Challenges and opportunities for proteomics and the improvement of bread wheat quality.

Wheat remains a critical global food source, pressured by climate change and the need to maximize yield, improve processing and nutritional quality and ensure safety. An enormous amount of research has been conducted to understand gluten protein composition and structure in relation to end-use quality, yet progress has become stagnant. This is mainly due to the need and inability to biochemically characterize the intact functional glutenin polymer in order to correlate to quality, necessitating reduction to monomeric subunits and a loss of contextual information. While some individual gluten proteins might have a positive or negative influence on gluten quality, it is the sum total of these proteins, their relative and absolute expression, their sub-cellular trafficking, the amount and size of glutenin polymers, and ratios between gluten protein classes that define viscoelasticity of gluten. The sub-cellular trafficking of gluten proteins during seed maturation is still not completely clear and there is evidence of dual pathways and therefore different destinations for proteins, either constitutively or temporally. The trafficking of proteins is also unclear in endosperm cells as they undergo programmed cell death; Golgi disappear around 12 DPA but protein filling continues at least to 25 DPA. Modulation of the timing of cellular events will invariably affect protein deposition and therefore gluten strength and function. Existing and emerging proteomics technologies such as proteoform profiling and top-down proteomics offer new tools to study gluten protein composition as a whole system and identify compositional patterns that can modify gluten structure with improved functionality.

Wheat Fusarium Protease Specificity and Effect on Dough Properties.

Fusarium infection is a worldwide agricultural problem of billion dollar proportions globally, and it has increasingly threatened entire regional food supplies. In addition to the toxin deoxynivalenol (DON), Fusarium species express digestive enzymes that degrade starch and protein, affecting the quality of infected grains, especially wheat processing performance which depends largely on gluten proteins. In this study, the impact of Fusarium protease on the functionality of Canada Western Red Spring (CWRS) wheat was assessed by adding Fusarium-damaged kernels (FDK) to a FDK-free base wheat sample. Digestion of beta-casein by extracts of flours, milled from sound and FDK-spiked wheat samples, demonstrated elevated cleavage in FDK-spiked flour extracts as follows: N-terminal to lysine (eight-fold), N- and C-terminal to isoleucine (four-fold and three-fold, respectively), N-terminal to tyrosine (three-fold) and C-terminal to arginine at P1' (five-fold). Comparison of abbreviated (45 min) and standard (135 min) extensigraph test results indicated that desirable increases in dough resistance to extension (Rmax) due to gluten re-polymerization after longer resting were partially to completely counteracted in FDK-spiked flours in a dose-dependent manner. Baking tests confirmed that while loaf volume is similar, proofed dough from FDK-spiked samples caused detectable loaf collapse at 3% FDK. Extensigraph Rmax and Fusarium protease levels were inversely related, and effected by both the extent and severity of infection. While the current FDK tolerances for grading Canadian wheat can effectively control protease damage, prevalence of deoxynivalenol (DON) weak- and non-producing Fusarium strains/species (e.g., F. avenaceum) in some growing regions must be considered to protect functionality if grading is solely based on DON content.

Development of optimized extraction methodology for cyanogenic glycosides from flaxseed (Linum usitatissimum).

A reference method (higher accuracy) and a routine method (higher throughput) were developed for the extraction of cyanogenic glycosides from flaxseed. Conditions of (essentially) complete extraction were identified by comparing grinding methods and extraction solvent composition, and optimizing solvent-to-meal ratio, extraction time, and repeat extraction. The reference extraction method consists of sample grinding using a high-speed impact plus sieving mill at 18 000 rpm with a 1.0 mm sieve coupled with triple-pooled extraction in a sonicating water bath (40 degrees C, 30 min) using 75% methanol. The routine method differs by the use of a coffee mill to grind samples and a single extraction. The 70 and 80% methanol solutions were equal and superior to other combinations from 50 to 100% aqueous ethanol or methanol. The extraction efficiencies of the routine method (relative to the reference method) was 87.9 +/- 2.0% SD (linustatin) and 87.6 +/- 1.9% SD (neolinustatin) using four composite samples that were generated from seeds of multiple cultivars over two crop years and locations across Western Canada. Ground flaxseed was stable after storage at room temperature, refrigeration, or freezing for up to 7 days, and frozen for at least 2 weeks but less than 2 months. Extracts were stable for up to 1 week at room temperature and at least 2 weeks when refrigerated or frozen.

Development of extraction and gas chromatography analytical methodology for cyanogenic glycosides in flaxseed (Linum usitatissimum).

The development of well-characterized rapid methodology for the extraction and gas chromatographic analysis of the cyanogenic glycosides linustatin and neolinustatin from flaxseed (Linum usitatissimum L.) is reported. Two quantitation methods using phenyl-beta-D-glucopyranoside as an internal standard are described: direct quantitation using linustatin and neolinustatin external standard curves [standard curve slope variabilities of 2.6 and 5.7% relative standard deviation (RSD), respectively, over 7 days] or by use of methyl-alpha-D-glucopyranoside as a surrogate external standard, with conversion factors to convert to linustatin and neolinustatin concentration [1.109 +/- 0.015 (SD) mg linustatin/mg methyl-alpha-D-glucopyranoside and 1.180 +/-0.067 (SD) mg neolinustatin/mg methyl-alpha-D-glucopyranoside]. The former method is direct, thereby contributing less uncertainty to the method, and the latter adds a small degree of uncertainty coupled with considerable cost savings. Limits of detection for all standards were in the low- to sub-nanogram level and were 10-100 times lower than the lower limit of quantitation (LOQ). Repeatability precision was performed on 2 separate days at the lower and upper LOQs, with the RSD in peak response being 1% or lower in all cases. Extraction methods were evaluated for their ability to extract linustatin and neolinustatin from flaxseed using several combinations of aqueous ethanol, and recoveries were determined against the highest yielding method. Recoveries were as low as 82%, indicating that optimized extraction methodology is critical for the accuracy of results.