Research article Institute of Brewing & Distilling

Received: 22 April 2012 Revised: 2 May 2012 Accepted: 2 May 2012 Published online in Wiley Online Library: 19 July 2012

(wileyonlinelibrary.com) DOI 10.1002/jib.21

The influence of steep regime and germination

period on the malting properties of some
hull-less barley lines
J. S. Swanston* and J. E. Middlefell-Williams
Following field trials in 2008 and 2010, six lines, derived by mutation in the hull-less barley cultivar Penthouse, were selected
to provide a range in grain b-glucan content. These lines, along with Penthouse and the hulled, malting variety Optic, were
then malted, using four different steeping regimes, with samples kilned after 3, 4 and 5 days of germination. The longest
steep regime provided samples with optimum modification after 5 days of germination. Samples from the other steep regimes
were under-modified to varying degrees. In particular, the steeps with a single immersion gave poorly modified samples with
low extracts and alcohol yields. One line, with low grain b-glucan, gave higher alcohol yields than either Penthouse or
Optic, following a regime comprising two short immersions and a single air-rest, but there was no clear association, within
the lines, between b-glucan content and malting properties. Copyright © 2012 The Institute of Brewing & Distilling

Keywords: alcohol yield; b-glucan; hull-less barley; malting; modification; steeping

Introduction
Hull-less (naked) barley results from a naturally occurring mutation
in a gene on chromosome 7 H of barley (1), which blocks the syn-
thesis of the lipid that permits adhesion of the husk (hull) to the
outer grain tissues (2). Hull-less barley was probably first domesti-
cated for human consumption about 6500 BC (3) and remains a
staple part of the diet in some more mountainous regions of the
developing world (4). Its major contribution to Western agriculture
over the last 40 years has, however, been in the production of feed
for monogastric animals (5), the husk being essentially indigestible
for non-ruminants.
The absence of a husk also increases the contribution of endo-
sperm components to grain composition and the potential for a
higher starch content, to increase both extract and alcohol yield,
has encouraged efforts to develop hull-less barley for alcoholic
beverage production. Hull-less barley has also been shown to
malt more quickly than hulled barley (6,7) and this could have
benefits in reducing the consumption of both water and energy.
However, the husk also has a protective function (8) and damage
to embryos could lead to poor or variable germination. This
would result in uneven modification, which is regarded as a
major cause of processing problems (9). Additionally, husk parti-
cles are important in forming a filter bed when wort is run off in
malt distilleries (10) or breweries that employ lauter tuns.
Recent work (10) has, however, highlighted the importance of
modification in affecting the rate of filtration, while it has also
been shown that an adequate supply of husk particles can be
obtained by the inclusion of a proportion of hulled malt (11).
Additionally, Edney and Langrell (12) suggested that hull-less
barleys could modify as well as their hulled counterparts. A
similar conclusion was reached by Sole (13), although it was noted
that wort viscosity levels were higher than in well-modified hulled
barley. Swanston et al. (14) suggested that selection for even lower
b-glucan levels in hull-less compared with hulled varieties could be
186
necessary to overcome this, but they subsequently did not detect
a significant correlation between grain b-glucan and either extract
or predicted alcohol yield. However they only used one steeping
regime, which was optimized for hulled rather than hull-less types.
By contrast, Agu et al. (6) compared hulled and hull-less geno-
types, using two different steeping regimes, one of which com-
prised a single immersion of short duration. They found that
the hull-less genotypes were still able to malt satisfactorily,
following this abbreviated steeping procedure. In the work
described here, a total of four steeping regimes were used
and quality parameters were assessed after 3, 4 and 5 days of
germination. In addition, the hull-less lines that were malted
were selected for differences in b-glucan content, with the
hull-less variety Penthouse and the hulled malting variety
Optic also included. The objective was to determine whether
differences in b-glucan content altered the rate at which
malting traits achieved peak expression and whether short-
ening either the steeping or germination phases would
improve the performance of hull-less lines in comparison to
the hulled control variety.
Materials and methods
A population of hull-less lines derived by mutation from the
variety Penthouse was included in a trial at The James Hutton
Institute, Dundee, Scotland in 2010. A number of these lines
had previously been in trial in 2008 and details of their deriva-
tion and of the plot dimensions and treatments, which were
* Correspondence to: J. S. Swanston, Cell and Molecular Sciences, The
James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK. E-mail:
Stuart.Swanston@hutton.ac.uk

The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK

J. Inst. Brew. 2012; 118: 186–191 Copyright © 2012 The Institute of Brewing & Distilling
Influence of steep regime and germination period on the malting properties Institute of Brewing & Distilling

similar in 2010, were described by Swanston et al. (14). At harvest, Results

grain weights and moisture contents were determined, with
weights corrected to a common moisture content of 15% to give Comparison of 2008 and 2010 grain data
a measure of plot yield. Grain samples from each plot were
Analysis of variance for plot yield (Table 1) showed a significant
cleaned and a sample retained by a 2.25 mm sieve was used for
effect of genotype, but no effect of season and there was no signif-
subsequent analysis on 31 of the genotypes, which were common
icant genotype  season interaction. By contrast, grain nitrogen
to trials in both 2008 and 2010, the parent cultivar, Penthouse, and
(N) showed a significant effect of season, with results slightly
the hulled malting variety, Optic. Grain nitrogen content was
higher in 2010 (mean value 1.71%) compared with 2008 (mean
determined by near infra-red transmission using an Infratec
value 1.67%), but there was no significant effect of genotype.
1241 Grain Analyser (Foss UK, Warrington, UK). Grain b-glucan
For both grain N and grain b-glucan, there was no significant
content was measured using an enzymatic kit procedure
genotype  season interaction, but for grain b-glucan there were
(Megazyme Europe, Bray, Ireland) based on the method of
highly significant effects of both genotype and season. For the
McCleary and Glennie-Holmes (15). Results derived from the
2010 data, there was a significant negative correlation between
two seasons were subjected to analysis of variance and six
plot yield and grain N content (data not shown), but no significant
lines were chosen for further study, to give a range of b-glucan
correlations were detected between b-glucan content and either
contents.
plot yield or grain N content. This supported results obtained in
Malting was as described by Swanston et al. (14), except that
2008 (14) and demonstrated that selection for particular levels of
four different steeping regimes were used and samples were
grain b-glucan should not impact on grain yield.
kilned after 3, 4 and 5 days of germination to give three different
b-Glucan data from 2010 were plotted against those from
germination periods. Steeping regime 1 was the standard method
2008 (Fig. 1). Although the range of values was fairly small, espe-
used by both Agu et al. (10) and Swanston et al. (14) of 8 h immer-
cially in 2010, and some lines showed considerable variation
sion, 16 h air rest, 24 h immersion, with the water drained and
between seasons, it was possible to pick out lines that showed
replaced after 8 h. Steep 2 was the short single steep (8 h) also
comparatively high, medium or low expression in both years.
used by Agu et al. (6). Two additional regimes were added.
Lines numbered 11, 13, 20, 21, 24 and 33, identified in Fig. 1,
Steep 3 comprised two 8 h immersions with a 16 h air rest
were therefore selected and, along with the parent variety
between, while Steep 4 was another single steep, but of 16 h.
Penthouse and the malting variety Optic, used for subsequent
This final steep regime was recently used in a comparison of
malting and malt analysis.
two hulled malting varieties (16). All steep and germination
regimes were carried out at 16  C.
As a previous study on samples from 2008 had shown no signif-
Effect of steeping regimes on mean values for
icant correlation between the extent of hydration during steeping
Penthouse lines
and key malt quality parameters in this population (Swanston and
Middlefell-Williams, unpublished data), water uptake was not Mean values for the six mutant lines, for both extract (Fig. 2a) and
determined. Malt samples were, however, assessed for hot water PSY (Fig. 2b) were plotted against mean SNR. For all steep regimes
extract and fermentability, using scaled-down versions of the and for both characters there was a progressive increase with
Institute of Brewing Recommended Methods (17), with predicted days of germination. However, it was clear that both the single
spirit yields (PSY) calculated using a formula provided by the immersion regimes gave very low levels of modification, particu-
distilling industry (18). In addition, to provide a measure of filtra- larly the shorter Steep 2, where, even after 5 days of germination,
tion rate, the volume of wort collected after a 10 min filtration the mean SNR value was less than 30%. Steep 4 showed a slightly
was recorded for each sample. Soluble nitrogen content of higher level of SNR than Steep 2, but after 5 days was only
the wort was determined by a spectrophotometric method (19). approaching the values for SNR, extract and PSY observed after
The percentage soluble nitrogen was then used to calculate 3 days for Steep 3 (Fig. 2 a and b) and did not come close to even
the soluble nitrogen ratio (SNR) as a proportion of the total the lowest values observed for Steep 1. The highest mean values
nitrogen content (16). for both extract and PSY were observed using Steep 1 followed by

Table 1. Analysis of variance for plot yield, grain nitrogen and b-glucan content, in 31 mutant lines from the barley variety
Penthouse grown in trial in two seasons

Between Degrees of freedom ms

Plot yield Grain nitrogen b-Glucan
Rep 1 8,739 0.232 0.161
Genotype 30 68,933* 0.009 0.128***
Season 1 33,135 0.057* 1.314***
Genotype  season 30 26,700 0.004 0.053
Residual 61 38,918 0.011 0.034
Total 123
* Significant at the 5% level; ** significant at the 1% level; *** significant at the 0.1% level.
187

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 Institute of Brewing & Distilling J. S. Swanston and J. E. Middlefell-Williams

4.3
the samples being able to achieve commercially relevant malting
performance, without significantly extending the germination
24
period. However, further analysis was undertaken to determine
11
whether there were significant differences between the samples
Beta-glucan % 2010

3.9 13 in their response to the various malting regimes.

21

20
Comparison of individual lines across malting regimes
3.5 Analysis of variance was carried out (Table 2) for extract, ferment-
33
ability, PSY, filtration rate and SNR. For extract, there was a signifi-
cant effect of genotype and highly significant effects of both steep
regime and germination time, but there were no significant
3.1 interactions. As shown in Fig. 2(a), the highest levels of extract
3.1 3.5 3.9 4.3
were associated with Steep 1, followed by Steeps 3, 4 and 2
Beta-glucan % 2008
and lengthening the germination period also increased the
Figure 1. Grain b-glucan contents from 2010 plotted against those from 2008 for extract. Over all steep regimes and germination times, the
31 barley lines derived from cv. Penthouse. The six lines selected for malting and lowest extracts were seen in lines 24 and 21, with line 11 being
malt analyses are indicated by their numbers. highest, although the highest individual extract was observed
in genotype 20, from Steep 1, after 5 days of germination.
Fermentability also showed significant effects of genotype,
steep regime and germination time, but there was also a
a
85 steep  germination time interaction. This resulted from a slight
5 reduction in values from day 3 to day 5 for Steep 1, whereas
4
5
80 3
values rose, with increasing germination time, for the other
4 steeps. However, despite the slight reduction with increasing
3 germination time, Steep 1 still had the highest overall levels of
Extract %

75 5 fermentability, with the other steeps showing the same ranking

4 order as for extract. Among the individual lines, the lowest
70 5
4 3 values, as for extract, were observed in lines 21 and 24, but the
highest fermentability was shown by line 33.
3
65 As PSY is derived from extract and fermentability, lines 21 and
24 were, again, the lowest, with highest values shown by lines 11
60 and 33. In addition to a significant effect of genotype, there were
20 26 32 38 44 also significant effects of steep regime and germination time.
SNR % Although ranking orders for steep regime were as for the other
Steep 1 Steep 2 Steep 3 Steep 4 traits and PSY increased with longer germination time (Fig. 2b),
there was a significant steep  germination time interaction.
b This resulted from the reductions in fermentability, described
430
above, which caused a much smaller increase in PSY between
5
4 days for Steep 1, compared with the other steeps.
5
400 3 The measure of filtration rate was fairly crude, but did produce
4 statistically significant data (Table 2). None of the samples was
PSY l/tonne

3
particularly problematic, although the values obtained after 3 days
370 5 of germination from Steep 2 were the lowest. Differences in steep
4
5 regimes and germination times were statistically significant, with
4 3 ranking orders similar to those observed for the other traits.
340
Samples from Steep 2 showed a much larger improvement in
3
filtration rate with increased germination time than samples from
310 the other steeps, leading to a significant steep  germination
20 26 32 38 44 time interaction. For SNR, there were significant effects of geno-
SNR % type, steep and germination time, but no significant interactions.
Steep 1 Steep 2 Steep 3 Steep 4 Lines 21 and 24 showed the lowest overall values and were the
only lines not to achieve 35% from Steep 4, even after 5 days of
Figure 2. Mean values for the six lines derived from Penthouse for (a) extract germination. All genotypes, however, reached 35% or more by
and (b) predicted spirit yield, after 3, 4 and 5 days of germination, following four day 3 from both Steep 1, which gave the highest values overall,
different steeping regimes, plotted against soluble nitrogen ratio (SNR).
and Steep 3. However, none of the lines even reached 30%
from Steep 2.

a 5 day germination period. However, after 5 days of germination,

Comparison of Penthouse mutant lines with control cultivars
mean values of samples from Steep 3 were similar to values from
Steep 1 after 4 days of germination. These data suggested that The mean values for the Penthouse lines, from Steeps 2 and 4,
neither of the single immersion regimes was likely to result in for extract and PSY after 5 days of germination, were lower than
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Influence of steep regime and germination period on the malting properties Institute of Brewing & Distilling

Table 2. Analysis of variance for malt characters in six mutant lines from the barley variety Penthouse from four steep regimes
and 3, 4 and 5 days of germination

Between Degrees of freedom MS

Extract Fermentability PSY Filtration SNR
Replicate 1 29.4 6.1 1,333 0.1 0.1
Genotype 5 17.7* 7.3*** 838** 6.9 20.7***
Steep 3 1,136.3*** 23.9*** 35,017*** 221.7*** 1,267.5***
Days of germination 2 235.5*** 22.6*** 8,814*** 49.0** 159.4***
Genotype  steep 15 2.4 2.1 164 6.8 3.2
Genotype  days 10 5.5 1.9 220 3.1 5.6
Steep  days 6 4.2 12.8*** 497* 50.4*** 3.5
Genotype  steep  days 30 4.6 1.6 193 5.9 3.0
Residual 71 6.1 1.6 211 7.8 4.3
Total 143
* Significant at the 5% level; ** significant at the 1% level; *** significant at the 0.1% level.
PSY, Predicted spirit yields; SNR, soluble nitrogen ratio.

Table 3. Analysis of variance for extract and germination over 3, 4 and 5 days of germination for steep regimes 1 and 3, for eight
genotypes, comprising the barley varieties Optic and Penthouse and six mutant lines from the variety Penthouse

Between Degrees of freedom Steep 1 Steep 3

MS MS
Extract Fermentability PSY Extract Fermentability PSY
Replicate 1 1.02 0.01 115.2 2.0 0.1 140.8
Genotype 7 5.02* 2.24 163.3 17.6** 7.1*** 601**
Days of germination 2 35.63*** 0.14 994.0*** 111.8*** 11.0*** 4,482***
Genotype  days 14 2.67 1.82 142.5 1.9 1.6 91
Residual 24 1.81 1.25 67.9 3.8 1.1 120
Total 47
* Significant at the 5% level; ** significant at the 1% level; *** significant at the 0.1% level.

those from Steep 3 after 3 days (Fig. 2). Consequently, compari- germination period, despite a reduced length of steeping, in a
sons with the varieties Optic and Penthouse were restricted to hull-less line derived from Penthouse.
Steeps 1 and 3, to ensure that the data would have commercial
relevance. The steeps were analysed separately for extract,
fermentability and PSY, using analysis of variance, with genotypes 440
comprising Optic and Penthouse, in addition to the six Penthouse
mutant lines. Results showed considerable differences between
the two steep regimes (Table 3). For Steep 1, there were significant
PSY (l/tonne)

400
effects of germination length for both extract and PSY, but not for
fermentability, and significant effects of genotype only for
extract, where lines 11 and 20 gave the highest values. By
contrast, for Steep 3 there were significant effects of both 360
genotype and germination length for all three traits. Line 11
gave the highest value for extract with the two varieties Optic
and Penthouse being the lowest. However Optic had one of
320
the highest levels of fermentability, along with line 33, whilst 3 4 5
lines 21 and 24 were the lowest. Lines 33 and 11 were highest Days of Germination
overall for PSY, with lines 21 and 24 lowest. Values for PSY, over
Line 21 Line 33 Optic Penthouse
3, 4 and 5 days of germination following Steep 3 are compared
for Optic, Penthouse and the best and worst of the Penthouse Figure 3. Predicted spirit yields for Optic, Penthouse and the highest and lowest
lines in Fig. 3. The high levels obtained, here, for line 33 showed of the Penthouse lines, after 3, 4 and 5 days of germination, following a shortened
that it was possible to obtain high levels of PSY within a standard
189

steeping regime, with two 8 h steeps and a 16 h air rest.

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 Institute of Brewing & Distilling
Discussion
All the genotypes here achieved their best malting performance
following the longest steep regime and 5 days of germination.
Bathgate et al. (20) observed that peak levels for fermentability
occur slightly before those for extract. At optimal levels of modifi-
cation, fermentable carbohydrate is still being released more
quickly than it is being absorbed by the embryo. However, as
the grain becomes over-modified, the extract contains increasing
quantities of nitrogenous material, which is not fermentable, so
alcohol yield will decline (20). Here extracts and PSY were still
increasing at 5 days of germination, but fermentabilities had
started to decline, indicating that the samples were around their
optimum levels of modification. Consequently, samples from
the other three steep regimes were probably under-modified to
varying degrees and would have given improved extract and
PSY had germination time been extended. However, a longer
germination phase could counteract some of the commercial
benefit from shorter steeping. It was also very unlikely that
adequate levels of modification could have been achieved
following a single 8 h immersion, as SNR levels were still below
30% after 5 days of germination. This was similar to observations
made for Optic, following a single 8 h immersion (16).
These results, therefore, do not agree with those of Agu et al. (6),
who were able to demonstrate high levels of alcohol yield from
hull-less barley lines following a steeping regime comprising a
single immersion of 8 h. However, the breeding lines with which
these authors worked were from a superior genetic background
to that of Penthouse (6) and with lower grain N content than
the Penthouse lines in 2008 (14). Nitrogen levels in 2010 were
slightly higher, giving a significant effect of season (Table 1)
and probably explaining why no samples from Steep 1 were
over-modified after 5 days of germination. In addition, malting
equipment and conditions can vary between laboratories in
factors such as aeration, to replenish the oxygen, which is likely
to be critical in a steeping regime with no air rests. Relative
concentrations of oxygen and carbon dioxide and pH are
among the main factors that influence the rate of hydration (21).
Bathgate (22) stated that the first immersion, during steeping, will
raise the moisture content to around 35%, a very similar figure
to that noted by Bryce et al. (16) for Optic following a single 8 h
immersion. This is, however, well below the moisture content, after
steeping, of between 42 and 46% generally required to achieve
adequate levels of modification (23).
This level was not achievable in Optic from a single immersion
even after 24 h (16), but steeping is also influenced by barley
variety (20) and Bryce et al. (16) noted a sample of Oxbridge to take
up water more readily than a sample of Optic. However, there are
two concurrent processes taking place during steeping, i.e. water
uptake and internal redistribution (24). Whilst both processes occur
during immersion, redistribution occurs more slowly than during
air rests (25). Redistribution is also strongly influenced by the
structure of the endosperm. Palmer and Harvey (26) noted lack
of compaction in the inner endosperm to be a contributory factor
to ease of hydration. This creates a structure that can contain
cracks (23) and in which the starch granules pack fairly loosely into
the protein matrix, making diffusion of water easier. This structure
is likely to be present in Optic, particularly in samples containing a
high proportion of mealy grain, from which starch granules can be
readily released into suspension, following fine milling (27).
As husks retard water uptake (7), hull-less barleys will absorb
water more rapidly during steeping than hulled varieties. However,
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J. S. Swanston and J. E. Middlefell-Williams
achieving adequate hydration from a single immersion is likely to
depend upon optimizing steeping conditions. Additionally it will
be necessary to optimize the endosperm structure of the barley to
maximize hydration within a short time period. The hull-less lines
described here were all derived from the same genetic background
and showed a limited degree of variation for grain quality
traits, confirming initial observations made in a previous season
(Swanston et al., 2011). That initial study also indicated that
grain b-glucan content might not be a particularly effective
predictor of the rate of endosperm modification. While Steep 3
exposed clear differences for both extract and PSY between lines
33 and 24, identified as low and high b-glucan, respectively, differ-
ences between the other lines for these characters did not appear
to relate to b-glucan content. Future work, therefore, will have to
consider further aspects of endosperm structure and composition
that are likely to impact on ease of hydration, e.g. the integrity
of the protein matrix. This should improve the likelihood of
identifying lines, from the wider Penthouse mutant population,
more suited to a much abbreviated steeping regime.
Acknowledgements
The James Hutton Institute receives funding from the Scottish
Government Rural and Environment Science and Analytical
Services Division. The authors also wish to thank the Scottish
Society for Crop Research for additional financial support and
Dr W. T. B. Thomas for helpful discussion of the manuscript.
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