Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph experiments through dough partial reduction and reoxidation to allow these exogenously added HMW-GS to incorporate into gluten polymers. Detailed results are given for seven mixing and two extension parameters. HMW-GS synthesized in bacteria behaved similarly under these conditions to the same HMW-GS extracted from wheat flour. These experiments initially focused on the HMW-GS of the D-genome of hexaploid wheat encoded at the Glu-D1 locus; e.g. the Dx2, Dx5, Dy10, and Dy12 subunits. Experiments used five different flours and results are shown to be consistent when normalized to results from Dx5. The incorporation of Dx-type subunits into the gluten disulfide bonded network has greater effects on dough parameters than incorporation of Dy-type subunits. When Glu-D1 x- and y-type subunits are incorporated together, there are synergistic effects greater than those with either subunit type alone. This synergistic effect was greatest with approximately equal amounts of Dx- and Dy-type subunits - implying a 1:1 stoichiometric relationship.     

Influence of baking and in vitro digestion on steryl ferulates from wheat

So far, data on absorption and metabolism of steryl ferulates from edible sources is scarce. Therefore, the impact of enzyme-aided baking and in vitro digestion was examined in this study. Wheat flours and wheat breads were subjected to a static in vitro digestion model and changes in the contents of steryl ferulates and free sterols (possible hydrolysis products of steryl ferulates) were monitored. Baking degraded steryl ferulates at a similar rate in all types of breads (43–47%) compared to the corresponding flours, while baking induced changes in free sterols showed no clear pattern. In vitro digestion provoked five folds lower content of steryl ferulates in flours than in breads and it also resulted in significant free sterol accumulation. Interestingly, bioaccessibility (0.01–0.25%) was not influenced by the cereal matrix. The four steryl ferulate species, which were detected in wheat, showed similar hydrolysis rates during digestion. As baking had a significant impact on the steryl ferulate content of wheat, we suggest that both raw and processed sources should be considered further in vitro, animal or human trials, when studying the metabolic fate of steryl ferulates.     

Effect of wheat bran addition on in vitro starch digestibility,physico-mechanical and sensory properties of biscuits

Biscuits contain high amount of fat and sugar thus having high calorie but low nutrient density. Wheat bran is a good source of dietary fibre (DF) and protein and is thus a good candidate for nutritional enrichment of cereal foods. The aim of this study was to understand the effect of bran incorporation and particle size reduction on biscuit microstructure, texture and in vitro starch digestibility. Five different biscuits containing 5–15% DF were produced. Two different particle sized wheat brans were used: coarse (450 μm) and fine (68 μm). Bran particle size reduction increased the elastic modulus and hardness of biscuits. Biscuits containing fine bran had visually more compact structure without any surface or internal defects than those with coarse bran. Fine bran containing sample had the highest hardness value. Sensory evaluation showed that roughness and breakdown of biscuits in the mouth was significant for the coarse bran with highest level of bran addition. The instrumental elastic modulus, stress and hardness were closely related to sensory hardness and strength to break. Increasing DF content from 5 to 15% increased hydrolysis index by 16%, from 32 to 37.     

New insights into high-molecular-weight glutenin subunits and sub-genomes of the perennial crop Thinopyrum intermedium (Triticeae)

Intermediate wheatgrass (Thinopyrum intermedium) is a perennial crop that possesses desirable agronomic traits and provides environmental services, e.g., reducing soil erosion, nitrate leaching and inputs of energy and pesticide. Thus, intermediate wheatgrass is currently being domesticated as a perennial grain crop. However, the genetic information for molecular breeding is quite limited. Here we report a molecular analysis of high-molecular-weight glutenin subunits (HMW-GS) in intermediate wheatgrass using gene cloning and protein biochemistry. Five HMW-GS genes were isolated from individual intermediate wheatgrass plants: two x-type genes TiHGS1 and TiHGS4, and three y-type genes TiHGS2, TiHGS3 and TiHGS5. Among them, TiHGS5 was novel and possessed an additional cysteine residue at the N-terminal domain or repetitive domain. Sequence alignments showed that TiHGS1 and TiHGS2 genes shared high identities (>96%) with the Glu-1Dx and Glu-1Dy genes, respectively, in common wheat and Aegilops species, TiHGS3 with HMW-GS genes from Dasypyrum or Pseudoroegneria, and TiHGS4 and TiHGS5 with HMW-GS genes from Thinopyrum elongatum. This work provides substantial new insights into the gene compositions and protein profiles of HMW-GS in intermediate wheatgrass, and also gives evidence about the genome components of intermediate wheatgrass.     

In vitro dephytinization and bioavailability of essential minerals in several wheat varieties

Twelve wheat varieties were studied for their phytic acid, calcium, iron, zinc, copper and phosphorus contents, which respectively ranged between 114–166, 25.1–53.5, 3.41–5.55, 0.71–3.00, 0.65–1.32 and 298–314 mg/100 g. The molar ratios as Ca:Phy, Phy:Fe, Phy:Zn, Phy:Cu were found to be 0.14–0.29, 1.96–3.86, 5.11–20.5 and 13.0–23.9, respectively. Significant dephytinization, ranging between 35.3 and 69.3% in different varieties, was achieved on exogenous enzymatic treatment. Enzymatic dephytinization in the in vitro simulated gastrointestinal digestion resulted in significant increase in the bioavailability of essential minerals. The increase in bioavailability of calcium, iron, zinc and copper on exogenous enzymatic dephytinization ranged between, respectively (x-folds): 1.30–1.96, 1.11–1.52, 1.22–1.78 and 1.11–1.73.     

Development of introgression lines with 18 alleles of glutenin subunits and evaluation of the effects of various alleles on quality related traits in wheat (Triticum aestivum L.)

In this report, we present a set of 104 ILs with 18 alleles for five glutenin loci. They were developed from crossing and backcrossing 64 varieties as donor parents to Yanzhan 1 as recurrent parent. The effects of the 18 alleles on nine dough quality parameters were evaluated in a similar background using these lines. The results showed that Glu-A1a produced the highest SDS-sedimentation volume (Ssd), midline time x=8 width (MTxW), mixing tolerance (MT) and the lowest weakening slope (WS). At the Glu-B1 locus, Glu-B1f produced the highest values for all quality parameters but WS. At the Glu-D1 locus, Glu-D1d was the best for Ssd, grain hardness (GH), midline peak width (MPW), MTxW and MT. The positive effects of Glu-B1f on GH and Glu-B3b on Ssd were mainly from the effect of GPC. Overall, 5 interactive loci and 13 interactive alleles were found to be significant. No negative interaction between high quality glutenin alleles was detected. The preferred allele combinations for breeding were recommended based on the additive and interactive effects. Our results suggest that the ILs with multiple alleles are ideal genomic stocks for evaluating the effects of alleles on some traits and for pyramiding favorable alleles in breeding wheat varieties.     

Starch properties, in vitro digestibility and sensory evaluation of fresh egg pasta produced from oat,teff and wheat flour

Specific dietary requirements, e.g. celiac disease, as well as increased consumer demand for products of high nutritional value, makes the production of pasta from alternative cereals interesting. Raw material characterisation showed that the utilisation of oat and teff flour is beneficial as these ingredients contain higher levels of fibre and mineral composition is superior to that of wheat. Starch properties significantly influence pasta quality and therefore damaged starch levels, amylase activity, pasting properties and gelatinisation temperatures of the flours were investigated. Fresh egg pasta based on wheat, oat and teff flour was produced. Sensory properties of oat spaghetti were found to be very close to that of wheat pasta but improvement of smoothness and aroma is necessary, while teff spaghetti showed reduced sensory quality. An in vitro enzymatic digestion was performed using a dialysis system to mimic the behaviour of pasta as eaten and make predictions on the glycemic index (GI). The predicted GI was highest for wheat pasta, followed by teff and oat. Ultra structure was studied using confocal laser scanning microscopy, allowing the visualisation of differences in starch granule size and shape as well as gelatinisation occurring during the cooking process.     

Characterization of HMW glutenin subunits in common wheat and related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The sample preparation method of high molecular weight glutenin subunits (HMW-GS) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, without a separation step, by high-performance liquid chromatography (HPLC) was established in this study. Three major factors influencing mass spectra—the ratio of components of the solvent, the resolving time, and the sample volume—were optimized using HMW-GS mixtures extracted from Chinese cultivar Jing 411. The results showed that the optimized method for sample preparation was to resolve HMW-GS from 20 mg in an hour with 50 μl solvent of 0.4% TFA, 30.0% ACN and 69.6% H₂O. The stable mass spectra and accurate molecular weights of 16 major HMW glutenin subunits from common wheat and related species were obtained using the optimized MALDI-TOF-MS method. Seven subunits, where each was from 2–5 cultivars, showed very similar molecular weights. The determined molecular weights of 11 subunits were close to those calculated from their coding sequences. In addition, no positive reaction between HMW-GS and GelCode^® Glycoprotein Staining Reagent was observed. These results suggested that HMW-GS lack extensive post-translational modifications (PTMs), but low levels of glycosylation or phosphorylation present in some subunits cannot be ruled out. Because of its ability to obtain a rapid, complete and precise profile of HMW glutenin subunits without purifying procedures, MALDI-TOF-MS is expected to be a powerful technique for structural and functional studies of HMW glutenin subunits as well as other cereal proteins.     

Proteolytic extraction enhances specific detection of the novel ‘S’-type low molecular weight glutenin subunit in wheat by monoclonal antibody

Specificity of monoclonal antibodies (MAbs) is required in diagnostic immunoassays. However, structural homology between a target antigen and other cellular proteins often causes promiscuous cross-reactivity of a MAb to proteins other than its target antigen, thereby hindering specificity. It is proposed in this study that specific proteolytic enzymes could be useful in the reduction or elimination of cross-reactive epitopes while retaining target epitopes for certain MAbs. The hypothesis was tested using a novel ‘S’-type low molecular weight glutenin subunit (LMW-GS-‘S’) and its antibodies. The results demonstrated that the protease, chymotrypsin, markedly reduced the cross-reactivity in samples that would otherwise obscure the specific detection of LMW-GS-‘S’ by MAb F8-14E6 in an enzyme-linked immunosorbent assay (ELISA). Further investigations revealed that the working mechanism for this proteolytic treatment was indeed due to the selective cleavage that destroyed cross-reactive epitopes whilst retaining the intact specific epitope. As more proteases are being discovered or engineered, the successful utilization of protease in immunoassays as reported here will benefit general immunodiagnostic assay development in both medicine and agriculture by targeting specific epitopes with tailored proteolytic treatment of antigens.     

Assessment of dermal wound healing and in vitro antioxidant properties of Avena sativa L.

Avena sativa L. (Poaceae) has been reported to have traditional utilization against skin diseases and inflammation. Therefore, in this study, the n-hexane, ethyl acetate, ethanol, and water extracts of A. sativa were investigated for their wound healing and antioxidant activities. Total phenol and flavonoid contents of the extracts were established spectrophotometrically. For the wound healing activity, linear incision and circular excision models on rats and mice were evaluated with a standard ointment Madecassol^®. Antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferrous ion-chelating, and ferric-reducing antioxidant power (FRAP) assays. Significant wound healing activity was observed with the ointment formulation of the ethanol extract at 1% concentration. The histopathological examination results also supported the outcome of both linear incision and circular excision wound models. All of the extracts exerted low antioxidant activity in the applied assays. The present study provides a scientific evidence for the traditional usage of A. sativa in the management of wound healing.     

Changes in bran structure by bioprocessing with enzymes and yeast modifies the in vitro digestibility and fermentability of bran protein and dietary fibre complex

Bran is a good source of dietary fibre, phytochemicals, and also protein, but highly insoluble and recalcitrant structure of bran hinders accessibility of these components for gastrointestinal digestion. In the present work, influence of bioprocessing on the microstructure and chemical properties of rye bran and wheat bread fortified with the rye bran were studied. In vitro protein digestibility, and release of short chain fatty acids (SCFA) and ferulic acid in a gut model were studied. Bioprocessing of rye bran was performed with subsequent treatments with cell-wall hydrolysing enzymes (40 °C, 4 h) and yeast fermentation (20 °C, 20 h). Bioprocessing of rye bran resulted in reduced total dietary fibre content, caused mainly by degradation of fructan and β-glucan, and increased soluble fibre content, caused mainly by solubilisation of arabinoxylans. Microscopic analysis revealed degradation of aleurone cell wall structure of the bioprocessed rye bran. Bioprocessing caused release of protein from aleurone cells, assessed as a larger content of soluble protein in bran and a higher hydrolysis rate in vitro. Bioprocessed bran had also faster SCFA formation and ferulic acid release in the colon fermentation in vitro as compared to native bran.     

In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions

Expression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full-length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues.     

In vitro starch digestibility and pasting properties of germinated brown rice after hydrothermal treatments

Germinated brown rice (GBR) recently has received renewed attention due to its enhanced nutritional value. Pasting properties and in vitro starch digestibility of GBR were examined before and after hydrothermal treatments. Steeping in water (30 °C, 24 h) raised the moisture content and germination percentage of brown rice. Pasting viscosity was substantially decreased but gelatinization temperatures and enthalpy were decreased only marginally by germination (30 °C, 48 h). However, annealing (50 °C, 24 h) and heat-moisture treatment (100 °C, 1 h at 30% moisture) after germination resulted in increased pasting viscosity and gelatinization temperatures. The hydrothermal treatments, however, induced browning reactions to darken the flour of GBR. The digestibility of starch in brown rice was increased by germination. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) in the cooked brown rice were 47.3%, 40.8%, and 11.9%, respectively, but changed to 57.7%, 39.1%, and 3.2%, respectively upon germination. The hydrothermal treatments, however, decreased the digestibility of starch in GBR. The heat-moisture treatment decreased the RDS content in GBR near to that of native brown rice. The digestibility and physical properties of brown rice can be controlled by germination and hydrothermal treatments.     

Effects of potato/wheat flours ratio on mixing properties of dough and quality of noodles

The effects of potato flour/wheat flour (PF/WF) ratio on the mixing properties of dough and the quality of noodles were investigated. Substitution of wheat flour with potato flour caused the increases in the water absorption and the protein weakening value (C1-C2) of formulated flours, as well as the decrease in the corresponding dough mixing parameters including dough development time (DDT), dough stable time (DST), initial mixing time (C2), maximum torque during heating (C3), minimum torque of hot paste (C4) and the amount of retrogradation (C5-C4), indicating that the addition of PF weakened dough strength but improved degradation resistance. The textural, cooking, sensory characteristics and microstructure of noodle samples were evaluated. The results indicate that adhesiveness, springiness, broken ratio and cooking lose increased progressively with increasing PF content while the score of sensory evaluation continued to decrease. However, hardness, cooking yield and optimum cooked time for the samples of PF content range from 10% to 20% showed a distinctive variation. Environmental scanning electron microscopy (ESEM) confirmed changes in noodle microstructure as PF addition affected gluten network development. In general, noodles with PF content below 40% were acceptable.     

Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots

Loop-mediated isothermal amplification (LAMP), a novel DNA amplification technique, has been used to detect a variety of pathogens including viruses, fungi, bacteria and parasites. However, diagnosis of sedentary plant-parasitic nematode (PPN) species has not yet been attempted. In this study, we developed a universal LAMP set (RKN-LAMP) for the diagnosis of four common Meloidogyne species (Meloidogyne incognita, M. arenaria, M. javanica and M. hapla), and M. incognita-specific LAMP set (Mi-LAMP). In both assays, a typical ladder-like pattern on gel electrophoresis was observed in all positive samples but not in the negative controls. Amplification products were further confirmed using restriction analysis of the Hpa II enzyme, detection by visual inspection using SYBR Green I and the lateral flow dipstick (LFD) assay. The two LAMP sets were specifically able to detect four common Meloidogyne species and M. incognita populations having several different geographical origins and pathotypes. No cross reaction with DNA of other PPNs was observed. Sensitivity of the RKN- and Mi-LAMP was 10 and 100 fg of pure genome DNA respectively. Both LAMP sets could also amplified crude DNA isolated from the galled root tissue and from soil containing juveniles of M. incognita. The RKN- and Mi-LAMP sets offer the advantages of simplicity, rapidity and cost effectiveness. Both LAMP sets will be instrumental for the diagnosis of Meloidogyne spp. by local extension and regulatory personnel.     

Calendar and degree-day requirements for emergence of adult Macroglenes penetrans (Kirby), an egg-larval parasitoid of wheat midge, Sitodiplosis mosellana (Géhin)

The chalcidoid wasp, Macroglenes penetrans (Kirby), is an important parasitoid of wheat midge, Sitodiplosis mosellana (Géhin), in Europe and western Canada. Emergence of adult wasps was evaluated at 12 sites in Saskatchewan in 1991-2000. Emergence was assessed in relation to calendar days and accumulated degree-days above five different threshold air temperatures. Male wasps emerged 1-2 days before the female wasps. Dates for 10%, 50% and 90% emergence of both wasp sexes were July 16, July 21 and July 30, respectively. Standard deviations (SD) indicated that emergence dates varied by 6.2-7.7 days. The variation in dates related to degree-day accumulations between March 1 and July 31. Wasps emerged 2-12 days earlier than expected at sites with the highest accumulation and 2-17 days later than expected at sites with the lowest accumulation. Accumulated degree-days above 9 °C provided the most accurate estimate of 10% emergence (450 DD, SD = 2.4 days) and 90% emergence (579 DD, SD = 5.0 days). Accumulated degree-days above 5 or 9 °C provided the most accurate estimate of 50% emergence (823 DD, SD = 3.0 days; 503 DD, SD = 3.1 days, respectively). Deviations between observed and expected emergence were greatest at sites with either low or high precipitation. In most instances, wasps emerged 1-8 days earlier than expected at sites that received 20-40 mm rain in May and 1-11 days later than expected at sites that received more than 145 mm rain in May and June. Degree-days above 5 or 9 °C minimized the variation at these sites. Emergence was re-assessed in 2008 and 2009. In both years, accumulated degree-days above 9 °C predicted 10%, 50% and 90% emergence within 0.1-2.2 days. Forecast maps, based on daily degree-day accumulations above 9 °C, would assist producers in monitoring their fields for the presence of wasps. Producers could also adjust the timing, rate and placement of sprays for control of wheat midge to protect and conserve the parasitoid.     

Effects of two novel Wx-A1 alleles of common wheat (Triticum aestivum L.) on amylose and starch properties

Common wheat (Triticum aestivum L.) has three Wx proteins (Wx-A1, Wx-B1, and Wx-D1), which are granule-bound starch synthases I and are responsible for the amylose synthesis of flour starch. The effects of two novel Wx-A1 proteins identified by gel electrophoresis on amylose content and starch properties were analyzed. The variant Wx-A1 protein coded by the Wx-A1i allele was present in smaller amounts and produced less amylose (7.3%) compared to standard lines with the Wx-A1a allele (21.5%). Wx-A1i generated altered starch properties; greater swelling power (SP), glucoamylase digestibility, starch paste clarity, and gelatinization enthalpy in differential scanning calorimetry. The starch from Wx-A1i also showed an altered pasting profile on a Rapid Visco-Analyzer, greater peak viscosity, smaller final viscosity, and lower pasting temperature. The Wx-A1i allele is a novel genetic resource for reducing amylose content in wheat. The other Wx-A1 protein coded by the Wx-A1j allele showed a more basic isoelectric point compared to Wx-A1a on an electrophoretic gel. The amylose content of Wx-A1j did not differ from standard Wx-A1a. Starch SP, paste clarity, and glucoamylase digestibility also suggested that Wx-A1j produced amylose as much as Wx-A1a.     

Pulping and papermaking properties of Tunisian Alfa stems (Stipa tenacissima)—Effects of refining process

The objective of this work is to study characteristics of chemical pulps (soda cooking process) and of paper obtained from Alfa, also known as Stipa tenacissima. For this purpose, Tunisian Alfa stems, and both unbleached and bleached pulps were characterized by determining their chemical composition as well as their morphological and physical properties. Through a detailed comparison with the other pulps obtained from various species, we show that the properties of Alfa stem fibres are intermediate between those of non-wood and wood plants, and most often close to those of Eucalyptus fibres. Refining process (PFI mill device) was then applied to the unbleached and bleached Alfa pulps. The modifications of the morphological properties of the fibres and the drainability and water retention values of the pulps were studied as a function of the refining degree. Here again, Alfa fibres exhibit a behaviour similar to that of Eucalyptus fibres, as the fibre shortening is very limited during the refining process. Finally, conventional handsheets with a basis weight of 65 g/m² were prepared from the unrefined and refined pulps. Their characterization showed that Alfa based papers present low density values and quite good mechanical properties, which are significantly enhanced by the refining treatment, particularly for the unbleached pulp. This study demonstrates the high potentiality of this non-wood species for papermaking applications.     

Effects of fungicides on eyespot, caused predominantly by Oculimacula acuformis, and yield of early-drilled winter wheat

The effectiveness against eyespot of nine fungicide treatments, applied at GS 31, was evaluated in early-drilled winter wheat crops. Visual assessments of stem-base diseases were made before fungicide application and at GS 39/55 and 69/70 of cultivars Consort, Savannah and Claire. Competitive PCR was used to quantify Oculimacula acuformis, O. yallundae, Microdochium nivale, Fusarium spp. and Rhizoctonia cerealis. PCR results indicated that O. acuformis was the predominant pathogen causing eyespot in all three crops. Treatments containing cyprodinil were most successful in reducing disease index and DNA of O. acuformis across cultivars. Treatments containing prochloraz were less consistent in eyespot control, significantly reducing the DNA of O. acuformis only in one instance at GS 70 of cultivar Claire. None of the fungicide treatments had any significant effect on brown foot rot or sharp eyespot, or DNA of M. nivale or R. cerealis. The potential for O. acuformis to cause yield loss was indicated by significant negative relationships between yield and eyespot incidence or DNA of O. acuformis at GS 69/70 for each wheat crop. Relationships between DNA of O. acuformis and eyespot incidence or severity at each growth stage were weak but generally improved at the later growth stages of the crops when disease symptoms were easily distinguished. Both molecular methods and visual assessment failed to predict eyespot incidence or pathogen DNA late in season using incidence or DNA of O. acuformis at GS 31. PCR assays, however, accurately identified the stem base pathogens at early growth stages of the crops when the symptoms of their respective diseases were not readily diagnosed.