PRACTICAL

METHODS
TO DETERMINE
THE PRESENCE
OF TANNINS
IN SORGHUM
RALPH D.WANISKA', LEDA E HUGO and LLOYD W ROONEY
Department of Soil & Crop Sciences, Faculty of Food Science and Technology
TexasA&M University, College Station, l'X 77845-2474
Phone: (409) 845-2925
F f l : (409) 845-0456

DESCRIPTION
OF PROBLEM
Some sorghums contain tannins [l] that
adversely affect the metabolizable ener [2]
and rotein utilization [3] of oultry. E r e n
and aniska [l]show that see color is not a
good measure of tannin content. Hence, a
qualitative means is needed to determine the
presence of tannins in sorghum. 0 timall ,
chemical analysis could be preformeJperioKically to determine tannin content; however,
only a few commercial laboratories determine
the tannin content of sor hum. Even then,
methods of extraction an of anal sis yield
different results [4,5,6]. Therefore, etermining the actual tannin content of a given shipment of sorghum is often not the most
practical alternative. Determining 'ust the
presence of tannins in sorghum provi es suffcient information for proper poultry ration
formulation [7,8,9].

' h o quick qualitative methods are available to determine whether tannins are present
in sorghum: (a) the "Scratch" test and (b) the
"Bleach" test [4,5,6]. These methods are
based on the assumption that if the kernels
contain a pigmented testa layer then condensed tannins are present. Thus, these methI sorghum (without tannins
ods se arate
from &es I1 and I11 sorghums (with tannins

'

me

To whom correspondence should be addressed

PI

MATERIALS
AND METHODS
The scratch test consists of nothing more
than scratching away the outer pericarp with a
sharp kniie or scalpel to determine the resence or absence of a pigmented testa ayer
[4,61.

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Primary Audience: Nutritionists, Quality Control Personnel, Purchasing

Agents

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WANISKA et al.

123

TABLE 1. Equipment and procedure of the bleach test

EQUIPMENT

PROCEDURE

Magnetic stirrerwith hot plate and timer

1. Set the heat dial of the ma etic stirrer to maintain

water temperature at 6 0 0 ~(14Pq.

Aluminum or glass pan (to boil water)

2. Fill n with water to a depth of approximately 3 mm

(1/8 i n E n d set on heated mametic stir date.

250 ml glass beaker or jar

3. In the 250 ml beaker lace a stir bar, 7.5 KOH, 15g

sor um, and 70ml N a C h solution (bleach5. Place the
bea er in the water pan.

f 05g

4. Adjust the speed of the magnetic stirrer for maximal

agitation. Allow it to stir for 7 minutes.

~~

Balance toweigh

5. After 7 minutes, r e m m the beaker, carefully pour off

the bleach solution, and rinse the sorghum w t h cold
water.

5.25% Sodium hypochlorite (NaOCI; commercial laundry 6. Kemelswhich have turned black contain tannin in some
bleach)
concentration (either Type I1 or 111). Those which have

been bleached to a light yellow to white are nontannin

(Type 1) sorghum.

The "Bleach test causes the pericarp to

dissolve, revealing the presence or absence of
a pigmented testa layei [4,5,6 Materials required and the procedure for t e "Bleach test
are provided in Table 1. The test is simple,
rapid (15 to 20 minutes [or less ), permits
screening of representative samp es, and is
relatively inexpensive.

A-

RESULTS AND DIscussIoN

The ap earance of sorghum is affected by
pericarp co or and thickness, Presence of a
p l p e n t e d testa, endosperm color and text u r e ~geonotWe and environment
* Sorghums containmg tannins ( w e I1 an I I have

41

FIGURE 1.whole kernels (left), "Scratched' kernels (center), and "Bleached" [black (E)] kernels of
sorghum (right) that contain tannins (Type 11/111). The genetic color of the pericarp of these kernels are
white (top) and red (bottom). The pigmented testa layer (T) which contains tannins is the darker layer
just inside the pericarp (P). The white area is the endosperm (E).

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Potassium hydroxide (KOH)

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TANNINS IN SORGHUM

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a pigmented testa layer between the pericarp

and the endosperm (Figure 1).m e 111 sorghums have the highest tannin content and are
commonly grown as bird-resistant sorghums.
w e I1 sorghums contain tannins in the
pigmented testa layer while m e 111sorghums

Bleached

Original

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contain tannins in the pericarp and the

pigmented testa 1aye.r.
Scrapin a portion of the outer pericarp
surface of kernels of sorghum can indicate
the presence of a pigmented testa layer (Figure 1).Then, suspect samplescan be evaluated

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125

WANISKA et al.

TABLE 2. Standard sorahum samples that should be analvzed regularly

No

Sorghum

No

Tannin Sorghum

Yes

IInII

I Light yellow to white

Dark yellow, brown, red, etcA

Black

*Pigments are formed by the sorghum kernel after insects or disease stress the kernel. These pigments are formed in
a nonuniform pattern throughout the pericarp and into the endosperm.Hence, the "Bleach"test is not able to remove
or lighten these pigments.

tip of the kernel (the hylar area) is black in

I sorghums.
The number of kernels that turn black
divided by about 600 kernels in the 15g sample
ves the % of tannin containing kernels
I or 111sorghums).
Standard samples of sorghum, tannin sorghum and weathered sorghum should be acuired and bleached regularly (Table 2).
heaching" of 'Ifipe IInII sorghums causes the
surface to become black in color Figure 3).
Heating for 7 minutes is require for most
commercial sorghums; however, the heating
time may need to be increased or decreased to
facilitate specific sorghum Sam les. Comparison of the test sample with thebeached stan-

(me

FIGURE 3. Effect of heating time on the "bleached"color of U.S. Tannin Sorghum (Type 11/111) with a
white (top) and red (bottom) pericarp. Kernels received 4 (left), 5, 7, 11 and 15 min (right) of heating.
Note excessive bleaching yielded light colored kernels.

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using the "Bleach" test. to determine the percents e of tannin-containin kernels.

f i e "Bleach test clear y reveals kernels
that contain a pigmented testa, provided care
is taken durin analysis and evaluation. Kernels that turn lack contain tannins at some
concentration, either 'I)lpe I1 or I11
(Figures 1 and 2). Kernels that
low or white do not contain

TANNINS IN SORGHUM

126

TABLE 3. Bleach test problems and their suggested solutions

~

PROBLEM

SUGGEFTED SOLUTION

Kernels have black tips

a. All kernels have a Black Hylar region where the kernel attaches to
the ulant.

Kernels are white

a. Analyze Standard Sorghums to provide definitive examples of

sorghum with tannins.
b. All kernels do not have tannins.
E.

~~

Samples were heated too long.

d. TemDerature mav be too hot: check temmrature.

____

a. Analyze Standard Sorghums to provide definitive examples of

sorghum with tannins.
b. Old bleach; purchase fresh laundry bleach.

c. Pericaro is not being removed: increase heatine time.

I d. Temperature may be too cool; check temperature.

Kernels have dark speckles or spots

a. Analyze Standard Sorghums to provide definitive examples of

sorehum with tannins.
b. Kernels damaged by insects, diseases or weathering. These kernels
may have black areas; but, they do not contain tannins. Darkness of
pigmented spots depends upon the extent of damage in the kernel,
glume color, and secondary plant color.
a. Analyze Standard Sorghums to provide definitive examples of
sorghum with tannins.

Kernels are black

b. All kernels have tannins.

dards (Table 2) should reliably indicate kernels with a pigmented testa.

Problemswith the "Bleach test arise from
four areas: standards, rea ents, reaction time,
and kernel deterioration ?Table 3). Solutions
to these problems are also suggested in Tab'e
3. The most common roblemwith reagents 1s
that the bleach is 01 and weak; always use
fresh bleach for best results.
Sorghum plants react to stress (insect
bites, molds, weathering, etc.) by secreting defensive compounds to limit deterioration of

the kernel. Some of these compounds (e.g.,

anthocyanidin) stain the pericarp and the endosperm. These pigmented spots or regions
can be confused wth tannins in both tests.
Anthocyanidin pi ents do not become as
dark as tannins in t e"B1each test, but they do
appear dark and can be confused with tannins
in the bleach test.
Advantages and disadvantages of these
qualitative tests to determine the resence of
tannins in sorghum are listed in TaYlle 4.

8"

TABLE 4. Advantages and disadvantages of qualitative tests for tannins

ADVANTAGES

1.Both tests are rapid and inexpensive.

2. The "Scratch" test determines the presence of a pigmented testa layer between the pericarp and the endosperm.

3. The "Bleach" test causes kernels containin tannins ype I1 and 111) to become black while kernels with no tannins
(Type I) are a light yellow or white (or slightfy coloreds
DISADVANTAGES

1.Neither method quantifies tannin content.

2. These methods cannot differentiate between Type 11and 111sorghums.
3. Suspect grainshould be followed with proper confirmatory tannin analysis when possible. Thevanillin-HCl method
with blanks is recommended.

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Kernels are red to brown

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FIGURE 4. Examples of U.S. Yellow Sorghum that contain insect damage and weathering (top photo)
and staining and disease damage (bottom photo). "Raw"kernels (top) and "Bleached"kernels (bottom)
are displayed in each photo.

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CONCLUSIONS
AND APPLICATIONS

1. The "Scratch"and "Bleach"tests uickly determine the presence of a pigmented testa layer
between the pericarp and the en osperm.
2. Kernels containingtannins ( q e I1 and 111) have a igmented testa (brown to purple color)
that becomes black after the 'Bleach test. Kerne with no pigmented testa (no tannins,
we I} become a li ht yellow or white (or slightly colored) during the "Bleach' test.
3. Questionable samp es should be followed wtth proper confirmatory tannin analysis. The
v d m - H C 1 method with blanks [4,6] is recommended.

REFERENCES
AND NOTES

5. Price,M.L, and LG.Butler, 1977.Rapid visual

estimation and spectrophotometric determination of tan-

nin content of sorghum grain. J. Agric. Food Chem.

2.5~1268-1273.
6. Rooney, LW., M.E Blakely, F.R Miller, and D.T.
Rosenow, 1980. Factors affectin the polyphenols of sorghum and their development anflocation In the sor hum
d s e , J.
ernel. In:
(ed.). IDRG14 3. Ottawa, Ont. 25-35.
7: Gualtieri, M., and S. Rapacclni, 1990. Sorghum
g i n i z u l t r y feeding. World's Poultly Science J.
24458. Butler, LG., 1989. Sorghum Pol henols,pp 95Vol
Phenolics. ed.
121. in:
P.R-hC

IT

9. Purchasing U.S.Sorghum as designated by the

Federal Grain Inspection SeMce (FGIS assures one of
1 receiving grain w t h a low tannin level! U.S Sorghum
cannot contain more than 3% kernels with a pigmented
testa.
l

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1. Bonn, B., and RD. Waniska, 1992. So um

seed color as an indicator of tannin content. J.Tpp1.
Poultry Res. 1:117-121.
2. Blakcly, M.E! LW. Rooney, RD. Sullins, and
F.R Miller, 1979. Microscopy of the pericarp and the
testa of different genotypes of sorghum. Crop Science.
19: 837.
3. Nelson, T.S., EL Stephenson, A. Burgos, J.
Floyd, and LO. York, 1975. Effect of tannin content and
dry matter digestion on energy utilization and average
amino acid availability of hybnd sorghum grains. Poultry
Science 591620-1623.
C.F., J.O. Akingbala, S.H. Ring, and LW.
4.
Rooney, 1 1. Evaluation of several methods to determine tannins in sorghums with varying kernel characteristics. Cereal Chem. 58224-238.