Effect of split applications of urea on protein size distribution, physical dough properties, and baking performance of five experimental bread wheat lines

doi:10.4236/as.2011.23025

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Vol.2, No.3, 181-190 (2011)

doi:10.4236/as.2011.23025

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

Camacho-Casas2

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.

ABSTRACT

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

1. INTRODUCTION

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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182

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. MATERIALS AND METHODS

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

-Arthur/7/2*Rabe/8/Irena

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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183183

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

Analyses

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-

ables.

3. RESULTS AND DIS CUSION

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

Distribution

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-

ues.

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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185185

(a) (b)

(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 ().

A. R. Islas-Rubio et al. / Agricultural Science 2 (2011) 1 81-190

186

(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,

16].

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

A. R. Islas-Rubio et al. / Agricultural Science 2 (2011) 1 81-190

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FUPP.

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.

4. CONCLUSIONS

The application of urea to all BWL increased PC, the

A. R. Islas-Rubio et al. / Agricultural Science 2 (2011) 1 81-190

188

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.

Component

number Eigen value Individual variance

(%)

Cumulative

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

PC1 PC2 PC3 PC4

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.

5. ACKNOWLEDGEMENTS

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

Mercado-Ruiz.

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