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J Sci Food Agric 79 :745–749 (1999)
Journal of the Science of Food and Agriculture
Quantifying cyanogenic glycoside production in
the acrospires of germinating barley grains
J Stuart Swans ton*
Scottis h Crop Res earch Ins titute , Mylnefield , Invergowrie , Dundee DD2 5DA , UK
Abstract : Ethyl carbamate is an undesirable trace component in distilled beverages and its content in
Scotch whisky is largely determined by the cyanogenic pre-cursor, epi-heterodendrin (EPH) from
malted barley. A rapid colorimetric procedure can identify cultivars that do not produce EPH and
attempts were made to quantify this test, to determine diþ ering levels of production. Using a given
fresh weight of acrospire tissue from germinated grain, it was possible to distinguish between genotypes but diþ erences between replicates were substantial. Acrospire length was not a reliable indicator of EPH production and genotypes diþ ered not only in the quantity of EPH produced but in the
rate and pattern of production.
( 1999 Society of Chemical Industry
Keywords : barley ; cyanogenic glycoside ; malting ; germination
Ethyl carbamate is an undesirable trace element of
many fermented foods and beverages, including
Scotch whisky, and, due to its reported carcinogenic
nature, acceptable limits are strictly enforced.1 Production of ethyl carbamate in whisky is largely a
function of the barley cultivar used to make the
malt.2 Most barley cultivars produce the cyanogenic
glycoside epi-heterodendrin (EPH)3 in the acrospires
of germinating or malting grain. EPH survives at the
temperatures of malt kilning and hot-water extraction and in distillation, unlike brewing, there is no
wort boiling stage. Consequently, EPH persists into
fermentation, where it is hydrolysed by bglucosidase from yeast, with the heat-labile product
further broken down during distillation, to produce
traces of hydrogen cyanide. Ethyl carbamate results
from the reaction of hydrogen cyanide with ethanol
in the presence of oxygen and copper.4
A number of barley cultivars lack the capacity to
produce the cyanogenic precursor and can be identiüed by a simple, qualitative colorimetric procedure.5
As ethyl carbamate formation is not a problem in
brewing, it is thus possible to select cultivars speciücally for the distilling industry. However, while the
distilling industry consumes the equivalent of 50% of
the barley malt produced in Scotland,6 distilling is of
far lesser importance to malt producers elsewhere, so
there is little incentive for multinational breeding
companies to include non-production of EPH as a
breeding objective. Maltsters and distillers could, in
future therefore, have to rely on minimising EPH
production, by modifying their processing,4 rather
* Corres pondence to : J Stuart Swans ton, Scottis h Crop Res earch
Ins titute Mylnefield, Invergowrie, Dundee, DD2 5DA, UK
Contract/grant s pons or : SOAEFD
than having access, as at present, to a few cultivars,
that do not produce EPH.
While EPH production is a varietal character, the
quantity produced is inýuenced by the environments
in which the grain is both grown and malted.2 The
malster can, therefore, exert some control, but he
would also wish to identify which samples of particular genotypes would produce the lowest quantities of
EPH under optimal malting conditions. Barley
breeders would not discard early generation lines on
the basis of EPH production. However, a readily
performed test could assist in identifying potential
new cultivars, with low levels of EPH production,
which might be targeted towards Scottish growing
Initial trials of a rapid procedure, using enzymic
extraction of malt followed by distillation, gave
limited reproducibility between laboratories.7 In
addition the requirement for 50 g of malted barley
would preclude the test being routinely applied in
barley breeding. In this paper, the possibility of
quantifying the rapid screening test proposed in Ref
5 is examined.
All barley (Hordeum vulgare) genotypes used in these
experiments were grown at the Scottish Crop
Research Institute (SCRI), Dundee. Following
harvest, grain was screened over a 2.25 mm sieve and
only the grain retained was used for subsequent
(Received 16 March 1998 , revis ed vers ion received 17 July 1998 ;
accepted 18 September 1998 )
( 1999 Society of Chemical Industry. J Sci Food Agric 0022-5142/99/$17.50
JS Swanston
Acrospire fresh weight and EPH production
Five barley genotypes, Derkado, Hart, Camargue,
Tyne and Triumph, which were included in a trial of
two replications in 1995, were selected for testing.
Samples of 500 grains from both replications of each
genotype were germinated in petri dishes, with each
sample divided equally between üve petri dishes.
The germination conditions were as described previously.5 At the end of the germination period, grain
from each set of üve dishes was combined and acrospires were carefully dissected from the grain.
Samples of 400, 500, 600 and 700 mg fresh weight,
accurate to 5 mg, were extracted in 4 ml of 35 mM
phosphate buþer (pH 6.0), in a boiling water bath,
for 3 min. After cooling rapidly to 20¡C, an aliquot of
0.8 ml was taken from each sample and the rest of the
testing procedure followed that previously outlined.5
In samples where colour developed following the
addition of cyanide test reagents A (0.5% aqueous
chloramine-T) and B (dimethyl barbituric acid suspended in distilled water), absorbances were determined in a spectrophotometer at 590 nm, 5 min after
the addition of test reagent B.2 Colour production
indicated the presence of a soluble alkali-metal
cyanide.5 For purposes of comparison, it was not
considered necessary to determine the actual quantity of cyanide produced in the test and the absorbance readings were regarded as indicative of EPH
from each dish and weighed. They were then
extracted in 0.4 ml of 35 mM phosphate buþer and
tested for cyanide production. Samples were ültered
through a 1 mm mesh to remove acrospires, prior to
determining absorbance at 590 nm.
Acrospire fresh weight and EPH production
Average absorbance values of the two replications of
four of the cultivars tested are shown in Fig 1. No
colour development in the cyanide test was observed
for cv. Derkado, conürming previous ündings.5
Analysis of variance (Table 1) indicated signiücant
diþerences between cultivars and between fresh
weights. The two cultivars classed as medium/low
producers,2 Camargue and Triumph, gave lower
values than Hart and Tyne which were classed as
medium/high, but cvs Triumph and Hart were fairly
close in values. There was no signiücant genotype ] weight interaction, so the relationship
between EPH production and acrospire fresh weight
appeared to be similar for all four genotypes. This
enabled discrimination between genotypes at each
weight, so it was considered that 400 mg should be
an adequate quantity for subsequent testing.
EPH determination on trial samples
Sixty inbred lines from a cross between the cultivar
Derkado and an SCRI breeders’ line, previously
shown to produce EPH, were included in a trial of
two replications, in 1995. Grain from each plot was
germinated and samples of 400 mg (fresh weight) of
acrospires were extracted and tested as described
above. All acrospires taken were in the recommended
range of 1.5–2.0 cm in length.5
Effect of individual acrospire fresh weight
Grain from three genotypes, Blenheim, Celt and
Golden Promise, previously shown to vary considerably in EPH production,2 were germinated in petri
dishes. Ten acrospires between 1.5 and 2.5 cm in
length were dissected out from each sample and
weighed separately. Individual acrospires were
extracted in 0.2 ml of 35 mM phosphate buþer and
quantities of all other reagents, except for cyanide
test reagents A and B were similarly reduced by
50%. Prior to reading at 590 nm, samples were ültered through a 1 mm mesh to remove acrospires.
Effect of germination time
For each of six genotypes – Blenheim, Celt, Golden
Promise, Kym, Triumph and Plaisant – üve samples
of 100 grains were germinated for 5 days, as previously described. In addition, a further 5 samples
were germinated for both 6 and 7 days. At the end of
germination, the 5 longest acrospires, were taken
Figure 1. Relations hip between acros pire fres h weight and
cyanide tes t colour development in four cultivars of s pring barley.
J Sci Food Agric 79 :745–749 (1999)
Quantifying cyanogenic glycoside in germinating barley
Table 1. Analys is of variance for cyanogenic
glycos ide production (cg) in four barley genotypes ,
over four different fres h weights of acros pires a
Source of
Mean s quares (MS )
Acros pire wt
Genotype ] wt
Res idual
a Level of s ignificance : ***, P [ 0.001.
EPH determination on trial samples
Absorbance values determined from both replicates
of 60 random inbred lines, from a trial are plotted in
Fig 2. A very wide range in values was obtained and
there was a highly signiücant correlation between
replicates (r \ 0.665, 0.001 [ P). Whilst this data
would prove adequate in dividing low from high producers of EPH, variation between replicates would
preclude discrimination between samples which produced moderate levels. However, although all assays
were based on the same fresh weight of acrospires,
diþerences in the extent of acrospire growth between
cultivars meant that the actual number of acrospires
in a given fresh weight might not be constant. In
addition, it was not possible to assay all trial samples
in one day so, although the quantity of tissue tested
was the same for all samples, germination time was
variable. Both acrospire number and germination
time could be sources of variation in EPH production.
Effect of individual acrospire fresh weight
The range and mean values for fresh weights of 10
acrospires from each of three cultivars are given in
Table 2. The correlation between fresh weight and
absorbance values was not signiücant for any of the
cultivars, so longer, heavier acrospires may not
produce higher levels of EPH. There is a direct
relationship between grain size and EPH production,2 so this may confound any eþects of acrospire length. Although grain below 2.25 mm was
excluded from testing, grading into diþerent grain
size distributions had not been done. Consequently,
samples in which a larger number of acrospires were
present in a given fresh weight would be expected to
Table 2. Range of acros pire
fres h weights and correlation
between acros pire weight and
cyanogenic glycos ide production
in three cultivars of s pring barley
J Sci Food Agric 79 :745–749 (1999)
Gold Prom
Figure 2. Cyanide tes t colour development in s ixty barley inbred
lines – relations hip between res ults from trial replicates .
give a higher absorbance reading. It is therefore
likely that more accurate testing would be eþected by
using a given number of acrospires, but ensuring
that the fresh weight was as near to constant as possible between samples.
Effect of germination time
The combined fresh weight and absorbance of üve
acrospires taken from each of six cultivars, after 5, 6
and 7 days germination is shown in Fig 3. As would
be expected, acrospire fresh weight increased with a
longer germination period for all six genotypes. In an
analysis of variance (Table 3) this was reýected in a
Acros pire weight (mg )
Correlation coefficient a (r ),
Acros pire wt vs cyanogenes is
a Correlations were bas ed on 10 acros pires per s ample and were not s ignificant at the 5% level.
JS Swanston
Figure 3. Fres h weight (mg) and res ults of cyanide tes t colour
development for five acros pires taken from each of s ix barley
cultivars , over 5, 6 and 7 days germination.
signiücant diþerence between days. Diþerences
between genotypes were also highly signiücant, but
the ranking order remained fairly constant and there
was no genotype ] day interaction. It should,
however, be noted that Plaisant, as a winter cultivar,
had not been grown with the other genotypes tested.
Whilst it is therefore valid to observe diþerences
between genotypes, as representing a range of
samples, diþerences may not be a purely genetic
The pattern of EPH production, as demonstrated
by absorbance data (Fig 3) was much less clear.
Analysis of variance (Table 3) indicated signiücant
diþerences between cultivars and between days of
germination, but there was also a highly signiücant
genotype ] day interaction. The cultivars Golden
Promise, Kym and Triumph showed a sequential
increase in absorbance, although Triumph was
always much lower than the other two. The other
cultivars showed little or no increase between 5 and 7
days of germination.
Table 3. Analys is of variance for acros pire fres h weight (wt)
and cyanogenic glycos ide production (cg) in s ix barley
genotypes over 5, 6 and 7 days germinationa
Source of
Days germination
Genotype ] day
Res idual
Mean s quares (MS )
165 70.4***
a Levels of s ignificance : *, 0.05 [ P [ 0.01 ; **,
0.01 [ P [ 0.001 ; ***, P [ 0.001.
It would appear possible to distinguish between
genotypes using a given fresh weight of acrospires.
Diþerences between trial replicates were higher than
desirable, but this could be improved by ensuring
equal numbers of acrospires and equal germination
times. This could create a problem for the plant
breeder since, with large populations, it is difficult to
carry out all tests in a single day and a range of start
and end times for germination may not be accommodated within a working week.
A relationship between germination time and EPH
production in cv Maris Otter has been indicated.2
However, they only considered one cultivar and used
malted grain, with no indication given as to any differences in amount of acrospire tissue between
samples. Here it was shown that cultivar diþerences
occurred not only in the quantity of EPH produced,
but in the rate and pattern of production. The
relationship between acrospire length and EPH production, particularly over several days of germination, also appeared to vary between cultivars.
Future research will be aimed at determining how
EPH production in germinating grain relates to that
in malt. The germination procedure encourages
acrospire growth and should, therefore, increase
EPH production.4 However, the rate of uptake and
distribution of water through the grain is also likely
to be diþerent between germinated and malted grain.
In malting experiments5 EPH production was
increased by steeping grain to higher moisture levels.
Of the samples tested here, cvs Celt and Plaisant did
not give particularly high levels of EPH despite
being classed as high producers in malt tests.2
However, the categories into which these authors
classed cultivars are wide and not discrete, suggesting that variation is continuous. Thus, while
environmental variation does not change ranking
order among cultivars of diverse EPH production,2 it
could cause certain genotypes to change categories
between seasons. Further, the inclusion of both
winter and spring cultivars in the comparison made
previously2 suggests that testing was made on
samples which were available rather than on those
which had been grown together in a replicated trial.
Environmental eþects could result from diþerences in grain nitrogen contents. It has been
reported8 that varying the supply of nitrogen caused
signiücant, though not large, diþerences in cyanogenic glycoside production in barley seedlings. Differences in nitrogen supply to the acrospires of
germinating grain would be dependent on grain
nitrogen content and the rate and extent of solubilisation during germination. Patterns of endosperm
modiücation diþer between malted barley and grain
germinated in petri dishes. In particular, modiücation of highly compacted grain structures may be
restricted to a small area of petri dish germinated
grain9 and, with less soluble nitrogen available, EPH
production may be lower than would be the case in
J Sci Food Agric 79 :745–749 (1999)
Quantifying cyanogenic glycoside in germinating barley
malting. This would be consistent with observations
made in this study.
The genetic control of EPH production in barley
has not been studied extensively, but cyanogenesis
occurs in a range of cereals3 and other higher plant
species. Studies in Trifolium have been reviewed10
and it is likely that similar principles apply in other
species, since the biosynthetic pathway from aminoacid to cyanogenic glycoside is common.11 A nonfunctional allele at the Ac locus in Trifolium disrupts
the synthetic pathway, with the allele for cyanogenesis showing incomplete dominance.12 However
the level of cyanogenic glycoside production varies
among Trifolium genotypes and a wide range is
observed in the progeny of parents with diþerent
levels. Previous work13 concluded that this was due
to the presence of several modifying genes.
If EPH production in barley is under a similar
type of genetic control, the modifying genes may be
treated in the same way as other quantitative quality
traits.14 Location of quantitative trait loci (QTL)
and the development of appropriate molecular
markers would facilitate direct selection of DNA
sequences, to identify genotypes with the potential to
produce lower levels of EPH. This approach could
overcome the eþects of environment and of diþerences in germination which led to variation in this
Funding by The Scottish Office Agriculture,
Environment and Fisheries Department (SOAEFD)
is gratefully acknowledged.
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Erb N, Zinsmeister HD, Lehmann G and Nahrstedt A, A new
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