PRICE, BUTLER

Seah, T. C. M., Hoben, D. J., Can. J. Biochem. 47, 557 (1969). Thurston, C. E., J. Am. Diet. Assoc. 36, 212 (1960).
Sidwell, V. D., Bonnet, J. C., Zook, E. G., Mar. Fish. Rev. 35,16 Thurston, C. E., J. Agrie. Food Chem. 9, 313 (1961a).
(1973). Thurston, C. E., J. Food Sci. 26, 1 (1961b).
Sidwell, V. D., Foncannon, P. R., Moore, N. S., Bonnet, J. C., Mar. Thurston, C. E., J. Food Sci. 26, 495 (1961c).
Fish. Rev. 36, 21 (1974). Watt, B. K., Merrill, A. L., Agriculture Handbook No. 8, U.S.
Stansby, . E., Hall, A. S., Fish. Ind. Res. 3, 29 (1967). Department of Agriculture, Washington, D.C., 1963.
Stansby, . E., Mar. Fish. Rev. 38, 1 (1976).
Thompson, . H., J. Assoc. Off. Anal. Chem. 47, 701 (1964). Received for review April 29,1977. Accepted July 5,1977. Oregon
Thurston, C. E., J. Am. Diet. Assoc. 34, 396 (1958). Agricultural Experiment Station Technical Paper No. 4547.

Rapid Visual Estimation and Spectrophotometric Determination of

Tannin Content of Sorghum Grain
Martin L. Price and Larry G. Butler*
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via CONRICYT EZPROXY ADMIN ACCT on August 29, 2018 at 22:25:41 (UTC).

A new technique was developed to quickly distinguish between zero, low, intermediate, and high tannin
varieties of sorghum by the development of shades of yellow, green, and blue colors. A spectrophotometric
method was developed which detects low concentrations of tannin and other polyphenolics by the
formation of the Prussian blue complex. This method plus a rapid extraction procedure enables
polyphenol content of grains to be quantitatively compared in 20 min. The vanillin test was shown to
give misleading results unless a suggested modification is included in the procedure; of 35 varieties of
grain studied, 20 that had been considered low in tannin have no tannin detectable by this test. Some
phenolics which are extracted in water are not extracted in 0.2 M NaCl. This suggests a method which
may distinguish between tannins and nontannin polyphenols.

In recent years it has been recognized that the tannins “analysis” used by grain elevators consists of soaking the
present in many varieties of sorghum diminish the nu- grain in bleach and alkali to remove the pericarp, so that
tritional quality of the grain. Several investigators have the testa (if present and colored) becomes visible. If a testa
reported lower weight gains in young rats and chickens fed is seen, the grain is assumed to contain high amounts of
high tannin grain compared with those fed low tannin tannin. The method is subject to error because of in-
varieties. Typically the weight gains are 30-50% less for terference in some varieties by plant pigments, the color
the high tannin varieties (Armstrong et al., 1973; Jam- of which may persist through the bleach test and make
bunathan and Mertz, 1973), though actual weight loss has identification of a testa ambiguous, and because there is
been reported in rats fed one high tannin variety (Jam- not an absolute correlation between the presence of a
bunathan and Mertz, 1973). Similar results have been colored testa and the deleterious nutritional effects as-
reported comparing the effects of high and low tannin cribed to tannins. For example, the grain of IS 2319 has
beans on weight gain in rats (Ronnenkamp, 1977) and on a clearly identifiable testa, yet results of a feeding trial
in vitro dry matter disappearance trials (Bond, 1976). The showed it to be superior to three low tannin varieties
increasing concern over the nutritionally harmful effects without a testa (Oswalt, 1975). The method also is un-
of tannins in sorghum is creating strong pressures for the satisfactory because it is not quantitative.
sorghum industry to provide grain of low tannin content. The method of quantitative analysis for tannins that has
However, there are economic incentives for the pro- become most widely used for sorghum grain in the lab-
ducers to grow high tannin varieties of sorghum. The oratory is the vanillin test (Burns, 1971). This test is not
presence of tannin makes grain less desirable to depre- convenient for use at the grain elevator because it involves
datory birds, so “bird resistant” (high tannin) varieties can an overnight extraction and at least minimal laboratory
be of great importance in some regions. Estimated losses facilities.
of 50% in Georgia (Harris, 1969) and 48-72% in Arizona In this paper, a new analytical procedure is described
(Voight, 1966) have been reported from bird depredation. which can be used, without instrumentation, to provide
Tannin also apparently is associated with decreased a rapid and convenient visual estimation of the quantity
susceptibility of the grain to preharvest germination of tannin present in sorghum grain. In variations of this
(Harris and Burns, 1970) and seed molding (Harris and test, which can be completed in 1-10 min, the yellow color
Burns, 1973). changes to shades of green and blue with increasing tannin
This conflict between the benefits of tannin to the content. For more precise determination of tannin content
grower and the deleterious effects of tannin for the con- by those without a laboratory, a simplified procedure
sumer should lead to a reflection of tannin content in the requiring only a fixed wavelength colorimeter, some basic
price paid for the grain. However, analytical procedures glassware, a grinder, and reagents has been devised.
to quickly and accurately determine tannin content have Several samples can be determined in an hour. An even
not been available. The current method of tannin more precise spectrophotometric procedure is described
which requires about 20 min for analysis in the laboratory.
A new method is suggested to differentiate between large
Department of Biochemistry, Purdue University, West polymeric tannins and simple flavanoids, anthocyanidins,
Lafayette, Indiana 47907. and small polymers. An essential modification of the

1268 J. Agrie. Food Chem., Vol. 25, No. 6, 1977

 TANNIN CONTENT OF SORGHUM GRAIN

Table I. Visual and Colorimetric Estimation of Tannin Content of Sorghum Grain

Standard
spectrophotometric
Estimation methods methods

Colorimetric Prussian
blue
minus Corrected (water
Visual (variatio nl)
Water NaCl blank vanillin, extract),
Grain Color Rank Tannin content ( ^ 72 0 ) ( 72 0 ) C.E. C.E.
Br-54 Deep blue 1 High 0.71 0.58 3.2 0.58
NK-300 Blue 2 High 0.60 0.48 2.2 0.56
IS-8164 Turquoise 3 Moderately 0.50 0.35 1.4 0.36
high
IS-15612 Dark green 4 Moderately 0.40 0.25 1.0 0.33
high
IS-15991 Green 5 Intermediate 0.15 0.09 0.4 0.04
RS-610 Lime green 6 Low 0.04 0.00 0.0 0.04
IS-954063 Lime green 6 Low 0.04 0.00 0.00 0.00

vanillin test is also suggested. was added 2 mL of 0.008 M FeCl3 in 0.008 N HC1 and 10
mL of 0.0015 M K3Fe(CN)6. Absorbance at 720 nm was
EXPERIMENTAL SECTION read 30 s after adding the final reagent. NaCl blanks, if
Reagents. All solutions were prepared using deionized desired, were prepared in the same manner, except ex-
or distilled water. The 0.10 M FeCl3 was prepared in 0.1 traction was in 50 mL of 0.2 M NaCl, and subtracted from
N HC1 and the solution filtered. D-Catechin was pur- the first reading.
chased from Sigma Inc. The label indicates 2.5 mol of Spectrophotometric Measurement. Ground grain (60
water of hydration was present. Reagent grade ferrous mg) was shaken constantly for 60 s with 3 mL of methanol
ammonium sulfate was used for the iron(II) standard in a test tube, then poured into a Buchner funnel with the
curves. Reagent grade catechol, phenol, gallic acid, suction already turned on. The tube was quickly rinsed
quercetin dihydrate, and cyanidin chloride were used with an additional 3 mL of methanol and the contents
without further purification. poured at once into the funnel. The filtrate was mixed
Sorghum Grain. Sorghum grain was obtained through with 50 mL of water and analyzed within an hour. For
the courtesy of Dr. John Axtell from the 1975 crop grown aqueous extractions, 5 mL of water was used for the ex-
in seed trials at Purdue University. Grain for the visual traction and for the rinse, and the filtrate was added to
or colorimetric estimations was ground 1 min in an electric, 50 mL of water. Other ratios of water and extract can be
hand-held Krups 75 coffee grinder. All data in Table I was used as convenient, as long as appropriate standard curves
obtained within 3 days of grinding. For spectrophoto- are prepared.
metric studies grain was ground to pass a 0.4-mm seive in Three milliliters of 0.1 M FeCl3 in 0.1 N HC1 was added
a cyclone mill. The ground grain was stored in paper to the extract, followed immediately by timed addition of
envelopes at room temperature. Data were collected 3 mL of 0.008 M K3Fe(CN)6. When methanol was present,
during the second and third months after grinding (except the FeCl3 had to be added at the same timed intervals
for data in Table I). Collection of data for graphs showing because of a small increase in OD with time of exposure
comparisons was completed within a few days whenever of FeCl3 to methanol. The optical density was read after
possible to minimize any changes in tannin content with 10 min in 1-cm glass cells at 720 nm on a Zeiss PMQ II
time. spectrophotometer which had been zeroed with water. Ten
Visual Estimation of Tannin Content. Variation I. minutes for color development was chosen because the rate
Ground grain, loosely scooped up in a 2-mL measuring of reaction was considerably slower after that time and
spoon with excess scraped off flat, was added to a 125-mL because a precipitate often formed after 15-20 min. A
flask containing 50 mL of water. Contents were swirled blank of identical composition, but omitting the sorghum
frequently for 3 min. At 4 min 1-mL aliquots from each extract, was analyzed and subtracted from all other
flask were mixed with 1 mL of 0.008 M FeCl3 in 0.008 N readings.
HC1, followed by 1 mL of 0.003 M K3Fe(CN)6. All volumes Results were expressed as catechin equivalents using
were measured with Pasteur pipets calibrated to a 1-mL standard curves prepared daily for the conditions used in
volume. Color developed immediately, but observations the analysis, from fresh solutions of commercial D-catechin.
were recorded after 1 min to allow color to become more Catechin equivalents are milligrams of “catechin”/100 mg
stable. of sorghum grain that would be required to give the ob-
Variation II. Two milliliters of ground grain was served absorbance. Standard curves relating A720 with
measured as in variation I and placed in a 250-mL flask, moles of ferrous ion produced during the oxidation of
followed by 200 mL of 0.0004 M K3Fe(CN)6 and 10 mL extracted tannin were prepared, using reagent grade
of 0.008 M FeCl3 in 0.008 N HC1. Color developed within ferrous sulfate.
seconds, then deepened slowly over the next few minutes General Aspects of the Prussian Blue Test.
as more tannin was extracted. Glassware and cuvettes are stained blue after a few de-
Variation III. Three grains were split in two longitu- terminations, especially if not rinsed immediately at the
dinally and placed in a small test tube, followed by 0.5 mL conclusion of each experiment. Cuvettes must be scrubbed
of 0.0015 M K3Fe(CN)6 and 0.008 M FeCL3 in 0.008 N with a cotton swab after each set of experiments before
HC1. Tubes were swirled occasionally, then the color noted the stain dries. Once the stain has dried on glassware, it
after 10 min. can be easily scrubbed off after soaking overnight in an
Colorimetric Estimation. The flasks prepared in aqueous oxalic acid solution.
variation I were swirled occasionally for 20 min. After A large excess of FeCl3 is added in all variations of the
settling for 10 min, 1-mL aliquots were removed. To this test. This is to ensure rapid and complete reaction. The

J. Agrie. Food Chem., Vol. 25, No. 6, 1977 1269

PRICE, BUTLER

blank is kept low by using low K3Fe(CN)6 concentrations. reagent. However, this mixture is stable for only a few
The absorbance increases with time. Formation of the hours unless kept in the dark or in a brown bottle. If the
complex appears to be rate limiting, the redox reaction visual test is used in the field it must be done in the shade.
apparently reaching completion in seconds. Direct sunlight causes color to develop within minutes once
Because of inherent instability of dilute solutions of reagents are mixed.
FeCl3, this reagent is not added until just before the so- Table I shows the results of a typical determination of
lutions are ready to be analyzed. The results do not seem tannin content for seven varieties of sorghum grain, using
to change noticeably when FeCl3 is added up to half an variation I of the visual estimation. The colors obtained,
hour before analysis of the samples, as long as no organic the relative ranking of the grains according to tannin
solvents are present. A solution containing 6 mL of content, and estimated tannin content are presented. The
methanol in 50 mL of water, however, showed a 30% amount of “tannin” present as determined by two different
increase in absorbance of the blank when the iron was spectrophotometric methods are shown for comparison.
added 30 min early. Although the absorbance due to If rapid completion of the analysis is more crucial than
tannin was not affected, this increase in background re- precision, reagents can be added directly to the ground
quires that ferric chloride be added at timed intervals when grain, as in variation II. Within a few seconds low, me-
organic solvents are present. All dilutions were made from dium, and high tannin varieties give shades of yellow,
0.1 M FeCl3 in 0.1 N HC1 (added to increase stability).
green, and blue. The main problem with this approach
This stock solution is stable for months. is that the amount of extracted tannin is changing rapidly
The A720 readings are extremely sensitive to the slit during the first minute or two. After a few minutes even
width of the spectrophotometer. Widening the slit width low tannin varieties become dark green. Such change with
from 0.08 mm to near maximum (0.2 mm) reduced the time is not a problem in the procedure involving a 4-min
absorbance by 46%. extraction, since over 90% of the tannin has been extracted
Vanillin Test. The vanillin test was performed as by that time (see next section).
described by Burns (1971). Ground grain (200 mg) was In many cases it may be desirable to obtain an estimate
extracted with 10 mL of methanol in screw-capped test of tannin content where grinding is not feasible, such as
tubes which were continuously rotated for 18 h. For the when only one or a few grains must be tested, or for work
“corrected” vanillin test (see Results) separate blanks were in the field, or when speed is of prime importance. In such
read for each sample and substracted from the results of cases one-three grains can be split with a razor blade and
the regular vanillin test. The blanks were run under a small amount of the color reagents added (variation III).
conditions identical with the regular vanillin test except
Assuming roughly equal sized grains, the tannin extracted
that vanillin was omitted from the 4% HC1 in methanol. in a given time will be proportional to the total tannin
RESULTS content. High, medium, and low tannin grains can easily
be distinguished in this way after a few minutes. Changes
Visual Estimation of Tannin Content. This test is
in color over several minutes are most pronounced for this
based on the reduction by tannin and other polyphenols
variation of the visual estimation method. The two high
of ferric ion to ferrous ion, followed by the formation of
tannin grains in Table I were blue and clearly identifiable
a ferricyanide-ferrous ion complex. The colored product
as high in tannin. The others could be distinguished but
(commonly known as Prussian blue) absorbs maximally with less certainty. IS-15991 became light blue, probably
at 720 nm. The same reagents in more concentrated form
because it was a much larger grain than the others.
are often used to visualize phenolic compounds on paper
The colorimetric estimation is designed for situations
chromatograms. Initially the solution is yellow, the color where greater precision is desired than can be obtained by
of the reagents. Increasing amounts of tannin result in the
visual estimation, but a laboratory is not available. A
production of increasing amounts of the blue pigment,
which absorbs the red end of the spectrum. The solution, simple colorimeter with a fixed wavelength above 700 nm,
however, appears green because the blue end of the flasks, test tubes, medicine droppers, an inexpensive coffee
spectrum is still masked by unreacted ferricyanide. If the grinder, and reagents could perhaps be sold as a package.
initial ferricyanide concentration is sufficiently low, it will Values obtained by this method are included in Table I.
become noticeably depleted with higher amounts of tannin.
Extraction times from 30-180 min gave essentially
The result is a deepening of the green color, followed by equivalent values.
a change to turquoise and blue. It should be emphasized Six determinations were made on Br-54 using this
that because these colors vary with conditions used, they procedure. A mean A72o of 0.79 and a standard deviation
only reflect relative tannin contents. A few grains of of 0.06 was obtained.
known tannin content should always be run for com- As will be discussed more fully in the following sections,
parison. However, once these precautions are taken, grains these methods make no distinction between tannin and
can be ranked as high, intermediate, and low in tannin with other polyphenols. But none of the sorghum varieties we
reasonable certainty. have studied seem to have enough nontannin phenolics to
The sensitivity of the visual estimation can be adjusted cause significant error. A low value by the visual esti-
to maximally distinguish differences between high tannin mation method is nearly certain to mean the grain is low
varieties of grain by using more water in the extraction step in tannin, and a high value that it is high in tannin. This
or by adding water to the aliquot taken for developing the may be more of a problem in other seeds which have larger
color. Adding more ferricyanide can serve somewhat the proportions of nontannin polyphenols. For example, we
same purpose by requiring more tannin to be present found in a related project that one variety of cowpea gave
before enough ferricyanide has been used up to allow the high readings, but contained little tannin.
solution to appear blue. If the amount of ferricyanide is This problem could be eliminated in the colorimetric
increased without also adding more water, however, the estimation method (at the expense of doubling the work)
green color becomes so intense that it is difficult to dis- by doing a duplicate extraction in 0.2 M NaCl. Supposedly
tinguish changes in shade of color. only nontannins are extracted in this “NaCl blank" (see
If many samples are to be analyzed, the FeCl3 and next section). Values after such a correction are also
K3Fe(CN)6 solutions can be combined into a single color included in Table I.
1270 J. Agrie. Food Chem., Vol. 25, No. 6, 1977
 TANNIN CONTENT OF SORGHUM GRAIN

Figure 1. A72o for Prussian blue method vs. concentration of Fe2+ and various phenols: quercetin (A), catechin (B), gallic acid (C),
cyanidin chloride (D), catechol (E), hydroquinone (F), phenol (G), and Fe(NH4)2(S04)2 (H). Slopes relative to (H), except for phenol,
are in order: 12.4, 7.3, 6.7, 4.3, 1.9, 1.8.

SPECTROPHOTOMETRIC MEASUREMENT
Method. The FeCl3/K3Fe(CN)6 system provides a
sensitive method for quantitative determination of dilute
concentrations of polyphenolics in any of several solvents.
Figure 1 shows standard curves for A72o vs. concentration
of F2+, catechin, catechol, gallic acid, p-dihydroquinone,
phenol, quercetin, and cyanidin. As can be seen, excellent
linearity is obtained well above one OD unit for each
compound except phenol. The sensitivity of the test
toward flavanoid compounds is sufficient to determine
concentrations less than 10“4 M. This can easily be in-
creased fivefold by reducing the volume of H20 added to
each sample to 10 mL or less.
An advantage to this method is that comparison of the
observed slopes to the Fe(NH4)2(S04)2 slope provides a
measure of how many moles of Fe3+ are reduced per mole
of phenolic. The ratios of these slopes to Fe(NH4)2(S04)2
are given in Figure 1. The reason that some of these are
not integers may be due to uncertainty as to the degree
of hydration of some polyphenolics and to use of com- Figure 2. A720 for 18 h extraction of “tannin” from 25 varieties
pounds without further purification. We are investigating of sorghum compared to A720 for 1-min extraction, measured by
the usefulness of this technique for determining the degree Prussian blue formation.
of oxidation of purified tannin fractions.
The wide variation in the degree to which each molecule of 1.16 with a correlation coefficient of 0.974. Catechin
is oxidized (e.g., a sevenfold difference between quercetin equivalents were determined spectrophotometrically by
and hydroquinone) should serve to emphasize the im- measuring the formation of Prussian blue. The slope of
portance of using caution in interpreting results when the line indicates that approximately 86% of the tannin
mixtures of polyphenols are analyzed by any redox me- that can be extracted in 18 h has already been extracted
thod. in 1 min. Results of the 1-min determination can be
The Prussian blue method should give good estimates compared directly with the 18-h determination by mul-
of relative polyphenol content for mixtures of antho- tiplying by a factor of 1.16.
cyanidins, since they are not likely to differ greatly in the To check reproducibility of this method, five deter-
extent to which they are oxidized by Fe3+. Likewise, minations were made, each using 50 mg of Br-54. A mean
different condensed tannins might be estimated fairly A72q of 1.084 and a standard deviation of 0.015 resulted.
accurately, depending upon just how great the differences If more coarsely ground grain were used, the percent of
are between them. These are the two most likely poly- tannin extracted in 1 min and reproducibility would be
phenols present in the sorghum extracts studied here lowered. Sampling might also then become a problem
(Strumeyer and Malin, 1975). It is presently not possible unless more grain were used.
to determine whether the same number of Fe3+ ions are Comparison with the Vanillin Test. The prescribed
reduced per molecule of proanthocyanidin as per “subunit” method for the vanillin test (Burns, 1971) is apparently
of tannin. based on the assumption that the background color in the
Justification of 1-Min Extraction. A requirement for absence of vanillin is too low to require the subtraction of
rapid analysis is that the amount of tannin extracted in a blank for each sample. This assumption is clearly not
a few minutes be proportional to the total amount of valid; the solvent extracts colored materials which absorb
tannin in the grain. Figure 2 shows the catechin equiv- at the same wavelength. Table II lists C.E. values obtained
alents (C.E.) extracted by methanol in 1 min from 25 by the regular method and by correcting for the blank.
varieties of sorghum plotted against the catechin equiv- Evaluated this way, over half of the grain contained no
alents extracted in 18 h. The points fit a line with a slope tannin at all. Therefore, several varieties of sorghum that
J. Agrie. Food Chem., Vol. 25, No. 6, 1977 1271
PRICE, BUTLER

Table II. Regular and Corrected Results for

the Vanillin Test
C.E. after
Grain C.E. subtracting the blank
Br-64 3.42 2.45
IS-8193 2.68 2.11
IS-8164 1.99 1.74
IS-15526 1.83 1.34
NK-300 1.71 1.15
IS-15612 1.50 1.22
IS-15526 1.42 1.01
IS-15346 1.11 0.70
IS-6881 1.10 0.81
19-8687 0.70 0.02
IS-15991 0.60 0.39
IS-2279 0.58 0.05
IS-10486 0.57 0.00
IS-8544 0.51 0.00 Figure 3. A720 for 1-min aqueous extraction as a function of ionic
IS-9950 0.50 0.09 strength of the NaCl solution, measured by Prussian blue for-
IS-9954 0.49 0.00 mation. Sorghum grains extracted: IS-8164 (0), IS-2279 (O),
121142 0.42 0.00 IS-9180 (O), IS-8687 (v), IS-2319 (O), IS-15991 ( ).
IS-0339 0.36 0.00
131161 0.35 0.00
IS-9528 0.35 0.10
19-0158 0.33 0.00
121168 0.33 0.00
IS-9180 0.32 0.00
121199 0.27 0.00
IS-0114 0.26 0.00
IS-1031 0.26 0.00
IS-10594 0.21 0.00 Figure 4. A720 per 50 mg of sorghum extracted in 1.0 M NaCl
IS-10562 0.20 0.00 vs. A72o per 50 mg of extracted in H20, measured by Prussian blue
IS-12279 0.19 0.01 formation, for 17 varieties of sorghum grain.
19-2057 0.17 0.00
IS-10523 0.16 0.00
10493 0.16 0.00
IS-2042 0.15 0.00
IS-12317 0.15 0.00
121180 0.13 0.00

have been considered to be low in tannin actually contain

no tannin that is detectable by the vanillin test. We have
found that errors caused by not subtracting blanks in the
modified vanillin test (Maxson and Rooney, 1972) are
considerably greater. Acid used in the vanillin test will
itself cause an increase in absorbance with time, but this
does not appreciably affect the blank in 20 min. For
example a Br-54 extract gave A500 of 0.82 in the vanillin CATECHIN EQUIVALENTS
test and a blank of 0.072. The blank was already 0.051 at (vanillin minus blank)
15 s. Figure 5. Prussian blue C.E. values for 1-min extraction of nine
Without further modification the Prussian blue test varieties of sorghum in H20 minus the value from 1-min extraction
cannot be compared to the vanillin test, even after the in 1.0 M NaCl vs. C.E. values for the corrected vanillin test.
latter is corrected for background color. This is because
redox methods, as we have just seen, measure the total of varieties appear to contain water soluble components,
tannins and other polyphenols. presumably tannin, which are not extracted in 0.2 M NaCl.
Extraction with Aqueous Salt Solutions. It is de- All sorghum varieties tested contain other components,
sirable to measure only the (polymeric) tannins by the presumably anthocyanidins, whose extractability is
spectrophotometric Prussian blue method, despite the unaffected by up to 1.0 M sodium chloride.
presence of anthocyanidins or other low molecular weight Figure 4 compares extraction with water and 1.0 M
phenols which respond in the test. Because salt has long NaCl. All the C.E. values in salt solution are low in
been used to precipitate condensed tannins (Quesnel, comparison to C.E. values for high-tannin grains extracted
1968), it seems likely that salt would even more effectively in water. The salt-extracted polyphenolics apparently vary
prevent their extraction. Anthocyanidins can be extracted only within a low range, their concentration never ap-
quantitatively with aqueous salt solutions (Goto et al., proaching the concentrations of polyphenolics in true high
1976). Thus the difference in solubility in water and in tannin lines. If this were not true, the visual tests de-
salt solutions might provide the basis of a Prussian blue scribed earlier, variations I—III, would not be valid. The
test which is specific for condensed tannins. procedure described in variation I of the visual estimation
Figure 3 shows the Prussian blue color per 50 mg of grain method has sufficient water and K3Fe(CN)6 added to cause
measured after extracting 1 min with aqueous solutions minimal color change by the relatively low amounts of
of increasing salt concentration. Before the color was nontannins that are usually present.
developed, all solutions were adjusted to the same salt Figure 5 shows the C.E. values for the 1-min aqueous
concentration. The great sensitivity of some varieties to extraction of ten varieties of grain corrected for “non-
quite low salt concentrations is striking. Some sorghum tannins” by subtracting the values of a 1-min extraction
1272 J. Agrie. Food Chem., Vol. 25, No. 6, 1977
 TANNIN CONTENT OF SORGHUM GRAIN

used. (4) The rapidity and simplicity of the test makes

it ideal for use at the grain elevator.
The formation of the Prussian blue complex offers a
sensitive, versatile method for spectrophotometric de-
termination of total polyphenols. The main disadvantage
of this or any other redox method is that no distinction
is made between tannins and other phenols. A measure
of actual tannins present in the grain is more accurately
measured by the aqueous Prussian blue test when cor-
rected by the salt extraction method or by the vanillin test
as corrected in this paper.
Even these modifications cannot be said to definitely
measure only tannins. Monomeric proanthocyanidins will
give a positive vanillin test, and it is not known what effect
polymerization will have on the extent of the reaction and
the extinction coefficient of the product. The Prussian
blue method always yields a complex with the same ex-
tinction coefficient, but some error is introduced when
Figure 6. Quantity of polyphenols extracted in water and in 1.0 polyphenolics with varying hydroxylation patterns, degrees
M aqueous NaCl, measured by A72o in the Prussian blue test, vs. of polymerization, etc., are mixed in unknown proportions.
time of extraction. Upper curve, H20 extraction; lower curve, It cannot be considered proven that the salt extraction is
NaCl extraction. removing only anthocyanidins or that it removes them
completely.
in 1.0 M NaCl, plotted against the C.E. values for the It must be remembered in all methods used that only
vanillin test corrected for “nontannins” by subtracting the polyphenolics extractable under the given conditions are
background color. being measured. Changing solvents after exhaustive ex-
When a larger quantity of grain was extracted with the traction with another solvent has been known to bring out
same volume of salt solution, a nearly proportional increase additional tannin (Hillis and Swain, 1959).
in A720 was found. This ruled out the possibility that the The long-known chemistry of the formation of the
salt simply reduces the total solubility of all polyphenols. Prussian blue pigment can thus be adapted to determi-
The salt could reduce the rate at which the polyphenols nation of tannins in sorghum and probably other plant
dissolved, so that 1-min extraction times were not com- products, employing either precise laboratory work or
parable with similar data from water extracts. A com- simple, visual estimations in the laboratory, the field, or
parison of the amount of phenols extracted by water and at the grain elevator.
by 1.0 M NaCl as a function of time is shown in Figure 6.
In both solvents, maximum extraction is obtained at about LITERATURE CITED
10 min and then declines, presumably due to air oxidation Armstrong, W. D., Featherston, W. R., Rogler, J. C., Poult. Sci.
of the extracted material. Differences in completeness of 52, 1592-1599 (1973).
extraction after 1 min in the two solvents can account for Bond, D. A., J. Agrie. Sci. 86, 561-566 (1976).
no more than a 4% difference between them. Clearly the
Burns, R. E., Agron. J. 63, 511, 512 (1971).
Goto, T., Moshino, T., Ohla, M., Agrie. Biol. Chem. 40,1593-1596
large differences between the curves in Figure 6 are not (1976).
caused by differences in rate of extraction in water and Harris, . B., Proc. Ann. Corn Sorghum Res. Conf. 24th 24,
NaCl solution. 113-122 (1969).
After dialysis of a 1 M NaCl extract for 24 h against 1 Harris, . B., Burns, R. E., Agron. J. 62, 835-836 (1970).
M NaCl, the outside concentration of polyphenol, assayed Harris, . B., Burns, R. E., Agron. J. 65, 957-959 (1973).
by the Prussian blue method, was 35% as high as the Hillis, W. E., Swain, T., J. Sci. Food Agrie. 10, 135-144 (1959).
inside concentration. The comparable figure for a water Jambunathan, R., Mertz, E. T., J. Agrie. Food Chem. 2l, 692-696
extract dialyzed against water was 11%. This is consistent (1973).
with a relatively smaller proportion of high molecular Maxson, E. D., Rooney, L. W., Cereal Chem. 49, 719-728 (1972).
Oswalt, D. L., “International Sorghum Workshop”, University
weight polymeric tannins extractable by salt solutions. of Puerto Rico, College of Agricultural Science, Mayoguez,
Extent of Oxidation of Tannins. The C.E. values Puerto Rico, 1975, pp 530-552.
obtained by the Prussian blue method are always lower Quesnel, V. C., Phytochemistry 7, 1583-1592 (1968).
than those obtained by the corrected vanillin test, as can Ronnenkamp, R. R., Ph.D. Thesis, Purdue University, West
be seen by the slope of only 0.175 in Figure 4. This is due Lafayette, Ind., 1977.
in part to an inherent overestimation when catechin is used Strumeyer, D. H., Malin, M. J., J. Agrie. Food Chem. 23, 909-914
as a standard for the vanillin test. Rate studies (Price and (1975).
VanScoyoc, unpublished data) show that the absorbance Voight, R. L., Prog. Agrie. Ariz. 18, 30-32 (1966).
with catechin decreases to a fraction of its peak in the 20
min allowed for color development, whereas the absorbance Received for review May 11, 1977. Accepted August 22, 1977.
is still increasing for sorghum extracts. The Prussian blue This is Journal Paper No. 6715 from the Purdue Agricultural
test may underestimate when using a catechin standard Experiment Station. Supported by the U.S. Agency for Inter-
national Development, Contract No. 1175.
if tannin is more oxidized or less reactive than monomers.
DISCUSSION Note Added in Proof: Bleach Test: Five grams KOH, 1 ta-
The advantages of the Prussian blue method for visual blespoon of grain, and 0.25 cup of house-hold bleach are shaken
estimation of tannin content in sorghum are: (1) The series in a jar until the KOH dissolves. After 20 min, contents are placed
in a tea strainer and rinsed with running water, then spread on
of colors produced can be much more easily distinguished a paper towel and examined. The pericarp is removed by the
visually than would be the case if only one color of varying process and the testa, if present, will be exposed and appear dark
intensity resulted. The latter would be the case, e.g., if in color. Otherwise the seed will appear bleached, either white
the vanillin reagent were used. (2) The results are a direct or yellow, depending on the genetic constitution of the endosperm.
measure of soluble polyphenol content. (3) The test is so [Based on Weak, E. D., Miller, G. D., Farrell, E. P., Watson, C.
sensitive that no interfering color is present at the dilutions A., Cereal Chem. 49, 653-663 (1972)].
J. Agrie. Food Chem., Vol. 25, No. 6, 1977 1273