Beruflich Dokumente
Kultur Dokumente
129140 (2007)
DOI: 10.1556/CRC.35.2007.1.15
The effects of sowing date, nitrogen application level and timing on barley protein components
and malt quality were investigated. There was a significant difference in total protein and its
protein fractions among the four barley genotypes. The protein component was changeable
over the different growing conditions, and the extent of change varied with protein fraction and
genotype. Marked variation in malt quality over the different environments (sowing date, N
fertilizer rate and applying time) was also observed. Increased N fertilizer application increased diastatic power (DP) value, but reduced malt extract. Grain protein content was significantly and positively correlated with albumin, globulin and hordein, but was not correlated
with glutelin. However, glutelin was significantly related to other malt quality parameters.
Keywords: barley (Hordeum vulgare L.), protein, protein fractions, malt quality
Introduction
Barley (Hordeum vulgare L.) suitable for malting should have grain protein content below 11.5%, as higher protein content will not only reduce malt extract, but
also reduce final beer quality (Rasmusson and Glass 1965). Conversely, higher
grain protein content is associated with desirable malt quality parameters (Eagles
et al. 1995; Molina-Cano et al. 1997; Zhang et al. 2006). It is well documented that
diastatic power (DP) is closely related to b-amylase activity (Arends et al. 1995;
Georg-Kraemer et al. 2001; Evans et al. 2003). b-Amylase is actually considered
as a barley storage protein as it comprises approximately 1% of total protein,
* Corresponding author; Fax: +86 571 86971498; E-mail: zhanggp@zju.edu.cn
130
which is beyond the apparent amount required for its physiological function
(Lauriere et al. 1986). However, no significant correlation was detected between
b-amylase activity and protein content among different cultivars planted in the
same environment (Zhang et al. 2006), although it was found that they were positively correlated for a given cultivar (Yin et al. 2002). Moreover, the barley
cultivars with similar protein content may differ greatly in malting quality. Therefore, it is suggested that protein composition may play a more important role in
malt quality than absolute total protein content.
Protein in barley grains can be divided into albumin, globulin, prolamin
(hordein) and gluten fractions according to Osborne (1924). The albumin, globulin and gluten fractions consist predominantly of structural and metabolic proteins, while hordein is the major storage protein. Protein content in barley grains is
quite variable between environments (Smith 1990), varying with available soil N
level (Weston et al. 1993; Eagles et al. 1995) and climatic factors (Coles et al.
1991; Grant et al. 1991). Similarly, the composition of the protein fractions is also
affected by environmental factors, including N level (Zhao et al., 2006), although
there is a distinct response between cultivars (Giese and Hejgaard 1984; Duffus
and Cochrane 1993). Qi et al. (2006) reported that N application rate had a significant effect on total protein and hordein content in barley grains. However, little is
known about the change in the composition of the protein fractions in barley
grains in response to agronomic factors, such as fertilization and sowing date,
which will alter the growing conditions for the growth and development of barley.
Although extensive research has been published on the relationship between
barley protein content and malt quality, the relationship between protein fraction
composition and malt quality is still not clear. Previous investigations were
mainly focused on the relationship between the hordein fraction and malt quality,
and the relationship has been found to be inconsistent (Howard et al. 1996;
Brennan et al. 1998).
Therefore, a comprehensive understanding of the relationship between malt
quality and protein fractions is imperative. The objectives of this study were to determine the effect of sowing date, N application rate and timing on protein components and also to illustrate the relationship between protein fractions and malt
quality.
Material and Method
Cultivars and field experiment
The field experiments were conducted at experimental farm of Huajiachi campus,
Zhejiang University in 2005. The soil type was silt-loam with medium fertility.
Cereal Research Communications 35, 2007
131
The experiment consisted of three trials: sowing date, N application level and timing. The experimental design used was a split-plot with three replicates. The main
treatments were sowing date, N rate and application timing, with sub-treatment
being cultivar. Plots were 2 3 m with 12 lines and 0.2 m between lines.
In the sowing date trial, 4 cultivars differing in malt quality were used,
namely Xiumai 3, 92-11, Zheyuan 18 and ZJU 3. There were three sowing dates:
7th, 11th and 17th of November. Other field management practices were the same
as did locally. In the N level trial, two commercial cultivars were used: Xiumai 3
and 92-11. There were three N application rates. All plots were supplied with 40
kg/ha N as base fertilizer before sowing. NL1, NL2 and NL3 treatments received
50, 75 and 100 kg/ha N at the 2-leaf stage and booting stage with same rate, respectively. In the N-timing trial, the same two cultivars were used as in the N level
trial. The total N application rate was 150 kg/ha N, with all treatments receiving a
40 kg/ha N application before sowing. Treatments NT1 and NT3 received the remaining 110 kg/ha N at 2-leaf stage or booting stage, respectively, while the NT3
treatment was top-dressed N fertilizer with 55 kg/ha at 2-leaf stage and 55 kg/ha
booting stage.
Measurement of grain protein and its compositions
Mature grains were ground in a Cyclotec 1093 sample mill (Tecator AB, Hoganas,
Sweden) to pass a 0.5 mm screen. Protein content was determined by near infrared
reflectance spectroscopy (NIRA, Matrix-1, Bruker Co., Germany) using a previously established calibration curve (Yin et al. 2002). Protein factions were sequentially extracted from samples (0.5 g) according to Shewry et al. (1983) with
some modifications. Albumin and globulin were extracted twice for 0.5 h each
with water and 0.15 M potassium phosphate (pH 8.0, 2.5 ml) at room temperature,
respectively. The residual material was collected by centrifugation at 2000 g for 5
min and the supernatants were combined. The hordeins were extracted three times
for 0.5 h each with 55% (v/v) propan-2-ol (2.5 ml), 1% (v/v) acetic acid (2.5 ml)
and 2% (v/v) 2-mercaptoethanol (2.5 ml) at 60 C. The glutens were extracted
three times for 0.5 h each with 0.05 M sodium borate (pH 10, 2.5 ml), 2% (v/v)
2-mercaptoethanol (2.5 ml); 1% (w/v) sodium dodecyl sulphate (2.5 ml) at room
temperature. Protein fraction was analyzed with bovine serum albumin (BSA) as
the standard protein.
Micro-malting and malt analysis
Barley grains were screened through a 2.2 mm sieve, and the grains remaining on
2.2 mm sieve was used for micro-malting. Samples (200 g) were micro-malted in
Cereal Research Communications 35, 2007
132
Sowing date
Albumin
Hordein
Glutelin
8.20a
9.14a
8.55a
12.77b
14.42a
9.96c
58.52a
57.65a
56.98a
12.43a
11.54a
9.38b
8.80a
9.08a
8.37a
13.82c
18.11a
16.35b
48.52b
48.45b
57.54a
D1
D2
D3
10.17a
10.74a
6.48b
7.28a
8.04a
7.32a
12.06b
16.56a
16.37a
38.65a
35.67a
36.98a
D1
D2
D3
8.45b
9.61b
11.64a
7.33a
7.38a
8.04a
14.69a
14.73a
11.88b
48.12b
52.27a
45.09b
(C)
(D)
Xiumai 3
D1
D2
D3
10.99a
9.33b
8.71b
92-11
D1
D2
D3
Zheyuan 18
ZAU 3
Globulin
mg/g
ns
The values within a column followed by different letters are significantly different at 95% probability.
s and ns represent significant and not significant at 95% and 99% probability levels, respectively. The follows are same.
133
mean of 37.10 mg/g. In the case of hordein content, 92-11 was the highest (16.09
mg/g), with Zheyuan 18 the lowest (7.54 mg/g). The influence of sowing date on
protein fractions varied with cultivars and protein fraction. There was a significant
difference in the composition of albumin, globulin and hordein among between
sowing dates for the cultivars but not for globulin content. The cultivars showed
different responses to sowing date as indicated by the change of protein fraction
composition. For instance, the highest albumin content was detected in the latest
sowing date for ZAU 3, while in the earliest sowing date for Xiumai 3 and 92-11.
Similarly, there was no significant difference among three sowing dates in
glutelin content for Xiumai 3 and Zheyuan 18, while for 92-11 and ZAU 3 the latest and the second sowing dates had significantly higher content than other sowing
dates, respectively.
The effect of N level on protein fraction composition in barley grains varied
with genotype and protein fraction (Table 2). Among the three N levels, there was
a significant differences in the globulin, hordein and glutelin contents for Xiumai
3 and in globulin and hordein contents for 92-11, while no significant difference in
albumin content for Xiumai 3 and in albumin and glutelin contents for 92-11. In
general, the content of each protein fraction increased with N level. There was a
significant difference between the two barley cultivars in protein fraction compoTable 2. The effect of N application level on protein fractions in two barley cultivars
Cultivar
Nitrogen
(C)
Level (NL)
Albumin
Globulin
Hordein
Glutelin
mg/g
Xiumai 3
NL1
NL2
NL3
10.14a
10.84a
11.77a
8.41b
10.07a
10.53a
12.05b
16.17a
18.19a
57.73c
74.03b
83.77a
92-11
NL1
NL2
NL3
12.56a
12.77a
13.87a
9.78b
10.29ab
11.23a
18.30b
19.80ab
21.59a
55.41a
55.43a
55.95a
ns
ns
ns
The same letter after data within a column means no significant at 95% probability.
ns and s, represent not significant and significant at 95% probability, respectively.
134
Nitrogen
(C)
Timing (NL)
Albumin
Globulin
Hordein
Glutelin
mg/g
Xiumai 3
NT1
NT2
NT3
10.63b
12.89a
8.88c
9.09ab
8.22b
10.35a
12.91a
14.29a
14.06a
67.52a
63.10a
71.78a
92-11
NT1
NT2
NT3
12.75a
11.51a
11.91a
9.83a
9.47a
8.91a
19.61a
19.41a
18.76a
51.15a
56.54a
56.73a
ns
ns
ns
The same letter after data within a column means no significant at 95% probability.
ns and s, represent not significant and significant at 95% probability, respectively.
Table 4. The effect of sowing date on malt quality parameters in four cultivars
Cultivar
(C)
Sowing
date
(D)
Malt extract
(%)
Viscosity
(CP)
Xiumai 3
D1
D2
D3
75.26a
74.92a
74.87a
1.46a
1.41a
1.42a
39.89a
36.01b
36.48b
338.24a
297.16a
324.38a
9.75a
10.90a
9.63a
92-11
D1
D2
D3
71.15a
70.84a
71.96a
1.63a
1.69a
1.67a
26.31a
26.45a
25.71a
347.51a
404.05a
363.69a
10.67b
11.66a
10.54b
Zheyuan 18 D1
D2
D3
74.79a
74.44a
75.29a
1.58b
1.67ab
1.75a
33.38a
31.69ab
29.65b
276.72b
362.99a
307.57ab
9.74b
11.17a
10.18b
ZAU 3
74.87b
75.66ab
77.41a
1.61a
1.61a
1.51a
30.26b
33.49b
36.38a
363.03a
289.85b
330.39ab
9.22a
9.32a
9.81a
D1
D2
D3
ns
Kolbach
index
(%)
Diastatic
power
(WK)
The same letter after data within a column means no significant at 95% probability.
ns and s, represent not significant and significant at 95% probability, respectively.
Grain protein
content
(%)
135
N Level
(NL)
Malt extract
(%)
Viscosity
(CP)
Xiumai 3
NL1
NL2
NL3
75.50a
72.51ab
70.41b
1.43a
1.45a
1.45a
36.89b
35.08b
41.92a
487.03b
541.66ab
562.71a
10.02b
10.70ab
11.74a
92-11
NL1
NL2
NL3
71.80a
69.34a
70.34a
1.59a
1.59a
1.59a
27.53a
28.49a
30.51a
415.22b
515.31a
460.89ab
11.42a
12.65a
11.80a
ns
ns
ns
ns
Kolbach
index
(%)
ns
Diastatic
power
(WK)
Grain protein
content
(%)
The same letter after data within a column means no significant at 95% probability.
ns and s, represent not significant and significant at 95% probability, respectively.
The effect of N application timing on malt quality for the two barley cultivars
was shown in Table 6. There were significant differences in DP and grain protein
Cereal Research Communications 35, 2007
136
Cultivar
(C)
Nitrogen
Timing
(NT)
Malt extract
(%)
Viscosity
(CP)
Xiumai 3
NT1
NT2
NT3
74.72a
73.31a
71.80a
1.48a
1.43a
1.47a
35.22a
38.60a
36.96a
351.55b
488.57a
419.09ab
10.14b
11.21a
10.69ab
92-11
NT1
NT2
NT3
72.43a
70.36a
70.72a
1.54a
1.57a
1.53a
32.08a
28.48a
27.86a
359.15b
485.42ab
499.53a
11.33c
12.10b
12.83a
ns
Kolbach
index
(%)
ns
ns
Diastatic
power
(WK)
Grain protein
content
(%)
ns
The same letter after data within a column means no significant at 95% probability.
ns and s, represent not significant and significant at 95% probability, respectively.
content between the timing of N application for the two cultivars, while no significant difference between the timing of N application in malt extract, Kolbach index
and viscosity was found. There were distinct differences in malt quality between
two cultivars, and the significant interaction between cultivar and N timing in
grain protein content.
Relationship between protein fraction composition and malt quality
Correlation analysis showed that hordein content was negatively correlated with
malt extract and Kolbach index, but positively correlated with diastatic power
(DP) and viscosity (Table 7). Albumin content was negatively correlated with
malt extract, and positively correlated with DP. Globulin content was negatively
correlated with malt extract, and positively correlated with DP. Glutelin content
was negatively correlated with malt extract, and positively correlated with DP. In
Table 7. Correlations among protein fractions and malt quality parameters
Albumin Globulin
Albumin
Globulin
Hordein
Glutelin
Grain protein
Malt extract
Viscosity
Kolbach index
Diastatic power
1
0.50*
0.45*
0.14
0.57*
0.51*
0.04
0.08
0.41*
1
0.50*
0.52*
0.55*
0.59*
0.15
0.01
0.47*
Hordein
Glutelin
1
0.04
0.70*
0.63*
0.35*
0.39*
0.42*
1
0.19
0.27*
0.56*
0.43*
0.54*
* Significant at P<0.05
Grain
protein
1
0.78*
0.17
0.36*
0.60*
Malt
extract
1
0.25*
0.46*
0.56*
Viscosity
1
0.69*
0.23*
Kolbach
index
1
0.09
137
addition, it was found that there was a positive correlation between grain protein
content and protein fraction composition.
Discussion
Protein composition in barley grains was dependent on growing conditions, including sowing date and N level in soil (Duffus and Cochrane 1993; Qi et al.
2005). Howard et al. (1996) also found that N fertilizer level greatly affected the
of protein fraction compositions in barley grains. While, Molina-Cano et al.
(2000, 2001) showed that there was a marked effect of N fertilization on protein
and hordein content in barley grains. In this investigation, significant differences
in total protein and its component content were found among four barley cultivars.
Meanwhile, the protein component was also sensitive to the growing conditions
(N level and sowing date), indicating that it was variable between environments.
On the whole, the content of each protein fraction composition increased with N
fertilizer level, although a distinct difference was found between genotypes in the
extent to which protein composition content changed, suggesting it is possible to
modify the N response of a protein fraction via a genetic approach. Moreover, it
was found that the response of protein fractions to agronomic factors, sowing date,
N level and application time varied with protein fraction type and genotype. In
general, albumin in barley grains was easily manipulated by sowing date and N
application time, but was less influenced by N level. Globulin showed the opposite response to albumin to these agronomic factors. The two primary storage protein fractions in barley grain, hordein and glutelin, were significantly affected by
sowing date and N level, but were less influenced by N application time. Qi et al.
(2005) also reported significant differences in hordein content between sowing
date treatments.
Protein concentration in barley grains is negatively correlated with malt extract and positively correlated with diastatic power (Bishop and Day 1993; Arends
et al. 1995; Howard et al. 1996). Molina-Cano et al. (1995) reported that grain protein concentration was significantly and negatively correlated with malt viscosity
and Kolbach index. The influence of N application timing on malt quality was also
studied by Chen et al. (2006). In that study, at equal N application rates, malt
Kolbach index and extract declined as top-dressed N ratio increased at later
growth stages, while DP and viscosity showed the opposite response, indicating
that more N application at later growth stage will reduce malt quality, although
there may be a slight quality improvement due to increase in DP.
In southern China, topdressing of N fertilizer at the booting stage is commonly recommended for barley production, as it may increase grains per spike
Cereal Research Communications 35, 2007
138
and delay leaf senescence during grain filling (Chen et al. 2004). In this study, we
found a marked variation in malt quality between environments for this treatment.
The results also showed that higher N application increased DP value, but reduced
malt extract. Moreover, the response of malt quality in response to these agronomic treatments varied with barley cultivar. Hence the significant interactions
were observed between genotype and sowing date or N treatments (level and application time). The results indicate the importance of adopting suitable sowing
date and N fertilizer application regimes that are specific for individual malting
barley varieties to improve resulting malt quality. The investigation also showed
that it was possible to improve the stability of malt quality performance between
environments by appropriate cultivar selection.
It has been reported that grain protein content was negatively and significantly correlated with malt extract, and the B hordein displayed the strongest negative correlation with malt extract (Howard et al. 1996; Molina-Cano et al. 2000;
Qi et al. 2005). In the present study, we found that grain protein was also highly
and negatively related to malt extract, Kolbach index, and significantly but positively correlated with DP. We also observed that grain protein was significantly
and positively correlated with albumin, globulin and hordein levels, but did not influence glutelin levels. However, glutelin was significantly and negatively correlated to all four malt parameters measured in this study. Overall, it is concluded
that it may be possible to improve malt quality while keeping grain protein concentration below the critical level of 11.5% by breeding for lower levels of
glutelin.
Acknowledgements
The authors are grateful to the Natural Science Foundation of China (30471022) for its financial support.
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