CLEGG-ESTIMATION OF STARCH I N CEREALS

Reference8
1 Phillips, J . D., Annu. Rep. agric. ltovt. Res. Sfa. 16 Feigl, F., ' Spot Tests. Vol. 11. Organic Applica-
Bri;tol,-1g52. p. 137 ~
tions '. 1954, 4th edn., p. 261 (London : Elsevier
*Beech, F. W., Burroughs, L. F., & Codner, R. C., Publishing Co.)
Annat. Rep. agric. hort. Res. Sta. Bristol, 1951, 1 7 Partridge, S. M., Nature. Lond., 1949. 1 6 4 , 443

P. I49 18 Hough, L., Jones, J . K. N., & Wadman, W. H.,

8 Beech, F. W., AnRu. Rep. agric. hort. Res. Sta.
Bristol, 1952, p. 125
4 Carr, J . G., Anwar. Rep. agric. hovt. Res. Sta. Bristol,
19.52, P. I44
5 Jacquin, P., & Tavernier, J., Zndustr. agric., 1949,
66, 5x3
II Jacquin, P., & Tavernier, J., Industr. agvic., 1952,
6 9 , 599
7 Hulme, -4. C., J . exp. Rot., 1951, 2 , 298
8 Rentschler, H., & Tanner, H., Mitt. Lebensm. Hyg.,
Bern, 1954. 4 5 , 142
0 Rentschler, H., & Tanner, H., Mitt. Lebensm. Hyg.,
Bern, 1954. 45, 305
10 Isherwood, F. A., & Hanes, C. S., Bioclrem. J . , 1953,
55, 824
11 Bryant, F., & Overell, B. T., Biochitn. biophys.
Acts, 1953, 10, 471
1* Phillips, J . D., & Pollard, A., Nature, Lond., 1953,
1 7 1 , 41
18 Hulme, A. C., Nalzrre, Lond., 1953, 1 7 1 , 611
1 4 Stark, J. B., Goodban, A. E., & Owens, H. S.,
Anal-yt Chem.. 1951, 2 3 , 413
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Znd., 1955. P. 230
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19 Carr, J . G . , Phillips, J . D., Pollard, A,, & Whiting,
G . C., Chem. Ci t n d . , 1954. p. 1515
9 0 Pollard, A , , & Whiting, G. C., unpublished work
e l Hulme, A. C., Biocliem. J., 1953, 53, 337
ee Burroughs, L. F., Annw. Rep. agrtc. hort. R P S .Sta.
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83 Challinor, S. W., & Burroughs, L. F.,.?n?lit. Rep.
agric. Irort. Res. S f a . Bristol, 1947, p. 1 7 2
$4 Winkler, W. O., J . Ass. off. agric. Chew. Wash.,
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*s Beech, F. W., unpublished work
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97 Williams, A. H., Science and Fruit ', edited by
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John Hopkins Press;

THE APPLICATION OF THE ANTHRONE REAGENT TO

THE ESTIMATION OF STARCH IN CEREALS
By K. M. CLEGG
,. ,
,,

A colorimetric method using anthrone is described for the estimation of starch and
soluble sugars in cereals, cereal by-products, and mixed poultry rations. The method is
rapid and requires no elaborate apparatus. An internal glucose standard is used. Results
for the starch content of these materials show a standard deviation of 2.2% of the mean
value.

Introduction
For a study of the relationship between the metabolizable energy values of cereals in poultry
feeding and their chemical composition, determinations had to be made of their content of
' available carbohydrate '-a term used in analytical tables' of human feeds to embrace starch,
dextrin and di- and mono-saccharides.
Of the possible methods for the estimation of starch in cereals, polarimetry presents difficul-
ties because of turbidity, and methods based on the reduction of alkaline copper sulphate after
.hydrolysis of the starch in test materials to glucose are lengthy.
Recently, more rapid methods for the estimation of carbohydrates in different classes of
material2$3s 4 v have been adopted which make use of the colour reaction between anthrone and
sugars.6 The present paper reports the successful application of the same reaction to the estima-
tion of carbohydrates in cereals.
For the preparation of extracts the method of McCready et al.7 has been adopted, simple
sugars being separated by alcohol extraction, the residual starch then being brought into
solution with perchloric acid.
J. Sci. Food Agric., 7, January, 1956
 CLEGG-ESTIMATION OF STARCH I N CEREALS 41
Experimental
Materials and methods
The method to be described has been applied to the analysis of the starch and sugar content
of pure starches, cereals, cereal by-products and experimental poultry rations.
Anthrone reagent.-One of the difficulties encountered with anthrone, which is usually
dissolved in sulphuric acid, is that on mixing with an aqueous extract containing carbohydrates
heat is liberated which may influence the course of the reaction unless strictly controlled. In the
present study, the technique described by Fairbairns of preparing the reagent in a more dilute
sulphuric acid solution has been adopted, which reduces the rise in temperature, and constant
conditions can be obtained working a t room temperature. Samples of anthrone (B.D.H. Ltd.)
were used without further purification.
Range of straight-line response.-In the final method, as described below, 10ml. of anthrone
reagent are added to 2 ml. of aqueous extract. Up to 300 pg. of either glucose or ' solubilized '
starch have been used (in 2 ml. of aqueous solution) with no deviation from a linear response in
the optical density (Ilford filter 607) after reaction with anthrone. At the highest levels the
optical densities were of the order of 0.7.
Internal standard-In order to compensate for possible variations in different batches of the
anthrone reagent, and for the possible presence of interfering agents in the extracts of the test
materials, an internal glucose standard has been used throughout. As shown below, the anthrone
reagent is added to I ml. of the test extract with I ml. of water and, in parallel, to I ml. of the test
extract with I ml. of a standard solution containing IOO pg. of glucose.

Method of determination
Extraction of sugars from test materials.-Weigh approximately 0.2 g. of finely ground
material into a 50-ml. centrifuge tube, add two drops of 80% alcohol to aid mixing, and then 5 ml.
of water and stir thoroughly. Add 25 ml. of hot 80% alcohol and stir thoroughly, set aside for
5 min. and centrifuge. Decant the alcoholic solution and repeat the alcohol extraction pro-
cedure by adding 30 mI. of hot 80% alcohoI to the residue. Combine the two alcohol extracts.
Alcohol interferes with colour development in the anthrone-sugar reaction and is removed from
the combined extracts by evaporation under reduced pressure in a boiling water-bath. Dilute
the remaining cloudy aqueous fraction with water ; a final concentration of sugars equivalent to
about IOO pg. of glucose/ml. is required ; the aqueous solution of sugars is then ready for analysis
with the anthrone reagent.
Grass products require a modification of the procedure as the plant pigments in the alcohol
extracts cause interference. A colourless solution is obtained by passing the extracts, after
diluting with water t o a known volume to reduce the alcohol content to less than 50%, through
a column of magnesium oxide (I cm. diam. x 10 cm. length). Remove the alcohol-from a n
aliquot of the eluate and dilute the solution of sugars as described above.
Extraction of starch from test materials.-The extraction of starch is carried out at room
temperature. Add 5 ml. of water to the residue of test material which has been extracted with
alcohol and, while stirring, add 6 5 ml. of 52% perchloric acid (prepared by adding 270 ml.of
72% perchloric acid to IOO ml. of water). Stir continuously for 5 minutes with a glass rod and
then occasionally for the next 15 minutes. Add 20 ml. of water and centrifuge. Pour the super-
natant liquid into a Ioo-ml. volumetric flask. Add 5 ml. of water to the residue and repeat the
extraction with perchloric acid, stirring occasionally for the next 30 minutes. Wash the contents
of the tube into the flask containing the first extract. Dilute the combined extracts to IOO ml.
with water and filter, discarding the first 5 ml. of the filtrate. Dilute an aliquot portion to give
a final concentration equivalent to about IOO pg. glucose/ml. which is then ready for analysis
with the anthrone reagent.
Analysis of the extracts.-A standard aqueous solution containing I mg. of DL-glucose/ml. is
prepared every two or three days and diluted daily to give a solution of IOO pg. of glucose/ml.
The anthrone reagent is made b y dissolving I g. of anthrone in I litre of sulphuric acid solution
containing 760 ml. of conc. H,SO, that has been left to cool to room temperature. The required
quantity of reagent is prepared daily.
J. Sci. Food Agric., 7, January, 1956
42 CLEGG-ESTIMATION OF STARCH I N CEREALS
Using 25 x 150 mm. boiling tubes prepare:
in duplicate (i) ' the blank '-containing 2 ml. of water ;
in triplicate (ii) test extract alone '-containing I ml. of diluted extract and I ml. of water :
in triplicate (iii) ' test extract +glucose standard '-containing I ml. of diluted extract and
I ml. (100pg.) of glucose standard.
To all tubes add 10ml. of the anthrone reagent and mix the contents thoroughly. Heat the
tubes (closedwith rubber stoppers containing a piece of capillary glass tubing) in a boiling water-
bath for 12 minutes and then cool to room temperature in a water-bath. Using an absorptio-
meter with an Ilford 607 filter, read the intensity of colour developed by the test extracts with
the reagent blank as'standard. (The anthrone-sugar complex has maximum absorption a t
630 mp.) The green colour is stable for at least 3 hours. Samples of anthrone reagent may be
checked by reading the blanks against distilled water.
CaEcuZation.-Take the average of the two sets of triplicate readings and calculate the
equivalent quantity of glucose in the test extract by comparison with the increased absorption
due to IOO pg. of glucose. For both extracts use this value and a dilution factor (and the con-
version factor 0.9 for the starch extract only since 0.9 g. starch yields approximately 1.0g. of
glucose on hydrolysis) to calculate the percentage of sugars and starch respectively in the original
material.
Results
Pure carbohydrates
Wheat, maize and potato starch analysed by the above procedure gave values in agreement
with their dry matter content (Table I). The colour intensity of the fructose-anthrone complex
was found to be equal to that of glucose on an equivalent weight basis, after heating for 12minutes,
as stated by Fairbairn.8 With fructose, some colour developed before the tubes were placed in

Table I
Starch and sugar content of cereals and other materials
Dry Starch sugars
Material matter No. of Mean Standard Mean
estimations value error value
on each
sample
% % %
Pure starches
Wheat starch 87.0 8 87.0 f 0.6 -
Maize starch 86.0 6 83.6 f 0.8 -
Potato starch 93'0 4 92.5 f 1'0 -
Cereals
Sorghum 87'4 6 61.3
Yellow maize meal 87'4 4 60.6
Plate maize 85.4 4 53'3
Wheat--' Guardsman ' 86.9 4 58.7
Wheat-' Hybrid 46 ' 87.0 I0 58.8
Scottish oats 85.9 4 47.8
Australian oats 88.8 5 36.7
Sussex ground oats 88.7 4 40.6
Barley I 89.6 5 534
Barley I1 91.0 4 48.1
Cereal by-products
Wheat offals-fine parings (middlings) 86.7 33'2 f 0.3 8.1
Wheat offals-French pollards 85.7 19'3 f 0'2 7'9
Wheat offals-coarse wheat bran 88.5 11.4 f 0'1 6.4
Maize gluten feed 88,5 13'9 f 0'2 0.8
Oat feed I 94'2 13'4 f 0'2 1'7
Oat feed I1 89.7 7.7 f 0'1 1 '4

Grass products
Grass meal 87.8 2 2'5 - 5'5
Leaf protein concentrate 94'2 2 1'2 - 1'4
J. Sci. Food Agric., 7, January, 1956
 CLEGG-ESTIMATION OF STARCH I N CEREALS 43

the boiling water-bath. Between 65 and 70% of a sample of dextrin was determined in the
alcohol extract and the remainder went into solution on treatment with perchloric acid. A
sample of a-cellulose gave no colour with anthrone as the extraction procedure of this method
was not strong enough to hydrolyse it. As examples of pentose sugars, arabinose and xylose
were tested and found to give 6.5% and 15% of the colour intensity, respectively, compared with
the same weight of glucose.
Cereals
Results for the starch and sugar content of cereals, cereal by-products and grass products are
given in Table I. For the starch estimations at least two samples were analysed in duplicate and
the results for the sugars given are the average of at least two values.
Statistical analysis of the starch results, not including grass products, indicated that the
variabilities within each set were homogeneous and that the standard deviation of a single value
was f 2.2% of the mean value.
Recovery experiments
The recovery of starch from various cereals averaged 98-5%,ranging from 96.7% to 101.0%
(Table 11), and was subject to similar limits of error as found for other materials.

Table I1
Recovevy of added starch and glucose fvom cereals
No. of Mean Standard
estimations recovery deviation

Wheat starch recovered from wheat-'

I ,

>.
I ,

I,
,,
,,
,,
Guardsman '
fine parings
,, French pollards
Maize starch recovered from wheat-' Guardsman '
Potato starch recovered from fine parings
4
4
4
7
4
g6.T
98.1 98-3
100'1
101'0
96.4*
f 2.7

*
f 2.6
1.9
Glucose recovered from
,, wheat-'
,, fine parings '
Guardsman 6 99'0
100'5}997 f 2'5
* Signhcantly less than 100% recovery.

Presence of non-carbohydrate material

The starch and sugar contents of four experimental poultry rations compoundedfrom cereals,
protein and mineral supplements, were in good agreement with the values calculated from average
starch values for the individual cereal components.
In a further experiment the starch content was determined of a mixture of equal parts of
maize, oats, barley and wheat, and found to be 51.7% compared with a figure of 50.7% calculated
from the values obtained with the individual cereals. When this cereal mixture was analysed
with 25% of supplements added which had a negligible starch content (equal parts of meat and
bone meal, white fish meal, grass meal and sunflower seed meal) the same value was obtained.
Discussion
The results for the starch contents of cereals gave good reproducibility and the presence of
protein and mineral materials did not interfere with the colour reaction with anthrone. The
possibility of interference by compounds such as sodium chloriden and tryptophanlO has been
reported, but only in quantities greatly in excess of their natural occurrence in plant materials
and the normal levels incorporated into rations for poultry.
Grass products which may be constituents of poultry rations were analysed as possible
refractory materials in the use of the anthrone reagent. It was found that a simple chromato-
graphic treatment of the alcohol extract removed the interfering pigments. In the analysis of
some of the experimental poultry rations containing 6% grass meal no interference was detected
at this level and the chromatographic treatment of the alcohol extracts was unnecessary in the
determination of the sugar content.
It has been established that different sugars form characteristic complexes with anthrone
with varying intensities of colour, Morris11 has given comparisons with glucose and shown that
J. Sci. Food Agric., 7, January, 1956
44 CLEGG-ESTIMATION OF STARCH I N CEREALS
fructose, sucrose and maltose could be determined with reasonable accuracy with glucose as
standard: Koch et a1.12 have demonstrated that these particular sugarsin the form of mono-, di- and
tri-saccharides constitute 72% of the sugars of wheat flour. Lactose differs from other sugars in
giving only 80% of the intensity of colour on reaction with anthrone as compared with glucose.
In a poultry ration containing as much as 5% dried milk, the reduced colour development by
lactose would have an insignificant effect on the total carbohydrate content of the ration.
Similarly, the cereals analysed had a low sugar content so that any variation due to the
+
nature of the sugars was insignificant in the total starch sugars ' and probably within the
error of the method. However, the percentage of sugars was higher in the wheat offals than in
whole cereals, as also reported by Fraps,13and a comparison was made with values obtained by the
method of Munson & Walker.14 The sugars of the samples of fine parings, French pollards and
coarse wheat bran determined with the anthrone reagent were S-Iyo,7.9% and 6.4% respectively,
and by the method of Munson & Walker, 6.1%, 5.3% and 4.8% respectively. I n view of the
small colour response obtained with arabinose and xylose it is unlikely that pentose sugars can
account for this discrepancy. Wheat bran may contain up to 10% ~ e l l u l o s ebut , ~ ~as cellulose
gives no colour in the procedure described it does not contribute t o the slightly higher values
found with the anthrone reagent ; all fibrous materials do not have high sugar contents, samples
of oat strawle containing 35-40% of cellulose analysed with the anthrone reagent contained
1.5-3.0% sugars. The results reported by Yemm & Willis5 offer an alternative explanation that
' the anthrone method yields results comparable with those obtained with the copper reagent,
but includes the sugars of stable glycosides which may constitute a large proportion of the
soluble carbohydrates in some plant tissues.'
The carbohydrate composition of a cereal is not necessarily constant as has been demon-
strated by the variability in the starch analyses of oats. Similar variations also occur in cereal
by-products, the composition of which is determined by the degree of milling ; wheat offals,
oat feed and Sussex ground oats are examples of ' manufactured ' materials for which a direct
determination of the starch content is of value.
A successful application of the starch and sugar results has been given in the following paper
where they have been used in the prediction of metabolizable energy values of cereals from the
analyses for protein, fat and ' available carbohydrates ' expressed in terms of glucose (other
workers have expressed i t in terms of starch1). A further refinement of the sugar analysis by
paper chromatographic separation is a possibility, but for the purpose of this work a compre-
hensive value was sufficient as was shown by the correlation with metabolizable energy values.
+
The application of the anthrone reagent to the estimation of ' starch sugars ' in cereals and
similar materials provides a method which is suitable for routine analysis in a non-specialist
laboratory.
Acknowledgments
This work was largely made possihle by the generosity of the Kellogg Foundation.

Rowett Research Institute

Aberdeen
Scotland
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J. Sci. Food Agric., 7, January, 1956