Vol.2, No.3, 181-190 (2011)
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Agricultural Scienc es
Effect of split applications of urea on protein size
distribution, physical dough properties, and baking
performance of five experimental bread wheat lines
Alma Rosa Islas-Rubio1*, Karla Chávez-Quiroz1, Francisco Vásquez-Lara1, Brenda
Silva-Espinoza1, María del Carmen Granados-Nevárez1, Humberto Gonzalez-Ríos1, Miguel
1Departamento de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, A.C., Carretera
a la Victoria Km 0.6, Hermosillo, Sonora, México; *Corresponding Author: aislas@ciad.mx
2Campo Experimental Valle del Yaqui, Centro de Investigación Regional del Noroeste, Instituto Nacional de Investigaciones
Forestales, Agrícolas y Pecuarias, Ciudad Obregón, Sonora, México.
Received 18 March 2011; revised 19 May 2011; accepted 9 July 2011.
Five experimental bread wheat lines (BWL) were
grown at the Yaqui Valley Experimental Station
in Sonora, México during two consecutive grow-
ing cycles. The effect of five nitrogen fertiliza-
tion (NF) treatments on protein size distribution
(PSD), physical dough properties (PDP), and
baking performance of the BWL was evaluated.
Polymeric and monomeric proteins were evalu-
ated by SE-HPLC. PDP were carried out by the
National Mixograph and the TA-XT2 Texture
Analyzer. Baking performance was evaluated
using the straight dough method. Protein con-
tent (PC), main protein fractions (glutenins, gli-
adins, albumins-globulins), and mixograph de-
velopment time (MDT) were significantly influ-
enced by NF trea tmen t a nd BWL but no t by th eir
interaction. On the other hand, PDP measured
by the Kieffer rig, and baking performance were
significantly influenced by the main factors (NF
and BWL) and their interaction. The amount and
timing of fertilizer applied to the BWL modified
the PC, PSD, PDP, and bread loaf volume. PDP
exhibited a larger variation in comparison to the
PSD of glutenins. The split application of 150 kg
of urea/ha (50-50-50) to all BWL showed a better
loaf volume response than the same amount of
urea applied at sowing (150-0-0). The applica-
tion of 300 kg of urea/ha to all BWL, either at
sowing or at three split applications of 100 k g of
urea/ha each, resulted in higher flour unex-
tractable polymeric p rote in ( FUPP ). On the other
hand, the split application of 100-100-100 kg of
urea/ha to three of the BWL represented the
higher total unextractable polymeric protein
(TUPP). Differences on PC and PSD were re-
flected on differences on PDP and bread loaf
volume observed among the BWL.
Keywords: Wheat; Nitrogen Fertilization; Protein
Composition; Dough Properties
Wheat quality is determined by genetic and environ-
mental factors. These factors affect protein content and
composition, therefore gluten functionality. Nitrogen
fertilization is an important management practice that
influences the amount of protein accumulated in the
grain. The amount of the most difficult to extract pro-
teins has been reported to be a major determinant of
gluten strength [1-3]. Grain protein composition is one
of the most important factors determining the bread-
making quality of wheat flour [4]. Protein accumulation
during grain filling has been investigated and found to
be influenced by irrigation and fertilizer rate [5,6]. Tem-
perature and nitrogen timing have been reported as the
main environmental factors leading to variation of a-
mount and size distribution of polymeric protein at grain
maturity [7-9]. Differences in amounts of all types of
proteins due to temperature have been reported [10]. The
influence of nitrogen fertilization on amounts and pro-
portions of different protein types in wheat flours was
reported [11]. Also, the effect of sulfur alone or in com-
bination with nitrogen fertilization on commercial-scale
quality, mixing requirements, protein composition and
dough strength has been investigated [12,13].
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The dependence of various quality parameters on pro-
tein composition has been determined [3,14-18]. Rela-
tionships reported in these studies are based on physical
dough measurements with instruments that require a
considerable amount of sample. Few studies have evalu-
ated the relationships of breadmaking performance of
wheat flours with rheological measurements at a micro-
scale level [19-21]. The use of small sample sizes and
the ability to measure protein composition related to
functional properties of wheat flours are required in
breeding programs. To our knowledge no report has been
made regarding the effect of nitrogen fertilization on
protein size distribution, micro-scale physical dough
measurements and baking performance of wheat flours.
Knowing the response of experimental wheat lines to
nitrogen fertilization may help to determine the nitrogen
level that results in an improved breadmaking quality.
Therefore, the aim of this study was to evaluate the ef-
fect of nitrogen fertilization (NF) treatments on protein
size distribution, micro-scale physical dough measure-
ments and baking performance of flours from experi-
mental bread wheat lines (BWL).
2.1. Wheat Cultivation
Five bread wheat lines were grown at the Yaqui Valley
Experimental Field in Ciudad Obregon, Sonora, Mexico,
during the 2002-2003 and 2003-2004 growing seasons
(Table 1). A randomized complete block design was
used in planting three replicate plots of each BWL under
the following nitrogen (urea) fertilization treatments: no
fertilization (0-0-0), application of 150 kg/ha at sowing
(150-0-0), 300 kg/ha at sowing (300-0-0), and three
split-application (at sowing-at the first auxiliary irriga-
tion-at the second auxiliary irrigation) of 150 kg/ha (50-
50-50) and 300 kg/ha (100-100-100). Wheat samples
from each replicate plot (8 m2) were separately harvested,
numbered, bagged and sent to the laboratory for milling
and subsequent quality evaluations.
Table 1. Origen of experimental bread wheat lines.
BWLa Pedigree
1 Vee/Koel/Siren/3/Ariv92
2 Rayon_F89
3 Irena/Babax/Pastor
4 Rabe/6/Wrm/4/Fn/3*Th//K58/2*N/3/Aus-6869/5/Pelotas
5 Weebil_35Y
aBread wheat line.
2.2. Wheat Milling and Quality Testing
Wheat samples from each replicate plot were tem-
pered (14% moisture content) overnight and milled in a
Brabender Quadrumat Jr. Mill (Hackensack, NJ) as de-
scribed in Approved Method 26-21A [22]. Milled flours
were evaluated for moisture and ash content using Ap-
proved Methods 44-19 and 44-08. Protein content (PC)
was determined according to the Kjeldahl method, using
a factor of 5.7 to calculate protein from nitrogen.
2.3. Protein Extraction and SE-HPLC
The total protein (TP) was extracted by sonicating
flour (10 mg) suspensions in 0.5% SDS in 0.05 M so-
dium phosphate buffer (1 mL), pH 6.9, for 15 s at power
setting of ~3 (output 6 Watts) in a membrane Dismem-
brator (model 100, Fisher Scientific, Pittsburg, PA) using
a stepped microtip probe (3 mm diameter) as described
by Batey et al. [23]. The extractable protein (EP) was
determined by collecting the supernatant, once the flour
suspension in SDS buffer was stirred for 5 min (no
sonication). The residue (pellet) from the previous ex-
traction was then sonicated for 25 s in the buffer (1 ml)
to solubilize the remaining protein, that is the unex-
tractable protein (UP). All extracts were filtered (0.45
mm filter), heated at 80˚C for 2 min in a water bath and
cooled with ice and water prior to injection in a BioSep-
SEC-S 4000 column (Phenomenex, Torrence, CA). The
HPLC profile was divided into peaks 1, 2 and 3 corre-
sponding to polymeric protein (TPP or glutenins), gli-
adins, and albumins/globulins and the absolute and rela-
tive peak areas were recorded. The ratio of EP peak 1 to
UP peak 1 or the percentage of UP peak 1 in TP peak 1
(EP peak 1 + UP peak 1), a measure of the relative size
distribution of the polymeric protein, was also calculated
and reported as total unextractable polymeric protein
(TUPP). The percentages of flour polymeric protein
(FPP) and flour unextractable polymeric protein (FUPP)
were also calculated. All SE-HPLC measurements were
made in duplicate and averaged.
2.4. Physical Dough Properties
Optimum water absorption and mixing time (MDT)
for each flour sample was determined in duplicate by a
10-g mixograph (National Mfg. Co., Lincoln NE). Mixo-
grams were determined using a modified AACC method
(54-40A) that included 2% NaCl w/w on flour and MDT
was recorded and used to mix the dough for the micro-
scale extension test.
The micro-scale dough extension test reported by
Kieffer et al. [19] was carried out in duplicate with the
Texture Analyzer (TA.XT2, SMS/Kieffer dough extensi-
bility rig, Stable Micro Systems, Surrey, England).
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Dough was optimally mixed in a 10-g National mixograph
and kept in a proofing chamber for 45 min at 30˚C af-
terwards, dough extensional properties were recorded.
Test speed for the micro-scale extension test was 3.3
mm/s. The maximum force (Rmax) and distance to rup-
ture (Ext) were considered as an indication of dough
resistance and extensibility, respectively. The area under
the curve, which represents the dough deformation work,
was also recorded. For the Kieffer rig extensibility test,
the average of at least five measurements was considered
a replicate.
2.5. Baking Test
All flour samples (35 g, 14% mb) were mixed with
salt (0.53 g), dry yeast (0.7 g), sugar (2.1 g), shortening
(1.05 g), and water in a 35-g mixograph (National Mfg.
Co., Lincoln, NE) and baked according to AACC
Method 10 - 10B. Doughs were sheeted with roller gaps
of 0.119” for pre-molding punch, and 0.143” for 1st and
2nd punches (M.D. Shogren, personal communication).
In this procedure, doughs were placed in 35-g baking
pans, proofed for 40 min at 30 ± 1˚C and 94% rh, and
baked for 17 min at 215˚C. On completion of baking,
each loaf was weighed and its volume determined by the
rapeseed displacement method. The specific volume of
each loaf (BSV) was calculated by dividing the loaf
volume (BLV) and weight (BWT). Two loaves of bread
were prepared from each flour sample per replicate.
2.6. Experimental Design and Statistical
A randomized complete block design with factorial
arranges was used in the experiment. The growing cycle
was considered as random effect of the block. BWL (5)
and NF treatments (5) were considered as factors and
three experimental units by treatment were used. The
effects of growing cycle, BWL, NF, and BWL x NF in-
teraction on the response variables at 5% level (p < 0.05)
were evaluated by the general linear model procedure
with the NCSS statistical package [24]. Three replicates
were analyzed for each BWL subjected to each NF
treatment. The BWL and NF were considered as fixed
effects. When significant effects were found, the mean
comparisons were done by the Tukey’s multiple range
test. Additionally, principal components analysis (PCA)
was conducted to obtain a small number of factors that
account for most of the variability in the response vari-
3.1. Flour Quality Testing
Moisture and ash content of flours (data not shown)
ranged between 11.4% - 13.7%, and 0.37% - 0.53%,
respectively. PC was significantly influenced by the
BWL and NF treatment (Table 2). The average PC of the
BWL was between 10.4% and 12.2% (Table 3). The
BWL 1 contained a significantly higher amount of pro-
tein than the rest. The lower PC corresponded to BWL 3
and 5. A high availability of nitrogen after anthesis gen-
erally leads to a high concentration in the kernel [25],
therefore it is worthwhile to know how long it takes for
the different BWL to reach maturity. Availability of ni-
trogen after anthesis is unknown since the amount of
nitrogen in the soil previous to sowing was not deter-
mined in this study. Furthermore, nitrogen applied in the
form of urea, which was the case, is susceptible to loss
through drainage. The application of nitrogen to all the
BWL had a significant effect on PC. Depending on the
NF treatment, the average PC fluctuated between 9.1 and
12.1% (Table 3); the lower PC value corresponded to
the unfertilized BWL (0-0-0). In general, the PC in-
creased as the amount of nitrogen applied increased as it
was previously reported [20]. The application of 300 kg
of urea/ha resulted in higher PC than the application of
150 kg of urea/ha (12% - 12.1% vs. 10.7% - 11%). This
finding is in agreement with other authors [11,26]. It has
been demonstrated that management of nitrogen fertili-
zation can increase grain protein in wheat and the in-
crease of protein caused by higher levels of nitrogen
fertilization is strongly dependent on the genotype.
3.2. Protein Composition and Size
The interaction of BWL and NF treatment was not
significant for PC, TPP (glutenins), gliadins, and albu-
Tabl e 2 . Effect of bread wheat line, nitrogen fertilization treatment and its interaction on protein composition, rheological measure-
ments and bread loaf volume of floursa.
Source PC TPP Gliad TUPP MDT Rmax Ext Area BLV
BWL 35.2*** 3.5** 35.3*** 5.3*** 10.2*** 6.7*** 8.3*** 6.2** 30.5***
NFT 93.7*** 2.9* 178.6*** 52.7*** 30.8*** 12.5*** 5.1*** 10.7*** 36.2***
BWL*NFT 0.8 1.2 0.9 3.7*** 1.2 2.5** 1.8* 2.2** 3.0***
aF-values and level of significance (*, **, and *** represent p < 0.05, p < 0.01, and p < 0.0001, respectively.). BWL = bread wheat line, NFT = nitrogen fertiliza-
tion treatment, PC = protein content, TPP = total polymeric protein or glutenins, Gliad = gliadins, TUPP = total unextractable polymeric protein, MDT =
Mixograph development time, Rmax = dough maximum resistance, Ext = extensibility, Area = dough deformation work, BLV = bread loaf volume.
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Ta b l e 3 . Mixograph development time, protein content and composition of flours recorded in every bread wheat line as well as in
every nitrogen fertilization treatment, after two growing seasonsa.
Main Effect MDT (min) Protein (%) TPP (%) Gliadins (%) Alb + Glob (%) FPP (%)
BWL: 1 4.7 ± 0.2c 12.2 ± 0.3a 42.9 ± 0.4ab 44.1 ± 0.4a 13.1 ± 0.4c 5.2 ± 0.1a
2 5.3 ± 0.2ab 10.9 ± 0.3bc 43.8 ± 0.3a 42.5 ± 0.4b 13.7 ± 0.4bc 4.7 ± 0.1b
3 5.1 ± 0.2b 10.4 ± 0.2c 43.0 ± 0.4ab 41.8 ± 0.4c 15.3 ± 0.6a 4.5 ± 0.1c
4 5.0 ± 0.2bc 11.0 ± 0.2b 43.6 ± 0.3a 41.4 ± 0.4c 15.0 ± 0.4ab 4.8 ± 0.1b
5 5.5 ± 0.2a 10.4 ± 0.2 c 42.3 ± 0.6b 43.0 ± 0.5b 14.6 ± 0.7ab 4.4 ± 0.1c
NFT: 0-0-0 5.0 ± 0.2b 9.1 ± 0.2c 42.9 ± 0.8ab 38.7 ± 0.3c 18.2 ± 0.7a 3.9 ± 0.1c
150-0-0 6.0 ± 0.2a 11.0 ± 0.2b 43.7 ± 0.3a 42.3 ± 0.3b 14.0 ± 0.2b 4.7 ± 0.1b
300-0-0 4.9 ± 0.1bc 12.1 ± 0.2a 43.0 ± 0.2ab 44.3 ± 0.2a 12.8 ± 0.2c 5.2 ± 0.1a
50-50-50 5.1 ± 0.2b 10.7 ± 0.1b 43.5 ± 0.2ab 42.7 ± 0.2b 13.7 ± 0.3b 4.7 ± 0.1b
100-100-100 4.6 ± 0.1c 12.0 ± 0.1a 42.4 ± 0.3b 44.6 ± 0.3a 13.0 ± 0.3c 5.2 ± 0.1a
aValues represent the mean (n = 30) ± standard error. Within a column and the same main effect, values followed by the same letter are not significantly differ-
ent at p = 0.05 level. BWL = bread wheat line, NFT = nitrogen fertilization treatment with urea (at sowing-first auxiliary irrigation-second auxiliary irrigation),
MDT = mixograph development time, TPP = total polymeric protein, Alb + Glob = albumins and globulins, FPP = polymeric protein in the flour, FUPP = unex-
tractable polymeric protein in the flour.
mins/globulins. The mean values of these parameters for
each BWL and NF treatment are shown in Table 3. The
averages of the percentage of polymeric protein (gluten-
ins), gliadins, and albumins/globulins among the BWL
varied from 42.3% to 43.8%, 41.4% to 44.1%, and
13.1% to 15.3%, respectively. Depending on the NF
treatment, these main protein fractions varied between
42.4% and 43.7% (glutenins), 38.7% and 44.6% (gli-
adins), and 12.8% to 18.2% (albumins/globulins). The
percentage increase of gliadins was higher than that of
glutenins; this means that the ratio gliadins/glutenins
was increased by a high nitrogen level which is in
agreement with other studies [11,17,27-29]. A significant
reduction in the amount of albumins/globulins was ob-
served when urea was applied to all BWL (18.2% vs.
12.8% - 14%). It has been reported that this protein
group is scarcely influenced by nitrogen nutrition
[11,14,30,31] and it does not play an important role in
breadmaking quality of wheat flours. [32-34].
The three split-application of 300 or 150 kg of urea/ha
to all BWL resulted in a slight decrease on the polymeric
proteins in comparison with the same amount of urea
applied at sowing, with a concomitant increase in gli-
adins. A higher availability of nitrogen has been reported
to increase gliadins synthesis and decrease of glutenins
[17]. Most studies described in the literature report that
the gliadins/glutenins ratio was significantly increased
by a high nitrogen level [11,17,29]. This could explain
the variation in the amounts of glutenins and gliadins
observed in this study.
The FPP was not affected by the interaction of BWL
and NF treatment, but it was by BWL and NF treatment.
The averages of FPP among the different BWL and NF
treatments are shown in Ta bl e 3. The BWL 1 had a sig-
nificantly higher percentage of FPP, whereas BWL 5 and
3 showed the lowest percentage (4.4% and 4.5%, respec-
tively). Similar values of FPP were observed for BWL 2
and 4. Differences in the amounts of glutenins and PC
between these BWL are responsible for these FPP val-
NF treatment, BWL, and their interactions signifi-
cantly influenced the TUPP (Table 2) and FUPP (Figure
1(e)). Depending on the BWL and the amount and tim-
ing of urea application, the averages of the TUPP were
between 35.7% and 53.7% (Figure 1(d)), whereas those
of the FUPP were between 1.28% and 2.91%. The split
application of 100-100-100 kg of urea/ha to each BWL
rendered the higher TUPP, whereas the application of
50-50-50 kg of urea to BWL 1, 2 and 5 produced the
lower TUPP values for these BWL. On the other hand,
the lower TUPP values of BWL 3 and 4 were obtained
when 150 kg of urea were applied at sowing. This pro-
tein fraction was suggested as an effective evaluation
parameter in wheat breeding programs targeting quality
[35]. The FUPP increased with the application of urea to
each BWL. The BWL responded differently to the
amount and timing of urea application. The amount of
300 kg of urea, either at a single application at sowing
(to BWL 1, 2 and 5) or at three split applications
(100-100-100) (to BWL 3 and 4) gave the higher FUPP.
Four of the BWL showed the lower FUPP when the split
application of 50- 50-50 kg of urea was used.
The amount and timing of urea application had a sig-
nificant effect on protein size distribution of most BWL.
Small differences in TUPP were observed when 150 kg
of urea/ha (150-0-0 or 50-50-50) were applied to all
BWL. Higher values of FPP and FUPP were observed
when 300 kg of urea/ha were applied to all BWL, either
at sowing (300-0-0) or at split applications (100-100-
100), but there was no significant difference between
these two NF treatments. Gupta et al. [3,17] found a
stronger relationship between the polymeric protein
content of flours and Rmax values of dough rested for 45
min than with the total protein content in flours. Other
uthors [16] reported that the glutenin macropolymer a
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(a) (b)
(c) (d)
(e) (f)
Figure 1. Effect of nitrogen fertilization treatments on dough maximum resistance (a), extensibility (b), deformation work (c), total
unextractable polymeric protein (d), flour unextractable polymeric protein (e), and bread loaf volume (f) of the experimental bread
wheat lines. Data are means ± standard error for n = 3 replicates. Different lowercase letters on the top of bars indicate significant
differences (p < 0.05). Nitrogen fertilization treatments (kg of urea/ha): 0-0-0(), 150-0-0 (), 300-0-0 (), 50-50-50
(), 100-100-100 ().
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(GMP) content of dough after 45 min rest was more
strongly related to Rmax, whereas the GMP content of
flour was strongly related to extensibility and loaf vol-
ume. Larger molecules form stronger network which
stands a higher force before rupturing. The application
of urea to all BWL increased the TUPP and FUPP (Fig-
ures 1(d) and (e)). An increase in TUPP has been re-
ported to shift the molecular weigth distribution of the
glutenin to higher molecular weigths [18] and this has
also contributed, in some cases, to decreased extensibil-
ity. The extensional properties of doughs in those studies
were measured with the Brabender Extensigraph.
3.3. Physical Dough Properties
The minimum-maximum water absorption and MDT
values were 57.7% - 64.1% and 4.3 - 6.3 min, respec-
tively. The application of urea differently affected the
MDT of the flours (Table 3). Longer MDT was observed
in flours from BWL 5, followed by BWL 2. An extended
MDT is not desirable as this is an added cost to baking.
A maximum MDT of 4.5 min is desirable in the bread
wheat breeding program in Mexico. The proportion of
polymeric protein and not the total amount in the grain
or flour might be responsible for the longer MDT [15,
Measurements reported in this study corresponded to
the 45 min rest dough. The extension parameters ob-
tained at 45 min of rest time are more reproducible com-
pared with longer resting time [36]. The application of
urea differently affected the extensional properties of the
flours. Rmax (Figure 1(a)) ranged from 20.3 to 112.9 g.
On the other hand, dough Ext (Figure 1(b)) varied be-
tween 44.9 and 74.2 mm. The deformation work or area
under the TA-XT2 curve (Figure 1(c)) varied between
1,193.6 and 5,879.6 g-mm. The Rmax of all BWL in-
creased with the application of urea and the increase
depended on the BWL and the amount and timing of the
application. The higher increases in Rmax were ob-
served with BWL 2 and 3. An amount of 300 or 150 kg
of urea/ha applied to BWL 2 (300-0-0 or 50-50-50) and
BWL 4 (100-100-100 or 150-0-0) resulted in doughs
with similar strength (Rmax 110 - 113 g and 83 - 84 g,
corresponding to BWL 2 and 4, respectively). The
strongest dough obtained from BWL 1 corresponded to
the application of 150 kg of urea/ha at sowing, whereas
the application of 100-100-100 kg of urea/ha to BWL 5
produced the strongest dough for this BWL. In addition
to strength, breadmaking requires dough that is extensi-
ble, allowing ease of handling and the rising of bread to
form a large loaf [37]. Regarding to Ext, the application
of 150 kg of urea at sowing (150-0-0) to three of the
BWL resulted in a slight decrease in dough Ext as com-
pared with the unfertilized counterparts. BWL 4 showed
a reduction in dough Ext with any of the applications of
urea. The opposite happened with BWL 5. The more
extensible doughs from BWL 1, 3 and 5 corresponded to
the NF treatment of 100-100-100 kg of urea/ha. The
balance of dough strength and extensibility are believed
to be the most important factors governing the suitability
of a flour to make good bread [38].
As expected, the application of urea to all BWL made
their flours stronger (Figure 1). The doughs that re-
quired more work to be deformed were those obtained
from flours of the BWL 2 (300-0-0 NF treatment), BWL
4 (100-100-100), BWL 3 (150-0-0), and BWL 1 (150-0-
0). The deformation work represented by the area under
the curve described by the texture analyzer TA-XT2 re-
sembles that described by the Brabender Extensigraph,
but the difference between these curves is that the tex-
ture analyzer curve is stopped once the dough ruptures,
in other words, it last to the peak which corresponds to
the maximum resistance. This area is not comparable to
that obtained by the Brabender Extensigraph (W value).
The value of the area under the curve described by the
texture analyzer was not a good predictor of breadmak-
ing quality among wheat flours, as it is the W value of
the Brabender Extensigram. It seems to be more impor-
tant the shape of the curve to explain differences in
breadmaking quality. Extensible doughs are preferred for
breadmaking, but not too weak neither too strong. In the
case of BWL 2, two of the NF treatments produced a
very strong dough (110.3 and 112.9 g) with good exten-
sibility (59.2 and 67.4 mm), whereas the dough obtained
from the same BWL when 100-100-100 kg of urea/ha
were applied to it presented a more balanced gluten
(Rmax = 75.1 g and Ext = 65.3 mm), which resulted in a
higher loaf volume (254.2 cm3). For BWL 4, it appears
that the application of 50-50-50 kg of urea/ha produced
the best balance between Rmax and Ext (71.4 g and 65.2
mm). Based on the dough Ext values obtained from the
set of samples used in this study, it appears that Rmax
values higher than 80 g did not favor the gluten balance;
therefore, increases in loaf volume were not expected for
those BWL subjected to the NF treatments that resulted
in very strong doughs. In general, BWL 4 presented the
strongest dough whereas BWL 5 produced the weakest
one. Bangur et al. [15] and Weegels et al. [16] found that
dough strength is related to the fraction of polymeric
protein with the highest molecular weight. Similarly,
other authors [3,39] reported that attributes related to
dough strength increase with a higher proportion of very
large glutenin polymers. Also, Extensigram height (Rmax)
positively correlated to the percentage of protein only
extractable into SDS-buffer after sonication [3]. There-
fore, the observed differences in dough strength can be
attributed, in part, to differences in TUPP, FPP, and
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3.4. Loaf Volume
Bread loaf volume varied between 133.1 and 296.8
cm3. In general, higher loaf volumes were obtained when
urea was applied to all BWL and different responses
among BWL were observed (Figure 1(f)). For BWL 1
and 5, the application of 300 kg urea/ha, either at sowing
or at three split applications of 100 kg of urea each, re-
sulted in higher loaf volume (~300 cm3 for BWL 1 and
~240 cm3 for BWL 5). On the other hand, BWL 2
showed the highest loaf volume when three split appli-
cations of 100 kg urea/ha were used. The lower increase
in loaf volume corresponded to BWL 3. The breadmak-
ing quality of this BWL can be marginally improved
through nitrogen (urea) management. None of the flours
from BWL 3 produced loaves of bread with a volume
higher than 200 cm3. The lowest loaf volumes of breads
made from this BWL could be due to the limited re-
sponse in PC (it showed the lowest average) to the ap-
plication of urea. High PC has been associated with high
loaf volume [40-42]. For BWL 3 and 4, the split applica-
tion of 50-50-50 kg of urea/ha produced the higher loaf
volume. The split application of 150 kg of urea/ha (50-
50-50) to all BWL resulted in significantly higher loaf
volume than the application of 150 kg/ha at sowing.
When 300 kg of urea were applied to BWL 1, 3, and 5,
either as split application (100-100-100) or 300 kg/ha at
sowing, no significant differences in bread loaf volume
were observed.
The differences in bread loaf volume among BWL
might be ascribed, in part, to the potential of each BWL
to synthesize protein and the availability of nitrogen
during grain filling. Also, these differences can be ex-
plained by looking at differences in physical dough
properties. It is well known that flours of good bread-
making quality are characterized by a suitable balance
between dough resistance to extension (Rmax) and ex-
tensibility (Ext) [43]. In general, BWL-5 produced
loaves of bread with the lower volumes and its doughs
showed low resistance to extension (27.3 to 60.2 g) and
intermediate extensibility (average 56.9 mm), which
confers a lower strength to the dough. Both, Rmax and
Ext, are important for retaining the gas produced during
fermentation. An excessive resistance to extension may
result in lower loaf volume. Dough with poor extensibil-
ity is not desirable for breadmaking. Previous studies
showed that total glutenin content in flour (TPP) largely
affected the dough strength [44,45], but this measure is
not as widely applicable as a selection criterion as TUPP
and % UPP [3].
It is worthwhile to mention that, in this work, the
dough extensional properties were measured with the
Texture Analyzer TA-XT2 using a smaller amount of
flour (10 g) than the amount required for the Brabender
Extensigraph. In addition, the standardized baking pro-
cedure using 35 g of flour as well as the small amount of
sample required for SE-HPLC analyses (<1 mg) make
this methodology suitable for wheat breeding programs
where the amount of sample is limited. A total of 130 g
of flour is enough to run duplicates for most measure-
ments, except protein, ash, and moisture analyses which
are run in triplicate. This methodology was used in a
previous study [21] and the generated data used to pre-
dict loaf volume. Most of the variation in bread volume
(87%) was explained by PC, MDT, Mixograph water
absorption, dough deformation work (area under the
TA-XT2 curve), and TUPP.
3.5. Principal Component Analysis
Factor analysis using principal components (PCA)
resulted in the extraction of four factors that had Eigen
values 1 and together accounted for more than 80% of
the variability in the fourteen response variables studied
(Tab le 4). The first factor (PC1) has an Eigen value of
5.88, that is, it absorbs the variability of almost 6 vari-
ables, showing a contribution of 42% of the variance.
The PC2 has an Eigen value of 2.49 and an explained
contribution of 17.8%. The PC3 and PC4 showed Eigen
values of 1.77 and 1.06, respectively. Analyzing the
composition and contribution (Eigen values) of the
original variables within each selected principal compo-
nent (Ta b l e 5 ), it is observed that PC1 is an average of
all variables, even if a greater negative contribution of
the variables PC, BLV, BSV, gliadins, FPP, TUPP, and
FUPP is shown. Therefore, this suggests that PC1 is re-
lated to the indicative variables of protein quality of the
wheat flours. The influence of TPP, MDT, and BWT was
small, since their loadings for PC1 is close to null. The
PC2 can be described like a contrast of the variables,
where TPP, Area, and Rmax participate with positive
charge and MDT, BLV, BSV, and albumins-globulins
with negative charge, which suggests that the dimen-
sionality of PC2 is related to the rheological properties
of the flours. In PC3, the Eigen vectors indicate a greater
contribution of Rmax, dough deformation work (area),
and MDT with negative charges, therefore, the relation-
ship of PC3 with the technological characteristics of the
wheat flours. In PC4, the Eigen vectors indicate a close
relationship between PC and gliadins. The influence of
Ext is large, whereas that of albumins-globulins is small.
TUPP is more closely related to BLV than dough Ext.
The application of urea to all BWL increased PC, the
A. R. Islas-Rubio et al. / Agricultural Science 2 (2011) 1 81-190
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Ta b l e 4 . Eigen values, contribution of individual and cumula-
tive variance of the principal components to the total variabil-
ity obtained by the principal component analysis in the four-
teen response variables studied.
number Eigen value Individual variance
variance (%)
1 5.880 42.00 42.00
2 2.493 17.81 59.82
3 1.776 12.69 72.51
4 1.067 7.62 80.13
5 0.957 6.84 86.97
6 0.788 5.63 92.61
7 0.463 3.31 95.91
8 0.279 2.00 97.91
9 0.247 1.76 99.68
10 0.019 0.14 99.81
11 0.013 0.10 99.91
12 0.008 0.06 99.97
13 0.002 0.02 99.99
14 0.000 0.01 100.0
Ta bl e 5. Contribution (Eigen vectors) of the original variables
in the selected principal components.
Principal Components
Va ri ab l ea
PC –0.365 –0.105 0.103 –0.255
MDT 0.023 –0.397 –0.389 –0.009
BLWT 0.088 –0.184 0.178 0.469
BLV –0.301 –0.351 0.003 0.214
BSV –0.308 –0.326 –0.016 0.158
Rmax –0.164 0.274 –0.570 0.111
Ext –0.204 –0.024 0.245 0.599
Area –0.184 0.248 –0.548 0.197
TPP –0.024 0.534 0.194 0.224
Gliadins –0.342 –0.099 0.036 –0.308
Alb-glob 0.291 –0.351 –0.199 0.050
FPP –0.365 0.088 0.167 –0.200
TUPP –0.286 0.023 –0.105 0.201
FUPP –0.387 0.032 0.030 –0.064
aPC = protein content, MDT = mixograph development time, BLWT =
bread loaf weigth, BLV = bread loaf volume, BSV = bread specific volume,
Rmax = maximum resistance, Ext = extensibility, Area = deformation work,
TPP = total polymeric protein, Alb-glob = albumins-globulins, FPP = flour
polymeric protein,TUPP = total unextractable polymeric protein, FUPP =
flour unextractable polymeric protein.
ratio gliadin to glutenin, dough strength (Rmax and de-
formation work), TUPP, and FUPP of the flours. The
increase depended on the BWL and the amount and tim-
ing of the application. Higher bread loaf volumes were
obtained when urea was applied to all BWL and differ-
ent responses among BWL were observed. For BWL 1
and 5, the application of 300 kg of urea/ha, either at
sowing or at three split applications of 100 kg each, re-
sulted in higher loaf volume. In the case of BWL 2, the
highest loaf volume was obtained when three split ap-
plications of 100 kg of urea/ha were used. For BWL 3
and 4, the split application of 50-50-50 kg of urea/ha
produced the higher loaf volume. Even other environ-
mental factors also exert their effect, it is possible to
change the protein composition of the flours and en-
hance the balance between dough Rmax and Ext in order
to improve the breadmaking quality through the man-
agement of urea.
We thank the National Council for Science and Technology of Mex-
ico (CONACYT) for funding this research (Project 40239-Z) and
providing scholarships for participating students. We also thank all
members of the Yaqui Valley Experiment Station who had to do with
the field and laboratory work necessary to provide us the flour samples.
We would like to acknowledge the editorial assistance of Dr. Finlay
MacRitchie as well as the technical assistance provided by Jorge
[1] Bietz, J.A. and Wall, J.S. (1975) The effect of various
extractants on the subunit composition and associations
of wheat glutenin. Cereal Chemistry, 52, 145-155.
[2] Huebner, F.R. and Wall, J.S. (1976) Fractionation and
quantitative differences of glutenin from wheat varieties
varying in baking quality. Cereal Chemistry, 53, 258-
[3] Gupta, R.B., Khan, K. and MacRitchie, F. (1993) Bio-
chemical basis of flour properties in bread wheats. I. Ef-
fects of variation in quantity and size distribution of
polymeric protein. Journal of Cereal Science, 18, 23-41.
[4] Wall, J.S. (1979) The role of wheat proteins in determin-
ing baking quality. In: Laidman, D.L. and Wyn-Jones,
R.G., Eds., Recent Advances in the Biochemistry of Cere-
als, Academy, London, 275-311.
[5] Panozzo, J.F., Eagles, H.A. and Wootton, M. (2001)
Changes in protein composition during grain develop-
ment in wheat. Australian Journal of Agricultural Re-
search, 52, 485-493. doi:10.1071/AR00101
[6] Altenbach, S.B., DuPont, F.M., Kothari, K.M., Chan, R.,
Johnson, E.L. and Lieu, D. (2003) Temperature, water
and fertilizer influence the timing of key events during
grain development in USA spring wheat. Journal of Ce-
real Science, 37, 9-20. doi:10.1006/jcrs.2002.0483
[7] Johansson, E., Nilsson, H., Mazhar, H., Skerrit, J.,
MacRitchie, F. and Svensson, G. (2002) Seasonal effects
on storage proteins and gluten strength in four Swedish
wheat cultivars. Journal of Science Food and Agriculture,
82, 1305-1311. doi:10.1002/jsfa.1185
[8] Johansson, E. and Svensson, G. (1998) Variation in
bread-making quality: Effects of wheater parameters on
protein concentration and quality in some Swedish wheat
cultivars grown during the period 1975-1996. Journal of
Science Food and Agriculture, 78, 109-118.
[9] Johansson, E., Prieto-Linde, M.L. and Svensson, G.
(2004) Influence of nitrogen application rate and timing
on grain protein composition and gluten strength in
Swedish wheat cultivars. Journal of Plant Nutrition Soil
Science, 167, 345-350. doi:10.1002/jpln.200320332
[10] Johansson, E., Kuktaite, R., Anderson, A. and Prieto-
A. R. Islas-Rubio et al. / Agricultural Science 2 (2011) 1 81-190
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Linde, M.L. (2005) Protein polymer build-up during
wheat grain development: Influences of temperature and
nitrogen timing. Journal of Science Food and Agriculture,
85, 473-479. doi:10.1002/jsfa.2006
[11] Wieser, H. and Seilmeier, W. (1998) The influence of
nitrogen fertilization on quantities and proportions of
different protein types in wheat flour. Journal of Science
Food and Agriculture, 76, 49-55.
doi: 10.1002/(SICI)1097-0010(199801)76:1<49::AID-JS
[12] Wooding, A.R., Kavale, S., Wilson, A.J. and Stoddard,
F.L. (2000) Effects of nitrogen and sulfur fertilization on
commercial-scale wheat quality and mixing requirements.
Cereal Chemistry, 77, 791-797.
[13] Wooding, A.R., Kavale, S., MacRitchie, F. Stoddard, F.L.
(2000) Effects of nitrogen and sulfur fertilizer on protein
composition, mixing requirements, and dough strength of
four wheat cultivars. Cereal Chemistry, 77, 798-807.
[14] Cuniberti, M.B., Roth, M.R. and MacRitchie, F. (2003)
Protein composition-functionality relationships of a set
of Argentinean wheats. Cereal Chemistry, 80, 132-134.
[15] Bangur, R., Batey, I.L., McKenzie, E. and MacRitchie, F.
(1996) Dependence of extensigraph parameters on wheat
protein composition measured by SE-HPLC. Journal of
Cereal Science, 25, 237-241.
[16] Weegels, P.L., Hammer, R.J. and Schofield, J.D. (1996)
Functional properties of wheat glutenin. Journal of Ce-
real Science, 23, 1-18. doi:10.1006/jcrs.1996.0001
[17] Gupta, R.B., Batey, I.L. and MacRitchie, F. (1992) Rela-
tionships between protein composition and functional
properties of wheat flours. Cereal Chemistry, 69, 125-
[18] Gupta, R.B., Paul, J.G., Cornish, G.B., Palmer, G.A.,
Bekes, F. and Rathjen, A.J. (1994) Allelic variation at
glutenin subunit and gliadin loci, Glu-1, Glu-3, and Gli-1,
of common wheats. I. Its additive and interaction effects
on dough properties. Journal of Cereal Science, 19, 9-17.
[19] Kieffer, R., Wieser, H., Henderson, M.H. and Graveland,
A. (1998) Correlations of the breadmaking performance
of wheat flour with rheological measurements on a mi-
cro-scale. Journal of Cereal Science, 27, 53-60.
[20] Uhlen, A.K., Sahlstrom, S., Magnus, E.M., Faergestad,
E.M., Dieseth, J.A. and Ringlund, K. (2004) Influence of
genotype and protein content on the baking quality of
hearth bread. Journal of Science Food and Agriculture,
84, 887-894. doi:10.1002/jsfa.1797
[21] Islas-Rubio, A.R., MacRitchie, F., Gandikota, S. and Hou,
G. (2005) Relaciones de la composición proteínica y
mediciones reológicas en masa con la calidad panadera
de harinas de trigo. Revista Fitotecnia Mexicana, 28,
[22] AACC International (2000) Approved methods of the
American Association of Cereal Chemists, 10th Edition,
Methods 10-10B, 26-21A, 44-19, 44-08, and 54-40A.
The American Association of Cereal Chemists, St. Paul.
[23] Batey, I.L., Gupta, R.B. and MacRitchie, F. (1991) Use
of size-exclusion high-performance liquid chromatogra-
phy in the study of wheat flour proteins: An improved
chromatographic procedure. Cereal Chemistry, 68, 207-
[24] NCSS (2007) Number Cruncher Statistical System for
Windows. Kaysville, Utah.
[25] Mitra, R. and Bhatia, C.R. (1973) Studies on protein
biosynthesis in developing wheat kernels. Nuclear tech-
niques for seed protein improvement. IAEA, Vienna.
[26] Kelley, K.W. (1995) Rate and time of nitrogen applica-
tion for wheat following different crops. Journal of Pro-
duction Agriculture, 8, 339-345.
[27] Prugar, J. and Sasek, A. (1970) Einflu β der organischen
und mineral-dungung auf die vertretung der eiweiβfrak-
tionen im weizenkorn. Getreide Mehl, 20, 27-29.
[28] Jahn-Deesbach, W. and Jürgens, U. (1973) Der einfluβ
variierter stickstoffgaben auf die ertragsmorphologie und
die stickstoffeinlagerung bei sommerweizen in einem
gefaβversuch. IV. Mitteilung: Stickstoffeinlagerung in die
verschiedenen proteinfraktionen des ganskornes und der
einzelnen mehlfraktionen. Acker-und Pflanzenbau, 138,
[29] Doekes, G.J. and Wennekes, L.M.J. (1982) Effect of ni-
trogen fertilization on quantity and composition of wheat
flour protein. Cereal Chemistry, 59, 276-278.
[30] Pechanek, U., Karger, A., Gröger, S., Charvat, B.,
Schöggl, G. and Lelley, T. (1997) Effect of nitrogen fer-
tilization on quantity of flour protein components, dough
properties, and breadmaking quality of wheat. Cereal
Chemistry, 74, 800-805.
[31] Johansson, E., Prieto-Linde, M.L. and Jonsson, J.O.
(2001) Effects of wheat cultivar and nitrogen application
on storage protein composition and breadmaking quality.
Cereal Chemistry, 78, 19-25.
[32] MacRitchie, F. (1999) Wheat proteins: characterization
and role in flour functionality. Cereal Foods World, 44,
[33] Shewry, P.R. and A.S. Tatham, A.S. (1997) Disulphide
bonds in wheat gluten proteins. Journal of Cereal Sci-
ence, 25, 207-227. doi.org/10.1006/jcrs.1996.0100
[34] Wrigley, C.W. and Békés, F. (1999) Glutenin-protein
formation during the continuum from anthesis to proc-
essing. Cereal Foods World, 38, 68-74.
[35] Zhang, P., He, Z., Zhang, Y., Xia, X., Chen, D. and
Zhang, Y. (2008) Association between % SDS-UPP (%
UPP) and end-use quality in Chinese bread wheat culti-
vars. Cereal Chemistry, 85, 696-700.
[36] Suchy, J., Lukow, O.M. and Ingelin, M.E. (2000) Dough
microextensibility using a 2-g mixograph and a texture
analyzer. Cereal Chemistry, 77, 39-43.
[37] Anderssen, R.S., Bekés, F., Gras, P.W., Nikolov, A. and
Wood, J.T. (2004) Wheat-flour dough extensibility as a
discriminator for wheat varieties. Journal of Cereal Sci-
ence, 39, 195-203. doi:10.1016/j.jcs.2003.10.002
[38] Bushuk, W. and Békés, F. (2002) Contribution of Protein
to Flour Quality. In: Salgo, A., Tomoskozi, S. and Lasz-
tity, R., Eds., Proc. Novel Raw Materials, Technologies
and Products-New Challenge for the Quality Control. In-
ternational Association for Cereal Science and Technol-
A. R. Islas-Rubio et al. / Agricultural Science 2 (2011) 1 81-190
Copyright © 2011 SciRes. http://www.scirp.org/journal/AS/Openly accessible at
ogy (ICC), Budapest, 14-19.
[39] Wrigley, C.W., Andrews, J.L., Bekes, F., Gras, P.W.,
Gupta, R.B., MacRitchie, F. and Skerrit, J.H. (1998) Pro-
tein-protein interactions-essential to dough rheology. In:
Hamer, R.J. and Hoseney, R.C., Eds., Interactions: The
Keys to Cereal Quality, The American Association of
Cereal Chemists, St. Paul, 17-46.
[40] Lang, C.E., Lanning, S.P., Carlson, G.R., Kushnak, G.D.,
Bruckner, P.L. and Talbert, L.E. (1998) Relationship be-
tween baking quality and noodle quality in hard white
spring wheat. Crop Science, 38, 823-827.
[41] Habernicht, D.K., Berg, J.E., Carlson, G.R., Wichman,
D.M., Kushnak, G.D., Kephart, K.D., Martin, J.M. and
Bruckner, P.L. (2002) Pan bread and raw Chinese noodle
qualities in hard winter wheat genotypes grown in wa-
ter-limited environments. Crop Science, 42, 1396-1403.
[42] Souza, E.J., Martin, J.M., Guttieri, M.J., O´Brien, K.M.,
Habernicht, D.K., Lanning, S.P., McLean, R., Carlson,
G.R. and Talbert, L.E. (2004) Influence of genotype, en-
vironment, and nitrogen management on spring wheat
quality. Crop Science, 44, 425-42.
[43] Southan, M. and MacRitchie, F. (19999) Molecular
weight distribution of wheat proteins. Cereal Chemistry,
76, 827-836.
[44] Uthayakumaran, S., Gras, P.W., Stoddard, F.L. and Bekés,
F. (1999) Effect of varying the protein content and glu-
tenin-to-gliadin ratio on the functional properties of
wheat dough. Cereal Chemistry, 76, 389-394.
[45] Wieser, H. and Kieffer, R. (2001) Correlations of the
amount of gluten protein types to technological proper-
ties of wheat flours determined on a micro-scale. Journal
of Cereal Science, 34, 19-27. doi:10.1006/jcrs.2000.0385