K. R. Preston

Fusarium Head Blight: Effect of Fungal Proteases on Wheat Storage Proteins

ABSTRACT The effect of proteolytic enzymes, associated with Fusarium head blight, on wheat storage proteins and dough functionality was studied. Fusarium damaged kernels (FDK) and sound kernels were hand-picked from F. graminearum Schwabe and F. avenaceum (Fr.) Sacc. infected samples of bread and durum wheat. Scanning electron microscopy revealed significant degradation of endosperm protein in FDK. Storage proteins from FDK and sound kernels were analyzed by SDS-PAGE, RP-HPLC, and SE-HPLC. Total storage protein was lower in FDK but no significant qualitative differences in protein were detected by either RP-HPLC or SDS-PAGE. SE-HPLC was used to follow the hydrolysis of wheat storage protein by proteolytic enzymes found in FDK and a pure culture of F. graminearum. Selective inhibition of proteolytic activity by p-chloromercuribenzoate, and not soybean trypsin inhibitor or iodoacetic acid, suggests that the F. graminearum protease is an alkaline protease. Farinograph and extensigraph curves showed that the ...

Analysis of wheat gluten proteins by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition in several common and durum wheat varieties. Mass spectra were obtained directly from crude and partially purified wheat gliadin and reduced glutenin subunit fractions. Mass spectra of the gliadins and the low molecular weight (LMW) glutenin subunits show a complex pattern of proteins in the 30–40 kDa range. The observed gliadin patterns may be suitable for differentiation between wheat varieties, but the complexity of the mass spectra precludes the use of MALDI/MS as a stand-alone technique for the identification of most individual gliadin components. The mass spectra of the high molecular weight (HMW) glutenin subunits are much simpler and the complete HMW subunit profile can be determined directly from a single mass spectrum. This may prove particularly useful in wheat breeding programs for rapid identification of lines containing subunits associated with superior quality. The correspondence between previously identified HMW subunits and the mass spectral peaks was established with MALDI measurements of HPLC-separated subunits. Delayed extraction proved effective in improving the mass resolution for the monomeric gliadins and LMW glutenin subunit fractions, with masses less than 40 kDa. However, it gave little improvement for the HMW glutenin subunits which have masses of ∽80 kDa.

Effects of Commercial Hydrolytic Enzyme Additives on Canadian Short Process Bread Properties and Processing Characteristics

ABSTRACT The effects of increasing levels of eight commercial enzymes representing four types of fungal hydrolytic enzymes (α-amylases, proteases, xylanases, and cellulases) on Canadian short process (CSP) bread quality and processing characteristics were studied. Addition of all enzymes types at optimum levels resulted in increased loaf volume and bread score and softer crumb. All four types of enzymes appeared to be equally effective in improving bread properties compared with the controls. At high levels, greater tolerance to the addition of xylanases and cellulases compared with the addition of α-amylases and proteases was apparent. Mixing requirements increased with increasing levels of α-amylase but no change was apparent with the other enzymes. Addition of all enzymes reduced sheeting work requirements, indicating a dough softening effect. Optimum bread properties for all enzymes were attained within a relatively narrow range of dough sheeting work values, which presumably correspond to optimum dou...

Characterization of Wheat Gluten Proteins by HPLC and MALDI TOF Mass Spectrometry

We have performed a detailed characterization and identification of wheat gluten proteins obtained from the Teal variety of Canadian hard red spring wheat. RP-HPLC separation of the sample into 35 fractions has reduced the spectral complexity; this was followed by MALDI mass spectrometry (MS), which showed the presence of six or fewer resolved protein components above 20 kDa in each RP-HPLC fraction, giving a total of 93 MS resolved peaks. These included 17 peaks in the ω-gliadin fractions (F1–4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5–8), 59 in the α- and β-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9–31) and 5 peaks in the γ-gliadin fractions (F32–35). Peptide maps of tryptic digests of HPLC fractions were obtained from a tandem quadrupole time-of-flight mass spectrometer (MALDI QqTOF MS) and were submitted to the ProFound search engine. HMW glutenin subunits including Ax2*, Dx5, Bx7, and Dy10 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type I, were identified with reasonable sequence coverage from HPLC fraction 5, 7, 17, and 18. The identities of the peptides attributed to selected gluten proteins were confirmed using MS/MS with BioMultiView to match the predicted and measured partial amino acid sequences. Because of incomplete wheat DNA databases, many wheat gluten proteins could not be identified. These results suggest that the combination of RP-HPLC with MS and MS/MS techniques is a promising approach for the characterization of wheat gluten proteins.

Investigation of a small-scale asymmetric centrifugal mixer for the evaluation of Asian noodles

ABSTRACT A high throughput centrifugal mixer capable of using smaller amounts of flour (50 g) was evaluated for the production of oriental alkaline noodles. The unit requires a small footprint on a laboratory bench and offers variable speed mixing (300–3,500 rpm) for 5–60 sec. Three different mixing bowls, plain, pin, and paddle, were evaluated for the small-scale production of alkaline noodles using straight-grade flour derived from Canada Western Red Spring (CWRS) and Canada Prairie White Spring (CPSW) wheat. Under optimized mixing conditions (3,000 rpm for 30 sec), the pin and paddle bowls produced noodle dough with crumb size distribution and adhesion characteristics consistent with commercial requirements. The plain bowl produced dough with larger undesirable dough chunks and showed excessive heat buildup. Noodle sheets produced from this dough were not comparable in color characteristics to conventionally produced noodle sheets. Noodles prepared using the paddle mixer also displayed some significant...

Separation and Characterization of Barley Starch Polymers by a Flow Field-Flow Fractionation Technique in Combination with Multiangle Light Scattering and Differential Refractive Index Detection

ABSTRACT Flow field-flow fractionation (flow FFF) with frit inlet and frit outlet mode (FIFO) was coupled online to multiangle light scattering (MALS) and refractive index (RI) detectors to investigate the molecular characteristics of normal and zero amylose barley starch polymers. Application of two different cross-flows, 0.35 mL/min followed by 0.1 mL/min, and constant channel and frit flows of 0.1 and 1.0 mL/min, respectively, permitted a complete separation of amylose and amylopectin. The improved signals from the detectors due to application of the FIFO mode enabled the proper characterization of the small molecular weight species, as well as significantly enhanced the reproducibility of the measurements. The weight-average molecular weight (Mw) and zaverage root-mean-square (RMS) radii of gyration (Rg) values for amylose and amylopectin in the normal starch samples were 2.3 × 106 and 280 × 106, and 107 and 260 nm, respectively. The Mw and Rg of amylopectin in the zero amylose starch samples were 360...

Relationship of Kernel Size to Flour Water Absorption for Canada Western Red Spring Wheat

ABSTRACT Canada Western Red Spring (CWRS) wheat exhibits consistent positive relationships between kernel weight and farinograph and baking water absorption. These relationships are sufficiently robust to be statistically significant (P < 0.05) for historical Canadian Grain Commission harvest survey data generated one year apart for 17 years, and for historical data on individual cultivars in advanced Canadian wheat breeding trials, also generated annually. Verification of the relationships were obtained by analyzing different kernel size fractions obtained by sieving CWRS harvest survey samples and pure CWRS cultivars from various origins. In all cases, highly significant positive relationships were observed between kernel size and water absorption. The relationships were evident for individual streams from pilot-scale millings of sized fractions from CWRS harvested in two different years. Strong correlations of kernel weight to farinograph and baking absorption also were shown for sized fractions from c...

Specificity of two isolated wheat carboxypeptidases

Abstract The substrate specificity of two purified carboxypeptidases from germinated wheat has been examined. Both enzymes were active on a wide variety of carbobenzoxy substituted peptides but inactive with unsubstituted dipeptides. Neither enzyme was active upon endoprotease or amidase substrates and only low levels of esterase activity were evident. In time course studies, both enzymes gave rapid non-specific sequential release of amino acids, including proline, from the carboxyterminal of proteins and polypeptides of known amino acid sequence.

Characterization of Polymeric Wheat Proteins by Flow Field‐Flow Fractionation/MALLS

Abstract Flow field‐flow fractionation (FFF) combined with multiangle laser light scattering (MALLS) was used to investigate the size properties of monomeric and polymeric wheat flour protein fractions from common and durum wheat flours obtained by sequential extraction with dilute acetic acid with and without sonication. The major FFF peak obtained for the monomeric protein fraction showed M w values in the 31,000–33,000 range plus some larger monomeric and smaller polymeric protein eluting later in the fractogram. The major peak in the polymeric protein fraction showed M w values in the 300,000 range. At later elution times, M w values for the polymeric proteins approached 10,000,000. Radius of gyration values showed relatively small changes for the polymeric protein fraction with increasing elution time, suggesting that the larger M w proteins tend towards a more compact structure than smaller M w polymeric proteins.

Simplified Dilute Acetic Acid Based Extraction Procedure for Fractionation and Analysis of Wheat Flour Protein by Size Exclusion HPLC and Flow Field-Flow Fractionation

ABSTRACT A simple, highly efficient and reproducible two-step extraction procedure using dilute acetic acid without (AN) and then with sonication (AS) has been developed for the fractionation of wheat flour protein. Approximately 97% of total protein was extracted from a Canadian hard red spring wheat flour; an additional 1.2% protein could be recovered by further extraction with 1% DDT and 50% 1-propanol (AR). Size-exclusion HPLC (SE-HPLC) and flow field-flow fractionation (flow FFF) showed that the AN extract, which accounted for most of the total extractable protein (AN + AS + AR), consisted primarily of monomeric protein. The AS extract was composed primarily of polymeric proteins. Flow FFF showed that AN polymeric protein, including that eluting at the SE-HPLC void volume, showed smaller Stokes diameters than AS polymeric protein. Flow FFF profiles of AS SE-HPLC subfractions showed that the void volume subfraction contained monomeric and small polymeric protein in addition to large polymeric protein,...