Electrophoretic Icentification of Pakistani Wheats for Glladin and HMW Glutenin Subunit Composition and Their Relationship with end use Quality

Abstract

Wheat is a staple food of Pakistani diet. It is grown in every nook and corner of the country but more than 72% contribution in the total national wheat production is by the Punjab province only. Wheat is the cheapest and principal source of carbohydrates and proteins in the daily diet of Pakistani people. It provides more than 60% protein and calories in the total daily requirements. In Pakistan about 80% of the total wheat produced is utilized in the form of an unleavened flat bread locally known as ‘chapati’ and its culinary variations, ‘tandoori roti’, ‘nans’, ‘prathas’ and ‘pooris’, while the rest 20% is used for other bakery products such as bread, cookies, cakes and pastries etc. Pakistani wheats have not been evaluated for various quality characteristics when grown in one set at one location under identical conditions, so that the quality characteristics related to their genetic make up can be ascertained with minimizing other factors, such as environmental and production conditions. This information will be useful for the breeders to know the variation in the quality characteristics among different wheats related to their inheritance, the processors i.e., millers and bakers can choose the best wheat cultivates which can be suitable for their need. The consumer will also know the information of wheat best suited for their need. 1. 2. 3. To find out the variation in the physio-chemical and rheological characteristic of different wheat varieties. 4. To catalogue by establishing electrophoretic identification procedure based on gliadin bands. 5. To find out the correlation between high molecular weight (HMW) glutenin subunit with other quality attributes. 6. To find out the environmental influence on electrophoretic identification pattern and other quality parameters. 7. To provide the information to the wheat breeders for screening good quality wheat in early generations based on identification correlation. 8. To evaluate the Pakistani wheat for amino acid composition. Methodology: The whole study is oriented on promising wheat varieties grown under similar set of condition at wheat Research institute, Faisalabad for two consecutive crop years 1995-96 and 1996-97. The work in this project during two years has been carried out on the following quality parameters. i. Physical Characteristics ii. Thousand Kernel Weight Thousand kernel weight is a function of kernel size and density and is recorded in grams per 100 kernels. Fifty-gram representative sample of each wheat cultivar was taken and thousand kernel weight was recorded by counting clean, unbroken and sound kernels. iii. Particle size index: The particle size index was determined by milling the samples in Udy cyclone Mill and then by sieving through sieve shaker (Model RX-86-2) using U.S. standard sieve no.70 as described in AACC (1983) METHOD NO. 55-30. 2. Chemical Characteristics • Moisture content: The moisture content in each whole wheat flour sample was determined by using an air forced draft oven at a temperature of 105± 50C by following the procedure given in AACC (1983) method no. 44-15 A. • Ash content: The ash content in flour samples was determined in a muffle furnace at 550 0C by using the procedure given in AACC (1983) method no. 08-01. • Crude protein: The nitrogen present in each whole wheat flour sample was estimated by using Kjeltec system II form Tecator AB, Hoganas, Sweden based on Kjeldhal’s method as described in AACC (1983) method no. 46-10. The protein percentage was calculated by multiplying nitrogen content with a factor 5.7. • Crude fat: The crude fat was determined by using Soxhlet apparatus according to AACC (1983) method no 30-10. • Crude fiber: Each flour was tested for crude fiber conent by the following procedure as described AACC (1983)Method NO.32-10 3. Rheological Properties : Farinographic studies : Fariographic studies were carried out by running flour samples through brabender farinographic equipped with 50 gram capacity bowl to determine the dough behaviour of ech wheat flour . The dough characteristics such as water absorption dough development time and dough stability were interpreted from each faringogra according to the ,ethod described in AACC 1983) Method no.54-21 . Water absorption Waer absorption capacity of the flour is the percentage of water required to reach at the centre of the curve on the 500 Brabender Unit (BU) lie with maximum consistency of the dough (peak) Dough development time (mixing time ) This is the time required for te curve to reach at its full development or maximu consistency possessing highest peak . High peak values are associated with strong wheats that having long mixing time . Doug Stabiity : This is the time at which top of the curve remains above the 500 BU line and is measured from the arrival time to the departure time . Mixographic studies : Mixographic studies were carried out by using a mixographby adding 60 %constant water in each sample according to the instructions described in AACC (1983) METHOD NO.54-40 a. Each mixogram was interpreted for dough development time and maxium peak height . 4. Electrophoretic Identification of Cultivars Through Acid -PAGE The wheat samples were ground with a Udy cyclone Mill . The mill was cleaned after grinding each sample . Acid polyacrylamide Gel Electrophoresis (acid -PAGE)was done to obtain gliadins . 250mg samles were extracted with 750 µ1 70 % ethanol for 1 hour at temperature . Samples were centrifuged at 5500 rpm for 15 minutes . To 350 µ1 supernatants , five drops of glycerol were added to increase the density of te protein solution and one drop of 1% methyl green solution to increase the density of the protein solutions as a tracking dye was added . The samples sampls were kept in cold storage for later use . The gel contained 6%(w/v) acrylamide , 0.25% (w/v) N, N-methylenebisacrylamide, 0.024% (w/v) ascorbic acid, 0.25% (v/v) of a 0.1% (w/v) FeSO4. 7H2O and 0.25% (w/v) aluminum lactate. The PH was adjusted to 3.1 using lactic acid. The gel solution was filtered and polymerized by adding 100 µl of H2O2 per ml of gel solution (Lookhart et al 1986). Combs of 3 mm thickness were used. The gel solution was poured immediately into gel chambers already assembled. The gel was allowed to polymerize for 15 minutes. The running buffer solution for acid= PAGE contained 5.625g aluminum lactate dissolved in 4500 ml distilled water, and the PH was adjusted to 3.1 using lactic acid. Gliadin exract (15µl) was loaded in each 3mm thick slot. The temperature was set at a constant 200C using a water bath. The electrophoresis was carried out at constant voltage of 500 v for 2 hours. The gel were removed from the glass plates and placed in plastic containers, and strained with 300ml straining solution composed of 9 ml of 1% (w/v) Coomassie Brilliant Blue R in ethanol and 61 ml of 50% (v/v) trichloroacetic acid (TCA) for two days. After straining the gels were destained in a 300ml solution containing 50% TCA and distilled water (Lookhart et al 1986), and photographed. 5. HMW Glutenin Unit Composition Through SDS-PAGE SDS-continuous system was used. The formulation of gel is given below as described by Anjum (1991). Sample preparation: Forty milligram of whole wheat flour samples were extracted with 450 µl of sample treatment buffer (0.5 ml sample treatment buffer contained 6.25 ml sample stock buffer 0.75 ml β-mercaptoethanol and 3 g sucrose) and 150 µl blue juice (39% of 20% SDS, 20% β-mercaptoethanol and 41% of 0.2% bromophenol blue in glycerol). The composition of sample stock buffer was 1.21 g tris dissolved in 10 ml distilled water, to PH 6.8 with HCI in this solution 6.25 ml of 5% pyronine Y was added, mixed and filtered. To these contents 2 g SDS will be added and diluted to 50 ml. The samples were centrifuged at 5000 rpm for 15 minutes. After centrifugation the supernatants were heated in a boiling water bath for 2-3 minutes. • Stock Solutions A. Monomer: 29.2% (w/v) acrylamide, 0.4% (W/V bisacrylamie) B. Running gel buffer: 18.15% (w/v) Tris 4% (v/v), 10% SDS C. Stacking gel buffer: 6.05% (w/v) Tris, 4% (v/v.10% SDS • Resolving gel Solution (2 gels) Running gel 24.38 ml, distilled water 33.8 ml, distilled water 33.8 ml, monomer solution: 39.03 ml, N, N, N, N tetramethylenealiamine (TEMED) 0.0975ml, 10% ammonium persulate 150 µl/ • Stacking gel solution (2 gels) Stacking gel buffer 8.25 ml, distilled water 20.59 ml, Monomer solution 4.21 ml, TEMED 0.033 ml and 250 µl 10% ammonium per sulfate. Tank buffer gel contained 0.3% (W/V) Tris, 0.1435% (w/v) glycine and 0.1% (w/v) SDS. The higher molecular weight glutenin subunits (HMW) were identified by comparing with standard samples of known bands. 6. Amino Acid Analysis: The amino acid profile with special reference to essential amino acid was estimated in each wheat variety by using High speed Amino Acid Analyzer (L-8500 A. HITACHI). Sample (0.1g) was taken in a test tube and 10 ml of 6 N HCI was added to it. The test tube was evacuated by nitrogen, sealed and placed in oven at 1000C for 22 hours. The test tube was allowed to cool at room temperature. The hydrolysate so farmed was evaporated under vacuum at 600C to dryness to remove HCI. The hydrolysate was dissolved in 5 ml 0.02 N HCL, centrifuged and filtered to remove the visible sediments. A known volume of the supernatant was injected into amino acid analyzer for the estimation of amino acid profile.