Composition of gluten proteins in spring and winter wheat grain cultivated under conditions of varied fertilization

Abstract

The composition of protein fractions, particularly gluten protein, determines the nutritional values and baking properties of wheat grain. The baking characteristics of grain are strongly related to the quantity of monomeric gliadins and polymeric glutenins. Their accumulation and proportions impact the viscosity, porosity, and degree of rising in baking. These properties determine the applications of wheat in the baking industry. The changes in protein composition are mainly influenced by the genotype, the environment, and the interactions between the genotype and the environment and fertilization. The presented results of the studies refer to the impact of fertilization using waste products (meat bone meals, MBMs) originating from the recycling industry, in comparison to organic and mineral fertilizers, on the technological quality of spring and winter wheat based on the proportion of gluten proteins. The content of gluten proteins, particularly high molecular weight (HMW) and low molecular weight glutenins, in the protein of the examined spring and winter wheat cultivars increased with manure and bone meat meal fertilization, both with and without the addition of effective microorganisms. The grain of the Tybalt spring wheat cultivar had a low content of the harmful α/β-gliadin subunits in relation to the examined wheat cultivars under the influence of fertilization. Fertilization with compost generated a beneficial ratio of the most desired HMW glutenins to the total content of gluten proteins in spring wheat. The evaluated fertilizers (MBM, organic fertilizers) and the addition of microbiological preparations to MBM have good fertilizing properties by making the soil nutrients available to plants.

Keywords:

• effective microorganisms
• HMW
• LMW
• meat bone meal
• organic fertilization

Introduction

The composition of protein fractions, particularly gluten protein, determines the nutritional values and baking properties of wheat grain (Wieser & Kieffer 2001; He et al. 2005; Wieser 2007; Zhang et al. 2007; Naeem et al. 2012). The changes in protein composition are mainly influenced by the genotype, environment, interactions between the genotype and environment and fertilization (Graybosch et al. 1996; MacRitchie 1999; Zhu & Khan 2002; Stępień & Wojtkowiak 2011). The type and volume of fertilizers, especially the N:P:K ratio, is essential to the chemical components of wheat protein.

Progressive specialization in agriculture and environmental pollution, due to the excessive use of chemical plant protection products and mineral fertilizers, has stimulated a search for pro-ecological solutions (Chen et al. 2011). Restrictions in using natural fertilizers (manure) have led to the implementation of different forms of organic fertilization. Their action is not only limited to providing the nutrients necessary for healthy plant growth but also contributes to maintaining the activity and stability of biological, physical, and chemical homeostasis of the cultivated layer of soil and, consequently, impacts on plant yield. In recent years, a number of alternative sources of organic matter have been discovered, such as products made of waste materials and recycled into composts or as MBM (Jeng et al. 2004; Vagstad et al. 2009; Chen et al. 2011; Konopka et al. 2012; Poulsen et al. 2013). MBM, a by-product of the meat industry, contains approximately 8% N, 5% P, 1% K, and up to 10% Ca, depending on the recycling process and the origin of organic waste material (Chen et al. 2011; Stępień 2011). The high fertilization value and use of MBMs to fertilize cereals have been confirmed by the results of field experiments, mainly conducted in Finland (Salomonsson et al. 1995; Chen et al. 2011). Their impact on the yielding and the content of protein in grain was comparable to the effects recorded using mineral fertilizers. According to Fredriksson et al. (1997), fertilization with MBM provides wheat plants cultivated under ecological conditions with the nitrogen necessary for good baking efficiency. The nutrients in MBM are in a biological form (Ylivainio et al. 2007). An increase in the efficacy of transition of macro- and microelements found in soil into available forms and their assimilation by plants may be improved with the application of effective microorganisms (EM) (Mondini et al. 2008; Poulsen et al. 2013).

The objective of the studies was to present the impact of fertilization with waste products originating from the recycling industry, compared to organic and mineral fertilizers, on the technological quality of spring and winter wheat measured with the content of gluten fractions.

Materials and methods

Site and experimental set-up

The presented results of studies on different systems of fertilization in crop rotation systems originate from a static field experiment using a randomized block model in four repetitions. The experiment was carried out in 2005–2009 at the Production and Research Centre in Bałcyny near Ostróda, Poland (53^°36′N, 19^°51′E) on soil classified according to the granule size composition as loamy sand in the third (very good rye-type) soil complex class, with a high abundance of phosphorus and a moderate abundance of potassium and magnesium. The surface of the experimental harvest plot was 24.75 m². Spring wheat (Nawra cultivar) was cultivated in 2005 after a cereal mixture, winter wheat (Oliwin cultivar) in 2007 in a crop rotation system after field bean, and spring wheat (Tybalt cultivar) was seeded in 2009 after winter rape.

The year 2005 was the least favorable for the vegetation of spring wheat in terms of water availability, which did not foster optimal humidity of the soil and the release of nutrients from the fertilizers. In 2007, the distribution of precipitation in combination with higher temperatures favored the cultivation of winter wheat and the stable release of nutrients from organic fertilizers. In terms of humidity and temperature, 2009 was the most optimal year.

On the experimental object fertilized with mineral fertilizer (NPK), spring and winter wheat were fertilized with nitrogen at 60 kg ha⁻¹ before seeding and at 30 kg ha⁻¹ at Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) 31–32. Phosphorus fertilizers were applied before seeding at 31 kg ha⁻¹ as triple superphosphate (46%) and fertilization with potassium was used at 83 kg ha⁻¹ as 60% potassium salt. The composition of organic fertilizers and MBM are given in Table 1.

Table 1. Diagram of the experimental model including the fertilization systems and introduction of nutrients (kg ha⁻¹ year⁻¹).

System of fertilization	Without fertilization	Mineral fertilizers (NPK)	Farm yard manure (FYM)	Compost	Meat and bone meal (MBM)	Meat and bone meal and effective microorganisms spraying (MBM + EM)
Dose of fertilizer	–	–	10 t ha^−1	10 t ha^−1	1.5 t ha^−1	1.5 t ha^−1
Content of mineral compounds
N	–	90.0	51.0	71.0	99.8	99.8
P	–	31.0	12.1	29.0	59.7	59.7
K	–	83.0	49.0	62.0	6.2	6.2
Mg	–	–	8.0	16.0	3.0	3.0
Ca	–	–	34.0	52.0	28.5	28.5
Cu	–	–	0.05	0.05	0.02	0.02
Fe	–	–	3.85	5.58	0.77	0.77
Mn	–	–	0.45	0.74	0.01	0.01
Zn	–	–	0.25	0.50	0.15	0.15

Manure and compost were introduced annually at 10 t ha⁻¹ in the pre-seeding system of summer-autumn (winter wheat) or spring (spring wheat) cultivations.

MBM (MBM + EM) was applied before seeding at 1.5 t ha⁻¹. MBM supplemented with EM was used on the object MBM + EM. This microbiological preparation was applied in the cultivation of all plants at 5 dm³ ha⁻¹ divided into two doses (the first dose at 3 dm³ ha⁻¹ before seeding and the second dose at 2 dm³ ha⁻¹ before the first procedure, i.e., harrowing). It contained the microorganisms described by Valarini et al. (2003). The preparation EM was prepared according to the instructions by activating the microorganisms through incubation with a proper dose of sucrose.

Seeding, cultivation procedures, and the harvesting of spring and winter wheat were carried out in accordance with the agricultural engineering requirements specific for a given plant species. Weeds were eradicated only by mechanical means. The elimination of diseases and pests was not performed.

Protein extraction and analysis

The samples of grain were purified of loosely bound contamination and fractions with grain widths below 2.2 mm. Next, the grain was comminuted in an IKA A10 laboratory mill (Labortechnik) to filter particles passing through a 400-µm sieve, with the majority composed of particles smaller than 250 µm. Such samples were weighed into Eppendorf test tubes at 100 mg and the protein fractions were extracted with the method of Wieser et al. (1998). Gliadins were extracted three times with 1 ml 60% ethanol and glutenins were extracted twice in 1 ml of mixture composed of 50% propanol-1, 2M urea, TRIS–HCl, and 1% DTE and saturated with nitrogen. The assays were performed in a Hewlett Packard series 1050 apparatus with the following parameters: RP-18 Vydac 218TPP54, 5 µm, 250×4.6 mm column, Zorbax 3000SB-C18 4.6×12.5 mm pre-column, column temperature of 45°C, flow of mobile phase at 1 ml/min, and an injection volume of 20 µl. The detection was carried out with a Hewlett Packard detector at the 210-µm wave length. The results were analyzed with high-performance liquid chromatography (HPLC) software in accordance with 3D Chem Station by HP and expressed in milliabsorbance units seconds (mAUs) units.

Statistical analyses

The results were statistically processed with STATISTICA 9.0 software (Statsoft). The statistical calculations were performed with a one-way analysis of variance. Apart from the basic parameters, standard deviation and statistically homogenous groups were determined with Duncan's test at α = 0.05. Coefficients of linear correlation (r Pearson's) were calculated.

Results and discussion

According to Stępień (2011), the grain of the spring wheat Nawra cultivar cultivated in 2005 had good quality parameters: high thousand grain weight (44.1 g) and the content of 13.8% protein and 29% gluten. Slightly worse parameters characterized the grain of the Tybalt cultivar: 33.2 g, 12.1%, and 24.9%, respectively. The grain obtained in 2007 had good parameters for winter wheat (Oliwin): 40.0 g thousand grain weight, 11.5% protein, and 23.1% gluten. The thousand grain weight depends mainly on the properties of a cultivar and may be modified by environmental and agrotechnical conditions. The results of studies on the relation of protein content and its fraction to the size and mass of grain are not unanimous. Some authors claim the positive relation between protein content, protein fraction, and grain size, whereas others deny its occurrence. According to Konopka et al. (2007a), the average protein content diminishes along with the decrease in grain size. But Daniel and Triboi (2002) demonstrated that in the case of the decrease in grain mass, the protein content increased significantly. In the present study, such unanimous relation was not found.

High grain yield and good protein quality are important features for the production and trade of wheat. The content and composition of gluten proteins are the main factors influencing its technological quality (Uthayakumaran et al. 2000; Zhu & Khan 2002; He et al. 2005; Anjum et al. 2007). Gliadins and glutenins are storage proteins with the latter being more important than gliadins for obtaining good dough properties (Wieser & Kieffer 2001; Zhang et al. 2007; Li et al. 2008). Both fractions are rich in glutamic acid, proline, and hydrophobic amino acids, whereas gliadins have a relatively low content of lysine, histidine, threonine, and tryptophan (Wieser 2007). It is thus thought that the nutritional value of gliadins and glutelins is lower than the structural proteins (albumins and globulins). The quality parameters of grain may be improved with proper agricultural engineering procedures (Labuschagne et al. 2006). The rheological properties of bread depend mainly on the method of wheat cultivation, with nitrogen fertilization being the most important factor influencing the content and quality of cereal grain protein (Johansson et al. 2004; Labuschagne et al. 2006).

Changes in the amino acid and fractional composition of protein, which determine its biological value, may be a consequence of intensive plant fertilization. The studies by Li et al. (2008) confirmed the impact of the content of protein and protein fractions on the baking properties of dough.

The results of our studies showed that α/β gliadins were the quantitatively predominant fraction of gliadin proteins in the grains of the examined wheat cultivars (Table 2). It was found that fertilization with manure and MBM (alone and supplemented with EM) contributed to a substantial increase in the content of harmful α/β and γ protein subunits in all wheat cultivars. Moreover, the content of ω-gliadins increased in the Oliwin cultivar of winter wheat under the impact of the same fertilizers. The comparison of wheat cultivars revealed that the Tybalt cultivar grain (of the smallest thousand grain weight) generally had the lowest content of ω-gliadin (1.1–1.4 thousands) and α/β (13.3–15.9 thousands) fractions with the highest content of γ-gliadins (12.0–13.3 thousands).

Table 2. The content of protein fractions (the surface of peaks expressed in thousands mAU s) in grain of the spring and winter wheat under different fertilization methods.

Specification		Without fertilization	NPK	FYM	Compost	MBM	MBM + EM
Spring wheat (2005, v. Nawra)
Gliadin sibunits	ω	2.3^b±0.22	2.5^b±0.13	2.6^b±0.14	1.4^a±0.67	2.7^b±0.03	2.7^b±0.08
	α/β	15.9^b±0.05	16.4^b±0.36	17.1^c±0.10	13.9^a±0.20	18.1^d±0.13	18.3^d±0.13
	γ	9.7^bc±0.35	9.6^ab±0.03	10.2^c±0.24	9.1^a±0.12	11.1^d±0.23	11.0^d±0.05
Glutenin sibunits	HMW	7.0^a±0.06	7.3^b±0.01	8.1^e±0.04	7.1^a±0.04	7.7^c±0.02	8.0^d±0.05
	LMW	16.8^a±0.04	17.5^b±0.13	19.2^d±0.27	16.3^a±0.30	18.5^c±0.03	18.6^c±0.12
Σ Gluten protein		51.7^b±0.64	53.2^b±0.31	57.1^c±0.71	47.8^a±1.09	58.0^cd±0.44	58.7^d±0.12
Winter wheat (2007, v. Oliwin)
Gliadin subunits	ω	2.2^a±0.11	2.3^ab±0.05	2.5^bc±0.08	2.3^ab±0.04	2.5^c±0.12	2.5^c±0.04
	α/β	15.7^a±0.30	16.5^b±0.08	16.5^b±0.21	16.1^ab±0.31	17.8^c±0.31	17.8^c±0.09
	γ	8.4^a±0.13	8.5^ab±0.04	8.7^bc±0.06	8.6^ab±0.07	9.0^cd±0.11	9.1^d±0.20
Glutenin subunits	HMW	4.8^a±0.15	6.3^d±0.07	5.6^c±0.05	5.3^b±0.10	6.3^d±0.10	5.7^c±0.04
	LMW	11.9^a±0.11	15.3^e±0.04	14.0^d±0.06	12.7^b±0.19	12.6^b±0.22	13.3^c±0.34
Σ Gluten protein		43.0^a±0.79	48.9^e±0.12	47.2^c±0.08	45.0^b±0.53	47.4^cd±0.66	48.5^de±0.06
Spring wheat (2009, v. Tybalt)
Gliadin subunits	ω	1.1^a±0.02	1.3^d±0.02	1.2^b±0.01	1.3^c±0,01	1.4^f±0.00*	1.3^e±0,01
	α/β	13.3^a±0,00	14.7^c±0.01	14.2^b±0.04	14.6^c±0.05	15.0^d±0.02	15.9^e±0.21
	γ	12.1^a±0.09	12.0^a±0.01	13.0^c±0.04	12.4^b±0.19	13.3^d±0.06	13.0^c±0.08
Glutenin subunits	HMW	7.0^b±0.01	6.9^a±0.03	7.5^c±0.06	7.5^c±0.03	7.7^d±0.07	7.6^cd±0.05
	LMW	16.9^a±0.07	17.4^ab±0.02	18.2^c±0.01	17.7^bc±0.06	17.8^bc±0.02	18.3^c±0.06
Σ Gluten protein		50.4^a±0.01	52.2^b±0.07	54.2^d±0.09	53.3^c±0.03	55.2^e±0.05	56.0^f±0.05

a – within each line means with the same letter are not significantly different (p < 0.05),±SD

* values below 0.01, Σ – sum, NPK – mineral fertilizers, FYM – farm yard manure, MBM – meat and bone meal, MBM + EM − meat and bone meal and effective microorganisms spraying.

High molecular weight (HMW) subunits of glutenins and low molecular weight (LMW) subunits of glutenins and gliadins are associated with dough energy and bread baking quality (Gupta et al. 1991; Wieser & Kieffer 2001; He et al. 2005). The number of glutenin subunits is positively correlated with dough energy and volume of bread, whereas the content of gliadins and a higher gliadin:glutenin ratio have a negative impact on these parameters (Uthayakumaran et al. 2000; Wieser & Zimmermann 2000; Singh & Skerritt 2001; Wieser & Kieffer 2001). Despite the content of HMW proteins being lower in the grain of the examined cultivars, according to Wieser and Kieffer (2001) they determine the properties of dough to a higher degree than LMW proteins.

The content of HMW and LMW glutenins in the grain of the tested wheat cultivars significantly increased under the influence of manure applied at 10 t ha⁻¹ and MBM applied alone at 1.5 t ha⁻¹ and with the addition of EM in comparison with the control object. Furthermore, in this experiment, the content of both glutenin fractions in winter wheat Oliwin cultivar cultivated in another year of crop rotation system (2007) increased under the influence of mineral fertilization (NPK) and fertilization with compost and in the Tybalt cultivar of spring wheat under the influence of compost. Konopka et al. (2007b), based on the studies with three wheat cultivars, found that the concentration of γ-gliadins and HMW and LMW glutenins decreased in the case of water deficit. The Nawra cultivar was the most susceptible to the changes, which was not confirmed in our studies.

The beneficial influence of organic fertilizers demonstrated in the current study results from an increase in soil fertility, among others. These fertilizers exert an impact on the increase of macro- and micronutrients in soil, including total nitrogen (Tenuta & Lazarovits 2003; Jeng et al. 2006; Ylivainio et al. 2007). Jeng et al. (2004) found that nitrogen administered to plants as MBM covered 80% of the nitrogen requirements for fertilizers in cereals. In our studies, the content of individual protein fractions in the grain could have been determined by cultivar features and weather conditions during plant growth. Numerous authors have emphasized their impact, in particular, on a deficit in water (Erekul & Köhn 2006; Konopka et al. 2007b).

The current study found that the experimental fertilization impacted on the proportion of gluten proteins in the grains of the examined cultivars of spring and winter wheat (Table 3). The Nawra cultivar of spring wheat cultivated at the beginning of a crop rotation (2005) reacted positively to the applied mineral fertilization and fertilization with manure and MBMs. The second cultivar (Tybalt) of spring wheat cultivated in the subsequent years of the crop rotation system had a higher percentage content of gluten proteins (gliadins + glutenins). The grain of this wheat cultivar had the highest accumulation of the least valuable gliadin and glutenin proteins, especially with the fertilization with MBM at 1.5 t ha⁻¹ combined with EM (82.3%). The investigated cultivars, similar to the studies by Konopka et al. (2007a), differed in their content and composition of protein.

Table 3. Percentage of gluten proteins in the grain in relation to total protein grain of the spring and winter wheat under different fertilization methods.

Specification	Without fertilization	NPK	FYM	Compost	MBM	MBM + EM
Spring wheat (2005, v. Nawra)	73.1^b±0.19	75.2^c±0.45	75.0^c±0.20	70.9^a±0.49	74.4^c±0.46	78.3^d±0.45
Winter wheat (2007, v. Oliwin)	78.8^b±0.18	79.8^c±0.21	79.9^c±0.33	79.0^b±0.17	75.7^a±0.11	72.8^a±0.11
Spring wheat (2009, v. Tybalt)	79.7^a±0.07	81.9^d ±0.11	80.9^c ±0.04	80.4^b±0.03	80.2^b±0.08	82.3^e±0.15

^a Within each line means with the same letter are not significantly different (p < 0.05), ±SD

NPK, mineral fertilizers, FYM, farm yard manure; MBM, meat and bone meal; MBM + EM, meat and bone meal and effective microorganisms spraying.

The content of gluten fractions in the protein in winter wheat (Oliwin cultivar) significantly increased with the standard doses of mineral fertilization (NPK) and manure. According to Górecka et al. (2009), the stimulating effect on the yielding of the solid fertilizers applied at a lower dose was comparable to the effects recorded after the administration of simple natural fertilizers.

The nutritional value of plant protein is influenced not only by the content of individual fractions of proteins with varied amino acid composition but also mainly by their ratio (Zhang et al. 2007). Stępień and Wojtkowiak (2011) suggested that the changes in the quantitative proportions between the protein fractions in wheat grain which lowered the nutritional value of the protein could have resulted from intensive nitrogen fertilization.

In addition to the presented results, a statistical analysis of the data also confirmed generally a positive correlation between the subunits of gliadin and glutenin and the sum of gluten protein. In the spring wheat of Nawra cultivar (2005), the coefficient of r Pearson's correlation ranged from 0.86 to 0.98, while in the case of Tybalt cultivar (2009) from 0.78 to 0.87. The exception was the lack of relationship between gliadin γ subunits and the sum of gluten protein of winter wheat (2007). For the other protein fractions of this wheat form the positive correlation ranging from r=0.66 to r=0.89 was found.

In our study, the ratio of individual protein subunits in the cereal grain cultivated in 2005–2009 changed in relation to the total content of complex proteins under the influence of applied fertilization (Table 4). The lowest parameters were recorded by comparing the fractions of α/β gliadins and LMW glutenins in relation to the total content of gluten proteins. The changes in the quantitative proportions between the subunits of protein fractions under the influence of fertilization may suggest a decrease in the nutritional value of protein. The significant increase in the proportions between adverse α/β gliadins was influenced by the fertilization with MBM in the grain of wheat cultivated in 2007 and 2009 and with MBM combined with EM in the last year of crop rotation (2009). This method of fertilization also exerted an impact on the adverse changes of parameters in the composition of ω-gliadins in relation to the total content of gluten proteins in the grain of spring wheat cultivated in 2009. The increase in the proportion of ω-gliadins was also detected following the application of nitrogen at 90 kg ha⁻¹ and compost at 10 t ha⁻¹ in the cultivation of Tybalt spring wheat cultivar. HMW and LMW glutenins and their proportions are responsible for the quality of bakery products (Wieser & Zimmermann 2000). By comparing the indices of glutenin subunits in relation to gluten proteins, the content of LMW glutenin proteins has higher values than the more desired HMW glutenins. The organic fertilization with manure and compost generated a significant increase in the proportions between these proteins in the wheat grain cultivated at the beginning of the crop rotation in 2005. The mineral fertilization and organic fertilization with manure influenced the changes in the composition of LMW protein in relation to the total content of protein responsible for the baking properties in the Oliwin cultivar of winter wheat.

Table 4. Relation of protein fraction to the total gluten proteins in the grain of the spring and winter wheat under different fertilization methods.

Specification	Without fertilization	NPK	FYM	Compost	MBM	MBM + EM
Spring wheat (2005, v. Nawra)
Gliadin ω:gluten protein	0.05^b	0.05^b	0.05^b	0.03^a	0.05^b	0.05^b
Gliadin α/β:gluten protein	0.31^c	0.31^c	0.30^b	0.29^a	0.31^c	0.31^c
Gliadin γ:gluten protein	0.19^bc	0.18^ab	0.18^a	0.19^c	0.19^c	0.19^bc
HMW:gluten protein	0.14^a	0.14^a	0.14^b	0.15^c	0.13^a	0.14^a
LMW:gluten protein	0.32^ab	0.33^bc	0.34^cd	0.34^d	0.32^a	0.32^a
Winter wheat (2007, v. Oliwin)
Gliadin ω:gluten protein	0.05^b	0.05^a	0.05^b	0.05^b	0.05^b	0.05^b
Gliadin α/β:gluten protein	0.37^d	0.34^a	0.35^b	0.36^c	0.38^e	0.37^d
Gliadin γ:gluten protein	0.20^d	0.17^a	0.19^b	0.19^c	0.19^bc	0.19^bc
HMW:gluten protein	0.11^a	0.13^c	0.12^b	0.12^b	0.12^ab	0.12^ab
LMW:gluten protein	0.28^b	0.31^d	0.30^c	0.28^b	0.27^a	0.28^b
Spring wheat (2009, v. Tybalt)
Gliadin ω:gluten protein	0.02^a	0.03^c	0.02^a	0.02^b	0.03^d	0.02^bc
Gliadin α/β:gluten protein	0.26^a	0.28^c	0.26^a	0.27^b	0.27^b	0.28^c
Gliadin γ:gluten protein	0.24^b	0.23^a	0.24^b	0.23^a	0.24^b	0.23^a
HMW:gluten protein	0.14^c	0.13^a	0.14^bc	0.14^c	0.14^c	0.14^b
LMW:gluten protein	0.34^c	0.33^c	0.34^c	0.33^abc	0.32^a	0.33^ab

^aWithin each line means with the same letter are not significantly different (p < 0.05).

NPK, mineral fertilizers; FYM, farm yard manure; MBM, meat and bone meal; MBM + EM, meat and bone meal and effective microorganisms spraying.

Based on the results of the current study, it is concluded that the evaluated fertilizers (MBM, organic fertilizers) and the addition of microbiological preparations to MBM have good fertilizing properties by making the soil nutrients available to plants, which was confirmed in the studies by Hessey (2003) and Mondini et al. (2008).

Acknowledgments

The study was financially supported by the Ministry of Scientific Research in Poland within the framework of grant No. N 310 082 32/3238.