Article pubs.acs.org/JAFC
Effect of Rising Atmospheric Carbon Dioxide Concentration on the Protein Composition of Cereal Grain Stefanie Wroblewitz,† Liane Hüther,†,* Remy Manderscheid,‡ Hans-Joachim Weigel,‡ Hermann Waẗ zig,§ and Sven Dan̈ icke† †
Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Bundesallee 50, 38116 Braunschweig, Germany ‡ Institute of Biodiversity, Thünen-Institute (TI), Federal Research Institute for Rural Areas, Forestry and Fisheries, Bundesallee 50, 38116 Braunschweig, Germany § Institute of Medicinal and Pharmaceutical Chemistry, Technical University Carolo-Wilhelmina Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany ABSTRACT: The present study investigates effects of rising atmospheric CO2 concentration on protein composition of maize, wheat, and barley grain, especially on the fractions prolamins and glutelins. Cereals were grown at different atmospheric CO2 concentrations to simulate future climate conditions. Influences of two nitrogen fertilization levels were studied for wheat and barley. Enriched CO2 caused an increase of globulin and B-hordein of barley. In maize, the content of globulin, α-zein, and LMW polymers decreased, whereas total glutelin, zein, δ-zein, and HMW polymers rose. Different N supplies resulted in variations of barley subfractions and wheat globulin. Other environmental influences showed effects on the content of nearly all fractions and subfractions. Variations in starch−protein bodies caused by different CO2 treatments could be visualized by scanning electron microscopy. In conclusion, climate change would have impacts on structural composition of proteins and, consequently, on the nutritional value of cereals. KEYWORDS: maize, wheat, barley, prolamin, glutelin, SE-HPLC, Osborne fractionation
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INTRODUCTION Due to human activity, an increase of the atmospheric CO2 concentration has been recognized. Within the last 260 years the CO2 concentration rose from about 280 ppm to a current value of 398 ppm.1 Predictions of a further increase up to 540 or even 970 ppm over the next 100 years were estimated by different emission scenarios.2 Numerous CO2 exposure techniques were developed to study this influence on plant growth and development.3 The FACE (free-air carbon dioxide enrichment) technique is one of the most important models to cultivate and study plants under realistic field conditions.4 A series of well-known effects of elevated atmospheric CO2 such as the stimulation of photosynthesis and enhanced plant growth were observed in C3 plants, but not in C4 plants.5 An elevated CO2 concentration is also known to affect the metabolism of carbon and nitrogen in many C3 plants, resulting in changes of chemical composition, especially of proteins and amino acids.6−8 However, the mechanism of CO2 enrichment affecting the protein content of cereal grain has not completely been elucidated until now.9 The concentration of cereal grain protein and its composition are important factors of the nutritional value10 and bread making quality.11 Cereals provide several billion tons of protein for the nutrition of human and livestock. Besides, cereal proteins influence the utilization in food processing, such as the bread making quality of wheat.12 In maize, the most common proteins are the prolamins called zeins with more than 50% of total seed proteins. Because of their lack in several essential amino acids, maize grain is nutritionally poor for monogastrics. © 2014 American Chemical Society
Therefore, mutants with lowered zein content were bred to improve nutritional quality characteristics and to develop quality protein maize for human and livestock consumption.13 The grain proteins can be separated into four fractions according to Osborne.14 He developed a classification based on the solubility in a series of solvents. Metabolic albumins are soluble in water and storage globulins in diluted saline. According to their solubility in aqueous ethanol, storage gluten can be divided into soluble prolamin and insoluble glutelin.15 Glutelins are the least well understood grain protein fractions. As a result of several studies, the glutelin fractions are renamed as polymeric forms of the prolamins whereby the prolamins represent the corresponding monomers.16−18 Gluten proteins, which are often used in animal nutrition as a supplement, represent the major protein fraction of the starchy endosperm and contain hundreds of components such as monomers or oligomers and polymers linked by interchain disulfide bonds. They are rich in glutamine and proline and have low contents of amino acids with charged side groups.19 Studies investigating the crude protein digestibility of maize grain describe partially significant increases in crude protein digestibility caused by crop cultivation at elevated atmospheric CO2 concentration.20 Furthermore, Wieser et al.21 found significant decreases in crude protein, prolamin, and glutelin Received: Revised: Accepted: Published: 6616
March 13, 2014 June 24, 2014 June 28, 2014 June 28, 2014 dx.doi.org/10.1021/jf501958a | J. Agric. Food Chem. 2014, 62, 6616−6625
Journal of Agricultural and Food Chemistry
Article
JKA Works Ic., Wilmington, NC, U.S.A.). All separations of supernatants and precipitates were performed by centrifugation for 10 min at 3000 rpm (Varifuge 3.OR, Heraus Sepatech, Osterode, Germany). All supernatants were dried to constant mass at 60 °C by centrifugal evaporator (Christ RVC 2-18HCL, Osterode, Germany). SDS-PAGE. Isolated protein fractions were treated with 7 mM SDS (>99%, Carl Roth GmbH Co., Karlsruhe, Germany) and 200 mM DTT prior to separation in 4−20% Mini-PROTEAN TGX gels (Bio− Rad, Hercules, CA, U.S.A.) using a modified method of Laemmli.24 Proteins were completely dissociated by boiling the samples at 95 °C for 5 min. Electrophoresis was carried out at 50 V for 30 min followed by 200 V for 1 h until the tracking dye, bromophenol blue (ACS, Carl Roth GmbH Co., Karlsruhe, Germany), had reached the bottom of the gel. Molecular weight markers were Roti-Mark 10−150 and RotiMark PRESTAINED (Carl Roth GmbH Co., Karlsruhe, Germany) to identify the individual protein groups on the gel. After electrophoresis, gels were stained in a Coomassie brilliant blue G-250 (Carl Roth GmbH Co., Karlsruhe, Germany) solution overnight and destained in water−methanol−pure acetic acid (40/10/50, v/v/v). SE-HPLC. SE-HPLC was performed by using a Shimadzu instrument (liquid chromatograph, LC-20AT; autosampler, SIL-20AC; column oven, CTO-10ASvp; diode array detector, SPD-M20A) and a Yarra 3 μ SEC-2000 column (300 × 7.8 mm, Phenomenex; Torrance, CA) with guard cartridges (Phenomenex; Torrance, CA). Data was collected and analyzed using the CLASS-VP-6.14 SP2A software (Shimadzu). Mobile phases (50 mM phosphate buffer containing 0.1% SDS) were prepared by mixing a 50 mM disodium hydrogen phosphate dihydrate solution and a 50 mM sodium dihydrogen phosphate monohydrate solution according to Henderson−Hasselbalch equation for required pH values of 6.60 (A) and 6.94 (B). Gliadin (from wheat, Sigma, Steinheim, Germany) and glutenin standards (Osborne fractionation of gluten, from wheat, Sigma, Steinheim, Germany) as well as grain protein samples (concentration: 2−35 mg mL−1) were weighed and dissolved in mobile phase B by using an ultrasonic bath (FB15051, Fischer Scientific, Singen, Germany). Standards and samples were filtered (syringe filters, 0.45 μm, PVDF, anchor GmbH) before injection (50 μL). The measurements were performed at 25 °C with a flow rate of 0.7 mL/min and a gradient of mobile phase (0 min 100% B, 16 min 0% B, 22 min 100% B for re-equilibration). The monitoring wavelength was 214 nm with a run time of 30 min. Calibration and evaluation of logarithm scale with the standard proteins bovine serum albumin, ovalbumin, myoglobin, and cytochrome c were carried out to determine the sizes of the single protein subfractions. SEM. For scanning electron micrographs of milled kernels of all three cultivars, an environmental SEM (EVO LS25, Carl Zeiss SMT GmbH, Oberkochen, Germany) was used. The voltage of the electron beam was 8.0 kV and a secondary electron detector was used. In preparation, kernels were fractured at ambient temperature at 1 mm. Statistics. Data were evaluated by analysis of variance (ANOVA) after testing for normal distribution and variance homogeneity using the Kolmogorov−Smirnov test and the Bartlett-χ-square test, respectively. A complete 3-factorial design of ANOVA was used to evaluate the results for wheat and barley with the cultivation year (2000 and 2003), the CO2 level (AMBI and FACE), the N-fertilization level (50 and 100% of adequate N-supply) and the corresponding interactions being the fixed factors. For evaluating the maize results the CO2 level was the only fixed factor. Significance was assumed at p < 0.05 and STATISTICA for Windows Operating System (Version 10.0., StatSoft, Inc., 2011) was used for statistical evaluations.25
of winter wheat grain due to an enriched atmospheric CO2 concentration. Therefore, the aim of the present study was to get more detailed information on the structural composition of the protein fractions prolamin and glutelin of winter wheat, winter barley and maize grains cultivated using the FACE technique. Osborne fractionation was performed to isolate the single protein fractions followed by proving the purity by SDS-PAGE. To characterize prolamins and glutelins in detail a size exclusion high performance liquid chromatography (SE-HPLC) method was developed. In addition, kernels were observed by scanning electron microscopy (SEM) to study differences in proteinstarch bodies. These investigations should also explain the observed variations in digestibility of crude protein. The study intended to assess the potential impact of a future atmospheric CO2 concentration expected by 2050 on the protein composition and quality of these important animal feedstuffs.
■
MATERIAL AND METHODS
Materials. The experiment with winter wheat and barley was conducted on a 22 ha experimental field site and the investigation on maize was performed on a 10 ha field site located at the experimental station of the Friedrich Loeffler Institute (FLI), Federal Research Institute for Animal Health, Braunschweig, Germany. A FACE system engineered by Brookhaven National Laboratory and previously described by Weigel and Manderscheid7 with experimental rings of 20 m in diameter was used to study CO2 enrichment effects on maize cv. Romario (silage maize), winter wheat cv. Batis (one of most frequently cultivated bread wheat varieties in Germany) and barley cv. Theresa (one of most frequently cultivated barley varieties in Germany) as well as the influence of N fertilization of winter wheat and barley. Therefore, two (barley and wheat) or three (maize) of the FACE rings were treated with blowers with air of ambient CO2 concentration (AMBI, 380 ppm atmospheric CO2) and the other two (barley and wheat) or three (maize) with blowers with CO2 enriched air (FACE, 550 ppm atmospheric CO2). The FACE rings were placed exactly at the same position in each growing season. For winter wheat and barley, two different N fertilization levels were applied in each half of the rings to study interactions between CO2 enrichment and N supply. One was an adequate N fertilization (N100) for each crop species and one was a reduced fertilization with 50% (N50) of the adequate N treatment as it was described by Weigel and Manderscheid.22 For both crops the space between the two fertilization levels was set to approximately 1 m. Varieties of all three cereals were chosen because of their general reliable yield forecasts. For analytical use, kernels of winter wheat and winter barley of the two cultivation seasons and kernels of maize cultivar were predried, milled at 1 mm and stored at −20 °C. Protein Extraction. A modified Osborne fractionation according to Wieser et al.23 was performed to separate the protein fractions of all three cultivars. 0.5 g milled (1 mm) flour was extracted two times with 0.5 mol L−1 sodium chloride (NaCl, ≥ 99%, Ph.Eur., USP, Carl Roth GmbH Co., Karlsruhe, Germany) for 10 min at room temperature (RT ∼ 23 °C). Supernatant (albumins/globulins) was dialyzed (ZelluTrans, regenerated Cellulose Tubular Membrane, 6000−8000 Da, 1.67 mL/cm, Carl Roth GmbH Co., Karlsruhe, Germany) against water to separate both protein fractions in water - soluble albumins and water insoluble globulins. Precipitate (prolamins/glutelins) was extracted twice with 70% ethanol (EtOH, Rotipuran, ≥ 99.8%, p.a., Carl Roth GmbH Co., Karlsruhe, Germany) for 30 min at RT. The supernatant consisted of EtOH-soluble prolamins. Precipitate was extracted twice with a solvent of 50% 1-propanol containing 1% dithiothreitol (DTT), 0.05 M trisHCl, and 2 M urea (all chemicals at least ≥99%, p.a. quality, Carl Roth GmbH Co., Karlsruhe, Germany) for 20 min at 60 °C. Each extraction step was initiated by vortexing until complete mixing and continued by vortexing at 600 rpm (MS1 minishaker,
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RESULTS Protein Extraction and SDS-PAGE. The relative amounts of the single protein fractions were obtained by gravimetric analysis after Osborne fractionation. Tables 1−3 summarizes the effect of different N fertilization, CO2 treatment and crop growing year on the percentages of the four fractions of winter barley, wheat, and maize. 6617
dx.doi.org/10.1021/jf501958a | J. Agric. Food Chem. 2014, 62, 6616−6625
0.083
Effect of Rising Atmospheric Carbon Dioxide Concentration on the Protein Composition of Cereal Grain Stefanie Wroblewitz,† Liane Hüther,†,* Remy Manderscheid,‡ Hans-Joachim Weigel,‡ Hermann Waẗ zig,§ and Sven Dan̈ icke† †
Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Bundesallee 50, 38116 Braunschweig, Germany ‡ Institute of Biodiversity, Thünen-Institute (TI), Federal Research Institute for Rural Areas, Forestry and Fisheries, Bundesallee 50, 38116 Braunschweig, Germany § Institute of Medicinal and Pharmaceutical Chemistry, Technical University Carolo-Wilhelmina Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany ABSTRACT: The present study investigates effects of rising atmospheric CO2 concentration on protein composition of maize, wheat, and barley grain, especially on the fractions prolamins and glutelins. Cereals were grown at different atmospheric CO2 concentrations to simulate future climate conditions. Influences of two nitrogen fertilization levels were studied for wheat and barley. Enriched CO2 caused an increase of globulin and B-hordein of barley. In maize, the content of globulin, α-zein, and LMW polymers decreased, whereas total glutelin, zein, δ-zein, and HMW polymers rose. Different N supplies resulted in variations of barley subfractions and wheat globulin. Other environmental influences showed effects on the content of nearly all fractions and subfractions. Variations in starch−protein bodies caused by different CO2 treatments could be visualized by scanning electron microscopy. In conclusion, climate change would have impacts on structural composition of proteins and, consequently, on the nutritional value of cereals. KEYWORDS: maize, wheat, barley, prolamin, glutelin, SE-HPLC, Osborne fractionation
■
INTRODUCTION Due to human activity, an increase of the atmospheric CO2 concentration has been recognized. Within the last 260 years the CO2 concentration rose from about 280 ppm to a current value of 398 ppm.1 Predictions of a further increase up to 540 or even 970 ppm over the next 100 years were estimated by different emission scenarios.2 Numerous CO2 exposure techniques were developed to study this influence on plant growth and development.3 The FACE (free-air carbon dioxide enrichment) technique is one of the most important models to cultivate and study plants under realistic field conditions.4 A series of well-known effects of elevated atmospheric CO2 such as the stimulation of photosynthesis and enhanced plant growth were observed in C3 plants, but not in C4 plants.5 An elevated CO2 concentration is also known to affect the metabolism of carbon and nitrogen in many C3 plants, resulting in changes of chemical composition, especially of proteins and amino acids.6−8 However, the mechanism of CO2 enrichment affecting the protein content of cereal grain has not completely been elucidated until now.9 The concentration of cereal grain protein and its composition are important factors of the nutritional value10 and bread making quality.11 Cereals provide several billion tons of protein for the nutrition of human and livestock. Besides, cereal proteins influence the utilization in food processing, such as the bread making quality of wheat.12 In maize, the most common proteins are the prolamins called zeins with more than 50% of total seed proteins. Because of their lack in several essential amino acids, maize grain is nutritionally poor for monogastrics. © 2014 American Chemical Society
Therefore, mutants with lowered zein content were bred to improve nutritional quality characteristics and to develop quality protein maize for human and livestock consumption.13 The grain proteins can be separated into four fractions according to Osborne.14 He developed a classification based on the solubility in a series of solvents. Metabolic albumins are soluble in water and storage globulins in diluted saline. According to their solubility in aqueous ethanol, storage gluten can be divided into soluble prolamin and insoluble glutelin.15 Glutelins are the least well understood grain protein fractions. As a result of several studies, the glutelin fractions are renamed as polymeric forms of the prolamins whereby the prolamins represent the corresponding monomers.16−18 Gluten proteins, which are often used in animal nutrition as a supplement, represent the major protein fraction of the starchy endosperm and contain hundreds of components such as monomers or oligomers and polymers linked by interchain disulfide bonds. They are rich in glutamine and proline and have low contents of amino acids with charged side groups.19 Studies investigating the crude protein digestibility of maize grain describe partially significant increases in crude protein digestibility caused by crop cultivation at elevated atmospheric CO2 concentration.20 Furthermore, Wieser et al.21 found significant decreases in crude protein, prolamin, and glutelin Received: Revised: Accepted: Published: 6616
March 13, 2014 June 24, 2014 June 28, 2014 June 28, 2014 dx.doi.org/10.1021/jf501958a | J. Agric. Food Chem. 2014, 62, 6616−6625
Journal of Agricultural and Food Chemistry
Article
JKA Works Ic., Wilmington, NC, U.S.A.). All separations of supernatants and precipitates were performed by centrifugation for 10 min at 3000 rpm (Varifuge 3.OR, Heraus Sepatech, Osterode, Germany). All supernatants were dried to constant mass at 60 °C by centrifugal evaporator (Christ RVC 2-18HCL, Osterode, Germany). SDS-PAGE. Isolated protein fractions were treated with 7 mM SDS (>99%, Carl Roth GmbH Co., Karlsruhe, Germany) and 200 mM DTT prior to separation in 4−20% Mini-PROTEAN TGX gels (Bio− Rad, Hercules, CA, U.S.A.) using a modified method of Laemmli.24 Proteins were completely dissociated by boiling the samples at 95 °C for 5 min. Electrophoresis was carried out at 50 V for 30 min followed by 200 V for 1 h until the tracking dye, bromophenol blue (ACS, Carl Roth GmbH Co., Karlsruhe, Germany), had reached the bottom of the gel. Molecular weight markers were Roti-Mark 10−150 and RotiMark PRESTAINED (Carl Roth GmbH Co., Karlsruhe, Germany) to identify the individual protein groups on the gel. After electrophoresis, gels were stained in a Coomassie brilliant blue G-250 (Carl Roth GmbH Co., Karlsruhe, Germany) solution overnight and destained in water−methanol−pure acetic acid (40/10/50, v/v/v). SE-HPLC. SE-HPLC was performed by using a Shimadzu instrument (liquid chromatograph, LC-20AT; autosampler, SIL-20AC; column oven, CTO-10ASvp; diode array detector, SPD-M20A) and a Yarra 3 μ SEC-2000 column (300 × 7.8 mm, Phenomenex; Torrance, CA) with guard cartridges (Phenomenex; Torrance, CA). Data was collected and analyzed using the CLASS-VP-6.14 SP2A software (Shimadzu). Mobile phases (50 mM phosphate buffer containing 0.1% SDS) were prepared by mixing a 50 mM disodium hydrogen phosphate dihydrate solution and a 50 mM sodium dihydrogen phosphate monohydrate solution according to Henderson−Hasselbalch equation for required pH values of 6.60 (A) and 6.94 (B). Gliadin (from wheat, Sigma, Steinheim, Germany) and glutenin standards (Osborne fractionation of gluten, from wheat, Sigma, Steinheim, Germany) as well as grain protein samples (concentration: 2−35 mg mL−1) were weighed and dissolved in mobile phase B by using an ultrasonic bath (FB15051, Fischer Scientific, Singen, Germany). Standards and samples were filtered (syringe filters, 0.45 μm, PVDF, anchor GmbH) before injection (50 μL). The measurements were performed at 25 °C with a flow rate of 0.7 mL/min and a gradient of mobile phase (0 min 100% B, 16 min 0% B, 22 min 100% B for re-equilibration). The monitoring wavelength was 214 nm with a run time of 30 min. Calibration and evaluation of logarithm scale with the standard proteins bovine serum albumin, ovalbumin, myoglobin, and cytochrome c were carried out to determine the sizes of the single protein subfractions. SEM. For scanning electron micrographs of milled kernels of all three cultivars, an environmental SEM (EVO LS25, Carl Zeiss SMT GmbH, Oberkochen, Germany) was used. The voltage of the electron beam was 8.0 kV and a secondary electron detector was used. In preparation, kernels were fractured at ambient temperature at 1 mm. Statistics. Data were evaluated by analysis of variance (ANOVA) after testing for normal distribution and variance homogeneity using the Kolmogorov−Smirnov test and the Bartlett-χ-square test, respectively. A complete 3-factorial design of ANOVA was used to evaluate the results for wheat and barley with the cultivation year (2000 and 2003), the CO2 level (AMBI and FACE), the N-fertilization level (50 and 100% of adequate N-supply) and the corresponding interactions being the fixed factors. For evaluating the maize results the CO2 level was the only fixed factor. Significance was assumed at p < 0.05 and STATISTICA for Windows Operating System (Version 10.0., StatSoft, Inc., 2011) was used for statistical evaluations.25
of winter wheat grain due to an enriched atmospheric CO2 concentration. Therefore, the aim of the present study was to get more detailed information on the structural composition of the protein fractions prolamin and glutelin of winter wheat, winter barley and maize grains cultivated using the FACE technique. Osborne fractionation was performed to isolate the single protein fractions followed by proving the purity by SDS-PAGE. To characterize prolamins and glutelins in detail a size exclusion high performance liquid chromatography (SE-HPLC) method was developed. In addition, kernels were observed by scanning electron microscopy (SEM) to study differences in proteinstarch bodies. These investigations should also explain the observed variations in digestibility of crude protein. The study intended to assess the potential impact of a future atmospheric CO2 concentration expected by 2050 on the protein composition and quality of these important animal feedstuffs.
■
MATERIAL AND METHODS
Materials. The experiment with winter wheat and barley was conducted on a 22 ha experimental field site and the investigation on maize was performed on a 10 ha field site located at the experimental station of the Friedrich Loeffler Institute (FLI), Federal Research Institute for Animal Health, Braunschweig, Germany. A FACE system engineered by Brookhaven National Laboratory and previously described by Weigel and Manderscheid7 with experimental rings of 20 m in diameter was used to study CO2 enrichment effects on maize cv. Romario (silage maize), winter wheat cv. Batis (one of most frequently cultivated bread wheat varieties in Germany) and barley cv. Theresa (one of most frequently cultivated barley varieties in Germany) as well as the influence of N fertilization of winter wheat and barley. Therefore, two (barley and wheat) or three (maize) of the FACE rings were treated with blowers with air of ambient CO2 concentration (AMBI, 380 ppm atmospheric CO2) and the other two (barley and wheat) or three (maize) with blowers with CO2 enriched air (FACE, 550 ppm atmospheric CO2). The FACE rings were placed exactly at the same position in each growing season. For winter wheat and barley, two different N fertilization levels were applied in each half of the rings to study interactions between CO2 enrichment and N supply. One was an adequate N fertilization (N100) for each crop species and one was a reduced fertilization with 50% (N50) of the adequate N treatment as it was described by Weigel and Manderscheid.22 For both crops the space between the two fertilization levels was set to approximately 1 m. Varieties of all three cereals were chosen because of their general reliable yield forecasts. For analytical use, kernels of winter wheat and winter barley of the two cultivation seasons and kernels of maize cultivar were predried, milled at 1 mm and stored at −20 °C. Protein Extraction. A modified Osborne fractionation according to Wieser et al.23 was performed to separate the protein fractions of all three cultivars. 0.5 g milled (1 mm) flour was extracted two times with 0.5 mol L−1 sodium chloride (NaCl, ≥ 99%, Ph.Eur., USP, Carl Roth GmbH Co., Karlsruhe, Germany) for 10 min at room temperature (RT ∼ 23 °C). Supernatant (albumins/globulins) was dialyzed (ZelluTrans, regenerated Cellulose Tubular Membrane, 6000−8000 Da, 1.67 mL/cm, Carl Roth GmbH Co., Karlsruhe, Germany) against water to separate both protein fractions in water - soluble albumins and water insoluble globulins. Precipitate (prolamins/glutelins) was extracted twice with 70% ethanol (EtOH, Rotipuran, ≥ 99.8%, p.a., Carl Roth GmbH Co., Karlsruhe, Germany) for 30 min at RT. The supernatant consisted of EtOH-soluble prolamins. Precipitate was extracted twice with a solvent of 50% 1-propanol containing 1% dithiothreitol (DTT), 0.05 M trisHCl, and 2 M urea (all chemicals at least ≥99%, p.a. quality, Carl Roth GmbH Co., Karlsruhe, Germany) for 20 min at 60 °C. Each extraction step was initiated by vortexing until complete mixing and continued by vortexing at 600 rpm (MS1 minishaker,
■
RESULTS Protein Extraction and SDS-PAGE. The relative amounts of the single protein fractions were obtained by gravimetric analysis after Osborne fractionation. Tables 1−3 summarizes the effect of different N fertilization, CO2 treatment and crop growing year on the percentages of the four fractions of winter barley, wheat, and maize. 6617
dx.doi.org/10.1021/jf501958a | J. Agric. Food Chem. 2014, 62, 6616−6625
0.083
