Purification and Characterisation of Ascorbate Oxidase from Flour and Immature Wheat Kernels

White flour from wheat was shown to contain basic-ascorbate oxidase (AOX) enzymes (pI 7·6–9·6) and acidic-AOX enzymes (pI 5·1–6·6) in a ratio of 0·4:1, based on chromatography data. Immature wheat kernels (two weeks post-anthesis) contained about 12 times more AOX activity (units/g dry weight) than flour from mature grain, and the ratio of basic- to acidic-AOX was 5:1. Acidic-AOX was purified 90-fold from flour by hydrophobic interaction, gel filtration and anion exchange chromatography. Basic-AOX was purified 20 000-fold from immature wheat by hydrophobic interaction, anion exchange, cation exchange and gel filtration chromatography in a yield of 5%. The acid-AOX had a M of 140 k, was optimally active at pH 6·3 and 40 °C, and was stable in the pH range 5–9 and at 30 °C for 0·5 h at pH 6·2. The Km values were 0·26 m for L-ascorbic acid and 0·93 m for D-iso ascorbic acid. The basic-AOX had a M of 139 k and subunit M of 72 k. The enzyme was optimally active at pH 6·2 and 50 °C, and was stable in the pH range 5–9 and at 40 °C for 0·5 h at pH 6·2. The Km values were 0·30 m for L-ascorbic acid and 0·53 m for D-iso ascorbic acid. The absorption spectrum of basic-AOX had absorption maxima at 280 nm and 607 nm of similar magnitude to those measured in AOX fromCucurbita species (squash). This indicates that wheat AOX contains protein-bound copper similar to other plant AOX.     

The Role of Dextrins in the Stickiness of Bread Crumb made from Pre-Harvest Sprouted Wheat or Flour Containing ExogenousAlpha-Amylase

Dextrins were extracted in water from bread made from pre-harvest sprouted wheat or standard flour supplemented with exogenousalpha-amylases. The dextrins were separated by gel permeation chromatography and the dextrin content (% of crumb weight) determined for different degree of polymerisation (DP) size classes; DP 1–2, DP 3–10, DP 11–50, DP 51–200 and DP >200. There were significant correlations between the dextrin content in each size class and crumb stickiness (r=0·84–0·91, 22 df ). The most significant correlation (r=0·96) was between total dextrin content and crumb stickiness. Addition of dextrins of various DP ranges from various sources to standard flour produced bread with sticky crumb. Again, the degree of stickiness was generally related to the amount of total dextrin in the crumb and not to size distribution of dextrins. In this instance, extensive enzymic hydrolysis of starch was not necessary to produce sticky crumb; the dextrins caused crumb stickiness directly. Addition of dextrins to reconstituted gluten–starch flour produced bread with unexpectedly low dextrin levels and correspondingly low stickiness scores. It is concluded that, to produce sticky crumb, high levels of dextrin of any size are necessary in the crumb; a sticky mass is produced when dextrins dissolve in the excess «free» water that is normally «bound» to starch, gluten and other insoluble components of bread crumb.     

Effects of Flour Particle Size on the Textural Properties of Flour Tortillas

Three wheat flours, hard red winter (HRW), hard white winter (HWW) and soft red winter (SRW), were fractionated by sieving into four different particle size fractions, <38, 38–53, 53–75, and >75 μm. The medium fractions, 38–53 and 53–75 μm, of the HRW and HWW wheat had higher protein contents than the finest and coarsest fractions. The finest fractions had the highest levels of damaged starch. Tortillas made from the medium fractions of HRW and HWW, especially the 53–75 μm fraction, had longer rupture distance and better foldability. The finest fraction yielded tortillas with shorter rupture distance and worse foldability. Fractionation by flour particle size slightly affected protein composition of the flour. Size exclusion-high performance liquid chromatography (SE-HPLC) analyses showed that the compositions of SRW wheat proteins were significantly different from those of HRW and HWW wheat proteins. SRW had more low molecular weight (LMW) and less high molecular weight (HMW) protein than HRW and HWW had. The <38 μm fractions of HRW and HWW had more LMW proteins than other fractions. The amount of LMW proteins of flours correlated negatively with the rupture distance and foldability of tortillas. Particle size of the flours was the major factor affecting the tortilla texture.     

Distribution of Water between Wheat Flour Components: A Dynamic Water Vapour Adsorption Study

The water adsorption properties of hard and soft wheat flours and flour components (starch, damaged starch, gluten, soluble pentosans, and insoluble pentosans) were determined at 25 °C using a controlled atmosphere microbalance. At different levels of relative humidity (from 10% to 95%), changes in sample mass (i.e., water gain) were continuously measured versus time and described using exponential models (R²≥0·994). Water adsorption isotherms were constructed for wheat flours and flour components and described using Guggenheim-Anderson-de Boer models (R²≥0·997). It was not possible to distinguish between the selected hard and soft wheat flours by their isotherms. The water-soluble pentosans had the highest water adsorption capacity. The theoretical distribution of water between the flour components (calculated using the Guggenheim-Anderson-de Boer parameters) was starch, 88%; gluten, 10%; and pentosans, 2%.     

Comparative Studies of HighMrSubunits of Rye and Wheat. I. Isolation and Biochemical Characterisation and Effects on Gluten Extensibility

The structural features of highM_rglutenin subunits of wheat were compared with those of analogous proteins from rye. Subunits of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction with 50% (v/v) aqueous propan-1-ol under reducing conditions at 60°C followed by precipitation using a 60% concentration of propan-1-ol. The yields of dialysed and freeze-dried subunits were 0·33% and 0·32% (w/w of flour), respectively (rye cultivars), and 0·91% (Rektor). SDS–PAGE revealed that the rye cultivars contained at least five subunits with mobilities corresponding to the x-type subunits of wheat. Separation by RP–HPLC indicated that the rye cultivars did not differ in the qualitative composition of subunits, but in their quantitative proportions. The surface hydrophobicities of the rye subunits were significantly lower than those of wheat subunits. The amino acid compositions of single rye subunits were characterised by high contents of Glx, Gly and Pro, and they were closely related to those of wheat subunits, except that the Glx content was generally lower and the Cys content higher. Notable differences between rye and wheat subunits were found in their contributions to gluten strength. Whereas wheat subunits, reoxidised with potassium bromate and mixed with a standard wheat flour, caused a significant increase in gluten strength, reoxidised rye subunits had the opposite effect.     

Structural Characterisation of Water-extractable and Water-unextractable Arabinoxylans in Wheat Bran

Water-extractable arabinoxylan (WE-AX) in wheat bran, of which only a minority originates from adherent endosperm, amounted to 6% of the total arabinoxylan (AX) content in such bran. WE-AX had an arabinose to xylose (A/X)-ratio of 0·45. Graded ethanol precipitation (20–80% range) yielded AX with an A/X-ratio increasing from 0·31 to 0·85. A population of molecular weight (MW) of 50 kDa precipitated between 0 and 40% ethanol and one with much lower apparent MW precipitated at higher ethanol concentrations. Wheat bran unextractable cell wall material (UCM) was obtained as the residue withstanding thermostable α-amylase and protease treatments and consisted mainly of AX (ca 45%) and cellulose (30–35%). Two consecutive extractions of UCM with alkaline hydrogen peroxide (AHP; 2·0%, pH 11·5, 4 h, 60 °C) resulted in a cellulose rich residue (CRR) containing 33% of the AX originally present in UCM. The average A/X-ratio of AX in CRR was lower than that in UCM. The extracted AX polymers (ca 45% of the AX originally present in UCM) had a high A/X-ratio (0·82). Their elution profiles showed two polydisperse peaks with apparent MW of respectively 100–120 kDa and 5–10 kDa. Graded ethanol precipitation resulted in a lowly substituted fraction (A/X=0·40; 0–40% ethanol) of high MW and a fraction of highly substituted AX (A/X>0·95; 40–70% ethanol) containing both low molecular weight (LMW-) as well as high molecular weight (HMW-) AX. At an ethanol concentration of 70% or more, only LMW-AX precipitated.     

Reliability of InGaAs focal plane array imaging of wheat germination at early stages

To assist our Kansas breeding program, we have developed nondestructive methods to test new lines of wheat for resistance to premature germination. The high sensitivity of subsurface imaging, compared with visual detection, α-amylase determination, or viscosity testing, permits germination detection at early stages. This report is concerned with detection reliability via chemical imaging of intact wheat kernels at early stages of germination by using an InGaAs focal plane detector array in the 1100–1700 nm range. Ninety kernels from each of six different cultivars, including HRW and HWW wheat, were exposed to moist conditions for 6, 12, 24, 36, and 48 h. Images of each 90 kernel group were examined, and images of those kernels exposed to moisture for 36 h were compared with images of kernels treated for 3 h as a control. Images of each were classified as sprouted or unsprouted. Criteria for classification included images of log 1/R at select wavelengths or images of select factors resulting from principle component analysis (PCA) treatment of reflectance intensity data from each pixel. Sprouted kernels determined by PCA factors 1 and 4 from 90 kernels tested in a 36-h moisture exposure numbered 87, 85, 80, 74, 70, 48 for six cultivars tested. Cultivar KS-2174 was shown to be distinctly more resistant to germination than the other cultivars. When KS-2174 was compared with Betty wheat, for all exposure times, Betty had approximately 45% more germinated kernels.     

Water Sorption and Dielectric Relaxations of Wheat Dough (Containing Sucrose, NaCl, and their Mixtures)

Dielectric relaxations of wheat doughs with different water contents and effects of sucrose, NaCl, and their mixture on relaxation temperatures were investigated using dielectric analysis (DEA). All ingredients were dissolved in distilled water used to prepare wheat flour doughs to optimum consistency. Before analysis, samples were stored at room temperature in vacuum desiccators over a_w range of 0·225–0·753. Dynamic DEA measurements were made at a heating rate of 2 °C/min from 40 °C below and above the observed relaxation zone. The frequencies used were 0·1, 0·5, 1, and 5 Hz. Steady state water contents varied from 3·21 to 10·89 g H₂O/100 g dm over a_w range used for the plain dough (flour+water). Added ingredients increased sorption of doughs. The tan δ of DEA showed an α-relaxation (glass transition) in all doughs at all frequencies used. The relaxation peak temperature, taken as T_g, increased with increasing frequency. Added sucrose decreased the T_g of doughs, as well as added NaCl. A dramatic depressing effect of NaCl on T_g was probably due to an increase in conductivity of doughs.     

Thermal, Dynamic-mechanical, and Dielectric Analysis of Phase and State Transitions ofFrozen Wheat Doughs

The physical state of wheat dough at sub-zero temperatures was investigated using Differential Scanning Calorimetry (DSC), Dynamic-mechanical Analysis (DMA) and Dielectric Analysis (DEA). Also, the water sorption properties of freeze-dried dough were investigated. DMA and DEA measurements were made at a heating rate of 1 °C/min from −150 to 10 °C. Before the measurements, samples were equilibrated at −25 °C for 15–20 min to allow maximum ice formation, and then cooled at 1 °C/min to −150 °C. The frequencies used were 0·1, 0·5, 1, 2·5, 5, 10, and 20 Hz for DMA and 0·1, 0·5, 1, 5, 10, 20, 50, 100 and 1000 Hz for DEA. The DSC thermograms were obtained for annealed samples by scanning from −80 to 20 °C at 5 °C/min, and they showed only ice melting starting at −18 °C. The tan δ of DMA and DEA showed an α-relaxation (glass transition), two low temperature relaxations (β and γ) and melting of ice. At the higher frequencies (>0·5 Hz), the α-relaxation coincided with melting of ice, and all relaxation temperatures (α, β and γ) increased with increasing frequencies as measured by DEA.     

Characteristics of enzymatic hydrolysis of thermal-treated wheat gluten

Effect of thermal treatment at 50–90 °C on wheat gluten hydrolysis by papain was evaluated in this study. Thermal treatment decreased the amount of sodium dodecyl sulfate (SDS) extractable protein. The treatments at 80 and 90 °C had a strong impact on protein extractability. Thermal treatment for 30 min resulted in a significant reduction in SDS extractable glutenin level in wheat gluten. A significant drop in free sulphydryl level was found in wheat gluten treated at 70 °C for 30 min. It indicated that cross-linking of glutenin through S–S occurred during thermal treatment. The treatments at 70–90 °C led to significant decreases in soluble and nitrogen level, while significant increases in peptide nitrogen amount in the hydrolysates from treated gluten were found. A time-dependent effect was observed for the changes in soluble forms of nitrogen and PN. Thermal treatment resulted in molecular mass distribution change according to gel permeation chromatography analysis. Thermal treatment significantly increased the amount of fractions with molecular mass beyond 10 K (67.2%) in the hydrolysates and greatly decreased the amounts of fractions with MM of 10–5 K and below 5 K in hydrolysates.     

Development of a calibration to predict maize seed composition using single kernel near infrared spectroscopy

The relative composition of protein, oil, and starch in the maize kernel has a large genetic component. Predictions of kernel composition based on single-kernel near infrared spectroscopy would enable rapid selection of individual seed with desired traits. To determine if single-kernel near infrared spectroscopy can be used to accurately predict internal kernel composition, near infrared reflectance (NIR) and near infrared transmittance (NIT) spectra were collected from 2160 maize kernels of different genotypes grown in several environments. A validation set of an additional 480 kernels was analyzed in parallel. Constituents were determined analytically by pooling kernels of the same genotype grown in the same environment. The NIT spectra had high levels of noise and were not suitable for predicting kernel composition. Partial least squares regression was used to develop predictive models from the NIR spectra for the composition results. Calibrations developed from the absolute amount of each constituent on a per kernel basis gave good predictive power, while models based on the percent composition of constituents in the meal gave poor predictions. These data suggest that single kernel NIR spectra are reporting an absolute amount of each component in the kernel.     

Mapping quantitative trait loci (QTLs) associated with dough quality in a soft × hard bread wheat progeny

Bread wheat (Triticum aestivum L.) quality is a key trait for baking industry exigencies and broad consumer preferences. The main goal of this study was to undertake quantitative trait loci (QTL) analyses for bread wheat quality in a set of 79 recombinant inbred lines (RILs) derived from a soft × hard bread wheat cross. Field trials were conducted over two years, utilizing a randomized complete block design. Dough quality was evaluated by sedimentation test, mixograph and alveograph analysis. Protein content was measured by near-infrared reflectance analysis and grain hardness was determined by the single kernel characterization system (SKCS).     

Interspecific and Intraspecific Variation in Grain and Groat Characteristics of Wild Oat (Avena) Species: Very High Groat (1→3),(1→4)-β- -glucan in an Avena atlantica Genotype

Grain characteristics and groat composition have been evaluated in 35 genotypes from nine taxonomic species of Avena, including three species (A. agadiriana, A. atlantica, A. damascena) for which no previous data are available. There was substantial interspecific and intraspecific variation for all characteristics measured. The proportion of groat in the grain ranged from 32·7–62·1%, and mean groat weight from 2·4–37·4 mg. Groat protein concentrations ranged from 13·9–41·3%, and exceeded 32% in one A. atlantica, two A. damascena and one A. murphyi genotype. Groat β-glucan concentration showed very wide variation (2·2–11·3%) are there were substantial interspecific and intraspecific differences. The highest β-glucan concentrations were found in genotypes of A. atlantica. Although there were interspecific and intraspecific variations in groat oil concentration (4·2–10·6%), and in fatty acid composition, data were within previously reported ranges for A. sativa. Overall these data indicate that some of the genotypes of the wild species studied may be of value for breeding oats with improved levels of β-glucan and protein, and that further studies are warranted into both interspecific and intraspecific variations in grain quality factors in wild oat species.     

Fatty Acid Composition of Three Rice Varieties Following Storage

The petroleum ether extractable lipids (PEE-L) and aqueous propan-1-ol extractable lipids (PWE-L) of three varieties of rice were determined gravimetrically and characterised by fatty acid profiles. The content of PEE-L (22·5–28·2 mg g⁻¹) was higher than that of PWE-L (7·4–11·5 mg g⁻¹) in brown rice with the situation reversed in milled rice (3·0–4·5 mg g⁻¹vs. 7·2–8·7 mg g⁻¹). The ratio of unsaturated fatty acid to saturated fatty acid was about two times higher in PEE-L than that in PWE-L for both brown and milled rice reflecting the selective complexation of saturated fatty acids. Rice storage at 37 °C resulted in some minor but statistically significant changes in the fatty acid profile. In the case of brown rice, the only notable changes were a reduction in the amounts of oleic and linoleic acids in the aqueous propan-1-ol extractable fatty acid fraction (PWE-FA) following storage at the higher temperature. Milled rice of all three varieties showed a decrease in linoleic acid content of PEE-L following storage at 37 °C for 4 and 7 months compared to storage at 4 °C. There was no change in fatty acid contents of PWE-L of milled rice when stored at 4 and 37 °C for 4 and 7 months. This implies that the PWE-L (or bound lipids) were more stable than PEE-L (or free lipids) during storage.     

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.     

Effects of drought and the presence of the 1BL/1RS translocation on grain vitreosity, hardness and protein content in winter wheat

Grain texture is an important component of end-use quality in wheat. The effects of water availability on the components of texture; vitreosity, determined using a Light Transflectance meter (LTm), grain hardness measured using the single-kernel characterisation system (SKCS), and protein content, were studied in field experiments of winter wheat in the UK in 2001/2002 and 2002/2003. Experiments were grown on a drought prone soil and employed a mapping population of 46 doubled haploid (DH) lines and their parents, Beaver (+1BL/1RS, soft wheat) and Soissons (1B, hard wheat). The results showed that drought increased hardness in both seasons, but the effect was never sufficient to move a line from the soft class into the hard class. Puroindoline (PIN)-a:b peak height ratio explained ca. 78% of the variation in hardness, and drought also appeared to increase the amounts of PINs in the grain. Minor quantitative trait loci (QTLs) were found for hardness on chromosomes 2A, 2D, 3A and 6D, also associated with QTLs for PINs. Vitreosity also increased in response to drought in both seasons. Variation in vitreosity explained 7–11% of the overall variation in texture within a hardness class, with hardness increasing on average by 2.2 SKCS units for each 10% increase in the proportion of vitreous grains. The relationship between vitreosity and protein content was poor, despite the fact that protein content also increased in response to drought. Minor QTLs associated with both protein content and vitreosity were found on chromosomes 1B, 4D and 5D. A minor QTL for vitreosity was also found on chromosome 2D. However, there appeared to be no direct relationship between alleles at the Ha locus, the gene which controls the difference between hard and soft wheats, and vitreosity. A positive relationship between the presence of the 1BL/1RS translocation and the proportion of vitreous grains was identified, suggesting that vitreosity was strongly linked to changes in protein quality.     

Phytase activity, phytate, iron, and zinc contents in wheat pearling fractions and their variation across production locations

Pearling is an effective method for evaluating the distribution of chemical components in wheat grain. Twelve pearling fractions (P₁–P₁₂) of wheat grain were obtained using two rice polishers for 10 cultivars (six soft red wheats and four hard white wheats) grown at two locations with different environmental conditions in Jiangsu Province, China. The results show that the effects of cultivar, location, and pearling on wheat flour phytase activity, phytate, iron, and zinc contents were all significant, with pearling having the greatest effect. All the four components showed a diminishing trend as pearling progressed from the outer layers to the inner part of wheat grain. Generally, the P₂ fraction (the outer 4–8% layer of wheat grain) had the highest phytase activity and phytate and iron contents, whereas the P₁ fraction (the outer 0–4% layer) ranked the highest for zinc content. Growing location had a large influence on grain phytase, phytate, and iron, but the differences between locations decreased as pearling level increased.     

Dielectric Relaxations of Frozen Wheat Doughs Containing Sucrose, NaCl, Ascorbic Acid and Their Mixtures

The effects of sucrose, NaCl, and ascorbic acid on dielectric relaxations of frozen wheat doughs were investigated using dielectric analysis (DEA). All ingredients were dissolved in distilled water used to prepare wheat flour doughs to optimum consistency using a 10 g bowl Micro-Mixer. DEA measurements were made at a heating rate of 1 °C/min from −150 to 10 °C. Before the measurements, samples were equilibrated at −30 °C for 15 min to allow maximum ice formation, and then cooled at 1 °C/min to −150 °C. The frequencies used were 0·1, 0·5, 1, 5, 10, 20, 50, 100 and 1000 Hz. The dissipation factor (tan δ) of DEA showed an α-relaxation (glass transition), two low temperature relaxations (β and γ) and ice dissolution. Added NaCl had a markedly depressed the glass transition temperature (T_g′) and onset of melting of ice temperature (T_m′), probably because of the higher conductivity of the frozen material, and the decreased transition temperatures of the unfrozen solute phase. At the higher frequencies, the α-relaxation coincided with melting of ice, and all relaxation temperatures (α, β and γ) increased with increasing frequencies.     

Hydrothermal Processing of Barley (cv. Blenheim): Optimisation of Phytate Degradation and Increase of FreeMyo-inositol

Optimal conditions were developed for hydrothermal processing of whole barley kernels (cv. Blenheim) to degrade phytate (myo-inositol hexaphosphate) and to increase the content of freemyo-inositol. The hydrothermal treatment comprised of two wet steeps, where lactic acid solution of different concentrations was used, and two dry steeps followed by successive drying. Experiments were performed as a central composite design and evaluated by multiple linear regression. The variables in the experiments were temperature in the first wet and dry steep (T₁), temperature in the second wet and dry steep (T₂) and lactic acid solution concentration in both wet steeps (C) and mathematical models were developed in these variables. Optimal conditions for maximal phytate degradation and for maximal increase of freemyo-inositol wereT₁=48 °C,T₂=48–50 °C andC=0·8%, at these conditions the amount of phytate was reduced by 95–96% and the freemyo-inositol concentration was increased from 0·56 to 2·45 μmol/g d.m. We conclude that this hydrothermal process can be used to produce a barley product (cv. Blenheim) with a low phytate content and a high level of freemyo-inositol.     

Field emission scanning electron and atomic force microscopy,and Raman and X-ray photoelectron spectroscopy characterization of near-isogenic soft and hard wheat kernels and corresponding flours

Secondary field emission scanning electron microscopy (FE SEM), atomic force microscopy (AFM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to investigate native near-isogenic soft and hard wheat kernels and their roller milled flours. FE SEM images of flat-polished interior endosperm indicated distinct differences between soft and hard wheats with less internal continuity in the soft wheat, whereas individual starch granules were much less evident in the hard kernel due to a more continuous matrix. AFM images revealed two different microstructures. The interior of the hard kernel had a granular texture with distinct individual spheroid features of 10–50 nm while the images obtained for the soft kernel revealed less distinct small grains and more larger features, possibly micro-structural features of starch granules. Raman spectra resolved identical distinct frequencies for both kernel types with slightly different intensities between types. Finally, the chemical surface compositions of flour for these two types of kernels obtained by XPS provided subtle insight into the differences between soft and hard wheat kernels. These combined advanced microscopic and spectroscopic analyses provide additional insight into the differences between the soft and hard wheat kernels.