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Variation in rheological properties of gluten from three biscuit wheat cultivars in relation to nitrogen fertilisation
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Journal of Cereal Science 46 (2007) 132–138 www.elsevier.com/locate/jcs
Variation in rheological properties of gluten from three biscuit wheat cultivars in relation to nitrogen fertilisation Lene Pedersena,, Johannes Ravn Jørgensenb a
Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Aarslev, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark b Department of Genetics and Biotechnology, University of Aarhus, Faculty of Agricultural Sciences, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark Received 24 May 2006; received in revised form 24 October 2006; accepted 12 January 2007
Abstract Rheological properties of gluten from three biscuit wheat cultivars (Triticum aestivum, L., cv. Reaper, Ritmo, Encore) were studied. The cultivars were grown in two seasons (1997–1999) with three different nitrogen levels, and nitrogen fertiliser was applied using three different strategies. Protein and gluten contents were significantly affected by the N level (Po0.001), but inter-cultivar differences were only significant in 1999, when growing conditions were restricted by environmental factors. The viscoelastic properties of gluten were characterised by creep recovery and oscillation testing. The results showed a significant inter-cultivar effect (Po0.001), with an additional effect from the N level (Po0.001). Increasing levels of nitrogen fertiliser increased the viscous properties of gluten, through an increase of maximum strain and recovery strain, and through a decrease of the storage (G0 ) and loss modulus (G00 ), whereas the phase angle, d, increased. This increase in viscous behaviour is suggested to be attributed to a higher gliadin/glutenin ratio in the gluten. The fertiliser application strategy did not influence the rheological properties significantly. Thus, high N fertiliser application in biscuit wheat cultivation may be beneficial to obtain rheological properties, which are suitable for biscuit making. r 2007 Elsevier Ltd. All rights reserved. Keywords: Biscuit wheat; N fertiliser; Gluten; Creep recovery; Oscillation
1. Introduction It has long been established that quantity and quality of protein both influence the end use quality of wheat, and bread making performance in particular (Finney and Barmore, 1948; Tipples and Kilborn, 1974; Booth and Melvin, 1979; Gupta et al., 1992). The protein content in the wheat grain is mainly determined by the genetic background and cultivar, but also to a large extent by environmental factors such as nitrogen application, water access and temperature during growth (McDonald, 1992; Abbreviations: G0 , storage modulus; G00 , loss modulus; d, phase angle; NIT, Near Infrared Transmission Corresponding author. Current address: Southern University of Denmark, Faculty of Engineering, Niels Bohrs Alle 1, DK-5230 Odense M, Denmark. Tel.: +45 65507469. E-mail addresses: [email protected], [email protected] (L. Pedersen), [email protected] (J.R. Jørgensen). 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.01.001
Daniel and Triboi, 2000; Luo et al., 2000; Tea et al., 2004). Both genetic background and amount of N fertiliser in addition to the application time strongly influence protein composition in the grain, as determined by the relative amount of different protein subunits (Peltonen and Virtanen, 1994; Wieser and Seilmeier, 1998; Johansson et al., 2001), with a subsequent influence on functional and baking properties (Baezinger et al., 1985; Johansson and Svensson, 1999; Wooding et al., 2000). The gliadin/glutenin ratio has been shown to be the most important factor influencing the functional properties and baking quality (Weegels et al., 1995; Vereijken et al., 2000; Daniel and Triboi, 2000; Veraverbeke and Delcour, 2002). This will also be the case in relation to biscuit making properties of wheat. Low protein has been correlated to good biscuit baking properties (Wade, 1972). However, protein composition is also of importance (Huebner et al., 1999; Pedersen et al., 2004). Functional properties of
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biscuit wheat are mainly governed by rheological characteristics of the biscuit dough and of the gluten network developed in the semi-sweet biscuit dough, which have a relatively low fat content. Therefore rheological characterisation of gluten from biscuit wheat may be useful for indicating the suitability of wheat for biscuit products, since the differences in gluten properties are more pronounced than between the corresponding flour/water dough in which starch–starch interactions may dominate the results of the rheological characterisation (Dreese et al., 1988; Kokelaar et al., 1996). Rheological studies of gluten using fundamental methods have been used to characterise the viscoelastic properties of gluten in relation to cultivar differences and protein composition (Khatkar et al., 1995; Janssen et al., 1996a, b; Tsiami et al., 1997). The influence of fertilising has been studied by characterising gel protein, gluten strength and mixing properties (Luo et al., 2000; Wooding et al., 2000; Vereijken et al., 2000). However, many of the studies included wheat cultivars with pronounced differences in bread making potential, whereas gluten from biscuit wheat cultivars, which are characterised by a weak gluten structure, has not been studied much. The objective of the present study was therefore to compare the fundamental rheological behaviour of gluten from different biscuit wheat cultivars, and to investigate the influence of N fertilisation on the gluten content and the rheological properties of gluten. Oscillation and creep recovery testing were performed on gluten isolated from flour, and frequency dependency and long time behaviour were evaluated. Results are interpreted in relation to N fertilisation with the aim to select optimum growing parameters for wheat cultivars suitable for biscuit manufacturing.
133
Table 1 Combination of N levels (130, 170 and 210 kg N ha1) and the three application strategies (a single (100%), a two (40%/60%) and a three split (40%/40%/20%) application) and timing of application (BBCH) N level
BBCH
Strategy 1
Strategy 2
Strategy 3
Applied N (kg ha1) 130 kg N ha1
170 kg N ha1
210 kg N ha1
22 25 31 51 22 25 31 51 22 25 31 51
52
52
78
52 26
68
68
102
68 34
84
84
126
84 42
130
170
210
BBCH: Decimal code for development of wheat.
in Table 1. Pesticides and growth regulators were used uniformly for all plots according to normal practice. Grain samples from each plot were harvested, milled and analysed separately and original seed material was used each year. The protein content of the grain samples was determined using near infrared transmission (NIT) on a Foss-Tecator, Infratecs 1241 (Buchmann and Runfors, 1995; Buchmann et al., 2001). Grain samples were stored for five months before milling. Milling was done on a Brabender Quadromat Juniors. Before milling the samples were conditioned to 15.0% moisture.
2. Experimental 2.2. Gluten isolation and rheological characterisation 2.1. Wheat material The three winter wheat cultivars in the present study were selected due to diversity in quality characteristics and biscuit-making properties. All cultivars are frequently used in biscuit manufacturing, and were included in a research project in the Danish Cereal Network concerning biscuit wheat quality. The cultivars used in the investigations were Ritmo and Reaper, which are hard endosperm cultivars, and Encore with soft endosperm. Field trials were carried out in 1997/1998 and 1998/1999 at Research Centre Flakkebjerg, Denmark (UTM 652360 Eastern, 6133550 Northern). Each cultivar was grown in four replicated plots of 18 m2 size in a fully randomised block-design. For each cultivar three different N levels (130, 170, 210 kg N ha1) were applied according to three different strategies (a single (100%), a two (40%/60%) and a three split (40%/40%/ 20%) application). The timing and uniformity of application were based on the BBCH plant development stage (Lancashire et al., 1991). An overview of the combination of the N levels and the application strategies are presented
Glutens were isolated from the flour according to ICC standard test No. 137 (ICC, 1980) with minor modifications. The flour/water dough rested after mixing for 15 min before the washing was started. This was done to facilitate the separation of gluten from the starch fraction. The gluten content was expressed as % wet gluten of flour weight. Gluten washing was done with four replications, and two of the gluten fractions obtained were placed in the rheometer immediately after washing, and two other fractions were used for determination of gluten dry mass (100 1C for 12 h). Rheological measurements were performed on the glutens from the gluten isolation. A Bohlin CVO (control stress) rheometer was used in oscillation and creep recovery testing. The gluten was placed between two parallel plates (25 mm diameter), which were serrated to prevent the gluten from slipping. The gap between the plates was 2.0 mm, and the edges of the gluten were sealed with a polymer resin (Plastybycol) to prevent drying. The gluten rested 3 min to obtain a constant temperature of 30 1C
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before measurements were started. Rheological testing included creep recovery and oscillation. Amplitude sweeps were done in a stress range from 1 to 500 Pa at a frequency of 1 Hz in order to determine the linear viscoelastic region. This was determined to be up to 3% strain, corresponding to a stress value of 60 Pa, and a value of 40 Pa was used in the creep recovery and oscillation testing. Creep and recovery time were 300 s and maximum strain after creep and recovery strain was calculated. Frequencies from 0.1 to 50 Hz were applied in the frequency sweep, and storage modulus (G0 ), loss modulus (G00 ), and d (tan1 G00 /G0 ) at 1 Hz were used in the statistical testing. Creep recovery and oscillation were done consecutively and in duplicate for each flour sample, and flour from two plots from each treatment was analysed. 2.3. Statistical analysis A general linear model (GLM) procedure in the statistical analyses system (SAS, version 8.0, 1999) was used for statistical analysis of the different wheat varieties in the field trials. Least significant difference (LSD) was used to compare mean values of protein and gluten content, and the rheological parameters measured by creep recovery and oscillation. Regression analysis was used to show correlation between protein and gluten content. The following notation *, **, *** refers to significance at Po0.05, Po0.01, and Po0.001, respectively. NS, non-significant. 3. Results and discussion 3.1. Yield and protein content of grain and gluten content of flour in relation to N management The grain yields of 1998 were approximately 2 ton higher per hectare than in 1999 for all three cultivars (Table 2). Ritmo yielded most in both years but only significantly more in 1998. Increased fertiliser application
Table 2 Yield and protein yield of grain from 1998 and 1999 Yield (t ha1)
Protein (%)
1998
1999
1998
Ritmo Encore Reaper
10.46b 9.99a 9.99a
8.59a 7.97a 8.03a
9.78aa 12.18a 10.16a 13.24b 10.11a 13.13b
Fertiliser application (kg N ha1)
130 170 210
9.62a 10.25b 10.59c
8.22a 8.26a 8.10a
9.25a 9.94b 10.84c
11.71a 13.05b 13.79c
Fertiliser strategy
Single split Two split Three split
10.12a 10.25a 10.05a
8.25a 8.07a 8.25a
10.18a 9.93a 9.94a
12.98a 12.58b 12.99a
Cultivar
a
1999
Mean values within a column followed by the same letter are not significantly different.
increased yield significantly in 1998 as expected but not in 1999. The fertiliser strategy did not influence yield notably. The grain protein content varied between the two years, mean values were 10.0% in 1998 and 12.9% in 1999. This variability is probably a result of differences in the climatic conditions during growth, leading to high yield/low protein level in 1998, and low yield/high protein level in 1999. Due to these differences, which mainly were caused by low rainfall in 1999, data were analysed separately for each year (Table 2). As expected, protein content of the grain was significantly influenced by N level (Po0.001) in both 1998 and 1999, and additionally cultivar and fertiliser strategy significantly influenced the protein content in 1999 (Po0.001 and Po0.01, respectively). This indicates that strategy and cultivar influenced the protein content of the grain only in 1999 when restrictions in growth were observed. In 1999, Encore and Reaper had significantly higher protein contents than Ritmo. Strategy 2 (two-split) resulted in lower protein content than strategy 1 (single) and 3 (three-split). There are no obvious explanations to the observation that a two-split application resulted in lower protein content than a one- or three-split application. Previous studies have shown that late application of N increased protein content in bread wheat (Pushman and Bingham, 1976; Darwinkle, 1983; Luo et al., 2000). However, this effect was not found in these trials with the biscuit wheat cultivars. Flour gluten content is shown in Fig. 1. The overall difference between the two years was mainly related to an increased level in % gluten in 1999 compared to 1998, which also was reflected in the grain protein content. The gluten was significantly (Pp0.001) different for the three cultivars, with a ranking of Reaper4Ritmo4Encore. Increasing N levels resulted in higher contents of gluten (Pp0.001), agreeing well with other studies (McDonald, 1992; Luo et al., 2000; Johansson et al., 2001). The effect of the N strategy was found to be significant in both years (Pp0.001 in 1998, and Pp0.01 in 1999). This was not the case for protein content, where the application of the N strategy had no significant effect. In both years the gluten content for strategy 2 was significantly lower than for strategies 1 and 3, which also was found for protein content. Cultivar, N strategy, and N level did not affect water content of the washed gluten, and mean values for the cultivars were in the range 34.7–35.8 w/w% (data not shown). 3.2. Rheological properties of gluten An example of gluten creep recovery behaviour is presented in Fig. 2. The gluten is characterised by a very fast elastic response followed by a viscous flow. The maximum shear strain for all samples measured varied between 10% and 90%, which means that the resistance to deformation varied considerably. Recovery strain, calculated as the difference between maximum strain and the final strain after 300 s of recovery, varied between 5% and 40%. This indicates a fairly high
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30 25 20 15 10 5 0 0
N strategy 3
100
200
300 400 Time [s]
500
40
10000
35 Modulus [Pa]
30 25 20
1000 G' G'' Delta 100 0.1
N strategy 3
N strategy 2
N strategy 1
170 kg N
N strategy 3
N strategy 2
N strategy 1
N strategy 3
N strategy 2
N strategy 1
130 kg N
Encore
700
Reaper
40 35 30 25 20 15 10 5 0
Ritmo
600
1.0 10.0 Frequency [Hz]
15
Delta [°]
N strategy 2
Encore
N strategy 1
N strategy 3
N strategy 2
N strategy 1
N strategy 3
N strategy 2
N strategy 1
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Gluten [%]
135
35
40 35 30 25 20 15 10 5 0
Strain [%]
Gluten [%]
L. Pedersen, J.R. Jørgensen / Journal of Cereal Science 46 (2007) 132–138
10 5
0 100.0
Fig. 2. Creep recovery and oscillation for cv. Encore, (strategy 3 ¼ three split, N level 3 ¼ 210 kg N ha1). Mean and standard deviation for four replications is indicated.
Reaper 210 kg N
Fig. 1. Gluten content (% of flour) from (a) 1998 and (b) 1999 at three N levels (130, 170, and 210 kg N ha1). Standard deviation for four replications is indicated.
variability in elasticity between the samples. After 300 s of recovery a part of the maximum strain is permanent, indicating that the gluten behaves like a viscoelastic liquid. It has been stated that gluten relaxation can be divided into two processes, the first occurs after a short time, 0.1–10 s, and the other occurs at longer times, 10–104 s (Funt BarDavid and Lerchenthal, 1975; Bohlin and Carlson, 1981). It was also found that gluten was able to recover completely (Funt Bar-David and Lerchenthal, 1975). In our measurements only a part of the deformation seemed to be recoverable, but due to a rather short time of recovery the gluten was not allowed to recover to a steady state. However, this short-time scale corresponds to real life conditions during processing of biscuit dough, and the method could easily distinguish between gluten from varying cultivars and growth conditions. Oscillation behaviour (Fig. 2) of the gluten showed that G0 and G00 were of the same level of magnitude, but that the value of G0 exceeded G00 for all samples measured, meaning that the solid-like structure is the dominating one. Both G0 and G00 increased with increasing frequency. However, as G00 increased more than G0 , the phase angle d increased with increasing frequency. The same frequency dependency was found in previous studies (Dreese et al., 1988; Khatkar
et al., 1995; Janssen et al., 1996a), and reflects the fact that the hydrated gluten may exhibits a more viscous behaviour when stress is applied on a short time scale. 3.3. Effect of N management on rheological properties of gluten Results from the statistical analysis of the rheological characteristics are shown in Table 3. All parameters from creep, recovery, and oscillation were significantly different for the cultivar types (Po0.001); mean values are shown in Table 4. In the creep recovery measurement, Reaper differed from the two other cultivars due to a low resistance to deformation measured by a high maximum strain and a high recovery strain, whereas Ritmo and Encore had the same values. With regard to oscillation, Ritmo had higher values of G0 and G00 , which indicates a more stiff structure of the gluten and a high degree of cross-linking compared to the other two cultivars. Phase angle d, which reflects the relative contribution of the elastic and viscous behaviour in the gluten, differed significantly between the three cultivars tested, where the gluten of Reaper was considerably more viscous in behaviour (high value of d). These differences in viscoelasticity are most likely explained by differences in the protein composition expressed as different ratios of gliadins to glutenins. Gluten from wheat with good and poor bread making quality was also found to be significantly different in oscillation (Khatkar et al., 1995; Janssen et al., 1996a), and several studies have demonstrated that differences in gliadin/glutenin ratio are reflected in the measurements of
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viscoelastic properties (Janssen et al., 1996b; Schropp and Wieser, 1996; Uthayakumaran et al., 1999; Tronsmo et al., 2003). Results from the present study indicate that gluten from biscuit wheat cultivars with a relatively low variability in protein content may exhibit large differences in viscoelastic properties measured by oscillation testing. The effect of N level varied between the two years, and between the rheological parameters measured (Table 3). In 1998 all parameters, except G00 were highly affected by the N level, but in 1999 only phase angle d was affected. Growing conditions in 1998 resulted in high yield and a relatively low protein, and in this year with optimum growing conditions, the N level significantly affected the viscoelasticity of the gluten. In creep recovery, maximum strain and recovery strain increased with increasing N level, and in oscillation G0 and G00 decreased, whereas phase angle d increased when N level was increased (Table 4). Thus, increasing N level seemed to alter the gluten making
Table 3 Variance ratios (F-values) and significance levels of cultivar, N level and N strategy for 1998 and 1999
1998 Cultivar N strategy N level Cultivar N strategy Cultivar N level 1999 Cultivar N strategy N level Cultivar N strategy Cultivar N level
Maximum strain (%)
Recovery strain (%)
G0 (Pa) G00 (Pa) d (3)
62.18*** 0.44NS 9.11*** 1.19NS
57.67*** 0.61NS 8.43*** 1.28NS
17.83*** 0.60NS 5.14** 3.37*
2.92*
3.05*
2.51*
72.07*** 1.86NS 2.90NS 2.97*
83.83*** 1.82NS 3.46* 3.24*
38.74*** 2.16NS 6.18* 1.84NS
1.49*
1.88NS
2.66*
Significance levels: NS, non significant, *, Po0.05, Po0.001.
38.75*** 1.07NS 0.64NS 3.84*
56.84*** 0.32NS 14.92*** 0.44NS
2.22NS 3.79**
17.17*** 2.07NS 2.42NS 0.99NS
58.58*** 0.57NS 5.06** 1.64*
1.50NS 2.45* **
, Po0.01,
***
,
it less elastic and more viscous likely due to changes in the protein composition. Khatkar et al. (1995) found that increasing gliadin/glutenin ratio decreased G0 and increased phase angle d. Thus, an increased level of gliadins in the gluten has a softening effect on the gluten properties. Increasing levels of gliadins at high N levels most likely can explain our observation, also due to the fact that the water content of the gluten was not altered in relation to N level. A relationship between N level and cultivar was observed (Fig. 3). The responses to the N level were equal for Reaper and Encore, whereas Ritmo differed. The gluten from Encore and Reaper likely changed in structure between N level one and two, but the structure of gluten from Ritmo was likely changed between N level two and three. These results demonstrate that the effect of the level of N application on the rheological properties of gluten varied among the three cultivars. Previous studies of the influence of N fertiliser showed that the amount of N fertiliser did not directly affect the amount of high molecular weight glutenins relative to the other storage proteins (Fullington et al., 1983). However, subsequent work of Wooding et al. (2000) demonstrated that an increase in gliadins was observed for increasing N level when combined with addition of S fertiliser, and they also showed a cultivar–nitrogen fertiliser relationship. The study of Daniel and Triboi (2000) also agreed with these observations, namely, that N level was affecting the amount of gliadins in the grain. Thus, if increased level of N fertiliser results in increased production of gliadins, it can be concluded that this change correlates well with increased viscous properties of gluten. The cultivars included in these trials are all characterised by a weak gluten structure, and increasing levels of gliadins may alter the viscoelastic properties more compared to cultivars suitable for bread making. 4. Conclusions The results of the study showed that protein and gluten contents were influenced by the genetic origin (cultivar), and the N strategy. However the effects were only significant for 1999, where growth was restricted due to limited rainfall. Two-split N application resulted in
Table 4 Mean values of rheological properties of gluten from different cultivars and fertiliser application Maximum strain (%)
Recovery strain (%)
G0 (Pa)
1998
1999
1998
1999
1998
1999
1998
1999
1998
1999
Ritmo Encore Reaper
38.6b 36.7b 59.4a
29.1b 24.5b 55.6a
20.0b 18.0b 32.2a
15.2b 13.0b 26.3a
1000a 829b 853b
1134a 1122a 810b
584a 454b 559a
627a 587b 530c
30.3b 29.0c 33.3a
29.1b 27.8c 33.1a
Fertiliser application (kg N ha1) 130 170 210
39.5a 46.3b 50.0b
33.4a 36.3ab 39.6b
20.1a 24.7b 26.2b
17.1a 17.9ab 19.6b
1044a 1081a 947b
543a 543a 531a
583a 598a 562b
29.9a 31.2b 32.2c
29.4a 29.8a 30.9
Cultivar
Means within a column followed by the same letter are not significantly different.
948a 904ab 846b
G00 (Pa)
d (3)
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Maximum strain [%]
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40 30
800 600
20
400
10
200 0
0 1
2
3
40 35 30 25 20 15 10 5 0
1
2
3
1
2 N level
3
40 35 [°]
Recovery [%]
137
30 25 20
1
2 N level
3
Ritmo
Encore Reaper
Fig. 3. Creep, recovery, and oscillation of three cultivars (1998) as a function of N level, 1 ¼ 130 kg N ha1; 2 ¼ 170 kg N ha1; 3 ¼ 210 kg N ha1.
significantly lower protein and gluten content than one or three-split. Protein and gluten content increased significantly with increasing N level in both years, and the cultivar Reaper attained the highest values. These results also demonstrate the capability of fundamental testing, since creep recovery and oscillation could discriminate the gluten among the three cultivars grown with different N levels. Inter-cultivar differences and N level account for the variability in viscoelastic properties of gluten. The N level mainly influenced the viscoelasticity of gluten in the year with optimum growing conditions (1998). In this year increasing N level resulted in gluten with a more viscous behaviour. The increase in viscous properties of gluten is suggested to be a result of a higher ratio of gliadins in the gluten network. Increased viscous properties of gluten will be an advantage in biscuit dough processing, and therefore it may be beneficial to grow biscuit wheat with higher than normal N applications, in order to obtain biscuit wheat with optimum biscuit-making properties. Acknowledgement The present work was funded by the First Framework Programme of The Cereal Network (The Danish Ministry of Food, Agriculture and Fisheries). References Baezinger, P.S., Clements, P.S., McIntosh, M.S., Yamazaki, W.T., Starling, T.M., Sammons, D.J., Johnson, J.W., 1985. Effect of cultivar, environment and their interaction and stability analyses on
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Pushman, F.M., Bingham, J., 1976. The effects of granular nitrogen fertiliser and foliar spray of urea on the yield and bread-making quality of ten winter wheats. Journal of Agricultural Science (Cambridge) 87, 281–292. Schropp, P., Wieser, H., 1996. Effects of high molecular subunits of glutenin on the rheological properties of wheat gluten. Cereal Chemistry 73, 410–441. Tea, I., Genter, T., Naulet, N., Boyer, V., Lummerzheim, M., Kleiber, D., 2004. Effect of foliar sulfur and nitrogen fertilization on wheat storage protein composition and dough mixing properties. Cereal Chemistry 81, 759–766. Tipples, K.H., Kilborn, R.H., 1974. Baking strength index and the relation of protein content to loaf volume. Canadian Journal of Plant Science 54, 231–234. Tronsmo, K.M., Magnus, E.M., Færgestad, E.M., Schofield, J.D., 2003. Relationships between gluten rheological properties and hearth loaf characteristics. Cereal Chemistry 80, 575–586. Tsiami, A.A., Bot, A., Agterof, W.G.M., 1997. Rheology of mixtures of glutenin subfractions. Journal of Cereal Science 26, 279–287. Uthayakumaran, S., Gras, P.W., Stoddard, F.L., 1999. Effect of varying protein composition and gliadin-to-glutenin ratio on the functional properties of wheat dough. Journal of Cereal Science 76, 389–394. Veraverbeke, W.S., Delcour, J.A., 2002. Wheat protein composition and properties of wheat glutenin in relation to breadmaking functionality. Critical Reviews in Food Science and Nutrition 42, 179–208. Vereijken, J.M., Klostermann, V.L.C., Beckers, F.H.R., Spekking, W.T.J., Graveland, A., 2000. Intercultivar variation in the proportions of wheat protein fractions and relation to mixing behaviour. Journal of Cereal Science 32, 159–167. Wade, P., 1972. Flour properties and the manufacture of semi sweet biscuits. Journal of the Science of Food and Agriculture 23, 737–744. Weegels, P.L., Orsel, R., van de Pijpekamp, A.M., Lichtendonk, W.J., Hamer, R.J., Schofield, J.D., 1995. Functional properties of low m(r) wheat proteins. 2. Effects on dough properties. Journal of Cereal Science 21, 117–126. Wieser, H., Seilmeier, W., 1998. The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour. Journal of the Science of Food and Agriculture 76, 49–55. Wooding, A.R., Kavale, S., MacRitchie, F., Stoddard, F.L., Wallace, A., 2000. Effects of nitrogen and sulfur fertiliser on protein composition, mixing requirements, and dough strength of four wheat cultivars. Cereal Chemistry 77, 798–807.
Journal of Cereal Science 46 (2007) 132–138 www.elsevier.com/locate/jcs
Variation in rheological properties of gluten from three biscuit wheat cultivars in relation to nitrogen fertilisation Lene Pedersena,, Johannes Ravn Jørgensenb a
Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Aarslev, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark b Department of Genetics and Biotechnology, University of Aarhus, Faculty of Agricultural Sciences, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark Received 24 May 2006; received in revised form 24 October 2006; accepted 12 January 2007
Abstract Rheological properties of gluten from three biscuit wheat cultivars (Triticum aestivum, L., cv. Reaper, Ritmo, Encore) were studied. The cultivars were grown in two seasons (1997–1999) with three different nitrogen levels, and nitrogen fertiliser was applied using three different strategies. Protein and gluten contents were significantly affected by the N level (Po0.001), but inter-cultivar differences were only significant in 1999, when growing conditions were restricted by environmental factors. The viscoelastic properties of gluten were characterised by creep recovery and oscillation testing. The results showed a significant inter-cultivar effect (Po0.001), with an additional effect from the N level (Po0.001). Increasing levels of nitrogen fertiliser increased the viscous properties of gluten, through an increase of maximum strain and recovery strain, and through a decrease of the storage (G0 ) and loss modulus (G00 ), whereas the phase angle, d, increased. This increase in viscous behaviour is suggested to be attributed to a higher gliadin/glutenin ratio in the gluten. The fertiliser application strategy did not influence the rheological properties significantly. Thus, high N fertiliser application in biscuit wheat cultivation may be beneficial to obtain rheological properties, which are suitable for biscuit making. r 2007 Elsevier Ltd. All rights reserved. Keywords: Biscuit wheat; N fertiliser; Gluten; Creep recovery; Oscillation
1. Introduction It has long been established that quantity and quality of protein both influence the end use quality of wheat, and bread making performance in particular (Finney and Barmore, 1948; Tipples and Kilborn, 1974; Booth and Melvin, 1979; Gupta et al., 1992). The protein content in the wheat grain is mainly determined by the genetic background and cultivar, but also to a large extent by environmental factors such as nitrogen application, water access and temperature during growth (McDonald, 1992; Abbreviations: G0 , storage modulus; G00 , loss modulus; d, phase angle; NIT, Near Infrared Transmission Corresponding author. Current address: Southern University of Denmark, Faculty of Engineering, Niels Bohrs Alle 1, DK-5230 Odense M, Denmark. Tel.: +45 65507469. E-mail addresses: [email protected], [email protected] (L. Pedersen), [email protected] (J.R. Jørgensen). 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.01.001
Daniel and Triboi, 2000; Luo et al., 2000; Tea et al., 2004). Both genetic background and amount of N fertiliser in addition to the application time strongly influence protein composition in the grain, as determined by the relative amount of different protein subunits (Peltonen and Virtanen, 1994; Wieser and Seilmeier, 1998; Johansson et al., 2001), with a subsequent influence on functional and baking properties (Baezinger et al., 1985; Johansson and Svensson, 1999; Wooding et al., 2000). The gliadin/glutenin ratio has been shown to be the most important factor influencing the functional properties and baking quality (Weegels et al., 1995; Vereijken et al., 2000; Daniel and Triboi, 2000; Veraverbeke and Delcour, 2002). This will also be the case in relation to biscuit making properties of wheat. Low protein has been correlated to good biscuit baking properties (Wade, 1972). However, protein composition is also of importance (Huebner et al., 1999; Pedersen et al., 2004). Functional properties of
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biscuit wheat are mainly governed by rheological characteristics of the biscuit dough and of the gluten network developed in the semi-sweet biscuit dough, which have a relatively low fat content. Therefore rheological characterisation of gluten from biscuit wheat may be useful for indicating the suitability of wheat for biscuit products, since the differences in gluten properties are more pronounced than between the corresponding flour/water dough in which starch–starch interactions may dominate the results of the rheological characterisation (Dreese et al., 1988; Kokelaar et al., 1996). Rheological studies of gluten using fundamental methods have been used to characterise the viscoelastic properties of gluten in relation to cultivar differences and protein composition (Khatkar et al., 1995; Janssen et al., 1996a, b; Tsiami et al., 1997). The influence of fertilising has been studied by characterising gel protein, gluten strength and mixing properties (Luo et al., 2000; Wooding et al., 2000; Vereijken et al., 2000). However, many of the studies included wheat cultivars with pronounced differences in bread making potential, whereas gluten from biscuit wheat cultivars, which are characterised by a weak gluten structure, has not been studied much. The objective of the present study was therefore to compare the fundamental rheological behaviour of gluten from different biscuit wheat cultivars, and to investigate the influence of N fertilisation on the gluten content and the rheological properties of gluten. Oscillation and creep recovery testing were performed on gluten isolated from flour, and frequency dependency and long time behaviour were evaluated. Results are interpreted in relation to N fertilisation with the aim to select optimum growing parameters for wheat cultivars suitable for biscuit manufacturing.
133
Table 1 Combination of N levels (130, 170 and 210 kg N ha1) and the three application strategies (a single (100%), a two (40%/60%) and a three split (40%/40%/20%) application) and timing of application (BBCH) N level
BBCH
Strategy 1
Strategy 2
Strategy 3
Applied N (kg ha1) 130 kg N ha1
170 kg N ha1
210 kg N ha1
22 25 31 51 22 25 31 51 22 25 31 51
52
52
78
52 26
68
68
102
68 34
84
84
126
84 42
130
170
210
BBCH: Decimal code for development of wheat.
in Table 1. Pesticides and growth regulators were used uniformly for all plots according to normal practice. Grain samples from each plot were harvested, milled and analysed separately and original seed material was used each year. The protein content of the grain samples was determined using near infrared transmission (NIT) on a Foss-Tecator, Infratecs 1241 (Buchmann and Runfors, 1995; Buchmann et al., 2001). Grain samples were stored for five months before milling. Milling was done on a Brabender Quadromat Juniors. Before milling the samples were conditioned to 15.0% moisture.
2. Experimental 2.2. Gluten isolation and rheological characterisation 2.1. Wheat material The three winter wheat cultivars in the present study were selected due to diversity in quality characteristics and biscuit-making properties. All cultivars are frequently used in biscuit manufacturing, and were included in a research project in the Danish Cereal Network concerning biscuit wheat quality. The cultivars used in the investigations were Ritmo and Reaper, which are hard endosperm cultivars, and Encore with soft endosperm. Field trials were carried out in 1997/1998 and 1998/1999 at Research Centre Flakkebjerg, Denmark (UTM 652360 Eastern, 6133550 Northern). Each cultivar was grown in four replicated plots of 18 m2 size in a fully randomised block-design. For each cultivar three different N levels (130, 170, 210 kg N ha1) were applied according to three different strategies (a single (100%), a two (40%/60%) and a three split (40%/40%/ 20%) application). The timing and uniformity of application were based on the BBCH plant development stage (Lancashire et al., 1991). An overview of the combination of the N levels and the application strategies are presented
Glutens were isolated from the flour according to ICC standard test No. 137 (ICC, 1980) with minor modifications. The flour/water dough rested after mixing for 15 min before the washing was started. This was done to facilitate the separation of gluten from the starch fraction. The gluten content was expressed as % wet gluten of flour weight. Gluten washing was done with four replications, and two of the gluten fractions obtained were placed in the rheometer immediately after washing, and two other fractions were used for determination of gluten dry mass (100 1C for 12 h). Rheological measurements were performed on the glutens from the gluten isolation. A Bohlin CVO (control stress) rheometer was used in oscillation and creep recovery testing. The gluten was placed between two parallel plates (25 mm diameter), which were serrated to prevent the gluten from slipping. The gap between the plates was 2.0 mm, and the edges of the gluten were sealed with a polymer resin (Plastybycol) to prevent drying. The gluten rested 3 min to obtain a constant temperature of 30 1C
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before measurements were started. Rheological testing included creep recovery and oscillation. Amplitude sweeps were done in a stress range from 1 to 500 Pa at a frequency of 1 Hz in order to determine the linear viscoelastic region. This was determined to be up to 3% strain, corresponding to a stress value of 60 Pa, and a value of 40 Pa was used in the creep recovery and oscillation testing. Creep and recovery time were 300 s and maximum strain after creep and recovery strain was calculated. Frequencies from 0.1 to 50 Hz were applied in the frequency sweep, and storage modulus (G0 ), loss modulus (G00 ), and d (tan1 G00 /G0 ) at 1 Hz were used in the statistical testing. Creep recovery and oscillation were done consecutively and in duplicate for each flour sample, and flour from two plots from each treatment was analysed. 2.3. Statistical analysis A general linear model (GLM) procedure in the statistical analyses system (SAS, version 8.0, 1999) was used for statistical analysis of the different wheat varieties in the field trials. Least significant difference (LSD) was used to compare mean values of protein and gluten content, and the rheological parameters measured by creep recovery and oscillation. Regression analysis was used to show correlation between protein and gluten content. The following notation *, **, *** refers to significance at Po0.05, Po0.01, and Po0.001, respectively. NS, non-significant. 3. Results and discussion 3.1. Yield and protein content of grain and gluten content of flour in relation to N management The grain yields of 1998 were approximately 2 ton higher per hectare than in 1999 for all three cultivars (Table 2). Ritmo yielded most in both years but only significantly more in 1998. Increased fertiliser application
Table 2 Yield and protein yield of grain from 1998 and 1999 Yield (t ha1)
Protein (%)
1998
1999
1998
Ritmo Encore Reaper
10.46b 9.99a 9.99a
8.59a 7.97a 8.03a
9.78aa 12.18a 10.16a 13.24b 10.11a 13.13b
Fertiliser application (kg N ha1)
130 170 210
9.62a 10.25b 10.59c
8.22a 8.26a 8.10a
9.25a 9.94b 10.84c
11.71a 13.05b 13.79c
Fertiliser strategy
Single split Two split Three split
10.12a 10.25a 10.05a
8.25a 8.07a 8.25a
10.18a 9.93a 9.94a
12.98a 12.58b 12.99a
Cultivar
a
1999
Mean values within a column followed by the same letter are not significantly different.
increased yield significantly in 1998 as expected but not in 1999. The fertiliser strategy did not influence yield notably. The grain protein content varied between the two years, mean values were 10.0% in 1998 and 12.9% in 1999. This variability is probably a result of differences in the climatic conditions during growth, leading to high yield/low protein level in 1998, and low yield/high protein level in 1999. Due to these differences, which mainly were caused by low rainfall in 1999, data were analysed separately for each year (Table 2). As expected, protein content of the grain was significantly influenced by N level (Po0.001) in both 1998 and 1999, and additionally cultivar and fertiliser strategy significantly influenced the protein content in 1999 (Po0.001 and Po0.01, respectively). This indicates that strategy and cultivar influenced the protein content of the grain only in 1999 when restrictions in growth were observed. In 1999, Encore and Reaper had significantly higher protein contents than Ritmo. Strategy 2 (two-split) resulted in lower protein content than strategy 1 (single) and 3 (three-split). There are no obvious explanations to the observation that a two-split application resulted in lower protein content than a one- or three-split application. Previous studies have shown that late application of N increased protein content in bread wheat (Pushman and Bingham, 1976; Darwinkle, 1983; Luo et al., 2000). However, this effect was not found in these trials with the biscuit wheat cultivars. Flour gluten content is shown in Fig. 1. The overall difference between the two years was mainly related to an increased level in % gluten in 1999 compared to 1998, which also was reflected in the grain protein content. The gluten was significantly (Pp0.001) different for the three cultivars, with a ranking of Reaper4Ritmo4Encore. Increasing N levels resulted in higher contents of gluten (Pp0.001), agreeing well with other studies (McDonald, 1992; Luo et al., 2000; Johansson et al., 2001). The effect of the N strategy was found to be significant in both years (Pp0.001 in 1998, and Pp0.01 in 1999). This was not the case for protein content, where the application of the N strategy had no significant effect. In both years the gluten content for strategy 2 was significantly lower than for strategies 1 and 3, which also was found for protein content. Cultivar, N strategy, and N level did not affect water content of the washed gluten, and mean values for the cultivars were in the range 34.7–35.8 w/w% (data not shown). 3.2. Rheological properties of gluten An example of gluten creep recovery behaviour is presented in Fig. 2. The gluten is characterised by a very fast elastic response followed by a viscous flow. The maximum shear strain for all samples measured varied between 10% and 90%, which means that the resistance to deformation varied considerably. Recovery strain, calculated as the difference between maximum strain and the final strain after 300 s of recovery, varied between 5% and 40%. This indicates a fairly high
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30 25 20 15 10 5 0 0
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100
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35 Modulus [Pa]
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1000 G' G'' Delta 100 0.1
N strategy 3
N strategy 2
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N strategy 3
N strategy 2
N strategy 1
N strategy 3
N strategy 2
N strategy 1
130 kg N
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700
Reaper
40 35 30 25 20 15 10 5 0
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600
1.0 10.0 Frequency [Hz]
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N strategy 1
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N strategy 2
N strategy 1
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N strategy 2
N strategy 1
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35
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Gluten [%]
L. Pedersen, J.R. Jørgensen / Journal of Cereal Science 46 (2007) 132–138
10 5
0 100.0
Fig. 2. Creep recovery and oscillation for cv. Encore, (strategy 3 ¼ three split, N level 3 ¼ 210 kg N ha1). Mean and standard deviation for four replications is indicated.
Reaper 210 kg N
Fig. 1. Gluten content (% of flour) from (a) 1998 and (b) 1999 at three N levels (130, 170, and 210 kg N ha1). Standard deviation for four replications is indicated.
variability in elasticity between the samples. After 300 s of recovery a part of the maximum strain is permanent, indicating that the gluten behaves like a viscoelastic liquid. It has been stated that gluten relaxation can be divided into two processes, the first occurs after a short time, 0.1–10 s, and the other occurs at longer times, 10–104 s (Funt BarDavid and Lerchenthal, 1975; Bohlin and Carlson, 1981). It was also found that gluten was able to recover completely (Funt Bar-David and Lerchenthal, 1975). In our measurements only a part of the deformation seemed to be recoverable, but due to a rather short time of recovery the gluten was not allowed to recover to a steady state. However, this short-time scale corresponds to real life conditions during processing of biscuit dough, and the method could easily distinguish between gluten from varying cultivars and growth conditions. Oscillation behaviour (Fig. 2) of the gluten showed that G0 and G00 were of the same level of magnitude, but that the value of G0 exceeded G00 for all samples measured, meaning that the solid-like structure is the dominating one. Both G0 and G00 increased with increasing frequency. However, as G00 increased more than G0 , the phase angle d increased with increasing frequency. The same frequency dependency was found in previous studies (Dreese et al., 1988; Khatkar
et al., 1995; Janssen et al., 1996a), and reflects the fact that the hydrated gluten may exhibits a more viscous behaviour when stress is applied on a short time scale. 3.3. Effect of N management on rheological properties of gluten Results from the statistical analysis of the rheological characteristics are shown in Table 3. All parameters from creep, recovery, and oscillation were significantly different for the cultivar types (Po0.001); mean values are shown in Table 4. In the creep recovery measurement, Reaper differed from the two other cultivars due to a low resistance to deformation measured by a high maximum strain and a high recovery strain, whereas Ritmo and Encore had the same values. With regard to oscillation, Ritmo had higher values of G0 and G00 , which indicates a more stiff structure of the gluten and a high degree of cross-linking compared to the other two cultivars. Phase angle d, which reflects the relative contribution of the elastic and viscous behaviour in the gluten, differed significantly between the three cultivars tested, where the gluten of Reaper was considerably more viscous in behaviour (high value of d). These differences in viscoelasticity are most likely explained by differences in the protein composition expressed as different ratios of gliadins to glutenins. Gluten from wheat with good and poor bread making quality was also found to be significantly different in oscillation (Khatkar et al., 1995; Janssen et al., 1996a), and several studies have demonstrated that differences in gliadin/glutenin ratio are reflected in the measurements of
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viscoelastic properties (Janssen et al., 1996b; Schropp and Wieser, 1996; Uthayakumaran et al., 1999; Tronsmo et al., 2003). Results from the present study indicate that gluten from biscuit wheat cultivars with a relatively low variability in protein content may exhibit large differences in viscoelastic properties measured by oscillation testing. The effect of N level varied between the two years, and between the rheological parameters measured (Table 3). In 1998 all parameters, except G00 were highly affected by the N level, but in 1999 only phase angle d was affected. Growing conditions in 1998 resulted in high yield and a relatively low protein, and in this year with optimum growing conditions, the N level significantly affected the viscoelasticity of the gluten. In creep recovery, maximum strain and recovery strain increased with increasing N level, and in oscillation G0 and G00 decreased, whereas phase angle d increased when N level was increased (Table 4). Thus, increasing N level seemed to alter the gluten making
Table 3 Variance ratios (F-values) and significance levels of cultivar, N level and N strategy for 1998 and 1999
1998 Cultivar N strategy N level Cultivar N strategy Cultivar N level 1999 Cultivar N strategy N level Cultivar N strategy Cultivar N level
Maximum strain (%)
Recovery strain (%)
G0 (Pa) G00 (Pa) d (3)
62.18*** 0.44NS 9.11*** 1.19NS
57.67*** 0.61NS 8.43*** 1.28NS
17.83*** 0.60NS 5.14** 3.37*
2.92*
3.05*
2.51*
72.07*** 1.86NS 2.90NS 2.97*
83.83*** 1.82NS 3.46* 3.24*
38.74*** 2.16NS 6.18* 1.84NS
1.49*
1.88NS
2.66*
Significance levels: NS, non significant, *, Po0.05, Po0.001.
38.75*** 1.07NS 0.64NS 3.84*
56.84*** 0.32NS 14.92*** 0.44NS
2.22NS 3.79**
17.17*** 2.07NS 2.42NS 0.99NS
58.58*** 0.57NS 5.06** 1.64*
1.50NS 2.45* **
, Po0.01,
***
,
it less elastic and more viscous likely due to changes in the protein composition. Khatkar et al. (1995) found that increasing gliadin/glutenin ratio decreased G0 and increased phase angle d. Thus, an increased level of gliadins in the gluten has a softening effect on the gluten properties. Increasing levels of gliadins at high N levels most likely can explain our observation, also due to the fact that the water content of the gluten was not altered in relation to N level. A relationship between N level and cultivar was observed (Fig. 3). The responses to the N level were equal for Reaper and Encore, whereas Ritmo differed. The gluten from Encore and Reaper likely changed in structure between N level one and two, but the structure of gluten from Ritmo was likely changed between N level two and three. These results demonstrate that the effect of the level of N application on the rheological properties of gluten varied among the three cultivars. Previous studies of the influence of N fertiliser showed that the amount of N fertiliser did not directly affect the amount of high molecular weight glutenins relative to the other storage proteins (Fullington et al., 1983). However, subsequent work of Wooding et al. (2000) demonstrated that an increase in gliadins was observed for increasing N level when combined with addition of S fertiliser, and they also showed a cultivar–nitrogen fertiliser relationship. The study of Daniel and Triboi (2000) also agreed with these observations, namely, that N level was affecting the amount of gliadins in the grain. Thus, if increased level of N fertiliser results in increased production of gliadins, it can be concluded that this change correlates well with increased viscous properties of gluten. The cultivars included in these trials are all characterised by a weak gluten structure, and increasing levels of gliadins may alter the viscoelastic properties more compared to cultivars suitable for bread making. 4. Conclusions The results of the study showed that protein and gluten contents were influenced by the genetic origin (cultivar), and the N strategy. However the effects were only significant for 1999, where growth was restricted due to limited rainfall. Two-split N application resulted in
Table 4 Mean values of rheological properties of gluten from different cultivars and fertiliser application Maximum strain (%)
Recovery strain (%)
G0 (Pa)
1998
1999
1998
1999
1998
1999
1998
1999
1998
1999
Ritmo Encore Reaper
38.6b 36.7b 59.4a
29.1b 24.5b 55.6a
20.0b 18.0b 32.2a
15.2b 13.0b 26.3a
1000a 829b 853b
1134a 1122a 810b
584a 454b 559a
627a 587b 530c
30.3b 29.0c 33.3a
29.1b 27.8c 33.1a
Fertiliser application (kg N ha1) 130 170 210
39.5a 46.3b 50.0b
33.4a 36.3ab 39.6b
20.1a 24.7b 26.2b
17.1a 17.9ab 19.6b
1044a 1081a 947b
543a 543a 531a
583a 598a 562b
29.9a 31.2b 32.2c
29.4a 29.8a 30.9
Cultivar
Means within a column followed by the same letter are not significantly different.
948a 904ab 846b
G00 (Pa)
d (3)
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40 30
800 600
20
400
10
200 0
0 1
2
3
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1
2
3
1
2 N level
3
40 35 [°]
Recovery [%]
137
30 25 20
1
2 N level
3
Ritmo
Encore Reaper
Fig. 3. Creep, recovery, and oscillation of three cultivars (1998) as a function of N level, 1 ¼ 130 kg N ha1; 2 ¼ 170 kg N ha1; 3 ¼ 210 kg N ha1.
significantly lower protein and gluten content than one or three-split. Protein and gluten content increased significantly with increasing N level in both years, and the cultivar Reaper attained the highest values. These results also demonstrate the capability of fundamental testing, since creep recovery and oscillation could discriminate the gluten among the three cultivars grown with different N levels. Inter-cultivar differences and N level account for the variability in viscoelastic properties of gluten. The N level mainly influenced the viscoelasticity of gluten in the year with optimum growing conditions (1998). In this year increasing N level resulted in gluten with a more viscous behaviour. The increase in viscous properties of gluten is suggested to be a result of a higher ratio of gliadins in the gluten network. Increased viscous properties of gluten will be an advantage in biscuit dough processing, and therefore it may be beneficial to grow biscuit wheat with higher than normal N applications, in order to obtain biscuit wheat with optimum biscuit-making properties. Acknowledgement The present work was funded by the First Framework Programme of The Cereal Network (The Danish Ministry of Food, Agriculture and Fisheries). References Baezinger, P.S., Clements, P.S., McIntosh, M.S., Yamazaki, W.T., Starling, T.M., Sammons, D.J., Johnson, J.W., 1985. Effect of cultivar, environment and their interaction and stability analyses on
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