lnfluence of different nitrogen application on flour properties,gluten properties by HPLC and end-use quality of Korean wheat

1. lntroduction

Gluten compositions and accumulation influence rheological properties of dough and end-use quality of bread, noodles,and cookies. Gluten accumulation is largely affected by environmental conditions, although gluten compositions in wheat are genetically determined (Malik et al. 2013).Gliadin/glutenin ratio and the ratio of high molecular weight glutenin subunits (HMW-GS)/low molecular weight glutenin subunits (LMW-GS) are also influenced by growing conditions (mainly N fertilization) and genotypes (Song and Zheng 2007). Gliadin and glutenin contents and gliadin/glutenin ratio are increased when application level of nitrogen is increased, although there are still contradictory results about the relationship among HMW-GS/LMW-GS ratio, amount and size distribution of polymeric proteins,and nitrogen application (Triboi et al. 2000; Labuschagne et al. 2006).

南京航空航天大学的金家楣等人研制了一种小型的摩擦惯性式压电驱动器，如图12所示[21]，该驱动器的定子为位移放大机构和轴(Shaft)，动子为一个环形块(Rubber ring)，其最大空载速度为11 mm/s，最大输出力为0.5 N。

Visco-elasticities of glutens are governed by glutenin compositions, gliadin/glutenin ratio, and HMW-GS/LMW-GS ratio. These properties also affect rheological properties and bread-making characteristics (Song and Zheng 2007). It is well known that protein content is positively correlated with bread loaf volume. The amount of specific glutenin and the total amount of glutenin are correlated with bread loaf volume because glutenin determines the strength and elasticity of dough (Veraverbeke and Delcour 2002). However, the effect of gliadins on bread loaf volume remains controversial,although glutenins are major components responsible for bread loaf volume. The texture of white salted noodles is positively correlated with glutenin content. However, there is no relationship among monomeric proteins, albumin,globulin, or gliadin (Hu et al. 2007). Information about the effect of gluten properties on the texture of Chinese fresh white noodles is still limited, although it has been verified that hardness of noodles is increased as the ratio of glutenin to gliadin is increased (Zhang et al. 2011). Among soft wheat cultivars, factors such as quantities of gliadin protein fractions, decrease in glutenin, and qualitative variation of HMW-GSs are significantly related to the quality of cookies(Huebner et al. 1999).

The objectives of this study were: 1) to establish the relationship among physicochemical properties of wheat flour, quantities and ratios of gluten, and end-use quality of Korean wheat and 2) to examine the effect of nitrogen application level on quantification of different gluten components in different wheat cultivars for the wheatbreeding program.

2. Materials and methods

2.1. Plant materials

Five Korean wheat cultivars, which are representative wheat cultivars originated in the 1990s, were sown in randomized complete blocks with 3 replicates in the Upland Crop Experimental Farm of National Institute of Crop Science,Rural Development Administration (Korea) in 2015/2016 on 50% of clay loam soil. The seeds were sown in late October and each plot consisted of three 4-m rows spaced 25 cm apart and plots were combine-harvested in mid-June in both years. Additonal nitrogen (N) fertilizer was applied with three different nitrogen levels: 2.5 kg 1 000 m–2 (I),5.0 kg 1 000 m–2 (II), and 7.5 kg 1 000 m–2 (III) in the end of February, and weeds, insects and disease were stringently controlled. No supplemental irrigation was applied. Grain from each plot was dried using forced air driers and bulked from replications to provide grain for quality analysis.

2.2. Analytical methods

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2.3. Mixolab analysis

Mixolab (Chopin, France) was conducted according to AACCI Approved Methods 54-60.01 (2010) and the manufacturer’s instructions (Dubat 2010). Flour (50 g, 14%moisture basis) added up to 75 g with distilled water. Mixing speed was 80 r min–1 and peak torque was maintained as(1.1±0.09) Nm for dough development. Mixolab parameters were analyzed using Chopin Mixolab software (Version 3.14,Chopin, France).

2.4. Reversed-phase high-performance liquid chromatography (RP-HPLC)

Flour (100 g, 14% MB) was mixed with a predetermined amount of sodium chloride solution in a pin mixer (National Manufacturing Co., USA) for 4 min, with a head speed of 86 r min–1. The concentration of sodium chloride solution for making noodles with 34% absorption was adjusted to have 2.0% sodium chloride in the noodle dough. The crumbly dough was passed through the rollers of a noodle machine(Ohtake Noodle Machine Manufacturing Co., Tokyo, Japan)running at 65 r min–1 with a 3-mm gap to form a dough sheet,and then the dough sheet was folded and put through the sheeting rollers. The folding and sheeting were repeated twice. The dough sheet was rested for 1 h and then put through the sheeting rollers three times at progressively decreasing gaps of 2.40, 1.85, and 1.30 mm. The dough sheet was cut by no. 12 cutting rolls to produce noodle strands of a 3 mm×2 mm cross-sectional dimension and about 30 cm in length. Raw noodles (20 g) were cooked for 18 min in boiling distilled water (500 mL) and then rinsed with cold water. Two replicates of cooked noodles were evaluated by texture pro file analysis (TPA) with a TA.XT2 texture analyzer (Stable Micro Systems, Godalming,UK) within 5 min after cooking. A set of five strands of cooked noodles was placed parallel on a flat metal plate and compressed crosswise twice to 70% of their original height with a 3.175 mm metal blade at a crosshead speed of 1.0 mm s–1. From force-time curves of the TPA, hardness,springiness, and cohesiveness were determined according to the description of Park et al. (2003).

2.5. Size-exclusion high-performance liquid chromatography (SE-HPLC)

SE-HPLC was performed to measure amount and size distribution of polymeric proteins. Soluble proteins by 0.5%SDS-phosphate buffer (pH 6.9) and insoluble proteins by sonicator (VCX 130, SONIC & MATERIALS, INC, USA)were extracted according to the modified methods by Gupta et al. (1993) and Johansson et al. (2004). For soluble proteins, flour (11 mg) was suspended in 1.0-mL 0.5% SDS-phosphate buffer and then vortexed for 10 s. At the room temperature, the solution with flour was reacted for 5 min with vibration and then centrifuged for 30 min at 10 000×g.The supernatant was used to analyze soluble proteins under filtering with 0.45 μm filters. The precipitated pellet was resuspended in the 0.5% SDS-phosphate buffer and sonicated using the sonicator with 25% pulse for 30 s. The sample was centrifuged at the same condition as above.The supernatant was used to analyze unsoluble proteins,which filtered using the same filter as above. SE-HPLC was conducted under 50% (v/v) acetonitrile and water including 0.1% (v/v) trifluoroacetic acid (TFA) through BioSepTM SEC-s4000, 500Å column (Phenomenex, Torrance, USA).The injection volume for SE-HPLC analysis was 20 μL under 0.8 mL min–1 flow rate. The proteins were detected at 210 nm UV absorbance.

2.6. End-use quality

Bread was baked according to the optimized straightdough bread-making method according to AACCI Approved Method 10-10.03 (2010). The ingredients of baking formula consisted of 100 g (14% moisture basis) of flour, 6 g of sugar,3 g of shortening, 1.5 g of salt, 5.0 g of fresh yeast, 50 mg of ascorbic acid, and 0.25 g of barley malt (about 50 DU g–1, 20°C). The optimum water absorption and mixing time were determined by the feel and appearance of the dough during the mixing. The dough was fermented in a cabinet at 30°C and 85% relative humidity for 70 min with two punches and a proof period of 60 min and then baked at 210°C for 18 min. After cooling for 2 h at room temperature, a slice 2.0-cm thick was cut from the center portion of the bread.Bread loaf volume was measured immediately by rapeseed displacement and weighted after the bread was taken out of the oven. White salted noodles were prepared according to our previous study (Park and Baik 2002).

RP-HPLC was performed to measure the proportion of glutenin components, including high molecular weight glutenin subunits (HMW-GSs), low molecular weight glutenin subunits (LMW-GSs), x- and y-type glutenin subunits (x- and y-type) in HMW-GSs and gliadin components including ω-,(α+β)-, and γ-gliadin. Extraction of gliadin was performed with 100 mg of wheat flour, which incubated in 400 μL of 70%ethanol at room temperature (RT) for 1 h under vortexing and then centrifuged at 13 000 r min–1 for 15 min (Zhou et al.2013). The supernatant was used to analyze gliadins. After extraction of gliadin, the pellet was used to extract glutenin according to the protocol of Yan et al. (2014). It was washed three times as follows: 500 μL of 55% isopropanol (v/v) for 30 min at 65°C and then centrifuging at 13 000 r min–1 for 10 min. After centrifuging, the pellet was incubated with 100 μL extraction buffer containing 50% isopropanol (v/v), 80 mmol L–1 Tris-HCl (pH 8.0), and 1% dithiothreitol for 30 min at 65°C and then centrifuged at 13 000 r min–1 for 10 min. For alkylation, the pellet was incubated with 100 μL extraction buffer, in which 1% dithiothreitol was replaced with 1.4%4-vinylpyridine (v/v), for 30 min at 65°C. After centrifugation at 13 000 r min–1 for 10 min, the supernatant was collected and added to cold acetone at –20°C to a final concentration of 80% and then stored at –20°C overnight. Precipitated glutenins were obtained by centrifugation at 13 000 r min–1 for 10 min and removal of acetone in a fume hood. The pellet was eluted in 200 μL of 20% acetonitrile with 0.1% trifluoroacetic acid. Extracted gliadins and glutenins were cleaned with a 0.2-μm polyvinylidene difluoride membrane filter (Pall Life Sciences, USA). The injection volume for RP-HPLC analysis was 20 μL. The condition for RP-HPLC analysis with an Agilent 1100 instrument with a ZORBAX 300StableBond C18 as a reverse-phase column (300Å,5 μm, 4.6 mm×250 mm, Agilent Technologies, USA) was followed as 65°C for column temperature, 0.8 mL min–1 flow rate for glutenin, and 1.0 mL min–1 flow rate for gliadin. For separation of glutenin, the initial solvent contained 25%acetonitrile and 75% water, each with 0.1% trifluoroacetic acid. The acetonitrile ratio gradually increased to 33% within 13 min, 43% within 54 min, and 50% within 62 min, and then returned to the initial condition within 64 min. For separation of gliadin, the initial solvent contained 25% acetonitrile and 75% water, each with 0.1% trifluoroacetic acid. The acetonitrile ratio gradually increased to 50% within 60 min,25% within 62 min and then returned to the initial condition within 70 min. The proteins were detected at 210 nm UV absorbance. Glutenin properties were measured according to the percentage of the area of total gluten or gliadin content.

1950年代云南民族民间文学的整理翻译工作，1953年的《阿诗玛》也是一个典范文本，其在全国范围的成功也是一个标志性事件。《阿诗玛》让外界见识了云南民族民间文化的魅力，也让云南的文化工作者看到民歌的力量——“可以促进各民族的‘伟大的精神解放’”（陆万美：《隽永的回忆》），而且也让很多少数民族知识分子唤起了自身的“民族文学”的意识以及对本民族文化的自豪感。陆万美说，云南其他兄弟民族的干部群众看到《阿诗玛》后，都说，“这样的歌，我们也有”。

With increasing nitrogen application level, protein content in Korean wheat cultivars was increased, thus increasing dry gluten and SDSSF. These characteristics are known to be affected by cultural environment rather than by genetic properties of cultivars (Rozbicki et al. 2015). However,SDSSP or flour milled was not influenced by protein content.SDS sedimentation test based on the constant content of protein was influenced by protein qualitative characteristics while flour milled was influenced by seed hardness. These results were similar to previous studies conducted in Japan with the similar cultural environment (Martin et al. 2001; Park et al. 2003; Takayama et al. 2006).

Wheat was milled using a Bühler experimental mill, based on the AACCI Approved Method 26-31.01 (2010). A total of 2 kg wheat under 15% moisture content was milled with a feed rate of 100 g min–1 and with roll settings of 8 and 5 in break rolls and 4 and 2 in reduction rolls. Protein content (%) of wheat flour was determined according to AACCI Approved Methods 46-30.01 (2010). Ash contents were determined according to AACCI Approved Methods 08-01.01 (2010).Damaged starch contents were determined according to the methods described by Gibson et al. (1992), by using enzymatic assay kits (Megazyme, Bray, Ireland). The flour particle size distribution was measured with an LS13320 Multi-Wavelength Laser Particle Size Analyzer (Beckman Coulter, Brea, CA, USA) according to AACCI Approved Method 55-40.01 (2010). An SDS sedimentation test was performed according to AACCI Approved Method 56-70.01(2010). Optimum water absorption and mixing time of wheat flour were determined using a 10-g mixograph (National Mfg. Co., USA) according to AACCI Approved Method 54-40.02 (2010). Dry gluten content was measured using a Glutomatic 2200 (Perten Instruments AB, Sweden) with a constant volume of Glutomatic wash solution (4.8 mL) based on AACCI Approved Method 38-12.02 (2010).

2.7. Statistical analysis

The ratios of x-, y-type, and x/y of HMW-GS measured by RP-HPLC were correlated with cookie diameter. Glutenin property was not correlated with bread loaf volume or cooked noodles texture. The ratio of ω-gliadin content showed negative correlations with bread loaf volume and texture of cooked noodles. It had a positive correlation with cookie diameter. However, the ratio of (α+β)/ω-gliadin showed an opposite tendency to the ratio of ω-gliadin content. The hardness of cooked noodles showed a positive correlation with the ratio of (α+β)-gliadin and γ/ω-gliadin. Cookie diameter showed a negative correlation with the ratio of γ/ω-gliadin. Soluble and insoluble protein contents except insoluble monomeric protein content showed a positive correlation with bread loaf volume. Soluble and insoluble protein contents showed a positive correlation with a hardness of cooked noodles but a negative correlation with cookie diameter. There was no correlation between soluble and insoluble protein contents and springiness or cohesiveness of cooked noodles.

3. Results

3.1. Flour properties and Mixolab parameters

With increasing N application level, the protein content of the flour, ash, damaged starch, size of flour particle, SDS sedimentation volume in flour (SDSSF), and dry gluten content were increased. However, flour milled or SDS sedimentation volume in protein (SDSSP) did not show significant variations (Table 1). The increase of protein content by increasing application level of fertilizer was high in Keumkang wheat cultivar for bread-making but low in Goso wheat cultivar for making cookies. Ash content ofGoso cultivar was not related to increase in protein content.However, ash content was increased in other cultivars when protein content was increased. The increase in the size of flour particle was large in Uri cultivar for making cookies but small in Baekjoong cultivar for making noodles when protein content was increased. The increase of SDSSF with increasing protein content was the highest in Suan cultivar but the lowest in Uri cultivar. Baekjoong, Suan, and Uri cultivars showed no significant SDSSP variations regardless of the increase in protein content. Goso cultivar showed a slight increase in SDSSP whereas Keumkang cultivar showed a slight decrease in SDSSP with increasing protein content. The increase of dry gluten content with increasing protein content was the highest in Keumkang cultivar but the lowest in Goso cultivar.

Table 1 Flour properties of five Korean wheat flours with different levels of nitrogen application1)

1) PSI, average of particle size; SDSSF, SDS sedimentation volume conducted with a constant flour weight (3 g); SDSSP, SDS sedimentation volume conducted with a constant protein weight (300 mg). 2) Additonal nitrogen (N) fertilizer was applied with three different nitrogen levels: 2.5 kg 1 000 m–2 (I), 5.0 kg 1 000 m–2 (II), and 7.5 kg 1 000 m–2 (III) in the end of February. Values followed by the same letters within each cultivar are not significantly different at P≤0.05.

Cultivar N treatment2) Protein(%)Dry gluten(%)Baekjoong I 8.7 c 71.4 a 0.39 b 5.6 c 79.5 ab 27.3 c 28.0 b 7.8 c II 11.0 b 71.3 a 0.41 b 5.8 b 79.1 b 39.5 b 29.5 a 8.8 b III 13.1 a 71.3 a 0.43 a 6.0 a 81.6 a 47.3 a 29.0 ab 11.0 a Goso I 9.2 c 66.7 a 0.36 a 2.4 c 51.6 b 11.8 c 26.5 b 7.4 c II 10.3 b 65.1 a 0.36 a 2.8 b 52.2 b 32.8 a 27.0 b 8.0 b III 13.0 a 66.1 a 0.35 a 3.3 a 55.2 a 31.0 b 29.0 a 9.3 a Keumkang I 10.2 c 72.5 a 0.42 b 5.4 c 69.9 c 46.0 c 32.0 a 7.1 c II 12.6 b 72.3 a 0.45 a 5.8 b 70.9 b 59.0 b 32.5 a 10.2 b III 15.7 a 72.2 a 0.47 a 6.2 a 73.0 a 72.3 a 30.5 b 13.6 a Suan I 8.9 c 71.0 a 0.41 b 5.5 b 68.8 b 21.6 c 29.0 b 6.6 c II 11.1 b 71.4 a 0.40 b 5.7 b 71.4 a 30.5 b 31.0 a 9.5 b III 13.2 a 71.2 a 0.43 a 6.1 a 72.4 a 48.3 a 30.5 a 11.9 a Uri I 7.6 c 62.8 a 0.36 b 1.9 c 48.5 c 11.8 c 27.5 a 4.6 c II 10.3 b 63.4 a 0.39 a 2.1 b 50.5 b 22.5 b 29.0 a 7.1 b III 12.0 a 63.4 a 0.39 a 2.5 a 52.9 a 30.5 a 29.0 a 9.4 a Flour Yield (%)Ash(%)Damaged starch (%)PSI(μm)SDSSF(mL)SDSSP(mL)

With increasing protein content, water absorption and dough development time of Mixolab were increased.Stability time of dough at constant temperature was different depending on cultivar. However, protein strength showed no significant variations (Table 2). When protein content was increased, starch retrogradation in the cooling phase was increased while starch gelatinization was decreased.Starch gel stability was different depending on cultivars.The increase of dough water absorption with increasing protein content was the highest in Keumkang cultivar but the lowest in Goso cultivar. Except for Keumkang cultivar,dough development time was increased two times for other Korean wheat cultivars when protein content was increased.With increasing protein content, stability (STB) time of dough stability at constant temperature was increased in Baekjoong and Uri cultivars but decreased in Keumkang cultivar. It was increased then decreased in Suan cultivar. However,there was no variation in Goso cultivar. Protein strength was increased in Uri cultivar as protein content was increased.However, there was no correlation between protein strength and protein content in other cultivars. Starch retrogradation in the cooling phase with increasing protein content was the highest in Uri cultivar. It was similar to each other for other cultivars. The decrease of starch gelatinization with increasing protein content was not significantly different among cultivars. However, starch gel stabilities of Goso and Suan cultivars were decreased. Those of other cultivars were decreased and then increased when protein content was increased.

3.2. Gluten properties by HPLC

Chromatograms of Korean wheat cultivars measured by RPHPLC and SE-HPLC are shown in Figs. 1 and 2. Regarding variation of glutenin ratio measured by RP-HPLC, the ratio of high molecular weight glutenin subunits (HMW-GS) was increased while the ratio of low molecular weight glutenin subunits (LMW-GS) was decreased in Baekjoong and Uri cultivars with increasing protein content. However, they were not significantly changed in other cultivars. When protein content was increased, the x-type ratio of HMW-GS was increased in Baekjoong and Suan cultivars but decreased in other cultivars. Therefore, the ratio of HMW-GS/LMW-GS and the ratio of x/y-type in HMW-GS by the increase of protein content showed the same tendency. Variation in the ratio of glutenin with increasing protein content was different depending on cultivars. Therefore, protein content and related characteristics were not correlated with the ratio of glutenin.

Table 2 Mixolab parameters of five Korean wheat flours with different protein contents

1) WA, water absorption (the amount of water required in dough development); DDT, dough development time; STB, stability time of dough stability at constant temperature; C2, protein strength; C3, starch gelatinization; C4, starch gel stability; C5, starch retrogradation in the cooling phase. Values followed by the same letters within each cultivar are not significantly different at P≤0.05.

Cultivar Protein(%)Mixolab parameters1)WA (%) DDT (min) STB (min) C2 (Nm) C3 (Nm) C4 (Nm) C5 (Nm)Baekjoong 8.7 c 56.5 c 1.0 b 5.1 b 0.43 a 1.96 a 1.59 a 2.57 c 11.0 b 59.2 b 1.3 b 7.3 a 0.45 a 1.80 b 1.45 b 2.70 b 13.1 a 62.0 a 3.6 a 7.0 a 0.47 a 1.73 c 1.68 a 2.86 a Goso 9.2 c 53.6 b 1.1 c 4.8 a 0.40 a 2.14 a 1.80 a 2.71 b 10.3 b 53.6 b 1.9 b 5.6 a 0.42 a 2.18 a 1.86 a 2.84 a 13.0 a 55.0 a 2.8 a 4.6 a 0.43 a 2.04 b 1.63 b 2.83 a Keumkang 10.2 c 53.0 c 4.0 c 9.8 a 0.55 a 2.01 a 1.75 a 2.84 b 12.6 b 56.1 b 4.5 b 9.8 a 0.53 a 1.97 ab 1.57 b 2.78 b 15.7 a 59.5 a 5.9 a 8.7 b 0.53 a 1.89 b 1.82 a 3.02 a Suan 8.9 c 56.0 c 1.0 c 5.0 c 0.48 a 2.07 a 1.67 a 2.78 b 11.1 b 58.5 b 2.8 b 7.5 a 0.48 a 1.91 b 1.48 b 2.60 c 13.2 a 60.0 a 3.3 a 6.7 b 0.50 a 1.90 b 1.50 b 2.93 a Uri 7.6 c 50.5 c 0.7 c 2.6 c 0.42 b 2.38 a 2.07 ab 2.74 c 10.3 b 52.0 b 1.3 b 5.9 b 0.46 ab 2.33 b 2.03 b 2.84 b 12.0 a 54.0 a 1.7 a 8.7 a 0.46 a 2.23 c 2.17 a 3.46 a

Fig. 1 Example of comparison of proportion of gluten composition by different N fertilizer application using reversed-phase highperformance liquid chromatography (RP-HPLC). A, glutenin composition in gluten. B, gliadin composition in gluten. HMW and LMW indicate high- and low-molecular weight.

With increasing protein content, the ratio of (α+β)-gliadin was increased while the ratio of ω- or γ-gliadin was decreased. However, there were no significant variations in the ratio of glutenin fractions (Table 3). Since the increase of(α+β)-gliadin content by the increase of protein content was higher than the decrease of ω-gliadin content, the ratio of(α+β)/γ and (α+β)/ω was increased with increasing protein content. On the other hand, since there were no significant variations in the decrease of ω- or γ-gliadin content with increasing protein content, there was no significant variation in the ratio of γ/ω. As a result, protein content showed positive correlation with the ratio of (α+β)-gliadin and γ/ωgliadin (r=0.87, P＜0.001 and r=0.58, P＜0.05, respectively).The ratio of ω-gliadin showed a negative correlation (r=0.88,P＜0.001) with protein content while that of (α+β)-gliadin showed a positive correlation (r=0.64, P＜0.01) with protein content. SDSSF which showed positive correlations with protein content, water absorption measured by dry gluten and Mixolab, dough development time, and STB also showed a negative correlation with the ratio of ω-gliadin.The decrease of γ-gliadin content with increasing protein content was remarkable in Suan and Baekjoong cultivars for making noodles while the decrease of ω-gliadin content was high in Uri and Goso cultivars for making cookies.

Fig. 2 Example of pro filing of protein depending on size with different protein contents using size-exclusion high-performance liquid chromatography (SE-HPLC). A, soluble proteins. B, insoluble proteins. P1 and P2 areas indicate total polymeric protein and gliadin as monomeric protein, respectively.

With increasing protein content, both soluble and insoluble protein contents measured by SE-HPLC were increased in Korean wheat cultivars. However, soluble polymeric protein content showed no variation in Uri cultivar with increasing protein content (Table 4). Increases of soluble and insoluble protein content in Suan and Baekjoong cultivars with increasing protein content were greater than those in other cultivars, with Keumkang cultivar showing the highest increase in soluble monomeric protein content.Since soluble and insoluble protein contents measured by SE-HPLC were increased with increasing protein content,flour characteristics that had a positive correlation with protein content also showed a positive correlation between soluble and insoluble protein contents. Soluble and insoluble protein contents measured by SE-HPLC showed positive correlations with Mixolab water absorption, dough development time, stability time of dough stability at a constant temperature. Protein strength showed positive correlations with soluble and insoluble protein contents except for soluble polymeric protein content. Starch gelatinization had a negative correlation with soluble protein content. Starch gel stability also had a negative correlation with soluble polymeric protein content. However, there was no correlation between starch retrogradation in the cooling phase and soluble polymeric protein content. The ratio of ω-gliadin content which had a negative correlation with the increase of protein content showed negative correlations with soluble polymeric content, monomeric protein content,and insoluble monomeric protein content (r=–0.68, P＜0.01;r=–0.64, P＜0.01; and r=0.53, P＜0.05, respectively). Soluble protein content showed a positive correlation with the ratio of (α+β)/γ-gliadin content.

Table 3 Gluten properties of five Korean wheat flours with different protein contents using reversed-phase high-performance liquid chromatography (RP-HPLC)1)

1) HMW, amount of high molecular weight glutenin subunits; x-type, amount of x-type in HMW; y-type, amount of y-type in HMW; LMW,low molecular weight glutenin subunits; H/L, ratio of HMW to LMW; x/y, ratio of x-type to y-type in HMW; ω, proportion of ω-gliadin;α+β, proportion of (α+β)-gliadin; γ, proportion of γ-gliadin; (α+β)/γ, ratio of (α+β)- to γ-gliadin; (α+β)/ω, ratio of (α+β)- to ω-gliadin; γ/ω,ratio of γ- to ω-gliadin. Values followed by the same letters within each cultivar are not significantly different at P≤0.05.

Cultivar Protein(%)Gliadin Proportion (%) Ratio Proportion (%) Ratio HMW x-Type y-Type LMW H/L x/y ω α+β γ (α+β)/γ (α+β)/ω γ/ω Baekjoong 8.7 c 44.6 a 65.4 b 34.6 a 55.4 b 0.80 a 1.89 b 17.6 a 53.0 c 29.4 a 1.80 c 3.01 b 1.67 c 11.0 b 43.0 b 66.5 a 33.5 b 57.0 a 0.75 b 1.99 a 13.5 c 56.8 b 29.7 a 1.91 b 4.20 a 2.20 a 13.1 a 42.7 b 67.0 a 33.0 b 57.3 a 0.75 b 2.03 a 14.5 b 57.9 a 27.5 b 2.11 a 3.99 a 1.89 b Goso 9.2 c 43.2 a 67.4 a 32.6 b 56.2 a 0.77 a 2.07 a 16.4 a 52.9 c 30.7 a 1.72 c 3.24 c 1.88 a 10.3 b 42.6 a 66.2 b 33.8 a 57.4 a 0.74 a 1.96 b 15.2 b 54.5 b 30.3 a 1.80 b 3.59 b 1.99 a 13.0 a 42.5 a 65.9 b 34.1 a 57.5 a 0.74 a 1.94 b 14.5 b 56.7 a 28.8 b 1.97 a 3.90 a 1.98 a Keumkang 10.2 c 40.8 a 69.8 a 30.2 c 59.2 a 0.69 a 2.31 a 16.4 a 49.3 c 34.2 a 1.44 c 3.00 c 2.08 b 12.6 b 41.2 a 69.6 b 30.4 b 58.8 a 0.70 a 2.29 b 14.1 b 52.5 b 33.4 a 1.57 b 3.75 b 2.38 a 15.7 a 41.8 a 67.7 c 32.3 a 58.2 a 0.72 a 2.09 c 13.1 b 55.7 a 31.2 b 1.79 a 4.26 a 2.38 a Suan 8.9 c 41.7 a 75.8 b 24.2 a 58.3 a 0.71 a 3.13 b 16.3 a 47.4 c 36.3 a 1.30 c 2.91 c 2.23 a 11.1 b 40.6 a 76.5 a 24.0 a 59.4 a 0.68 a 3.19 b 14.8 b 51.3 b 33.9 b 1.51 b 3.49 b 2.30 a 13.2 a 42.3 a 76.8 a 23.1 b 57.7 a 0.73 a 3.33 a 13.7 b 53.5 a 32.7 c 1.64 a 3.90 a 2.38 a Uri 7.6 c 41.0 a 63.4 a 36.6 b 59.0 b 0.70 a 1.73 a 17.8 a 49.5 c 32.7 a 1.51 c 2.78 c 1.84 b 10.3 b 41.2 a 62.4 b 37.6 a 59.1 b 0.70 a 1.66 b 15.5 b 52.5 b 32.1 b 1.64 b 3.39 b 2.07 a 12.0 a 39.8 b 62.2 b 37.4 a 60.2 a 0.66 b 1.66 b 14.2 c 55.5 a 30.3 c 1.83 a 3.93 a 2.14 a Glutenin

Table 4 Protein characteristics depending on size in five Korean wheat flours with different protein contents using size-exclusion high-performance liquid chromatography (SE-HPLC)

Values followed by the same letters within each cultivar are not significantly different at P≤0.05.

Cultivar Protein (%) Soluble proteins (mAU) Insoluble proteins (mAU)Polymeric Monomeric Polymeric Monomeric Baekjoong 8.7 c 4 686.8 c 4 894.2 c 2 292.6 b 400.3 c 11.0 b 5 143.2 b 5 845.8 b 2 496.7 b 449.4 b 13.1 a 5 922.1 a 6 909.0 a 3 019.8 a 539.3 a Goso 9.2 c 4 907.3 a 4 889.4 b 1 640.1 b 308.7 b 10.3 b 4 090.7 b 4 346.1 b 1 520.6 b 259.9 b 13.0 a 5 576.9 a 6 138.9 a 2 505.0 a 466.1 a Keumkang 10.2 c 4 343.1 c 6 928.3 c 3 360.2 c 549.0 c 12.6 b 5 272.9 b 9 245.5 b 4 210.8 a 844.5 a 15.7 a 6 525.9 a 10 583.7 a 3 665.4 b 641.6 b Suan 8.9 c 4 273.8 c 5 391.6 b 2 041.5 b 462.9 c 11.1 b 5 437.8 b 6 801.6 a 2 449.3 b 519.1 b 13.2 a 6 674.7 a 7 439.7 a 3 195.2 a 627.3 a Uri 7.6 c 3 908.2 a 4 171.1 b 2 356.1 b 406.3 b 10.3 b 4 156.6 a 4 592.6 ab 2 433.3 b 438.3 ab 12.0 a 4 280.0 a 5 024.8 a 2 751.5 a 518.4 a

3.3. End-use quality

With increasing protein content, bread loaf volume was increased. Keumkang wheat cultivar for bread-making showed a remarkable increase in bread loaf volume with increasing protein content. However, bread loaf volumes of other cultivars were similar to increasing protein content(Table 5). The hardness of cooked noodle, springiness, and cohesiveness were also increased with increasing protein content. Baekjoong cultivar showed a remarkable increase in the hardness of cooked noodles with increasing protein content. However, with increasing protein content, cookie diameter was decreased. The degree of this decrease was remarkable in Goso and Uri cultivars for making cookies.With these tendencies, the flour properties related to the protein content showed correlated tendencies such as bread loaf volume, cooked noodles texture and cookie diameter (Table 6). Bread loaf volume showed positive correlations with dough developing time, dough stability,and protein strength of Mixolab. However, it showed a negative correlation with starch gelatinization. The hardness of cooked noodles showed positive correlationswith water absorption and dough developing time while the springiness of cooked noodles showed positive correlations with dough developing time and starch retrogradation properties. The cohesiveness of cooked noodles showed a positive correlation with starch retrogradation. Cookie diameter showed a negative correlation with properties related to the protein of Mixolab parameters. It also showed negative correlations with starch gelatinization and starch gel stability.

内源激素 将1. 0 g甘薯样品于液氮中速冻,用80%的甲醇溶液(含二叔丁基对甲苯酚(BHT)1 mmol/L)匀浆,4 ℃提取8 h,4 000 r/min离心15 min,80%甲醇溶液沉淀,并重复提取3次,合并上清液,氮气吹干,PBSTG溶液定容,ELISA法测定．

Table 5 End-use quality of five Korean wheat flours with different protein contents

Values followed by the same letters within each cultivar are not significantly different at P≤0.05.

Cultivar Protein(%)Cooked noodles Cookie diameter(mm)Hardness (N) Springiness (ratio) Cohesiveness (ratio)Baekjoong 8.7 c 625.0 c 2.63 c 0.92 b 0.61 b 84.7 a 11.0 b 700.0 b 3.47 b 0.95 a 0.62 b 77.8 b 13.1 a 775.0 a 4.91 a 0.95 a 0.65 a 76.3 c Goso 9.2 c 575.0 c 2.60 c 0.94 b 0.61 b 92.5 a 10.3 b 625.0 b 3.86 b 0.94 b 0.62 b 90.9 b 13.0 a 725.0 a 4.52 a 0.96 a 0.65 a 83.8 c Keumkang 10.2 c 750.0 c 3.43 c 0.94 b 0.61 b 82.4 a 12.6 b 850.0 b 4.53 b 0.96 a 0.63 a 79.9 ab 15.7 a 900.0 a 4.82 a 0.96 a 0.64 a 78.7 b Suan 8.9 c 550.0 c 3.22 c 0.93 b 0.62 b 80.8 a 11.1 b 625.0 b 3.60 b 0.95 a 0.63 b 78.7 b 13.2 a 700.0 a 4.55 a 0.95 a 0.65 a 76.2 c Uri 7.6 c 575.0 c 2.99 c 0.92 c 0.62 b 95.8 a 10.3 b 625.0 b 3.74 b 0.95 b 0.64 a 92.6 b 12.0 a 675.0 a 4.12 a 0.97 a 0.65 a 88.1 c Bread loaf volume (mL)

Statistical analysis of data was performed by SAS software(SAS Institute, NC, USA) using Fisher’s least significant difference procedure (LSD), analysis of variance (ANOVA)and Pearson correlation coefficient. Differences were considered significant at P＜0.05, unless otherwise specified.All data were determined at least in duplicate and all were averaged.

4. Discussion

Sugar-snap cookie was baked to measure diameter following the AACCI Approved Method 10-52.01 (AACCI 2010). The ingredients for cookie baking were 40 g of flour with 14.0% moisture basis, 24.0 g of sugar, 12.0 g of shortening, 1.2 g of nonfat dry milk solid, 0.4 g of sodium bicarbonate, 4 mL of solution with 0.32 g of sodium bicarbonate, 2 mL of solution with 0.2 g of ammonium chloride, 0.18 g of sodium chloride and 2.7 mL of deionized water. The sifted sugar, nonfat dry milk solid, and sodium bicarbonate were combined with the shortening and then creamed by Kitchen Aid Professional KPM5 mixer (Kitchen Aid, MI, USA) equipped with a flat beater mixing arm(type K45AB) for 4 min. The creamed mass, 37.6 g was blended with water, a solution of sodium bicarbonate and ammonium chloride. The mixture was mixed again with flour by a National Cookie Dough Micromixer (National Mfg. Co., Lincoln, NE) at 172 r min–1 for 3 min. The cookie dough with 7 mm thickness was divided into a scrap using a cookie dough cutter (60 mm inside diameter). The scrap was baked at (205±2)°C for 10 min. Cookie diameter was measured after cookies were cooled at room temperature for 20 min. Four cookies were baked for each flour.

第五，巩固党的群众路线教育实践活动的成效，广泛开展精神文明创建和文化建设，落实中央八项规定，改进作风，力戒心浮气躁，倡导脚踏实地、真抓实干；落实党风廉政建设责任制，严格干部管理；加学习党组织建设，完善民主决策机制，充分发扬党内民主，营造团结干事的浓厚氛围。

计算上述四个结果的关键点在于如何推算出泵效水平、理想沉没率和安全下泵深度三个基准值，就很容易计算出上述结果。

Mixolab is widely used to evaluate bakery and confectionery properties because it can examine the process of dough forming during the mixing of wheat flour and water,combination of protein in the dough, variation of dough properties by heating, the degree of starch gelatinization,and retrogradation at the same time (Dubat 2010). Protein and dough properties of Mixolab have high correlations with sedimentation, dough properties of Mixograph, and Graph parino. They are also highly correlated with bread and cookie quality (Ozuturk et al. 2008; Koksel et al. 2009;Caffe-Treml et al. 2010). The amount of water absorption and dough development time of Mixolab were increased with increasing of protein content in Korean wheat cultivars.They also showed positive correlations with soluble and insoluble protein content measured by SE-HPLC. It is known that the degree of starch gelatinization of Mixolab has highly correlated the peak viscosity of amylograph(Dapčević et al. 2009). In Korean wheat cultivars, starch gelatinization was decreased but starch retrogradation in the cooling phase was increased with increasing protein content. Starch gelatinization properties of Mixolab had a negative correlation with soluble protein content and bread loaf volume, and a positive correlation with cookie diameter.The degree of starch retrogradation also had positive correlations with the springiness and the cohesiveness ofcooked noodles. In Korean wheat cultivars, soluble protein content was mainly increased with increasing protein content while the relative starch content was decreased which affected starch gelatinization and retrogradation.Variations in protein and starch properties appeared to affect the increase of bread loaf volume, the texture of cooked noodles, and the decrease of cookie diameter.

Table 6 Correlation coefficients for physicochemical properties of flour, gluten properties and end-use quality in five Korean wheat flours with different protein contents

1) SDSSF, SDS sedimentation volume conducted with a constant flour weight (3 g); SDSSP, SDS sedimentation volume conducted with a constant protein weight (300 mg); HMW, amount of high molecular weight glutenin subunits; x-type, amount of x-type in HMW;y-type, amount of y-type in HMW; LMW, low molecular weight glutenin subunits; H/L, ratio of HMW to LMW; x/y, ratio of x-type to y-type in HMW; α+β, proportion of α+β gliaidin; γ, proportion of γ gliaidin; ω, proportion of ω gliaidin; (α+β)/γ, ratio of (α+β)- to γ-gliadin;(α+β)/ω, ratio of (α+β)- to ω-gliadin; γ/ω, ratio of γ- to ω-gliadin. ＊, ＊＊, and ＊＊＊ are significant at P≤0.05, P≤0.01 and P≤0.001, respectively. –, no correlation.

Parameter1)Bread loaf volume Cooked noodles Cookie diameter Hardness Springiness Cohesiveness Flour properties Protein 0.85＊＊＊ 0.91＊＊＊ 0.72＊＊ 0.73＊＊ –0.61＊Flour yield 0.49 0.20 –0.14 –0.15 –0.87＊＊＊Ash 0.58＊ 0.51 0.41 0.44 –0.45 Damaged starch 0.53＊ 0.33 –0.08 – –0.93＊＊＊Particle size 0.46 0.22 –0.24 –0.08 –0.88＊＊＊SDSSF 0.94＊＊＊ 0.75＊＊ 0.41 0.35 –0.69＊＊SDSSP 0.66＊＊ 0.48 0.34 0.25 –0.66＊＊Dry gluten 0.77＊＊＊ 0.80＊＊＊ 0.58＊ 0.62＊ –0.70＊＊Mixolab parameters Water absorption 0.48 0.52＊ 0.09 0.32 –0.89＊＊＊Dough developing time 0.88＊＊＊ 0.72＊＊ 0.58＊ 0.32 –0.57＊Dough stability 0.72＊＊ 0.48 0.40 0.19 –0.55＊Protein strength 0.69＊＊ 0.49 0.34 0.19 –0.62＊Starch gelatinization –0.55＊＊ –0.40 0.02 –0.08 0.93＊＊＊Starch gel stability –0.21 –0.09 0.21 0.12 0.77＊＊＊Starch retrogradation 0.29 0.50 0.67＊＊ 0.62＊ 0.05 Gluten parameters HMW –0.07 –0.21 –0.42 –0.34 –0.16 x-Type 0.01 0.11 0.03 – –0.66＊＊y-Type –0.01 –0.11 –0.03 – 0.66＊＊LMW 0.10 0.25 0.41 0.37 0.11 H/L –0.05 –0.21 –0.38 –0.33 –0.14 x/y –0.06 0.10 0.05 0.06 –0.61＊α+β 0.49 0.58＊ 0.35 0.51 –0.26 γ–0.22 –0.25 –0.08 –0.24 –0.04 ω–0.67＊＊ –0.80＊＊＊ –0.58＊ –0.68＊＊ 0.60＊(α+β)/γ 0.34 0.42 0.20 0.37 –0.13(α+β)/ω 0.67＊＊ 0.78＊＊＊ 0.52＊ 0.66＊＊ –0.52＊γ/ω 0.46 0.52＊ 0.43 0.42 –0.56＊Size-excluded protein parameters Soluble polymeric protein 0.63＊ 0.65＊＊ 0.41 0.51 –0.76＊＊Soluble monomeric protein 0.88＊＊＊ 0.66＊＊ 0.45 0.32 –0.71＊＊Insoluble polymeric protein 0.86＊＊＊ 0.64＊ 0.45 0.38 –0.57＊Insoluble monomeric protein 0.76 0.63＊ 0.48 0.42 –0.63＊

With increasing nitrogen application level and increasing protein content, gluten components in Korean wheat cultivars varied. Such variation might be influenced by nitrogen application level, the temperature of growth duration, and characteristics of cultivars (Daniel and Triboi 2000; Triboi et al. 2000). The variation in the ratio of glutenin with increasing protein content was different depending on cultivar. In German wheat cultivars, the ratio of HMW-GS is increased but the ratio of LMW-GS is decreased with increasing protein content (Wieser and Seilmeier 1998).In American soft wheat cultivars, both ratios of HMW-GS and LMW-GS are increased with increasing protein content(Pierre et al. 2008). In British wheat cultivars, HMW-GS ratio is not changed whereas LMW-GS ratio is increased with increasing protein content (Kindred et al. 2008). With increasing protein content, the ratio of HMW-GS content to LMW-GS content showed was increased or not changed in Korean wheat cultivars. In addition, with increasing protein content, variations of x-type and y-type ratios of HMW-GS were dependent on cultivar because compositions of x- and y-type glutenin subunit were different. The ratio of ω-gliadin is increased while the ratio of γ-gliadin is decreased in German wheat cultivars. However, both ratios of ω- and y-gliadin are increased in French wheat cultivars (Weiser and Seilmeier 1998; Daniel and Triboi 2000). In the present study, both ratios of ω- and γ-gliadin were decreased in Korean wheat cultivars. With increasing protein content, the ratio of (α+β)-gliadin is decreased in French wheat cultivars (Daniel and Triboi 2000). This was the opposite in Korean wheat cultivars.In Korean wheat cultivars, variations in glutenin content and gliadin content were dependent on cultivar when protein content was increased. Therefore, more studies are needed in the future using more cultivars to determine whether this phenomenon is a characteristic of Korean wheat cultivars or unique to cultivars used in this study.

With increasing protein content due to increased N application level, variation in the amount of protein fraction is known to be affected by cultivars (Weiser and Seimeier 1998). For Swedish wheat cultivars, soluble and insoluble polymeric and monomeric protein contents are increased with increasing N application level. However, polymeric and monomeric protein contents are increased in German wheat cultivars (Weiser and Seilmeier 1998; Johansson et al.2001). In the present study, all fractions were increased in Korean wheat cultivars. Such increases in soluble and insoluble polymeric and monomeric protein contents were correlated with increases of bread loaf volume and hardness of cooked noodles and decreases of cookie diameter and protein content.

With increasing protein content, it has been reported that bread loaf volume and hardness of cooked noodles are increased but cookie diameter is decreased (Baik et al.1994; Souza et al. 1994; Graybosch et al. 1996). Such results were also found in Korean wheat cultivars. However,variations in glutenin content and the ratio of glutenin composition with increasing protein content were dependent on cultivars. These properties were not correlated with processing quality. The effect of gliadin on the increase of bread loaf volume remains controversial (Park et al. 2006;Wang et al. 2007; Ohm et al. 2010). In Korean wheat cultivars, the ratio of ω-gliadin was decreased in every cultivar when protein content was increased. This affected bread loaf volume, the texture of cooked noodles, and cookie diameter. Therefore, how to decrease ω-gliadin content should be determined in the future in order to improve the quality of Korean wheat cultivars.

5. Conclusion

In this study, it was demonstrated that different amounts of nitrogen fertilizer influenced flour and gluten properties, and end-use quality in Korean wheat. With increasing protein content by an increase in nitrogen fertilizer, the decreasing of ω-gliadin ratio was positively correlated with bread loaf volume and hardness of cooked noodles and negatively with cookies diameter. In addition, increasing of the amount of all protein fraction showed the same correlation tendency with the end-use quality. However, the ratio of glutenin composition was not different although the protein was increased. Hence, Korean wheat breeding program should focus on the extension of genetic diversity for glutenin composition, and more study is needed to identify the effect of not only amount but also a time of nitrogen fertilizer application for improvement of wheat quality.

文献[6]中的Dirichlet过程即为DP(α,P0),α为正实数,记F0(x)=P0{X ≤x}是先验过程的期望(均值函数).则在样本为x1,x2,···,xn时,F(x)的贝叶斯估计为

Acknowledgements

This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development, Rural Development Administration, Republic of Korea (PJ011009).

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