Prediction of end-use quality of Indian wheat varieties by using SECAbstractThe eminence of molecular weight distribution (MWD) of gluten proteins has been elucidated by correlation studies in context with flour characteristics, dough rheological properties and textural attributes of finished product. Size Exclusion Chromatography (SEC) chromatogram of wheat varieties was segregated into five domains depending on the molecular weight of the eluting protein fractions as peak 1: glutenins along with minor amount of omega gliadins, peak 2: gliadins with slight portion of low molecular weight glutenin subunits (LMW-GS), peak 3: low molecular weight (LMW) monomeric proteins, lastly peak 4 and 5: albumins and globulins. Absorbance area percentage (AA %) of peak 1 and 2 exhibited divergent influence on the flour characteristics, dough rheology and gluten protein quality characteristics; reflected the alliance of MWD patterns with their contrasting performance in noodle, bread, cookie and chapatti making. This investigation was subjected to utilization of SEC to indicate the varietal variation in terms of their peculiarities for a specific type of product. Moreover, high molecular weight glutenin subunits (HMW-GS) which is considered as prime determinant for varietal variation is not the sole root cause, but the discrepancy in the proportion of gluten protein fractions play an imperative role in deciding the technological and functional characteristics of a wheat variety. 1. IntroductionWheat (Triticum aestivum L.) is considered the worlds most domesticated cereal crop and possesses a great economic importance for India. The distinct rheological property of wheat is due to the viscoelastic complex called gluten which lets it to be processed into a plenitude food as well as non-food products (Dong, Hao et al. 2009). Gluten is a complex of heterogeneous protein components that form a three-dimensional network of linearly cross-linked glutenin subunits and gliadin components through hydrogen, hydrophobic, and disulfide bonds (Korableva and Kasymova 2010, Ferreira, Ruiz et al. 2012). Quantity and quality of wheat proteins is considered substantial in relation to baking quality of wheat (He and Hoseney 1992).The molecular size of wheat gluten proteins has been demonstrated extensively by SEC. Gliadin was first fractionated by SEC after extraction with 70% ethanol by Jones, Babcock et al. (1963) and Beckwith, Neilsen et al. (1966). Similarly Huebner and Wall (1974) revealed that reduced and alkylated glutenin resulted in three types of subunits differing in size and associative tendencies, followed by improvements of chromatographic procedures by Beitz (1984), Batey, Gupta et al. (1991) and Larroque and Bekes (2000). Numerous researches had emphasised on the relation of differences in molecular size of native flour proteins with wheat quality parameters. Ohm, Ross et al. (2006), Ohm, Ross et al. (2008), Ohm, Ross et al. (2009b) and Tsilo, Ohm et al. (2010) have demonstrated distinct associations of protein fractions with specific molecular weight with various quality parameters and wheat products related characteristics. Previous studies has mentioned the significant associations of protein fractions with wheat quality characteristics such as the prominent effects of HMW-GS (Uthayakumaran, Stoddard et al. 2000), LMW-GS (Verbruggen, Veraverbeke et al. 2001), gliadins (Uthayakumaran, Tmskzi et al. 2001, Khatkar, Fido et al. 2002a, Khatkar, Barak et al. 2013), the glutenin-to-gliadin ratio (MacRitchie 1987, Gupta, Batey et al. 1992, Uthayakumaran, Newberry et al. 2000) and overall protein content (MacRitchie 1992, Uthayakumaran, Gras et al. 1999) on end-product quality, but much emphasis has not been laid down on the relationships of variations in MWD with unique end use quality attributes of wheat variety. The present study was undertaken to understand the association of MWD patterns of gluten proteins with functional specialty of different Indian wheat varieties. The influence of quantitative and qualitative protein composition, as measured by SEC, on dough baking and cooking quality was investigated. The main objective of this research was to examine fundamental advantage of SEC of wheat proteins, in conjunction with physicochemical, rheological and textural analysis and to predict the finished product specificity of diverse background wheat varieties. Thus AA % of each particular protein fraction could be adapted as a successful marker in suggesting end use quality of wheat varieties and hence SEC could hold a potential for rapid assessment of wheat genotypes in breeding programmes. 2. Material and methodsFour wheat samples commonly cultivated in India HI 977, HW 2004, DBW 16 and C 306 were elite among the choices. The samples were tempered at 15.5 g/100 g moisture for 24 h and milled to 65% extraction rate. Physical characteristics of grains i.e. thousand kernel weight, test weight, kernel length and width, hardness of all the varieties were determined using single kernel characterization system (model 4100, Perten Instruments, Huddinge, Sweden) according to standard AACC procedures. Milling was carried out using Chopin laboratory mill (Model CD1, Villeneuve la Garenne, France). Moisture, ash and crude protein content were determined by standard AACC methods (2000). SDS Sedimentation values of flours were determined according to Axford, McDermott et al. (1979). Damaged starch was evaluated by the Chopin SDmatic. Gluten yield and gluten index were estimated by Glutomatic (Model GM 2200, Perten Instruments). 2.1 Gluten Extensibility TestMeasurements were performed with a TA-XT 2i texture analyzer (Stable Micro System) using Kieffer dough/gluten extensibility rig with the test speed of 3.3 mm/s and data acquisition rate of 200 pps. The test mode of the instrument used was force in tension.2.2Dough Rheological PropertiesRheological characteristics of dough were investigated using Chopin Mixolab by adopting Standard Chopin S protocol (Kahraman, Sakyan et al. 2008). It determines a comprehensive qualitative profile of the wheat flour and plots, in real time, the torque (expressed in Nm) produced by mixing dough between two kneading arms with a constant mixing speed of 80 rpm. The information recorded from the curves contained the percentage of water required for dough to produce a torque of 1.10.07 Nm i.e. water absorption (%); the time to reach maximum torque at 30o C i.e. dough development time (min); the slip away time at which the torque produced is maintained at 1.1 Nm i.e. dough stability (min); the difference between the maximum torque at 30o C and the torque at the end of holding time at 30o C i.e. mechanical dough weakening recorded in Farinograph Units (FU), was used to evaluate the rheological characteristics of various flours.2.3Fractionation of wheat gluten into gliadin and gluteninWheat flours were defatted by successive extraction with chloroform according to MacRitchie (1987). Flour (100g) was defatted using 200ml of chloroform, filtered through filter paper at room temperature and process is repeated thrice. The defatted flour was dried at room temperature. Gluten was isolated from defatted flour using glutomatic instrument as per standard ICC method. Dry gluten, wet gluten and gluten index were determined.2.4Product preparation and analysis2.4.1Instant NoodleInstant noodles were prepared by using the standardized formulation and processing conditions (Gulia and Khatkar 2013). The ingredients in noodle recipe (on 100 g flour basis) comprised of water (30.97%), alkaline salt (0.23%) (Potassium carbonate and sodium carbonate, 1:1), guar gum (0.28%) and salt (1.54%). Noodle dough was thoroughly mixed by incorporating wheat flour with all other ingredients dissolved in water using mixer (Kitchen Aid Inc., Michigan, USA) for 4 min. Crumbled dough was then sheeted using noodle machine (ATLAS, Marcato, Italy) by passing it four times through roller no. 1 and compounding it after every pass. A resting period of 10 min was given to the dough sheet in zip lock pouch to prevent surface moisture loss and later passed over through roller unit attachment five times to get a final thickness of 1.2 mm with a regulating knob set at position no. 2, 3, 4 and 5, respectively. Resting period of final sheeted dough was anew for 30 min and dough sheet was sent to cutter attachment to get the desirable shape of noodles. The culminated noodles were stowed uniformly on a sieve and steamed into a preheated (100C) steamer (Rice Cooker-Ultimate, UL-255, China) for 6.4 min. Steamed noodles were fried in soybean oil at 142C for 2 min in deep fat fryer (Friendz, FZ-591, China) and cooled for 15 min. The samples were stored for further analysis.Oil Uptake: Oil uptake was calculated according to the approved AACC (2000) method. Instant fried noodles were ground evenly and oil was extracted with petroleum ether (60-80C) using a solvent extractor (SER148, Velp Scientifica, Usmate, Italy). Oil uptake was expressed in terms of percentage on dry basis.Cooking quality: As per the procedure of Oh, Seib et al. (1983), fried noodles (10 g) were put into 400 ml of boiling water, cooked to the optimal cooking time and subsequently cooled for 1 min under running tap water. Ultimately, the noodles were reweighed and stocked in a capped petriplate at room temperature for texture analysis. The total water remaining after cooking in addition to that utilized for rinsing was collected to estimate cooking loss. An aliquot of 50 ml was evaporated in an oven at 100C for 4 h and outcome was recorded as per cent weight loss during cooking. The cooked weight was measured as exhibited by Wang, Huang et al. (2011) as per cent enhancement in weight of noodles after cooking.Texture analysis: Texture profile analysis of cooked noodles was demonstrated using Texture Analyzer TA-XT 2i within 15 min after cooking. Pre-test speed, test speed and post-test speed used were 2.0, 3.0 and 3.0 mm/sec, respectively with the compression plate probe of 45 mm 30 mm. Five noodle strands were placed closer to each other in flat position. The texture analysis results were presented as noodle hardness, springiness, adhesiveness, cohesiveness and chewiness.2.4.2Bread Bread making method of Finney (1984) was followed to compute the bread making efficiency of different wheat flours by performing optimized baking tests. The test baking formulation for 30 g bread was: refined flour (30 g, 14 % moisture basis), compressed yeast (1.59 g), salt (0.45 g), sugar (1.8 g), fat (0.9 g), malted barley flour (0.075 g) and ascorbic acid (100 ppm, flour basis). Salt, sugar and ascorbic acid were added in solution form and yeast was added as a suspension, which was mixed well each time before dispensing.Farinograph was utilized to prepare dough. After adequate mixing, dough was placed in a bowl, capped with a wet muslin cloth and proved for 90 min at 30oC and 98 % R.H. Punching of dough was rendered after 52, 77 and 90 minutes in a machine moulder by passing through a set of rollers with a gap setting of 9 mm. Dough was placed in a lightly greased baking tin after the ultimate punching and later proved at 30oC and 98 % R.H. Bread loaves were placed in a preheated baking oven at 230o C for 25 min and allowed to cool for 2 h on a wire mesh. Specific loaf volume of bread was measured by rapeseed displacement method. The breads were baked in triplicate for each cultivar.Texture analysis: The bread firmness was evaluated using AACC approved (74-09) standard method on Texture Analyzer TA-XT 2i with 25 mm cylindrical probe (P/36 R). The pre test speed, test speed and post test speed were 1.0, 1.7 and 10.0 mm/s, respectively, with data acquisition rate of 250 pps.2.4.3CookiesCookies were prepared according to AACC approved method 10-50D (2000) with slight modifications. The ingredients formulated were flour (225 g), sugar (130 g), shortening (64 g), dextrose solution (33 ml), sodium bicarbonate (1.6 g), ammonium bicarbonate (0.9 g), sodium chloride (2.1 g) and distilled water (16 ml). The dough was prepared and sheeted to a thickness of 10 mm on a dough sheeter and round shaped cut was given using cutter of 60 mm diameter. Cookies were baked in a lightly greased tray in baking oven at 205o C for 15 min followed by cooling and consecutively measured for diameter and thickness (six cookies) and average was calculated. The spread ratio was calculated by dividing diameter (mm) with thickness (mm). Cookies were prepared in triplicate.Texture analysis: The texture of cookies was persuaded via textural analyzer (Stable Micro Systems TA-XT 2i, Godalming, U.K.). The probe used was Knife Edge Insert (HDP/BS) to apply braking force required to fracture the cookies with 5 kg load cell Heavy Duty Platform (HDP/90) at pre test speed, test speed and post test speed of 1.5, 2.0 and 10.0 mm/s respectively and data acquisition rate of 400 pps. 2.4.4 ChapattiChapatti was prepared using method of Haridas Rao, Leelavathi et al. (1986) with minor modifications. Whole wheat flour (200 g) was mixed with adequate amount of salt and water in farinograph for 3 min to obtain dough of moderate stiffness. About 40 g of dough was rounded and sheeted to 2 mm thickness using rolling pin and desired shape was achieved using chapatti circular sharp edged die of 15 cm diameter. The shaped chapatti was baked on a hot plate at 210o C from both the sides, puffing was performed at 290-320o C for 15-20 s followed by cooling and packaging. Puffing height was immediately measured after puffing using a specifically designed system having a ruler attached to it in centimetres. Chapatti quality score was analysed via physical and sensory quality parameters which were assessed by trained panel.Texture analysis: The biaxial extensibility (softness) of wheat flour chapatti was determined on Texture Analyzer TA-XT 2i with tortilla/ pastry/ bursting (HDP/TPB) and heavy duty platform (HDP/90). The ball probe SMSP/1SP was used with the pre-test speed, test speed and post-test speed of 1.0, 1.0 and 10.0 mm/s respectively. Data acquisition rate was 200 pps during test.2.5Sample Preparation and Size Exclusion ChromatographyGluten was extracted with solvent 3M urea as suggested by Huebner and Wall (1974) with minor modifications. Acetic acid was added to maintain buffer pH 4.6. Sample was sonicated (Power Sonic 410, Hwashin Technology) for 10 min, centrifuged at 12,000 x g for 30 min (Remi Cooling Centrifuge) and filtered through syringe filter (0.22 m HV Millipore, DuraPore). SEC was performed on Sephacryl S-200 column (HI PrepTM 16/60 Sephacryl S-200 HR, GE Healthcare). The mobile phase was 3M Urea, 0.15 M NaCl, pH 4.6 with a flow rate of 0.5ml/min. Selected eluted fractions were then assayed by SDS-PAGE as described by Laemmli (1970).2.6Data AnalysisData was analysed using SPSS software version 16.0 (SPSS Inc.). Correlation among various quality characteristics of kernel, flour, dough, gluten, AA % and finished product were derived using Pearsons test (p