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J Sci Food Agric 79 :3746 (1999)

Journal of the Science of Food and Agriculture

The structure of barley endosperm An

important determinant of malt modification
G Sachin Chandra,* Michael O Proudlove and E Denis e Baxter
Brewing Res earch International , Lyttel Hall , Nutfield , Surrey RH1 4HY , UK

Abstract : Structural di erences in barley grains have been classied as either mealy or steely and
their relative proportions have been determined using a light transectance method in three barley
samples varying in the degree of steeliness, Target being the most steely and Chariot most mealy with
Blenheim being intermediate. These structural di erences were found to be associated with di erences in the concentration of endosperm components, particularly proteins and b-glucan. Analysis of
nitrogen within the endosperm showed that protein was mainly concentrated in the embryo and distal
regions with the inner, mid-endosperm containing lowest levels. As the total nitrogen (TN) of the
grain increased, the mealier samples accumulated nitrogen mainly in the embryo whereas the steely
sample had higher levels in the central endosperm. SDS-PAGE showed no di erences in the protein
banding pattern at di erent TN levels. Electron microscopy using immuno-gold labelling demonstrated that c-hordeins were present in sub-aleurone and outer endosperm whereas the C-hordeins
were found throughout the central endosperm. However, steely areas of central endosperm contained
c-hordeins. During malting, protein modication in Chariot was more extensive than in Target with
34kD and 97kD hordeins being completely degraded. In Chariot and Blenheim, level of b-glucan was
low and it was evenly distributed throughout the endosperm. In the steelier Target, however, the
amount of b-glucan was higher and was concentrated in the proximal and distal areas of the endosperm. Steely grains (containing high concentrations of protein and b-glucan) displayed slower water
distribution during steeping and later development and distribution of b-glucanase during germination. As a consequence, the steely sample achieved a lower degree of modication during malting.
The structure of the endosperm, therefore, has a prime inuence on the evenness of distribution of
moisture and enzymes which is crucial for homogeneous modication during malting.
( 1999 Society of Chemical Industry

Keywords : mealy ; steely ; moisture ; protein ; b-glucan ; b-glucanase

INTRODUCTION
It is evident from a visual examination of barley
grains that structural dierences can occur in the
endosperm. These have been described as mealy and
steely areas. Under the microscope mealy areas
appear to contain loosely packed cells with air spaces
between the starch granules. Steely areas, on the
other hand, appear densely packed with small starch
granules in a dense protein matrix. The causes of
these structural dierences are not fully understood
but are thought to be related to climatic conditions
such as water and nutrient availability during grain
lling.
Barleys with a high proportion of steely grains are
often, but not invariably, associated with higher
protein and b-glucan contents and uneven modication of the endosperm during malting. This
uneven modication of endosperm results in poor
extraction during brewery mashes and process difficulties relating to high wort viscosities, slower beer
ltration and formation of gels and hazes in the nal
product.1h3 However, the mechanism by which

endosperm structure can aect modication during

malting is poorly understood. Structural dierences
might be expected to have signicant eect on water,
and consequently enzyme movements within the
endosperm, thus limiting hydrolytic activity during
malting.
Dierences in the amount and distribution of bglucan and protein, and in the development and
activity of b-glucan degrading enzymes have therefore been investigated in samples of Chariot, Blenheim and Target that contained dierent proportions
of mealy and steely grains.

* Corres pondence to : G Sachin Chandra, Brewing Res earch

International, Lyttel Hall, Nutfield, Surrey RH1 4HY, UK.

(Received 8 Augus t 1997 ; revis ed vers ion received 2 March 1998 ;

accepted 15 April 1998 ).

EXPERIMENTAL
Materials
Barley samples (Blenheim and Chariot) were
obtained from the National Institute of Agricultural
Botany (NIAB), Cambridge, UK, and were from
their National List Trials. Plots were either
untreated or treated with fungicide at dierent stages
of stem elongation : growth stages 31 (Corbel 1 l

( 1999 Society of Chemical Industry. J Sci Food Agric 00225142/99/$17.50

37

GS Chandra, MO Proudlove, ED Baxter

hectare~1) and 39 (Tilt Turbo 1 l hectare~1) according to a typical routine for plant protection.
Untreated samples were used only to establish the
proportion of mealy and steely grains in each variety,
all other work being carried out on treated samples.
A sample of Target was supplied by Usborne Grain
Ltd, Andover, UK, and was a standard commercial
sample treated with fungicide. All chemicals used
were obtained from Sigma Chemical, UK, and were
of the highest purity available, unless otherwise
stated.

METHODS
Scanning electron microscopy
Transverse sections of barley grains were goldcoated and examined using a Phillips 505 scanning
electron microscope (SEM) operating at 25 kV.
Analysis of wet barley samples was carried out using
the Oxford CT1000 cryo unit. Samples were placed
on a stub and plunge frozen in liquid nitrogen/
nitrogen slush at [180C and transferred under
vacuum to the cryo-stage in the SEM. The sample
was then etched by raising the stage temperature to
[130C. When the sample was sufficiently etched, it
was coated with either gold or carbon.
X-ray microanalysis
Elemental analysis was carried out using an EDAX
PV9900 X-ray microanalysis system attached to the
SEM.
Light transflectance method4
Whole corns of barley were dehusked, using a small
scale pearling machine5 which incorporated an electric motor. The main chamber of the machine was
lined with an abrasive metal mesh and grains were
pearled against the abrasive surface with the help of a
wire brush for 58 min depending on the variety.
Grains were placed on a glass plate, and a Koch
Light bre optic light source (model KLS 85) diffused through a Whatman-105 Lens paper was used
to illuminate the grains from below. Under these
conditions the loosely packed starch in the mealy
endosperm absorbed light, causing the grain to
appear dark and opaque, whereas the densely packed
starch and protein steely grains transected light and
appeared translucent (Fig 1).
Micromalting
Micromalting was carried out using either the BRIs
2 kg malting unit or on a 350 g scale in glass
jars, both at 16C. Two interrupted steeping schedules at 16C of wet(w) or air(a) periods were used as
follows : (a) 8w/16a/24w or (b) 7w/17a/7w/17a/1w.
Germination was for ve days. In the case of ale malt
0.1 lg g~1 m gibberellic acid (GA) was added at the
end of steeping. Green malts were dried in an airow oven for 8 h at 45C, followed by 16 h at 65C.
38

Figure 1. Light trans flected barley grains s howing the differences

in endos perm morphology which have been identified as mealy
and s teely areas . The mealy areas (M) abs orb light and appear
dark and the s teely areas (S) trans flect light and appear
trans lucent.

Total grain moisture

This was measured by oven drying using the IOB
recommended method of analysis.6
Determination of water distribution
Grains from each variety were stained with iodine
vapour, using the method of Davies.7 The grains
were divided into two main populations : mealy and
steely. The steely population also included grains
which contained small patches of mealy endosperm
(M/S) and therefore classied as steely for calculation of hydration.
For both the mealy and steely populations an
iodine vapour score (IVS) was calculated using Fig
2:
IVS \ sum (number of grains in each group ] score).
Therefore, for a given sample (n \ 100) the overall
hydration value (OHV) after 16 h was calculated
from :
OHV \ [(M/100) ] IVS(M)] ] [(S/100) ] IVS(S)]
where M \ number of mealy grains in sample and
S \ number of steely grains in sample.
Total nitrogen
Total nitrogen in whole grains and sections of grain
was analysed by the Dumas method [Leco Instruments (UK) Ltd, UK] according to the IOB recommended methods of analysis.8
Total protein extraction
Total proteins were extracted from ground samples
of barley endosperm in Tris (0.7% w/v), containing
SDS (2% w/v), 2-mercaptoethanol (ME) (5% v/v)
and then precipitated with trichloroacetic acid
(TCA) (10% w/v) and analysed by SDS-PAGE.
J Sci Food Agric 79 :3746 (1999)

The structure of barley endosperm

Figure 2. Dis tribution of water in barley

endos perm meas ured as the area s tained
by iodine vapour. Each kernel is then
clas s ified us ing the s coring s ys tem
illus trated in this diagram.

Hordein extraction
Hordeins were extracted sequentially for 1 h using
the following : (a) Hordein I :-Propan-1-ol (50% v/v,
60C) and (b) Hordein II :-Propan-1-ol (50%
v/v) ] ME (2% v/v) at 60C. After each extraction
the samples were centrifuged at 10 000 ] g for
10 min, dried in a rotary evaporator and resuspended
in SDS-PAGE sample buer. Protease inhibitors
PMSF (200 mM), EDTA (100 mM), Leupeptin
(1 mM) and Pepstatin (1 mM) were added to stop any
protease activity during extraction.
SDS-PAGE analysis
Discontinuous gel electrophoresis was performed
under reducing conditions by the method of
Laemmli9 using the instrument supplied by Hoefer
Scientic Instruments, USA.
Immuno-gold labelling of barley and malt hordeins
Barley and malt grains were cut in half and blocked
in phosphate-buered saline (PBS) containing
Tween-20 (PBST): 140 mM NaCl, 1.5 mM KH PO ,
2 4
8 mM Na HPO , 2.7 mM KCl, pH 7.4, and 0.05%(v/
2
4
v) Tween-20) and 1%(w/v) skimmed milk powder.
The grains were then washed three times in PBST
for 15 min and xed in 205 buer [2%(v/v) paraformaldehyde and 0.05%(v/v) glutaraldehyde]. Grains
were incubated for 30 min with primary antibodies
specic for c and C hordeins (anti-hordein IgG),
diluted 1 : 200 with blocking buer, then washed
ve times for 15 min in PBS and incubated for
30 min with secondary antibody (goat anti-mouse
IgG attached to 10 nm gold particles, from BioCell
Research Laboratories, UK), diluted 1 : 1000 in
PBS. Grains were then washed ve times for 15 min
in PBS before silver enhancing using the BioCell
Silver Enhancing kit and visualized using the cryoscanning electron microscope.
Determination of b-glucan
b-Glucan was analysed in extracts of barley or malt
by ow injection analysis10,11 using a Tecator 5700
b-glucan analyser, supplied by Perstorp Analytical,
UK. The instrument was calibrated using puried
barley b-glucan standards (provided with the
instrument), which were reported to be of a relatively low molecular weight. The following extraction method was used.
J Sci Food Agric 79 :3746 (1999)

Barley our (100 mg) or malt our (250 mg) was

added to 10 ml of 1%(v/v)Termamyl a-amylase
(Novo Nordisk, Denmark) and placed in a boiling
water bath for 1 h. The samples were cooled and
10 ml of 75 mM sulphuric acid was added. The
extract was mixed and heated in boiling water for
10 min. Samples were cooled and centrifuged at
10 000 ] g for 10 min. A second extraction was
carried out to partially degrade b-glucans into
soluble fractions. It is important not to degrade the
b-glucans into units that are too small, since they will
not complex with Calcouor. Accordingly, acid
extraction is carried out for exactly 10 min.
b-Glucan solubilase
b-Glucan solubilase was measured as described by
Moore et al.12
Determination of b-glucanase
b-Glucanase was measured using the dye labelled
barley b-glucan as described by McCleary and
Shameer,13 using the kit supplied by Megazyme
(Australia).

RESULTS
Endosperm structure
Examination of barley endosperm sections by light
and scanning electron microscopy showed that steely
areas were densely packed with cell walls, protein
and starch granules when compared with mealy areas
of the endosperm which were loosely packed with air
spaces between the starch granules [Fig 3(a)]. Both
Table 1. Endos perm characteris tics of barley grains in each
s ample

Cultivar

Chariot
Blenheim
Target

U
T
U
T
T

TN
(%)

1.47
1.60
1.64
1.81
2.03

31
5
30
9
19

Population (%)
S /M
M
26
10
19
15
51

43
85
51
76
30

T Plants treated with fungicide

U Untreated plants
S , s teely ; M , mealy ; S /M , s ingle grains having both s teely and
mealy areas

39

GS Chandra, MO Proudlove, ED Baxter

Figure 3. Characteris ation of mealy and

s teely areas in barley endos perm :
(a) s canning electron micrographs ;
(b) light micros copy analys is . M, mealy ;
S, s teely, M/S, mixture of both mealy and
s teely endos perm ; Cw, cell wall ;
Ss , s mall s tarch granules ; Ls , large
s tarch granules .

mealy and steely patches could occur randomly

across the endosperm thus making the estimation of
the extent of steeliness difficult due to variation
between the sections [Fig 3(b)]. Therefore, a method
to identify the predominant endosperm structure was
developed using light transectance through whole,
dehusked barley corns (see Methods), with the
results shown in Table 1. Chariot and Blenheim had
a higher proportion of mealy grains, whereas approximately half of the corns of the Target sample contained both steely and mealy regions in a mainly
steely endosperm. Fungicide treatment of the
growing plant was one of the factors which increased
the proportion of mealy grains, possibly by extending the time available for transport of photosynthetic
products into the ear.
Moisture uptake and distribution
The rate of moisture uptake diered only slightly
between samples, and all reached the total moisture
level of around 43% by the end of steeping.
However, redistribution of moisture across the endosperm (overall hydration value, OHV) was very
much aected by endosperm structure (Table 2).

Cultivar

Chariot
Blenheim
Target
Table 2. Hydration of cultivars
with different proportions of
mealy and s teely grains

40

M
S
M
S
M
S

When mealy and steely grains were separately

steeped, it was found that water penetrated further
into the mealy endosperm than into the steely endosperm. This resulted in higher OHVs for Chariot
and Blenheim samples, which generally had a more
mealy endosperm. Mealy grains of Target hydrated
better in terms of the extent of water distribution
than the mealy grains in Blenheim but this was oset
by a much higher proportion of poorly hydrating
steely grains. Clearly, the population of mealy and
steely grains within a sample can profoundly aect
the extent of endosperm hydration.
Protein content and distribution
Figure 4 shows the distribution of nitrogen in transverse sections of grain, illustrating the distribution
(a) lengthwise from the embryo to the distal end
along the length of the corn and (b) radially from the
aleurone through to the central endosperm. For a
particular TN level the nitrogen was mainly concentrated in two areas : the embryo and the distal region.
Within the endosperm, the outer layers contained
more protein than the inner mid-endosperm. As the
protein content of the mealy samples increased, the
extra protein tended to be deposited in the embryo,

Population
(%)

Iodine vapour
s core

85
15 (S-5, M/S-10)
76
24 (S15, M/S-9)
30
70 (S-19, M/S-51)

98
67
75
50
90
29

Overall hydration
value a

93
69
47

M, mealy ; S s teely ; M/S, s ingle grain having both mealy and s teely areas
a Calculated us ing iodine vapour s core after s teeping for 16 h. Values account for proportion of
mealy and s teely grains in each variety. Steeping s chedule 7wet/17air/7wet/17air/1wet

J Sci Food Agric 79 :3746 (1999)

The structure of barley endosperm

Figure 4. Dis tribution of nitrogen in (a) trans vers e s ections and

(b) pearled endos perm fractions of barley grain. The differences
between mealy Chariot s amples at different total nitrogen values
and a s teely s ample of Target are s hown.

leaving the central endosperm still relatively low in

protein. In contrast, signicantly more of the protein
in the Target sample (1.5% compared with 0.7
0.9%) was located in the central endosperm.
Proteins were further characterised using SDSPAGE. The banding pattern (Fig 5) in barley grains
from a single variety was not signicantly aected by
the nitrogen content. Similar results were found by
Angelino et al.14 However, the distribution of
hordein in the endosperm may vary with the increase
in nitrogen as it has been suggested that the proportion of hordeins increases with the grain total nitrogen and can account for up to half the nitrogen of the
grain.15
Therefore, an immuno-gold labelling method was
used to locate specic hordeins within the grain
endosperm. Figures 6(a) and (b) are electron micrographs showing hordein distribution in barley endo-

Figure 5. Effect of total nitrogen on protein compos ition. Lane

number : S, molecular weight markers ; 1, Blenheim barley at TN
1.54 ; 2, Blenheim barley at TN 1.60 ; 3, Blenheim barley at TN
1.91 ; 4, Blenheim barley at TN 1.93.

J Sci Food Agric 79 :3746 (1999)

Figure 6. Scanning electron micrographs of hordein dis tribution

in barley endos perm : (a) general dis tribution ; (b) s pecific binding
to matrix protein without binding to s tarch. Ls , large s tarch
granules ; Ss ; s mall s tarch granule ; P, protein ; Cw, cell wall.

sperm. It shows the binding of antibody to hordeins

surrounding the starch granules. Figures 7(a) and (b)
are X-ray microanalysis maps illustrating the distribution of c and C-hordeins in Target barley endosperm. The c-hordeins were mainly present in the
outer endosperm and sub-aleurone areas whereas the
C-hordeins were evenly distributed throughout the
central endosperm. C-hordeins were found in both
mealy and steely areas, while c-hordeins were absent
in the mealy endosperm and concentrated in the
steely areas of central endosperm.
Protein modification
Total protein extracts of malt showed that certain
high molecular weight proteins were degraded
during the malting process. SDS-PAGE was used
therefore to examine hordein degradation during
malting in more detail [Figs 8(a) and (b)]. A comparison was made between barley, whole malt and
friable and non-friable malt fractions from a friabilimeter. Hordeins were extracted in the presence
of protease inhibitors to check if protein breakdown
was occurring during extraction. No changes were
seen in the protein prole on addition of the protease
inhibitors, thus conrming that the degradation of
41

GS Chandra, MO Proudlove, ED Baxter

Figure 7. Digital mapping of protein in barley endos perm us ing

X-ray microanalys is . (a) Dis tribution of c-hordeins . (b) Dis tribution
of C-hordeins . P, protein ; Ad1, digital image of grain ; AgL, digital
map of s ilver enhanced immuno-gold labelled proteins .

hordeins occurred during malting and was not an

artefact of extraction. Hordein I and II extracts from
barley, malt and friabilimeter fractions showed that
hordeins with molecular weights of 34kD and 97kD
(c- and D-hordeins, respectively) were only found in
barley and non-friable fractions, being absent from
the friable fraction. These hordeins are therefore
important since their degradation is associated with
malt friability.

Figure 8. Degradation of hordeins during malting : (a) hordein I,

degradation of low molecular weight hordein I fraction extracted
in propanol with and without proteas e inhibitors ; (b) hordein II,
degradation of high molecular weight hordein II fraction extracted
in propanol ] ME with and without proteas e inhibitors . B, barley ;
M, total malt ; F, friable fraction ; NF, non-friable fraction from
friabilimeter.

Immuno-gold assay conrmed that there was

better overall hordein modication in Chariot malt
when compared to Target (Table 3). Generally, the
hordein modication in the proximal endosperm for
both the varieties was good but the central and distal
endosperm (Table 3) of Target were less modied
than in Chariot.

Table 3. Dis tribution of hordeins in the barley grain and its breakdown in malt
Variety and Proximal
Aleurone Sub -aleurone
antibody
endos perm

Central
endos perm

Dis tal
end

Mealy

Steely

Proximal
Aleurone Sub -aleurone
endos perm

Central
endos perm

Dis tal
end

Broad
c-hordein
C-hordein

Chariot barley
]]]
]]
]]]
]]
]
]

]]]
]]]
]

]]]
]
]]]

]]] ]]] ]]]

]]]
[
]]]
]]] ]]] ]]]

Chariot malt
]
[
[

]
]
[

]
]]
[

]]
[
]

]]
]]

Broad
c-hordein
C-hordein

Target barley
]]]
]]]
]]]
]]
]]]
]

]]]
]]]
]

]]]
]]
]]]

]]]
]]
]

Target malt
]]
]
[

]]
]
]

]]]
]]]
]]

]]]
]]
]]]

]]]
]]
]]]

]]] ]]]
]
]]]
]]] ]]]

]]] High level

]] Medium level
] Low level
[ Not detected

42

J Sci Food Agric 79 :3746 (1999)

The structure of barley endosperm

b-Glucan
Steely grains in each sample contained higher levels
of b-glucan than did mealy grains (Fig 9) but the
extent of the dierence varied. Chariot had the
lowest levels at approximately 2% (w/w) with bglucan contents of steely grains being only slightly
higher than the mealy grains. In Blenheim, overall
level of b-glucan was higher at 2.5% and steely
grains contained signicantly more than mealy
grains. Mealy grains of Target had similar amount of
b-glucan, but steely grains contained almost twice as
much b-glucan as the mealy grains.
Figure 10 shows the distribution of b-glucan
within single grains of Target and Chariot. Although
b-glucan levels in the central endosperm were similar
for both samples, it is apparent that both proximal
and distal endosperm of Target contained signicantly more b-glucan than did comparable areas in
Chariot.
b-Glucan modification
Figure 9 shows that, during malting, the b-glucans
were well degraded in both lager and ale malts of
Chariot and Blenheim. However, a large amount of
b-glucan remained in Target, even when the malting
protocol included gibberellic acid (which stimulates
the production of b-glucanase).
A key determinant of malt quality is the level of
b-glucan present in the nal product. This is mainly

dependent on : (a) the initial levels and distribution

of b-glucan in the ungerminated grain and (b) capacity of the grain to produce enough cytolytic enzymes
to degrade this b-glucan. The main cytolytic
enzymes in barley are b-glucan solubilase and b 1-3,
1-4 glucanase.
b-Glucan solubilase
Most of the b-glucan degradation occurs during germination although, since some b-glucan solubilase is
present in raw barley, the process of cell wall attack
is presumed to start as soon as barley is steeped.16
Table 4 shows that the level of solubilase was much
lower in Target barley when compared with Chariot
and Blenheim although the level in all three malts
were similar. Therefore, the level of solubilase in
barley would be expected to promote the initial solubilization of b-glucan in Chariot and Blenheim and
since this is believed to be the rst step in the solubilization of b-glucan,17 low levels in Target barley
Table 4. b-Glucan s olubilas e

Cultivar

Chariot
Blenheim
Target

Barley
Malt
(mg b-glucan s olubilis ed min 1)
0.7
1.1
0.3

0.9
1.4
0.9

Figure 9. Concentration of b-glucan

in barley and malt s amples . For barley
s amples , the contribution made by
mealy and s teely grains to each
s ample is als o s hown.

Figure 10. b-Glucan dis tribution in the

proximal, central and dis tal areas of
barley endos perm of s amples with
differences in their morphological
s tructure. Each area repres ents an
average of 50 corns .

J Sci Food Agric 79 :3746 (1999)

43

GS Chandra, MO Proudlove, ED Baxter

Figure 11. Dis tribution of b-glucanas e in different areas of (a)

Target and (b) Chariot endos perms during germination. The
areas are : embryo ; proximal endos perm ; central endos perm,
and dis tal endos perm.

may contribute to the poor rate of b-glucan breakdown.

b-Glucanase
Unlike b-glucan solubilase, b-glucanase is not
present in raw barley but is synthesised during germination. Figures 11(a) and (b) show the changing
distribution of this enzyme across the endosperm as
germination proceeds. Activity remained very low in
Target [Fig 11(a)] during the rst four days of germination, then appeared to develop rst primarily in
the proximal area followed by an increase in the
central endosperm. Very little enzyme activity was
measured in the distal end of the grain. In Chariot
[Fig 11(b)], b-glucanase activity developed earlier at
day 3, and was found primarily in the central endosperm. Thus, the length of time for which the
enzyme was eective in this area was signicantly
greater for Chariot, resulting in better b-glucan degradation.
The viability of the aleurone layers was conrmed
by measuring the production of alpha amylase activity in aleurones isolated from Target and Chariot
grains. Enzyme production was the same for both
varieties.

DISCUSSION
It has been known for a long time that the barley
endosperm structure is an indicator for malting
quality of barley,18h22 hence grain cutters (eg a
farinater) are frequently used in selecting barley for
malting. Most of the work carried out on endosperm
structure has used single sections of the grain.
However, since the endosperm is not structurally
homogenous, it is important to evaluate the whole
44

kernel. Using light transectance we have been able

to analyse the structural dierences across the whole
of the barley corn.
In order to achieve uniform modication, it is
important that the endosperm becomes evenly
hydrated so that hydrolytic enzymes can uniformly
degrade the structural components of cell wall and
surrounding protein. During malting, maltsters aim
for total moisture content of around 4346%, but
even at this moisture level some parts of the endosperm can be relatively dry.23 Several factors, such
as variety, kernel size, total nitrogen, have been
reported to cause this, however, the current work
suggests that the structure of the endosperm is the
most important factor which ultimately determines
the distribution of moisture within the endosperm.
This is supported by the fact that steely samples
were able to reach cast moisture of about 44% but
showed slower penetration of water into the endosperm as identied by an iodine vapour method.
The dierences in grain morphology are due to
variations in the proportion and distribution of
protein and b-glucan within the endosperm. It is
generally thought that high nitrogen barleys are
steely, however high nitrogen does not necessarily
correlate with endosperm steeliness. Recent studies
on nitrogen content of individual kernels have shown
that there are dierences within an ear which were
attributed to the dierences in growing conditions.24
It has also been reported that under controlled nitrogen dressings and shading regimes employed during
plant growth, there were no clear dierences
between the nitrogen content of individual kernels of
a single spike, however, large variations between
spikes were found.14 The current work has shown
that dierences in the deposition of nitrogen within a
grain could have a profound aect on its breakdown.
Steelier samples such as Target store more nitrogen
in the endosperm where it is more likely to be
present as hordein. Proteins stored in the embryo, as
in samples with a high number of mealy grains, will
be more readily degraded and utilised for the manufacture of new enzyme proteins, whereas hordeins in
the endosperm will interfere with water and enzyme
distribution and thus reduce the homogeneity of
modication.
The breakdown of hordeins during malting has
been linked to malt quality by various authors.
Baxter25 has shown that a well-modied malt contains less than half the amount of hordein present in
the original barley with a pronounced decrease in the
B-hordein fraction. Similarly, it has been suggested
that gel proteins containing both B and D hordein
fractions can interfere with wort ltration and
analysis.26,27 The distribution of hordein in the
endosperm and its degradation during malting is,
therefore, an important aspect. Immuno-gold microscopic analysis reported here indicates that hordeins
with molecular weights of 34kD and 97kD are
associated with steely endosperm, and degradation of
J Sci Food Agric 79 :3746 (1999)

The structure of barley endosperm

these fractions is particularly important for attaining

a friable malt.
In addition to protein, the other principal dierence between mealy and steely grains is the amount
of b-glucan and the extent of its degradation. A
microuoro spectrophotometry method has been
used to show that b-glucan in oats is concentrated in
aleurone and sub-aleurone areas whilst in barley it
was mainly in the central endosperm.28 The current
investigation shows that although most of the bglucan in barley is present in central endosperm
there are dierences in its concentration from proximal to the distal region which can aect its degradation.
Using calcouor staining of b-glucans in half
corns, it has been reported that the normal pattern of
cell wall modication is for hydrolysis to occur rst
in the crushed cell layer adjacent to the scutellum,
proceeding from the ventral to the dorsal side, continuing under the aleurone layer near the scutellum
and nally towards the distal end of the grain.29 The
current investigation suggests that the presence of
areas rich in b-glucan adjacent to the scutellum in
the proximal endosperm may disrupt subsequent
degradation of b-glucan in the rest of the endosperm
by reducing the eectiveness of cell wall degrading
enzymes and thus aecting the extent of modication.

CONCLUSIONS
A method by which mealy and steely grains can be
identied using light transectance has been developed. Steely areas can be randomly distributed
within the endosperm and are associated with higher
concentrations of b-glucan and protein. Steeliness
appears to be a major factor restricting the even distribution of water across the endosperm during
steeping. This is supported by the observation that
the mealy grains of Target are capable of hydrating
as readily as those in Chariot and Blenheim.
However, the high proportion of steely grains in
Target resulted in much poorer endosperm hydration overall.
Uneven distribution of water across the endosperm can itself aect the distribution and activity of
key enzymes. The location of steely areas is therefore
crucial. For example, high levels of b-glucan associated with steely areas in the proximal endosperm of
Target are thought to have restricted the distribution
and activity of b-glucanases, restricting the enzyme
to the proximal end of the endosperm and preventing
it from reaching the central endosperm in time to
have any real eect in malting.
Steely areas of the endosperm are also associated
with a specic protein such as c-hordein, which is
found in the mealy areas of central endosperm. Complete degradation of both 34kD (c) and 97kD (D)
hordein would appear to be necessary in order to
obtain a friable malt. Observations also suggest that
J Sci Food Agric 79 :3746 (1999)

steely samples are more likely to store nitrogen

within the endosperm as hordeins rather than as
soluble proteins within the embryo, thus adding to
the difficulty in modifying higher nitrogen samples.
The morphological structure of the endosperm,
therefore, impacts on malt quality by aecting water
distribution. This will aect the release and extent of
activity of various enzymes, thus changing the
pattern of protein and cell wall breakdown. The proportion and location of steely areas within grains are
thus crucial factors impinging on malt quality.
ACKNOWLEDGEMENTS
The authors thank Professor CW Bamforth for critically reviewing this article. The Director General of
Brewing Research International is thanked for allowing us to publish this work. Dr J M Thornton and
Professor GH Palmer of Heriot Watt University are
thanked for their help during the project. Dr ENC
Mills of IFR Norwich is thanked for providing the
anitibodies. We also acknowledge the Home-Grown
Cereals Authority for partly funding the project.
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