SDS-Protein Gel Test for Prediction of Bread Loaf Volume

Flour dispersed in aqueous solutions of sodium dodecyl sulfate (SDS) forms a proteinaceous gel when centrifuged at high speed. The conventional methodology for SDS gel testing was modified to develop a small-scale (<1 g of flour or wheat meal) screening test for evaluation of the protein quality of wheat for breadmaking. The principal modification involved centrifugation with a swinging-bucket rotor to facilitate direct measurement of gel height, which is the primary test parameter. The effects of suspension temperature and time, centrifugation speed, sample size, and sieving of ground wheat or flour on the efficacy of the test were examined. Gel height, wet weight, and protein content were assessed as test parameters. In the standard test procedure that was developed, 0.67 g of flour or ground whole wheat was dispersed in 13.5 mL of 1.5% SDS solution for 15 min at 20°C, followed by centrifugation at 80,000 × g for 30 min. The test was evaluated using seven Canadian commercial wheat flours with diverse breadmaking quality. For the samples, gel height was strongly related to loaf volume (R² = 0.89 and 0.95 for flour and ground wheat, respectively). Sieving flour through a 75-μm sieve slightly increased the predictive power of the test (R² = 0.94). SDS gel height gave better discrimination of samples for prediction of loaf volume than did the traditional SDS sedimentation test. The performance of the sedimentation test improved when sieved ground wheat was used. The relationship between gel height or protein content and flour protein content was comparatively poor (R² = 0.25). The SDS gel test appears to primarily measure the effects of flour protein quality.     

Effects of Wheat Bug (Eurygaster maura) Protease on Glutenin Proteins

Proteolytic degradation of 50% 1-propanol insoluble (50PI) glutenin of six common wheat cultivars by wheat bug (Eurygaster maura) protease was investigated using reversed-phase HPLC. Wheat at the milk-ripe stage was manually infested with adult bugs. After harvest, bug-damaged kernels were blended (2:1, kernel basis) with undamaged grain of the same cultivar. Samples of ground wheat were incubated in distilled water for different times (0, 30, 60, and 120 min). The incubated whole meal samples were subsequently freeze-dried and stored until analysis. The degree of proteolytic degradation of 50PI glutenin was determined based on the quantity of total glutenin subunits (GS), high molecular weight GS (HMW-GS), and low molecular weight GS (LMW-GS). For ground wheat samples incubated for ≥30 min, 50PI glutenin was substantially degraded as evidenced by a >80% decrease on average in total GS, HMW-GS, and LMW-GS. Some cultivars showed different patterns of glutenin proteolysis as revealed by differences in the ratios of HMW-GS to LMW-GS between sound and bug-damaged samples; a significant decrease in this ratio was found for four cultivars. This evidence, combined with other observations, indicated that there were intercultivar differences in polymeric glutenin resistance to the protease of the wheat bug Eurygaster maura. While the nature of this resistance is unknown, it should be possible to select and develop wheat cultivars with improved tolerance for wheat bug damage. Propanol insoluble glutenin, which corresponds to relatively large glutenin polymers, appears to be an excellent quantitative marker for this purpose.     

Salt-Induced Disaggregation/Solubilization of Gliadin and Glutenin Proteins in Water

The effect of salt concentration used in preparing gluten, on the subsequent dissolution of gluten in water, was examined. Flour from a Canadian hard red spring wheat cultivar, Katepwa, was used to prepare glutens using three different solvents, i.e. distilled deionized water (DDW), 0·2% NaCl solution and 2% NaCl solution. The isolated wet glutens were extracted sequentially with DDW, providing four water soluble fractions and an insoluble residue. The amount of protein in each fraction was determined and respective compositions were assessed electrophoretically under reducing and non-reducing conditions. Surprisingly, DDW extracts of gluten prepared with 2% NaCl contained almost all the gliadins, except some ω-gliadin components, and most of the polymeric glutenin. For the gluten prepared with 0·2% NaCl, most of the gliadin, but only a small portion of glutenin, was extracted. For gluten prepared with DDW, only part of the gliadins and almost no glutenin was extractable with water. The DDW solubilities of gluten proteins prepared in DDW, 0·2% NaCl and 2% NaCl were 27, 52, and 85%, respectively, after four sequential extracts with DDW. The large increases in the solubility of gliadin and glutenin proteins in DDW when the gluten is prepared in salt solution (after removal of most of the salt) can be explained on the basis of a salt-induced conformational change of the proteins, which renders water a more effective solvent.     

Mechanical Properties of Bread Crumb Prepared from Flours of Different Dough Strength

Bread quality depends in part on the textural properties of the crumb; softness and strength being two important textural attributes. This study examined differences between instrumentally-measured textural properties of the crumb of bread made from two flours; one possessing extra strong dough mixing characteristics and a second of moderate strength (red spring wheat). Bread crumb specimens, notched and un-notched, were subjected to tensile loading and the crumb's initial (elastic) modulus, stress at failure (crumb strength) and tear resistance were determined. The same mechanical parameters were determined on bread crumb that had been compressed approximately five-fold in order to destroy crumb structure. For un-notched specimens, stiffness and strength were of the order of 11 and 1 kN/m², respectively, whereas after compression they were 230 and 10 kN/m², respectively. For CWRS breadcrumb, toughness increased from 4·1 J/m²to 12·3 J/m²following crumb compression. Bread crumb made from a flour possessing extra strong dough properties was stronger than bread crumb made from a more conventional red spring wheat flour, and there was an indication that extra strong flour bread crumb specimens were stiffer. Compression of the bread crumb lessened the difference between the mechanical properties of the two bread types, particularly for strength and tear resistance. The results indicated that bread crumb structure plays a predominant role in the textural properties of bread crumb.     

Durum wheat breadmaking quality: Effects of gluten strength,protein composition,semolina particle size and fermentation time

The effects of particle size of granulars (semolina and flour combined), gluten strength, protein composition and fermentation time on the breadmaking performance were compared for eleven durum wheat genotypes of diverse strength from North America and Italy grown in the same environment. All genotypes were γ-gliadin 45 types (low-molecular weight glutenin subunit 2 patterns) associated with superior pasta-making quality. Three cultivars with high-molecular weight glutenin subunit 20 exhibited relatively weak gluten, confirming that this subunit is associated with weakness in durum wheat. Gluten strength as measured by a range of technological tests was directly and strongly related to the proportion of insoluble glutenin (IG) in granulars protein as determined by a spectrophotometric procedure. Reducing the particle size of granulars by gradual reduction shortened development time in both the farinograph and mixograph. Reducing granulars also increased starch damage and, accordingly, farinograph water absorption, but remix-to-peak baking absorption was unaffected due to increased fermentation loss for finer granulars. Neither loaf volume, nor remix-to-peak mixing time were affected by the particle size of the granulars indicating that regrinding is not an asset for baking provided there is adequate gassing power. Loaf volume was directly related to gluten strength and IG content, and inversely related to residue protein, a non-gluten containing fraction. When fermentation time was reduced from the standard 165 to 90 min and 15 min, all genotypes exhibited a progressive increase in loaf volume. Therefore, regardless of strength, short fermentation time is preferred when high volume durum wheat bread is desired. Some of the stronger durum genotypes exhibited remix-to-peak bread volume comparable to that expected of good quality bread wheat, indicating that there is potential to select for genotypes with improved baking quality in conventional breeding programs by screening for high content of insoluble glutenin.     

Ultra-fast separation of wheat glutenin subunits by reversed-phase HPLC using a superficially porous silica-based column

A relatively new, unique column packing material for reversed-phase high-performance liquid chromatography (RP-HPLC) was evaluated for rapid separation of wheat glutenin protein subunits. The product named “Poroshell” by the manufacturer consists of a solid core and a porous coat instead of solid silica spheres used in conventional RP-HPLC column packing. This architecture favours rapid mass transfer, facilitating faster reversed-phase separations of biomolecules compared to conventional silica columns. The main objective of this study was to evaluate the quality of separations of glutenin subunits (GS), as well as to optimize conditions to produce the fastest possible run times without sacrificing resolution using a Poroshell 300SB-C₈ 2.1×75 mm column. The stability of GS separations over time was also assessed. Two different bread wheat genotypes were used for optimization of separation conditions and six more common and durum wheat genotypes possessing different subunit combinations were used for further evaluation. Glutenin protein was extracted with 0.08 M Tris–HCl buffer (pH 7.5) containing 50% 1-propanol under reducing conditions after pre-extraction of soluble proteins with 50% 1-propanol. Optimization of GS resolution and sample throughput by RP-HPLC was assessed in response to variation in eluent flow rate, acetonitrile (ACN) gradient, and column temperature. The best resolution of both HMW- and LMW-GS was obtained in 13 min using a 23–44% ACN gradient with a flow rate of 0.7 mL/min at 65 °C. Subunit elution times and integrated areas were highly repeatable even after several hundred injections. Highly satisfactory separation of HMW-GS and quantification of ratio of HMW- to LMW-GS were achieved in less than 4 min per sample using a modified HPLC gradient. Ratio of HMW- to LMW-GS was unaffected by the speed of the separations. As well, the elution order of HMW- and LMW-GS was unaffected by the rapid analysis, compared to conventional RP-HPLC separations, so no new learning was required for interpreting chromatograms and classification of subunits. The rapid RP-HPLC method using the Poroshell column appears to be very well suited for routine quantification of HMW-GS and LMW-GS especially for purposes of wheat quality screening and wheat cultivar development activities where large numbers of samples are typically encountered.     

Immunisation against ovine caseous lymphadenitis: efficacy of monocomponent Corynebacterium pseudotuberculosis toxoid vaccine and combined clostridial-corynebacterial vaccines

Sheep were immunised with Corynebacterium pseudotuberculosis toxoid formulated as a monocomponent vaccine with aluminium adjuvant or in combination with 5 clostridial antigens, and also in the combined form with sodium selenate. Immunised and control sheep were experimentally infected 16 days after vaccination and slaughtered and inspected after a further 3 months to determine their resistance to infection. All 3 vaccines afforded an equal and high level of protection; 91% of vaccinated sheep exhibiting no lesions of caseous lymphadenitis compared with 51.5% affected sheep in the control group. Average lesion counts were 1.2 per affected vaccinated sheep and 4.5 per affected control sheep. Antitoxin responses to the clostridial toxoids incorporated in the combined vaccines were not affected by inclusion of the C pseudotuberculosis toxoid or the sodium selenate.