EFFECTS OF GLIADIN/GLUTENIN AND HMW-GS/LMW-GS RATIO ON DOUGH RHEOLOGICAL PROPERTIES AND BREAD-MAKING POTENTIAL OF WHEAT VARIETIES

bs_bs_banner Journal of Food Quality ISSN 1745-4557 EFFECTS OF GLIADIN/GLUTENIN AND HMW-GS/LMW-GS RATIO ON DOUGH RHEOLOGICAL PROPERTIES AND BREAD-MAKING POTENTIAL OF WHEAT VARIETIES VANDANA DHAKA and B.S. KHATKAR 1 Department of Food Technology, Guru Jambheshwar University of Science and Technology, Hisar 125001, India 1 Corresponding author. TEL: +91-1662-263313; FAX: +91-1662-263313; EMAIL: bhup2009@hotmail.com Received for Publication November 16, 2013 Accepted for Publication August 16, 2014 10.1111/jfq.12122 ABSTRACT The gliadin/glutenin ratio of 15 diverse wheat varieties ranged from 0.75 to 1.16, whereas the high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS) ratio of these varieties ranged from 0.31 to 0.93. Gliadin/glutenin ratio showed a significant negative relationship with specific loaf volume (r = 0.73), dough development time (DDT; r = 0.73), dough stability (r = 0.79) and positive associations with LMW quantity (r = 0.72). Mixolab classified the wheat varieties into two groups on the basis of HMW-GS located at Glu-A1 and Glu-D1. Wheat varieties HI 977 and DBW 16 with subunits 2* and 5 +10 at Glu-A1 and Glu-D1, respectively, exhibited the characteristics of extra strong doughs with longer DDT of 8.3 and 7.2 min, and higher dough stability values of 9.1 and 8.6 min, respectively. Wheat varieties C 306, HW 2004 with null allele at Glu-A1 and 2 + 12 at Glu-D1, in contrast, were weak as they developed quickly, with low dough stability ( 4 min). PRACTICAL APPLICATIONS Bread has been one of the principal forms of food for man from the earliest times. Bread quality is determined by the composition and molecular structure of gluten which in turn controls the interactions of gluten subfractions during processing. Rheological properties, microstructure of gluten, gliadin/glutenin ratio and HMW-GS/LMW-GS ratio have been found to be associated with bread-making quality of wheat varieties. The research implications may be utilized both by industry personnel as well as researchers to assess the bread-making quality of wheat varieties. INTRODUCTION Gluten quality is a very intricate topic. Significance of gluten and its subfractions to the bread-making quality of wheat flour has been a subject of considerable debate in the literature. It is widely accepted that gliadins confer viscous properties to gluten required for dough development while glutenin imparts strength and elasticity which is essential to hold gases produced during the process of fermentation. Hoseney et al. (1969) proposed that gliadin governs the loaf volume potential of wheat varieties while Orth and Bushuk (1972) reported that glutenins are the prime determinants of bread-making quality of wheat varieties. Some researchers have also concluded that the dough properties, especially dough strength and bread-making potential of wheat varieties, can be ascribed to both the quality and quantity of gluten as well as compositional and quantitative differences in the glutenin polypeptides (Khatkar et al. 1995; Shewry and Halford 2002; Khatkar 2006). On the contrary, some scientists (Kasarda 1989; Singh et al. 1990) reported that it is not the subunit composition but the quantity of high molecular weight glutenin subunits (HMW-GS) which is accountable for the bread-making potential of wheat varieties. In addition, determination of rheological properties of wheat flour dough is also essential for the successful manufacturing of bread because they affect the behavior of dough during mechanical handling, thereby influencing the quality of the finished product (Khatkar et al. 2002a,b). Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc. 71

GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY V. DHAKA and B.S. KHATKAR A large number of studies have been conducted to relate the quality of gluten proteins with the presence or absence of specific gluten fractions, but very little has been emphasized on the importance of gliadin/glutenin and HMW- GS/LMW-GS ratio on the baking quality and rheological properties of wheat varieties. Therefore, in this study, gluten fraction, i.e., glutenins, gliadins, HMW-GS and LMW-GS, was isolated and their ratios were calculated. Efforts were made to comprehend the effects of gluten protein subfractions on rheological and bread-making performance of wheat varieties by establishing the correlations between these parameters. MATERIAL AND METHODS Materials Grains of 15 wheat varieties, namely WH 542, PBW 550, PBW 443, PBW 373, PBW 343, C 306, DBW 16, WH 147, HW 2004, WH 1025, WH 1021, WH 283, HS 490, HI 977 and WH 542, were procured from IARI Regional Centres; agriculture universities; DWR, Karnal and Central State Farm, Hisar. These varieties were selected mainly on the basis of their wide diversity for bread-making performances. The grains were cleaned manually to remove soil particles and broken and foreign seeds, and were stored in deep freezer ( 18C) until further use. Wheat varieties were tempered to 15.5% moisture content at room temperature for 48 h. Extra 0.5% moisture was added 30 min prior to milling. The grains of individual varieties were milled on a Chopin laboratory mill (Model CD1, Villeneuve la Garenne, France) into refined flour. The flours of all wheat varieties were stored under refrigeration conditions and thawed for 3 h before any analysis. Methods Physicochemical Analysis of Flour. The flour samples of all wheat varieties were analyzed for moisture, protein, ash, falling number, wet gluten (WG), dry gluten (DG) and gluten index (GI). These were determined according to standard AACC (2000) methods. The sodium dodecyl sulfate (SDS) sedimentation volumes of flour samples were estimated according to the method of Axford et al. (1978). Triplicate measurements were carried out for the chemical analysis and the results were averaged. Fractionation of Gluten into Glutenins and Gliadins. Modified Osborne method was used to separate gluten from gliadins and glutenins. Gluten was separated from dough by manual washing at a temperature of 15C. The gluten was then freeze-dried and ground to a uniform powder. Freeze-dried gluten powder (50 g) was suspended in 1 L of 70% (v/v) ethanol and stirred on magnetic stirrer for 3 h at room temperature ( 22C) followed by centrifugation at 1,000 g for 30 min in cooling centrifuge at 4C. The extraction was repeated. The precipitant was collected as glutenins and the supernatant was subjected to rotary evaporator at 30C to remove ethanol to recover the gliadins. Rheological Analysis. Mixolab determines a comprehensive qualitative profile of the wheat flour and plots, in real time, the torque (expressed in Nm) produced by the passage of the dough between two kneading arms when submitted to both shear stress and a temperature constraint. Key parameters derived from the Mixolab curve are water absorption (%), dough development time (DDT) and dough stability. Extensional Properties of Gluten. Uniaxial extensibility of gluten of different wheat varieties was assessed by the Kieffer dough and gluten extensibility rig developed by Stable Micro Systems for the TA-XT plus Texture Analyser. Gluten was extracted from the standard procedure and was rolled into a cylindrical shape and placed over three or four channels of the Teflon-coated block. Prior to the placement of gluten, the Teflon-coated block was prepared by placing nonadhesive Teflon strips which were coated with silicon oil in the channels. Once the gluten was placed in the Tefloncoated block, the upper half of the block was placed in position and tightly clamped, which distributed the gluten over three to four channels, to yield gluten strips of uniform geometry. The gluten was rested for 40 min at 25C prior to the test. The gluten strips were then separated from the Teflon strips, positioned across the Kiefferrig dough holder, and immediately tested on the TA-XT plus at a hook speed of 3.3 mm/s and a trigger force of 1 g (Ktenioudaki et al. 2010). The resistance to extension (g) and extensibility (mm) were determined in tension mode by recording the peak force and the distance at the maximum and the extension limit. Extraction of HMW-GS and LMW-GS Glutenin Subunits. Total glutenins were extracted according to Khatkar (2006). NaCl solution (0.5 M) was added to flour (1 g) sample to remove albumins and globulins. The pellet was dispersed in distilled water three times to remove the residual salt. And then the flour was suspended in 70% ethanol and stirred on a magnetic stirrer to remove gliadins. The above sample solution was then centrifuged at 15,000 g for 30 min. The procedure was repeated twice and the residue was further used for glutenin extraction. The residue obtained was then suspended in 5 ml of 50% (v/v) propan-2-ol, 0.08 M Tris HCl (ph 8.0) and 1% (w/v) 72 Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc.

V. DHAKA and B.S. KHATKAR GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY dithiothreitol. Then the samples were kept in water bath at temperature of 60C for 90 min with intermittent shaking and the tubes were vortexed after every 15 min. The tubes were centrifuged (15,000 g for 30 min, 20C) and supernatant was recovered. To the supernatant, 20 ml pure acetone was added to achieve a final concentration of 80% (v/v) to precipitate total glutenins. The precipitate was centrifuged (15,000 g for 30 min, 20C). The total glutenin residue was dried in an oven at 60C for 5 10 min, and solubilized in 2 ml of SDS sample buffer. Electrophoretic Analysis. Electrophoresis was run using slab gel apparatus of M/S ATTO (Tokyo, Japan). Glass plates (with 1-mm-thick spacers) were thoroughly cleaned with ethanol, dried and assembled in gel casting assembly with a 12% polyacrylamide separating gel containing 1.35% bis-acrylamide cross-linker according to the procedure of Laemmli (1970). Flours (40 mg) and proteins (4 mg) were suspended in SDS sample buffer (1.0 ml) containing 62.5 mm Tris/HCl (ph 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue and 5% (v/v) 2- mercaptoethanol. The flour/protein buffer mixtures were vortexed for 2 min and allowed to stand at room temperature for 3 h. The flour buffer mixtures were centrifuged, and the supernatants were heated for 3 min in a boiling water bath. The protein buffer samples were heated directly for 3 min in a boiling water bath. After cooling to room temperature, 15 μl of the sample was loaded into the wells. Two gels were run simultaneously at constant current of 40 mafor4horuntilthesample dye reached the end of the gel. Electrophoresis was stopped and gel was stained overnight in the staining solution (60% distilled water, 30% methanol, 10% acetic acid and 0.1% G-250 Coomassie Brilliant Blue, Sigma Aldrich, Spruce St., St. Louis). After staining, gels were transferred to destaining solution (60% distilled water, 30% methanol and 10% acetic acid). Quantitative Analysis of Glutenin Subfractions. Relative amounts of high and low molecular weight glutenin subunits were determined by densitometric scanning of SDS-PAGE gels of reduced proteins. First, the gel was positioned in a sample holder which was further filled with water up to the surface of gel to minimize the scattering of light. After that, the gel was covered with holding plate and placed on the scanner. The proteins in the individual lane were analyzed by using analysis software lab works version 1.4. Scanning Electron Microscopy. Microstructure of the glutenin fractions was analyzed using scanning electron microscopy (SEMTRAC Mini, Nikkiso, Germany). The glutenin samples were mixed with an optimum amount of water and the dough was formed and freeze-dried. The structure obtained after freeze-drying was fractured with a sharp knife to expose the inner structure and placed onto the stubs using double-sided sticky tape. The exposed surface was coated with gold using a sputter coater to make the sample conductive and then the inner surface was scanned at 5 kv potential and 100 magnifications. Preparation and Quality Evaluation of Bread. The bread-making performances of wheat flours were determined using the procedure described by Finney (1984) with little modifications. The baking formula was: flour (100 g, 14% moisture basis), compressed yeast (5.3 g), salt (1.5 g), sugar (6.0 g), fat (3.0 g), malted barley flour (0.075 g) and ascorbic acid (100 ppm, flour basis). Salt, sugar, ascorbic acid and yeast were added in solution form. Yeast was added as a suspension, which was mixed well each time before dispensing. Malted barley flour was added to adjust the falling number of wheat flours of different wheat varieties to 250 s. Doughs were mixed in farinograph having bowl of 100 g capacity (Promylograph T6 Farinograph, Max Egger, St. Blasen, Austria). Water absorption and the development time were determined using the Chopin + protocol of Mixolab. Additional 2 ml of water was added and mixing time with 1 min longer than DDT was used for baking. After mixing, doughs were placed in bowls, and covered with a wet muslin cloth and fermented for 90 min at 35C and 98% relative humidity (RH). Doughs were molded after 52, 77 and 90 min in dough molder. After the final molding, the dough was divided into four equal proportions and placed in lightly greased tins (internal dimensions for 30 g bread pan: bottom, 24.6 52.8 mm; top, 32.1 61.2 mm; height, 23.5 mm) and proved for 36 min at 35C and 98% RH. After adequate proofing, doughs were baked in baking oven (FG 156, Mono Equipment Ltd., Queensway, Fforestfach, Swansea, West Glamorgan SA5 4EB, U.K) for 13 min at 232C. After removing from the oven, loaves were placed on a wire grid for about 2 h for cooling and then weight and volume were determined. Loaf volumes were measured by rapeseed displacement to calculate specific loaf volume (SLV) by dividing volume of bread by its weight. Statistical Analysis. All determinations were made at least in triplicate. Data were analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL) and Microsoft Office Excel 2007 (Microsoft Incorporation). Means and standard error were derived with Microsoft Office Excel 2007 whereas correlations between various parameters were assessed by Pearson s test (*, ** significant levels at P < 0.01 and P < 0.05) in all cases using SPSS software. Relationships among the gluten yield and quality parameters were studied by multivariate method principal component analysis (PCA). In PCA, the information in the data is projected down to a Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc. 73

GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY V. DHAKA and B.S. KHATKAR small number of new variables called principal components (PCs), which are linear combinations of the original data. RESULTS AND DISCUSSION Flour Quality The protein content of wheat varieties ranged from 8.6 to 14.7% with an average value of 12.2% (Table 1). Flours suitable for bread making are those made from hard wheat and generally have high protein content in the range of 11 14% (Ktenioudaki et al. 2010). The highest protein content was noted in HI 977 and the lowest in HW 2004. Protein content and composition of wheat is the most important criteria in determination of wheat quality (Bilgin and Korkut 2005). Protein content was positively correlated with the SDS sedimentation value (r = 0.72). A similar correlation coefficient between protein content and SDS sedimentation value had been reported by Faergestad et al. (2000). It can be examined from Table 1 that wheat varieties HW 2004 and PBW 373 had lower protein content and SDS volume, implying poor protein quality and content; whereas varieties HI 977, PBW 550 and DBW 16 had higher protein content in conjunction with high SDS volume signifying good protein quality and quantity. The flour protein content correlated well with SLV of bread (r = 0.69). Sedimentation values varied from 31.3 to 62.0 ml. The highest sedimentation volume was observed for wheat variety HI 977 (62.0 ml), whereas wheat variety PBW 373 exhibited the lowest SDS sedimentation volume (31.3 ml). SDS sedimentation volume is one of the most important tests used to discriminate wheat genotypes based on their gluten quality and quantity, the two most important factors influencing bread-baking quality (Axford et al. 1978; De Villiers and Laubscher 1995; Carter et al. 1999), especially in genotypes with a protein content of up to approximately 13%, where high SDS sedimentation volume has been associated with stronger gluten and good quality (Carter et al. 1999). On the basis of SDS sedimentation test, wheat variety HI 977 could be classified as very good bread-making variety and the varieties PBW 373 and HW 2004 could be classified as being poor bread-making quality, with the remaining varieties possessing good bread-making quality. There has been evidence to suggest that the SDS sedimentation test singularly gives the best prediction of bread-baking potential and strength for hard wheat (Greenaway et al. 1966; Moonen et al. 1982). The SDS sedimentation volume was associated with the amount of HMW-GS (r = 0.82). It was inferred that the differences in SDS sedimentation volume indicated the differences in the amount of HMW-GS in flour samples. The SDS sedimentation volume was also positively correlated with the GI and resistance to extension (R) to extensibility (E) ratio of gluten. TABLE 1. FLOUR, DOUGH AND GLUTEN, AND BREAD QUALITY PARAMETERS OF WHEAT VARIETIES Wheat varieties PC SDS volume WA DDT DS WG DG GI R/E BF SLV HI 977 14.7 ± 0.1 a 62.0 ± 0.6 a 54.3 ± 0.1 j 8.3 ± 0.1 a 8.6 ± 0.1 b 26.4 ± 0.4 h 9.6 ± 0.2 ef 99.3 ± 0.2 a 1.5 ± 0.1 a 135 ± 0.2 a 4.4 ± 0.2 a DBW 16 14.2 ± 0.1 b 58.0 ± 0.6 b 53.9 ± 0.2 k 7.2 ± 0.1 b 9.1 ± 0.2 a 30.0 ± 0.4 f 10.7 ± 0.1 a 97.6 ± 0.2 a 1.5 ± 0.1 a 153 ± 1.6 c 4.3 ± 0.1 a PBW 550 12.9 ± 0.1 e 57.0 ± 0.6 b 55.5 ± 0.1 i 6.7 ± 0.1 c 8.7 ± 0.1 b 33.6 ± 0.4 bc 12.3 ± 0.3 cd 91.9 ± 0.3 b 1.4 ± 0.1 a 144 ± 1.1 b 4.1 ± 0.1 b WH 542 12.7 ± 0.1 ef 48.0 ± 0.7 c 59 ± 0.3 d 7.3 ± 0.1 b 7.2 ± 0.3 c 31.5 ± 0.3 def 11.1 ± 0.1 ab 80.8 ± 0.2 d 0.8 ± 0.1 d 158 ± 0.1 d 4.0 ± 0.2 bc WH 147 13.5 ± 0.1 d 45.8 ± 0.6 d 61.6 ± 0.1 b 7.0 ± 0.2 b 5 ± 0.2 f 27.9 ± 0.2 g 9.9 ± 0.2 e 80.5 ± 0.4 d 0.8 ± 0.2 cd 177 ± 0.3 f 3.9 ± 0.1 cd WH 283 12.6 ± 0.1 f 49.7 ± 0.9 c 55.2 ± 0.3 i 4.4 ± 0.3 e 6.7 ± 0.1 d 30.5 ± 0.3 f 10.6 ± 0.1 a 85.7 ± 1.4 c 1.0 ± 0.1 b 166 ± 0.4 e 3.9 ± 0.1 cd PBW 443 11.7 ± 0.1 h 46.0 ± 0.6 d 58.5 ± 0.1 e 6.0 ± 0.1 d 4.8 ± 0.2 f 34.9 ± 0.2 ab 11.4 ± 0.1 b 61.2 ± 1.7 gh 0.5 ± 0.3 ef 188 ± 1.3 h 3.8 ± 0.3 de PBW 343 13.9 ± 0.3 c 42.0 ± 0.3 f 59.1 ± 0.3 d 4.3 ± 0.1 e 6.4 ± 0.1 de 32.7 ± 1.5 cd 10.8 ± 0.4 ab 64.8 ± 1.6 g 0.8 ± 0.1 d 181 ± 0.6 g 3.8 ± 0.1 de HS 490 10.8 ± 0.1 j 49.0 ± 0.9 c 48.5 ± 0.1 m 1.9 ± 0.2 h 4.2 ± 0.1 g 26.3 ± 0.1 h 9.2 ± 0.2 f 77.6 ± 2.2 de 0.5 ± 0.2 ef 208 ± 0.7 j 3.7 ± 0.3 ef WH 711 12.8 ± 0.2 ef 49.0 ± 0.3 c 56.9 ± 0.1 h 3.5 ± 0.1 f 6.1 ± 0.1 e 35.5 ± 0.4 a 11.9 ± 0.2 c 74.5 ± 1.6 e 0.8 ± 0.2 d 200 ± 3.3 i 3.6 ± 0.2 f WH 1025 11.3 ± 0.1 i 49.0 ± 0.6 c 57.5 ± 0.1 g 3.4 ± 0.2 f 4.2 ± 0.3 g 30.9 ± 0.4 ef 10.9 ± 0.3 ab 69.8 ± 0.4 f 0.9 ± 0.1 bcd 246 ± 0.7 k 3.5 ± 0.3 g WH 1021 12.1 ± 0.1 g 42.7 ± 0.9 ef 52.8 ± 0.2 l 3.0 ± 0.1 fg 4.4 ± 0.1 g 32.3 ± 0.3 cde 11.4 ± 0.3 b 75.7 ± 2.7 e 0.9 ± 0.1 bc 256 ± 0.2 l 3.4 ± 0.2 gh PBW 373 8.9 ± 0.2 k 31.3 ± 0.3 h 57.9 ± 0.1 f 3.3 ± 0.1 f 3.9 ± 0.1 gh 21.6 ± 0.7 cd 8.4 ± 0.2 g 59.2 ± 0.4 h 0.6 ± 0.1 e 272 ± 0.6 m 3.4 ± 0.1 gh HW 2004 8.6 ± 0.2 l 36.0 ± 0.6 g 59.6 ± 0.1 c 2.0 ± 0.3 h 3.1 ± 0.1 i 25.6 ± 0.5 h 8.4 ± 0.1 g 62.6 ± 0.3 gh 0.8 ± 0.1 d 289 ± 0.2 n 3.3 ± 0.3 hi C 306 12.8 ± 0.3 e 41.0 ± 0.9 f 62.7 ± 0.2 a 2.9 ± 0.1 fg 3.5 ± 0.2 h 35.1 ± 0.2 ab 12.4 ± 0.1 d 65.1 ± 1.2 g 0.4 ± 0.3 f 301 ± 1.3 o 3.2 ± 0.2 i Note: The values are mean ± SD of determinations made in triplicate. Values followed by different superscripts are significantly different at P < 0.05. PC, protein content (%), SDS, sodium dodecyl sedimentation volume (ml); WA, water absorption (%); DDT, dough development time (%); DS, dough stability (min); WG, wet gluten (%); DG, dry gluten (%); GI, gluten index (%); R/E, resistance/extensibility (g/mm); BF, bread Firmness (g); SLV, specific loaf volume (cm 3 /g). 74 Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc.

V. DHAKA and B.S. KHATKAR GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY Gluten Content and Composition The gluten proteins impart unique bread-making properties to wheat. Wheat varieties varied significantly (P < 0.05) for their wet and DG contents as shown in Table 1. The values for WG ranged from 21.6 to 35.5%. The highest amount of WG was found in straight grade flour of wheat variety WH 711, whereas the lowest was found in PBW 373. The mean values for DG ranged from 8.4 to 12.4%. Wheat variety HW 2004 showed the lowest DG content, i.e., 8.37%, whereas variety C 306 reported the highest, i.e., 12.43%. A very significant positive correlation (r = 0.95) was observed between WG and DG contents (Table 3). DG is about onethird of the weight of WG. This is due to the evaporation of water during drying to obtain the dry content of gluten (Zaidel et al. 2010). The proportions of gluten subfractions from different wheat varieties are presented in Table 2. Gliadin fraction ranged from 43.1 to 53.2%, the highest for variety HW 2004 and the lowest for variety PBW 550. Glutenin proportion varied significantly among the varieties with HI 977 (57.2%) showing the highest content followed by DBW 16 (55.7%), whereas the variety HS 490 (45.7%) showed the lowest value of this subfraction followed by C 306 with 45.9% glutenin content. The gliadin/glutenin ratio ranged from 0.75 to 1.16 (Table 2). The variety HI 977 had the lowest gliadin/glutenin ratio of 0.75 while the variety C 306 showed the maximum ratio of 1.16. Gliadin/glutenin ratio showed higher negative correlation with the DDT (r = 0.73) and dough stability (r = 0.79), which are important indicators of dough strength and dough s tolerance to mixing. Relative proportions of glutenin subfractions from different wheat varieties are presented in Table 2. HMW-GS fraction ranged from 23.5 to 48.6%, the highest for variety PBW 550 followed by HI 977 with 48.1% HMW-GS content, while the variety HW 2004 had the lowest. LMW-GS also showed a significant variation among the wheat varieties with variety HW 2004 having the highest LMW-GS proportion followed by C 306, and the variety HI 977 demonstrating the lowest value of this subfraction. The HMW-GS to LMW-GS ratio ranged from 0.31 to 0.93. The variety HW 2004 had the lowest HMW-GS/LMW-GS ratio of 0.31, whereas the variety HI 977 and PBW 550 showed the highest ratio of 0.93. Gluten Extensional Properties R/E ratio of gluten affects dough structure. During the proofing and baking stages, dough should be sufficiently extensible to enlarge in response to gas pressure, yet strong enough to resist collapse of gas cells to produce a loaf with large volume (Dhaka et al. 2012). R/E ratio of the gluten of TABLE 2. GLUTEN SUBFRACTIONS QUANTIFICATIONS AND THEIR RATIO IN WHEAT VARIETIES HMW-GS/ LMW-GS Gliadin/ glutenin ratio HMW-GS LMW-GS Glu- 1 scores Gliadin Glutenin High molecular weight glutenin subunits Glu-A1 Glu-B1 Glu-D1 Wheat varieties HI 977 2* 17 + 18 5 + 10 10 43.5 57.2 0.75 48.1 51.7 0.93 DBW 16 2* 7 + 8 5 + 10 10 43.7 55.7 0.78 46.2 54.3 0.84 WH 542 2* 7 + 9 5 + 10 9 43.8 53.9 0.81 42.8 54.7 0.78 PBW 550 2* 7 + 9 5 + 10 9 43.1 55.4 0.77 48.6 52.2 0.93 PBW 443 2* 7 + 9 5 + 10 9 49.9 50.7 0.96 40.2 58.6 0.69 PBW 373 1 7 + 9 5 + 10 9 50.0 49.6 1.01 25.7 75.1 0.34 PBW 343 1 7 + 9 5 + 10 9 47.4 52.5 0.90 41.3 58.7 0.70 HS 490 2* 7 + 8 2 + 12 8 51.6 45.7 1.13 39.1 60.7 0.64 WH 1021 2* 7 + 8 2 + 12 8 46.3 53.1 0.87 33.8 67.9 0.50 WH 283 2* 7 + 8 2 + 12 8 43.5 55.7 0.78 41.4 58.3 0.71 WH 711 2* 17 + 18 2 + 12 8 46.7 54.8 0.85 44.7 57.1 0.78 WH 147 2* 7 + 8 2 + 12 8 45.4 54.8 0.83 46.1 53.1 0.87 WH 1025 2* 7 2 + 12 6 44.9 53.3 0.84 32.4 68.5 0.47 C 306 Null 20 2 + 12 4 53.1 45.9 1.16 26.6 74.1 0.36 HW 2004 Null 20 2 + 12 4 53.2 49.5 1.07 23.5 74.9 0.31 HMW-GS, high molecular weight glutenin subunit (%); LMW-GS, low molecular weight glutenin subunit (%); HMW-GS/LMW-GS, high molecular weight glutenin subunit/low molecular weight glutenin subunit. * Denomination of glutenin subunit. Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc. 75

GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY V. DHAKA and B.S. KHATKAR FIG. 1. SEM IMAGES OF INTERNAL STRUC- TURE OF GLUTEN FROM EXTRA STRONG (HI 977, A) AND WEAK (C 306, B) BREAD- MAKING WHEAT VARIETIES different wheat varieties ranged from 0.41 to 1.49 (Table 1). Varieties HI 977 and DBW 16 exhibited the highest R/E ratio of 1.5 each. The lowest R/E ratio of 0.41 was recorded for variety C 306. Several authors have also highlighted the need to assess dough extensibility when screening for enduse quality among wheat breeding lines (Suchy et al. 2000; Anderssen et al. 2004; Nash et al. 2006; Treml et al. 2006). R/E ratio was correlated with GI (r = 0.85), gliadin/glutenin ratio (r = 0.79) and HMW-GS (r = 0.59), and then negatively correlated with LMW-GS (r = 0.56). The gluten composition and R/E ratio results suggest that R/E ratio is primarily governed by relative proportion of HMW-GS and gliadin/glutenin ratio in wheat varieties (Table 2). There was a general trend which indicated that the R/E value decreased with increase in gliadins proportion from 43.1 to 53.2% in gluten samples of wheat variety which is in agreement with Wieser and Kieffer (2001) and Pena et al. (2005). Relationship of Gliadin/Glutenin Ratio with Dough Rheological Properties and Bread-Making Quality The quantity and quality of gluten proteins largely determines the dough rheological characteristics of wheat flour. Furthermore, they are also involved in the gas retention properties of the fermented dough, which are held responsible for the loaf volume and crumb structure of the bread (Goesaert et al. 2005). Gliadin/glutenin ratio is believed to largely affect the bread making of wheat varieties. Within the viscoelastic gluten protein network, the glutenins and gliadins play a different role. Because of its large size, glutenin polymers form a continuous network that endows elasticity and strength to the dough. The gliadins on the other side accomplish the role of plasticizers of the glutenin polymeric network. Hence, an appropriate balance between dough viscosity and elasticity/strength is essential. In the present study, the strength of each wheat variety could be adjudged from the Mixolab data on the basis of DDT and dough stability (Table 1). Wheat varieties HI 977 and DBW 16 exhibited the characteristics of extra strong wheat varieties with longer DDT of 8.3 and 7.2 min and higher dough stability values of 9.1 and 8.6 min, respectively. Wheat varieties HW 2004, PBW 373 and C 306, in contrast, were weak as they developed quickly, with low dough stability ( 4 min) indicating that these doughs were less tolerant to mixing as compared to the other wheat varieties. The gliadin/glutenin ratio showed a strong negative relationship with DDT (r = 0.73) and dough stability (r = 0.79), implying that flours containing relatively less gliadin and more glutenin protein require longer mixing times and suggesting that the DDT of flour is strongly influenced by its gluten protein composition. It is evident from the results that as the gliadin/glutenin ratio increased from 0.75 to 1.16, there was a significant decrease in DDT and dough stability which may be due to plasticizing effect of gliadin and the interference of gliadin with glutenin-glutenin interaction. DDT correlated positively with dough stability (r = 0.79) highlighting that wheat varieties that take longer time to mix also have high values of dough stability as exhibited by varieties HI 977 and DBW 16. High dough stability values are usually related to the strength of flours (Moreira et al. 2011). In a study reported by Lundh and MacRitchie (1989), differences in DDT between excellent bread-making quality wheat and moderately good breadmaking quality wheat were attributed to difference in proportion of glutenins. A significant negative correlation of gliadin/glutenin ratio with R/E ratio (r = 0.79) was also established. Hence, variation in gliadin/glutenin ratio appears to be a critical factor in determining the intervarietal differences in gluten viscoelasticity and rheological properties. This finding was further reinforced by nature of the gluten matrix obtained from SEM which clearly demonstrated that the gluten from the extra strong variety had a sheetlike structure, while from the weak variety open structure was obtained (Fig. 1). The differences in the structures may be attributed to glutenin/gliadin ratio. The matrix of 76 Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc.

V. DHAKA and B.S. KHATKAR GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY SLV (cm3/g) 4.6 4.4 4.2 4 3.8 3.6 3.4 3.2 R² = 0.5861 3 0.7 0.8 0.9 1 1.1 1.2 1.3 Gli/Glu ra o FIG. 2. THE RELATIONSHIP OF GLIADIN/GLUTENIN RATIO WITH SPE- CIFIC LOAF VOLUME (SLV) OF BREAD variety HI 977 with higher glutenin was probably stabilized by glutenin macro polymers. The relationship of gliadin/glutenin ratio and SLV is shown in Fig. 2. It was highlighted from the results obtained that the varieties with high gliadin/glutenin ratio had poor SLV while varieties with lower values of gliadin/ glutenin ratio had good SLV. Gliadin/glutenin ratio exhibited a negative relationship with SLV (r = 0.73) and also a positive relationship with the bread firmness (r = 0.74). This appeared due to the fact that insufficiently elastic gluten (having high gliadin/glutenin ratio) leads to low bread loaf volume and increased elasticity leads to higher SLV and lower bread firmness. The gliadin/glutenin ratio of wheat varieties 1 resulted in lower SLV reflecting the importance of glutenin quantity in explaining the variation in bread SLV. The results reveal the difference in the functionality of gluten fractions, i.e., glutenin subunits form high molecular weight aggregates, and a higher quantity in flour favors dough and gluten strength. Monomeric gliadins act as a solvent for glutenins; a higher gliadin/ glutenin ratio leads to an increased viscous and sticky doughaswellasgluten. Association of HMW-GS/LMW-GS Ratio with Dough Rheological Properties and Bread-Making Quality Another factor that affects the bread quality is the composition of its glutenin subfraction. It can be concluded that the divergence in functionality of glutenin results from either the composition of glutenin subunits (more than 20 HMW-GS and around 40 LMW-GS have been identified) or size distribution of glutenin polypeptides (HMW/LMW ratio) and/or the structure of glutenin subunits (Khatkar and Schofield 1997). Mixolab classified the wheat varieties under study into two groups on the basis of HMW-GS located at Glu-A1 and Glu-D1. Wheat varieties HI 977, DBW 16, PBW 550 and WH 542 with subunits 2* and 5 +10 at Glu-A1 and Glu-D1 correspondingly exhibited the characteristics of extra strong wheat varieties with longer DDT (8.3 7.2 min) and higher dough stability (9.1 8.6 min). Wheat varieties C 306, HW 2004 with null allele at Glu-A1 and 2 + 12 at Glu-D1, in contrast, were weak as they developed quickly, with low dough stability ( 4 min) indicating that these doughs were less tolerant to mixing as compared to the other wheat varieties. Popineau et al. (1994) reported that the removal of subunits at Glu-A1 and Glu-D1 resulted in loss of elasticity. It has also been reported that 5 + 10 subunit had greater influence on the elastic properties of dough than that of subunits 17 + 18 (Gupta et al. 1995). However, some scientists (Kasarda 1989; Singh et al. 1990) also reported that it is not the subunit composition but the quantity of HMW-GS which governs the bread-making potential of wheat varieties. DDT showed pronounced correlations with HMW-GS (r = 0.76), LMW-GS (r = 0.79) and HMW-GS/LMW-GS ratio (r = 0.81) as shown in Table 3. Wieser and Zimmermann (2000) have also reported that levels of HMW-GS are highly correlated to DDT. The ratio of HMW-GS/LMW-GS was higher for some varieties particularly HI 977 and PBW 550 (Table 2). Higher HMW- GS/LMW-GS ratio exhibited dough strengthening effect and longer mixing time as discussed above and also concluded by Anderson and Bekes (2011) and Veraverbeke et al. (1998). High correlation coefficients between HMW-GS/ LMW-GS ratio and dough stability (r = 0.85) were also noticed in this study as depicted in the correlation matrix (Table 3). A likely explanation for the dough strengthening effect caused by high HMW-GS/LMW-GS ratio might be linked to the fact that the HMW-GS proportion helps in forming the backbone of glutenin network. Based on the results of this study, it is conceivable that the glutenin subfraction, especially the HMW-GS, plays a positive role in dough rheological properties by contributing more to dough strength than the LMW-GS. Densitometric traces of SDS-PAGE pattern of glutenin subunits of extra strong and weak bread-making wheat varieties are shown in Fig. 3. The results demonstrate that the good bread-making wheat variety HI 977 contained higher amounts of HMW-GS than the poor PBW 373 bread-making wheat. It is widely accepted that there is a direct relationship between the relative amounts of HMW-GS and bread-making quality for a large number of wheat varieties (Jin et al. 2011). Figure 4 illustrates the dependence of SLV of bread on HMW-GS/LMW-GS ratio, specifying that higher HMW-GS/LMW-GS ratio would lead to a higher proportion of large size polymers and thus possibly a higher SLV. Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc. 77

GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY V. DHAKA and B.S. KHATKAR TABLE 3. CORRELATION COEFFICIENTS AMONG FLOUR, GLUTEN AND BREAD QUALITY PARAMETERS OF WHEAT VARIETIES PC SDS WA DDT DS WG DG GI R/E GLI GLU GLI/GLU HMW-GS LMW-GS HMW-GS/ LMW-GS BF SLV PC 1 SDS 0.72** 1 WA 0.02 0.45 1 DDT 0.69** 0.67** 0.08 1 DS 0.73** 0.82** 0.28 0.79** 1 WG 0.48 0.25 0.21 0.09 0.18 1 DG 0.54* 0.33 0.16 0.19 0.28 0.95** 1 GI 0.66** 0.89** 0.47 0.67** 0.84** 0.01 0.14 1 R/E 0.53* 0.76** 0.33 0.63* 0.84** 0.05 0.07 0.85** 1 GLI 0.64** 0.70** 0.22 0.71** 0.80** 0.16 0.29 0.76** 0.77** 1 GLU 0.60* 0.63** 0.08 0.71** 0.77** 0.14 0.20 0.69** 0.81** 0.92** 1 GLI/GLU 0.61* 0.66** 0.14 0.73** 0.79** 0.17 0.25 0.70** 0.79** 0.97** 0.98** 1 HMW-GS 0.79** 0.82** 0.29 0.76** 0.84** 0.29 0.33 0.76** 0.59* 0.75** 0.71** 0.74** 1 LMW-GS 0.76** 0.80** 0.25 0.79** 0.82** 0.25 0.27 0.74** 0.56* 0.72** 0.69** 0.72** 0.99** 1 HMW-GS/ 0.77** 0.82** 0.24 0.81** 0.85** 0.24 0.29 0.77** 0.62* 0.74** 0.71** 0.73** 0.99** 0.98** 1 LMW-GS BF 0.70** 0.79** 0.30 0.80** 0.87** 0.18 0.18 0.76** 0.63* 0.74** 0.71** 0.74** 0.94** 0.96** 0.94** 1 SLV 0.69** 0.83** 0.31 0.87** 0.91** 0.01 0.06 0.83** 0.75** 0.74** 0.71** 0.73** 0.88** 0.90** 0.90** 0.94** 1 **Correlation is significant at 0.01 level; *Correlation is significant at 0.05 level. PC, protein content; SDS, sodium dodecyl sedimentation volume; DDT, dough development time; DS, dough stability; WG, wet gluten; DG, dry gluten; GI, gluten index; R/E, resistance/extensibility; GLI, gliadin; GLU, glutenin; GLI/GLU, gliadin/glutenin; HMW-GS, high molecular weight glutenin subunit; LMW-GS, low molecular weight glutenin subunit; HMW-GS/LMW-GS, high molecular weight glutenin subunit/low molecular weight glutenin subunit; BF, bread firmness; SLV, specific loaf volume. 78 Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc.

V. DHAKA and B.S. KHATKAR GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY FIG. 3. DENSITOMETRIC PATTERN OF GLUTENIN SUBUNITS OBTAINED FROM EXTRA STRONG (HI 977, A) AND WEAK (C 306, B) BREAD- MAKING WHEAT VARIETIES USING 80% ACETONE PRECIPITATION METHOD SLV (cm 3 /g) 4.6 4.4 4.2 4 3.8 3.6 3.4 3.2 R² = 0.8146 3 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 HMW-GS/LMW-GS ra o FIG. 4. RELATIONSHIP OF THE RATIO OF HMW/LMW SUBUNITS OF GLUTENINS WITH SPECIFIC LOAF VOLUME (SLV) OF BREAD The wheat varieties used in this study varied in flour and rheological properties and, therefore, bread produced differed in the SLV and bread firmness (Table 1). Wheat variety HI 977 yielded the highest SLV of bread compared with the other varieties. The variety HI 977 had the highest protein content, HMW-GS/LMW-GS ratio, SDS volume and GI. Furthermore, good SLV but significantly lower than that of variety HI 977 was obtained when bread was produced from varieties DBW 16, PBW 550 and WH 542. The poor SLV of bread was achieved when using flour of wheat variety C 306. It has long been known that the protein content of flour has a direct effect on the volume of bread produced from it within wide protein range. However, when the protein range of samples is narrow, protein percentage does not clearly discriminate wheats for end-use quality. It is noteworthy that the SLV had significant correlations with GI (r = 0.83), HMW-GS (r = 0.88) and HMW-GS/LMW-GS ratio (r = 0.90). The study evidently suggests that higher SLV was obtained from wheat varieties having lower gliadin/glutenin and higher HMW-GS/LMW-GS ratio. R/E also exhibited a significant correlation (r = 0.81) with the SLV. This observation is consistent with the widely held view that a balance of elasticity and viscosity is essential for good bread-making performance of wheat varieties. The SDS-PAGE patterns of glutenin proteins of 15 wheat varieties are shown in Fig. 5. In the present study, it was observed that wheat varieties having subunits 2 + 12 at Glu-D1 had weaker doughs and lower SLV. However, some wheat varieties such as WH 283, WH 147 although having subunits 2 + 12, produced stronger doughs with good SLV (Table 1). Another noteworthy observation was that wheat varieties PBW 343 and PBW 373 (Glu-1 score 9) both having same HMW-GS composition 1, 7 + 9 and 5 + 10, but PBW 373, produced weaker dough (confirmed by Mixolab as well as gluten quality parameters) and poor SLV, which may be attributed to its higher gliadin/glutenin ratio of 1.01 as compared to gliadin/glutenin ratio of 0.90 in case of variety PBW 343. It was a general observation that varieties with higher Glu-1 score (Table 2) and having high molecular weight subunits (HMW-GS) 5 + 10, 2*, 17 + 18 and FIG. 5. SDS PAGE PATTERNS OF TOTAL GLU- TENIN SUBUNITS OBTAINED FROM 80% ACETONE PRECIPITATION METHOD FROM DIF- FERENT WHEAT VARIETIES The varieties are in lanes from 1 to 15. 1 C 306;2 PBW373;3 HI977; 4 DBW 16; 5 WH542;6 PBW343;7 WH1025; 8 PBW 550; 9 PBW 443; 10 HW 2004; 11 WH 283; 12 WH 711; 13 WH 147; 14 HS 490; 15 WH 1021; M Marker. Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc. 79

GLUTEN PROTEINS, DOUGH RHEOLOGY AND BREAD QUALITY V. DHAKA and B.S. KHATKAR TABLE 4. PRINCIPAL COMPONENT ANALYSIS OF FLOUR QUALITY CHARACTERISTICS* PC1 (66.3%) PC2 (13.2%) Quality trait Loading factor Quality trait Loading factor DDT 0.83 WG 0.94 DS 0.93 DG 0.91 SDS 0.88 GI 0.90 R/E ratio 0.82 GLI 0.87 GLU 0.84 GLI/GLU 0.87 HMW-GS 0.92 LMW-GS 0.91 HMW-GS/ LMW-GS 0.93 BF 0.93 SLV 0.95 * Principal components. Loading factors (eigenvalues). DDT, dough development time; DS, dough stability; SDS, sodium dodecyl sedimentation volume; GI, gluten index; R/E, resistance/ extensibility; GLI, gliadin; GLU, glutenin; GLI/GLU, gliadin/glutenin ratio; HMW-GS, high molecular weight glutenin subunit; LMW-GS, low molecular weight glutenin subunit; HMW-GS/LMW-GS, high molecular weight glutenin subunit/low molecular weight glutenin subunit; BF, bread firmness; SLV, specific loaf volume; WG, wet gluten; DG, dry gluten. 7 + 9 were found to have higher SLV while varieties with lower Glu-1 score having HMW-GS 2 + 12, 6 + 8, 20 or null alleles had inferior bread-making quality which is in agreement with the study of Khatkar (2006). PCA The relationships among the gluten quality and quantity parameters were measured with rheological and breadmaking quality for 15 wheat varieties and evaluated by PCA to determine the source of the underlying variability. The PCA revealed an approximate orthogonal variation in gluten and glutenin quality and quantity, as expressed by the GI, gliadin/glutenin ratio, HMW/LMW ratio, WG, DG, dough stability and DDT. The first two PCs accounted for 79.5% of the variability in terms of bread SLV, gluten characteristics and glutenin quantity parameters (Table 4). From Table 4, it can be seen that PC1 representing 66.3% of the variability was positively related to SLV, quantity and quality of gluten and glutenin subfractions, dough stability and DDT. These parameters were closely related at the right side of the loading plot as represented in Fig. 6. It was also observed that gliadin, gliadin/glutenin ratio, LMW-GS and bread firmness were negatively related which align themselves on the left side of the loading plot (Fig. 6). These results are consistent with the highly significant correlations among baking parameters and their close relationship to protein quality as discussed above. The second PC accounted for 13.2% of the variability. Very high loading factors were obtained for wet and DG contents, again strengthening the view that the gluten quantity is a major contributor to the bread-making quality of wheat. CONCLUSIONS The importance of gliadin/glutenin ratio and HMW-GS to low molecular weight glutenin subunits ratio on dough rheological properties and bread-making quality was assessed by using correlation approach. The 15 wheat varieties used in this study showed wide variations in dough rheological properties bread-making potential. SDS sedimentation volume, DDT, dough stability, gliadin/glutenin and HMW-GS to low molecular weight glutenin subunits FIG. 6. LOADING PLOT FOR THE FIRST TWO PCS IN PCA OF QUANTITATIVE AND QUALITA- TIVE PARAMETERS OF GLUTEN PROTEINS PC, protein content; SDS, sodium dodecyl sedimentation volume; WG, wet gluten; DG, dry gluten; GI, gluten index; RE, resistance/ extensibility; GLI, gliadin; GLU, glutenin; GLI/GLU, gliadin/glutenin ratio; WA, water absorption; DDT, dough development time; DS, dough stability; HMW-GS, high molecular weight glutenin subunit; LMW-GS, low molecular weight glutenin subunit; HMW-GS/ LMW-GS, high molecular weight glutenin subunit/low molecular weight glutenin subunit; BF, bread firmness; SLV, specific loaf volume. 80 Journal of Food Quality 38 (2015) 71 82 2015 Wiley Periodicals, Inc.

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