J. Sci. Fd Agric.

1972, 23, 161-169

A Colorimetric Bran Pigment Method for

Determining the Degree of Milling of Rice
K. R. Bhattacharya and C . M. Sowbhagya
Central Food Technological Research Institute, Mysore-ZA, India
(Acceptedfor publication July 1971)

The alkaline alcohol bran-pigment extraction method has been developed into a
simple and reliable quantitative colorimetric method. A requisite quantity
(0.1-1.0 g, depending on the degree of milling of the sample) of rice powder is
soaked overnight with a 1:2 (v/v) mixture of 2 % aqueous KOH-n-propanol(1 ml
for each 0.1 g powder). Next day, the balance of 10 ml of the solvent is added, the
mixture vigorously shaken and centrifuged.The yellow-brown solution is read in a
Spectronic 20 (filter colorimeters did not give linear readings) at 400 nm. On
plotting the absorbance/g/lO ml data expressed as percentage of that of the
corresponding brown rice against the degree of milling, the values in three raw
and one parboiled rice varieties fell into a single smooth curve. The extent of
milling of an unknown sample could, therefore, be determined by testing its
absorbance and that of the corresponding brown rice.
The extractant chosen, selected from different solvent combinations, gave a
clear extract and a negligible difference in reading between normal and red rice
kernels. The intensity of the colour was maximum at about pH 10.5 and above,
and its A,,,,, was at 360 nm (but 400 nm was used for convenience). The colour
was rather unstable, but was more stable in presence of excess rice flour. Hence
it was necessary to use a constant rice-to-solvent ratio during the extraction
for all analyses.
The concentration of the extractable pigment in successive layers of bran de-
creased linearly from the outermost to the innermost layer. The fat content, on
the other hand, first increased and then decreased.

1. Introduction
The present status of different methods for determining the degree of milling of rice has
been reviewed. Of the three approaches-dye staining, estimation of a constituent,
3
'

and optical methods-the latest optical methods* have been claimed to give satisfactory
results; but, in spite of truly numerous attempts, none of the other methods has met the
test of simplicity, accuracy and versatility.
A simple method based on the estimation of a constituent that decreases progressively
with milling would undoubtedly be of the greatest advantage. Analysis of crude fat3 is
probably the most reliable among methods in this category. However, a study showed
that it is only the total crude fat (determined by Soxhlet extraction of the ground rice)
that progressively decreased with increasing degree of milling; the surface fat of the
161
162 K. R. Bhattacharya and C. M. Sowbhagya

kernels, unlike earlier report^,^ first increased and then decreased with milling.4
Determination of total fat, however, is time-consuming and rather erratic. Moreover,
as much of the fat is on the surface of the kernels4 it is doubtful how far the fat content
would give a reliable indication of the degree of milling after the rice has undergone
some handling.
Desikachar5 had shown earlier that certain bran pigments of rice could be extracted
with alkaline alcohol as a yellow-brown solution, and its colorimetric determination
could be the basis of a quantitative test. Tentative application of this method has also
been reported.6* Careful studies, reported here, have now led to the development of a
simple and reliable colorimetric method for determining the degree of milling of rice
based on this principle.

2. Materials and methods

2.1. Rice
Three varieties of rice, two indica (Bangar Sanna, Coimbatore Sanna) and onejaponica
(Tainan 3), were used. One variety (Bangar Sanna) was also parboiled by the method
described.8
2.2. Milling
Paddy was shelled with a miniature commercial rubber roller sheller (the paddy being
separated and reshelled) to obtain brown rice with minimum scratches.
The brown rice was milled to different degrees of polish (d.p.-percentage by weight
of brown rice lost as bran on milling) with a modified McGill miller No. 1 having
attachments as in the McGill miller No. 3. The plastic pressure cover of the miller was
replaced with a stainless steel pressure cover, and over it was mounted a MS pressure
cover saddle and a weight lever rod with arrangements for hanging weights at its right
hand side end as in the No. 3 miller.9 The pressure cover weighed 155 g and the pressure
cover saddle 245 g, the lever rod weighed 325 g and extended 6.6 cm to the left and
22.6 cm to the right, and the weight holder weighed 340 g.
Different 120-g portions of the brown rice (freed from brokens) were milled in the
above with application of different weights (0-2.25 kg) and for different time intervals
(5-120 sec). The rice and the bran were seived in an I 8-mesh screen (1 6 mesh for par-
boiled rice) and the throughs were weighed to give the d.p. Very high degrees of milling
(8 ”/, and more) led to the production of some fine broken rice (up to 1 %) that passed
through the 18-mesh screen and hence introduced appreciable error in the calculation
of d.p.; this was deducted from the bran weight by approximately recovering it on a
22-mesh screen and weighing.
The milled rice was preserved in polyethylene bags. It was aspirated and its brokens
removed with a sizing device before use.
For analysis, the rice was ground in a hand mill or in a Braun Waring disintegrator to
pass a 30-mesh screen.
2.3. Extraction and colorimetry
The following basic procedure was adopted in the preliminary investigations the steps
of which were systematically varied and the effects noted.
Determining the degree of milling of rice 163

The extracting solvent was a suitable mixture of an aqueous alkali solution and an
alcohol. An aliquot (0.1-1.0 g) of theground rice was extracted with 10 ml of this solvent
in a glass-stoppered 100-ml conical flask for 2 h with occasional shaking. The mixture
was centrifuged at low speed, the centrifuge tubes were covered with aluminium foil
caps to prevent loss of solvent and absorption of carbon dioxide (corks gave some
brown colour). The yellow-brown solution was read in a photoelectric colorimeter with
a blue filter.

3. Results and discussion

3.1. Investigations
3.1.1. Clarity of extract
Desikachar5had used a 1:1 mixture of 2 % aqueous sodium carbonate and methanol as
the extractant. This gave no difficulty with fully milled rice,6 but with brown or under-
milled rice, it was found to give an opalescent extract that could not be directly sub-
jected to colorimetry. Previous defatting of the rice powder, or treatment of the extract
with hyflosupercel, eliminated the turbidity; but these steps were not considered
conducive to simplicity.
Efforts were therefore made to overcome this by using different solvent combinations.
Aqueous KOH was employed in place of carbonate because it gave a more intense
colour than the latter and was also miscible with many alcohols. It was found that extract
clarity improved in the following order.
1. Alcohol-methanol, ethanol, isopropanol/n-propanol.
2. Aqueous alkali: alcohol ratio-1 :1, 1:2 (v/v).
3. Alkali-1 % KOH, 2 % KOH, 4 % KOH.
Based on the above, 2 % K0H:n-propanol (1:2, v/v) was finally chosen as the
extracting solvent, which gave a clear extract even with brown rice (in the rice-to-
solvent proportion necessary to obtain a reasonable reading). n-Propanol was preferred
to isopropanol because of its higher boiling point, while 2 % KOH was preferred to 4 %
KOH because the latter led to gelatinisation and clumping of the rice powder in
varieties with low gelatinisation temperatures (such as Tainan 3) thus interfering with
the colour extraction.
3.1.2. Red grains
Another difficulty was that the red kernels, which were natural admixtures in most
commercial lots in India, gave a redder and deeper coloured extract with the original
carbonate-methanol solvent which introduced a source of inaccuracy. Fortunately,
this difference between the extract colours of red and normal kernels decreased with
various solvents in the same order as the extract turbidity above. With the final solvent
mentioned, the two extracts gave practically identical readings.
3.1.3. Optical absorption curve
Determination of the absorption curve with the spectrophotometer (Figure I ) showed
that the maximum absorption was at about 360 nm after which it fell quite rapidly, and
164 K. R. Bhattacharya and C. M. Sowbhagya

that the curves for red and normal kernels (for the same concentration) were only
slightly different. A setting of 400 nm was chosen for the test as this gave only a very
small difference between readings of red and normal kernels, but was still sufficiently
close to the peak.

I I I 1 I ! I 1
400 4 80 560 640
Wave length (nm)

Figure 1. Optical absorption curves of pigment extracts of normal and red kernels of CS brown rice.
Solvent, 4 % KOH-isopropanol (I :2); concn, 0.5 g rice powder:lO ml solvent; extracts clarified with
hyflosupercel. Read in Beckinan model DU spectrophotometer against solvent. +-C, Red ;
- ?--o-, normal.

3. J.4. Colour and p H

The extracted pigment was an acid-base indicator and changed colour in the approxim-
ate pH range of 8-9. The colour in acid p H was pale yellow and in alkaline pH more
intense brownish yellow. The maximum colour intensity was obtained at about p H
10.5 and above---which was well assured by the above solvent even after considerable
exposure to the air.

3.1.5. Choice of colorinzeter

Klett-Summerson photoelectric colorimeter (with filter 40,38 or 42) did not give linear
readings at all. Bausch and Lomb Spectronic 20 (with a grating monochromator) gave
linear readings at all wavelengths; at 400 nm, the readings were linear up to an absorb-
ance value of about 0.7. This was used in all subsequent work.
Determining the degree of milling of rice 165

3.1.6. Colour stability

The extracted colour was somewhat unstable and decreased in intensity with time.
Reducing the alkali strength, dilution with water, or keeping in the dark did not prevent
this slow deterioration. It was therefore desirable to work to a reasonably fixed time
schedule.
3.1.7. Optimum time of extraction
It was observed that the colour reading was maximum at a certain time of extraction,
which depended on the alkali concentration in the solvent as well as on the rice-solvent
ratio used (Table 1).

TABLE1. Effect of rice weight, time of extraction and solvent composition on colour reading.
Coimbatore Sanna variety used. Total volume of extracting solvent, 10 ml

Wt of rice Time of Absorbance/g/lO ml

d.p., % powder taken, g extraction, h 4 % KOH solvent 2 % KOH solvent

6.2 1.o 0.5 0.430 0.432

1.o 0.444 0.463
1.5 0.445 0.500
2.0 0.452 0.520
2.5 0.445 0.525
3.O 0.442 0.531
3.5 - 0.536
4.0 - 0.540
5.0 - 0.541
20.0" 0.402 0.540
0.8 0.2 1.o - 0.425
1.5 - 0.453
2.0 0.408 0.459
2.5 - 0.480
3.0 - 0.479
3.5 - 0.460
4.0 - 0.462
5.0 - 0.448
20.0" 0.340 0.405
20.0b 0.391 0.462

a Overnight. Powder left undisturbed with solvent overnight, shaken vigorously for 10 min and
centrifuged.
In this case only 2 ml of the solvent was added first and left overnight. Next morning another 8 ml
solvent was added, shaken and centrifuged.

It was clear from the data that on extraction for longer than the optimum time the
colour was being destroyed, the destruction being greater with higher alkali concentra-
tion, and that the presence of excess rice flour reduced or prevented this destruction.
It was therefore necessary to keep the rice flour-solvent ratio constant in all tests.
However, for optimal reading, the amount of rice powder for a test had to be varied
depending on its d.p. It was therefore decided to use a fixed rice-solvent ratio (1 ml
solvent for every 0.1 g of powder) during the major part of the extraction period, adding
the balance of the 10 ml solvent, if any, just before centrifuging (see last experiment in
166 K. R. Bhattacharya and C. M. Sowbhagya

Table 1). Overnight extraction under this condition gave stable readings with the
2 % KOH solvent and was also very convenient.
3.2. The final method
Based on the above results, the following final method was selected.

3.2.1. Reagent and test material

Solvent-2 %, aqueous KOH solution :n-propanol, 1 :2 (v/v).
Rice-ground to pass 30-mesh.
The requisite amount of the sample to be weighed for each test is indicated in the
table below; this enables an optimal absorbance value to be obtained with each sample.

d.p., X, Sample wt, g

0-1 0.2
1-2 0.3
2-3 0.4
3-4 0.5
4-5 0.6
5-6 0.8
over 6 1.o

3.2.2. Method
The requisite amount of the sample, based on an approximate visual appraisal of its
degree of milling, was weighed in a 100-ml glass-stoppered Pyrex conical flask. Extract-
ing solvent (1 ml for every 0.1 g of the powder) was added gently and left undisturbed
overnight (e.g. 2 ml solvent to 0.2 g sample, 5 ml to 0.5 g, etc.).
The balance of 10 ml solvent, if any, was added next morning (i.e. 8 ml for 0.2 g
powder, 5 ml for 0.5 g powder, etc.). The solution was shaken vigorously for 10-15 min
and centrifuged for a few minutes in a centrifuge tube covered with an aluminium foil
cap. The supernatant was decanted and the absorbance of the extract was read in a
Spectronic 20 colorimeter (or a spectrophotometer) at 400 nm.
The absorbance for each sample per gram of rice per 10 ml solvent was calculated.
All absorbance results thus obtained were expressed as a percentage of that for the
corresponding brown rice (with no or negligible scratches).
Note-in view of non-linearity of readings, filter colorimeters are not suitable.
However, in absence of appropriate equipment, they can be used if all the readings are
adjusted to fall within a close range.

3.3. Application
The above method was applied to three varieties of raw rice and one parboiled rice.
It was found that the actual absorbance value varied appreciably from variety to variety
as shown on the next page (average of 2-3 readings each).
Determining the degree of milling of rice 167

Absorbance/g/lO ml at
Variety 0.0 % 4% 8 % d.p.

Bangar Sanna 2.60 1.13 0.38

Coimbatore Sanna 2.61 1.18 0.38
Tainan 3 3.35 1.66 0.52
Parboiled BS 2.53 1.20 0.45

However, when all the values were expressed as a percentage of the absorbance of the
respective brown rice, results of all varieties fell approximately into a single smooth
curve (Figure 2). By knowing the absorbance value of the brown rice and of an unknown
sample, the degree of milling of the latter can be read from the graph or from a table
prepared from the graph (Table 2).

I I I I I I I
2 4 6 8 10
Degree of polish (%)

Figure 2. Fall in relative absorbance of pigment extract of rice with progressive milling. Rice:
0 , BS raw; 0, CS raw; x, Tainan 3; A, BS parboiled.

TABLE
2. Percentage absorbance of bran pigment extract
against degree of polish of rice

d.p., % 0.0 0.2 0.4 0.6 0.8

0 100.0 96.5 93.5 90.5 87.5

1 85.0 82.0 79.0 76.5 74.0
2 71.0 68.5 66.0 63.5 61.O
3 * 58.5 56.0 53.5 51.0 49.0
4 47.0 45.0 42.5 40.5 38.5
5 36.5 34.5 33.0 31.0 29.0
6 27.5 26.0 24.0 22.5 21.5
7 20.5 19.5 18.0 17.0 16.0
8 15.0 14.0 13.5 13.0 12.0
9 11.5 11.0 10.5 10.0 10.0
10 9.5 9.5 9.0 9.0 8.5
168 K. R. Bhattacharya and C. M. Sowbhagya

The data showed that the method was quite sensitive up to about 6 % d.p., after which
the sensitivity progressively decreased. It was also clear that for a rice to be considered
as fully milled (8 d.p. or more), its absorbance should be not more than 15 % of that
of the corresponding brown rice.
The method would be of general applicability and maximum advantage only if the
percentage absorbance data of all rice varieties fall into a single curve. Although this
has been found true here for three raw and one parboiled rice tested, further tests with
many more varieties are desirable.

3.4. Colour reading and fat content in different layers of bran

It was thought that by knowing the colour reading of the brown rice, of the highly
milled rice and of a sample of the milled bran, the content of true bran in a variety could
be calculated. But this expectation was belied as the colour reading of the pigment
extracted from the bran was not found to be constant in successive layers. Progressive
partial milling of rice and determination of the absorbance of colour extract from the
successive samples of bran revealed a linear decrease in the reading [Figure 3(a)].

40 I I I I I I 32
- (b)
-

+
30
52
,\“” I+- \ _ - I -

t
I I
2
I
4
I
6
I I
2 4
I I
6
I
Degree of polish (%)

Figure 3 . Colour intensity of pigment extract (a) and fat content (b) of successive layers of rice
bran (CS & BS varieties).

Apparently, the extractable pigment content of the bran decreased linearly from the
outermost to the innermost layer. That this result was unlikely to be due to dilution
of the “true bran” with endosperm particles, was shown by the fact that this decrease in
the absorbance occurred even from the initial layers. This differential pigment concen-
tration in the successive bran layers also explained the curvature in the curve of Figure 2.
Contrary to the above, the crude fat content in the successive bran layers was found
to increase first and then decrease [Figure 3(b), Ananthachar and Desikachar]. This
result was in agreement with data reported earlier by Raghavendra Rao.’O
References
1. Hogan, J. T.; Deobald, H. J. Rice J . 1965, 68 (lo), 10.
2. Stermer, R. A.; Schroeder, H. W.; Hartstock, A. W., Jr.; Kingsolver, C. H. Cereal Chem. 1968,
45, 358.
3. Autrey, H.; GrigorieR, W. W.; Altschull, A. M.; Hogan, J. T. J . ugric. Fd Chem. 1955, 3, 593.
Determining the degree of milling of rice 169

4. Bhattacharya, K. R.; Sowbhagya, C . M.; Indudhara Swamy, Y . M. J. Sci. Fd Agric. 1972,23,

171.
5. Desikachar, H. S . R. Cereal Chern. 1955,32,71.
6. Subba Rao, P. V.; Bhattacharya, K. R. J. ugric. Fd Chem. 1966,14,479.
7. Desikachar, H. S. R.; Bhashyam, M. K.; Parpia, H. A. B. J. Fd Sci. Technol. 1967,4, 156.
8. Bhattacharya, K. R.;Indudhara Swamy, Y . M. Cereal Chem. 1967,44,592.
9. McGill, H. T. Operating Instructionsfor McGill Miller No. 3, P.O. Box 18206, Houston, Texas,
U.S.A.
10. Raghavendra Rao, S. N.; Ananthachar, T. K.; Desikachar, H. S. R.J. Fd Sci. Technol. 1965,
2, 115.