–35 min for 4–6 samples done
simultaneously and 25 min for a single sample. This is
a lot faster compared with the AC analysis done in the
laboratory of at least 3 h. The accuracy of the method
using a set (n = 61) of PSB/NSIC Rc 2005 dry season
samples grown in Los Baños was 95% for AC when
AC Type Scoresb of Check with
Intermediate AC Low AC
VL VL Wx
H I IL
HI I L
H I L
H I L
I I L
H I L
231
 Field Test for Amylose Type of Milled Rice
compared with the iodine colorimetric measurement
(Table 2). Repeatability of the test between the two
laboratory technicians of the Rice Chemistry and Food
Science Division, PhilRice Los Baños was 100%. The
field AC type correlated positively with laboratory AC
values (r = 0.93**, n = 61). Some of the problem
samples identified have borderline AC of 20% between
low and intermediate, and 25% between intermediate
and high, which made the range of intermediate AC
wider. The samples with AC of 18–19% were scored
as intermediate since the color of the stain was more
similar to that of the intermediate AC than to the stain
color of the low AC check. In addition, some varieties
also showed AC variation as released and in the 2005
dry season crop at Los Baños (Table 2).
Preliminary test of the applicability of the method
further revealed its potential use in institutions and res-
taurants that purchase rice in bulk. The method is also
useful in verifying doubtful result of iodine colorimetry
and in the early breeding stage, wherein only informa-
tion on the amylose type is needed. Recently, the field
test was tried with the 1,600 sacks of rice delivered for
UPLB employees. The result showed that the delivered
batch of rice was of cluster 1 type, identical to IR64
(intermediate AC and intermediate GT). The test also
showed some mixtures as seen in the alkali spreading
value. The results coincided well with the feedbacks
from selected rice recipients.
CONCLUSION AND RECOMMENDATION
The iodine staining method of Kongseree et al. (2002)
for distinguishing low-AC from higher-AC milled rice
was successfully modified for classifying aged Philip-
pine milled rices into low-AC, intermediate-AC and high-
AC types. With distilled water in place of tap water,
there was no need for pH adjustment. By contrast, a
shorter method for GT on raw milled rice was less
reliable than the standard alkali spreading value through
23 h digestion in 1.7% KOH. These field tests for AC
and GT types will allow implementation of rice cluster-
ing based on AC and GT and texture of cooked rice by
Roferos et al. (2006). Its introduction in the field would
be directed to rice classification inspectors and bulk
purchasers of milled rice. The field test will also be
useful to laboratory analysts, researchers and breed-
ers. Some training on its use and application may be
necessary.
232
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Arvin Paul P. Tuaño et al.
ACKNOWLEDGMENTS
Ngamchuen Kongseree provided the idea for the
method. Technical assistance was provided by A
Peñaloza, A Carandang and A Agarin. Field test trial at
UPLB was coordinated by AdR Felix, Institute of Food
and Nutrition, College of Human Ecology, UPLB. The
preliminary draft of the paper was reviewed by TM
Masajo, HM Corpuz and VC Lapitan.
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The Philippine Agricultural Scientist Vol. 92 No. 2 (June 2009)