Molecular Weight Distribution of Proteins in Hard Red Spring Wheat: Relationship to Quality Parameters and Intrasample Uniformity

Molecular weight distribution (MWD) of proteins extracted from hard red spring wheat was analyzed by size‐exclusion HPLC to investigate associations with wheat and breadmaking quality characteristics. Certain protein fractions were related to associations between wheat and breadmaking parameters, specifically when effect of quantitative variation of protein on those parameters was statistically eliminated by partial correlation analysis. SDS‐unextractable high molecular weight polymeric proteins had positive partial correlations with percent vitreous kernel content and breadmaking parameters, including mix time and bread loaf volume. SDS‐extractable protein fractions that were eluted before the primary gliadin peak had positive partial correlations with kernel hardness and water absorption parameters. The proportion of main gliadin fractions in total protein had a negative partial correlation with bread loaf volume and positive correlations with kernel hardness and water absorption parameters. Intrasample uniformity in protein MWD and kernel characteristics was estimated from three kernel subsamples that were separated according to single kernel protein content within individual wheat samples by a single‐kernel near‐infrared sorter. Wheat subsamples were significantly different in protein MWD. Intrasample uniformity in protein MWD did not differ greatly among wheat samples.     

Fate of (14)C-labeled carbonyl sulfide on grains and grain fractions

14C-Labeled carbonyl sulfide (COS) was used to measure the amount of sorbed fumigant and alteration products on grains. Wheat, paddy rice, polished rice, mungbean, and safflower were exposed to a 60 mg L(-)(1) of (14)COS for 7 days and then aired for 5 days. Carbonyl sulfide and/or alteration products in sugars, protein, starch, amino acids, protopectines, and hemicelluloses were undetectable. The total uptakes of radioactivity determined after fractionation and also by extraction were in the range of 36-53 ng g(-)(1) COS equiv. The total radioactivity determined by autoradiography was below the detection limit of 70 ng g(-)(1) COS equiv. Radioactivity in the commodities was less than 0.003% of all the radioactivity applied. The amount of retained radiolabel was measured in three ways. First, biochemical fractions such as lipids and amino acids were separated by chromatography, and the activity was determined in each component. Second, commodities were crushed and extracted in aqueous acetone until the maximum amount of radiolabel was extracted. Third, autoradiography was carried out on commodity kernels.     

Sorghum Protein Extraction by Sonication and Its Relationship to Ethanol Fermentation

The objectives of this research were to develop a rapid method for extracting proteins from mashed and nonmashed sorghum meal using sonication (ultrasound), and to determine the relationships between the levels of extractable proteins and ethanol fermentation properties. Nine grain sorghum hybrids with a broad range of ethanol fermentation efficiencies were used. Proteins were extracted in an alkaline borate buffer using sonication and characterized and quantified by size‐exclusion HPLC. A 30‐sec sonication treatment extracted a lower level of proteins from nonmashed sorghum meal than extracting the proteins for 24 hr with buffer only (no sonication). However, more protein was extracted by sonication from the mashed samples than from the buffer‐only 24‐hr extraction. In addition, sonication extracted more polymeric proteins from both the mashed and nonmashed samples compared with the buffer‐only extraction method. Confocal laser‐scanning microscopy images showed that the web‐like protein microstructures were disrupted during sonication. The results showed that there were strong relationships between extractable proteins and fermentation parameters. Ethanol yield increased and conversion efficiency improved significantly as the amount of extractable proteins from sonication of mashed samples increased. The absolute amount of polymeric proteins extracted through sonication were also highly related to ethanol fermentation. Thus, the SE‐HPLC area of proteins extracted from mashed sorghum using sonication could be used as an indicator for predicting fermentation quality of sorghum.     

Use of Size‐Exclusion High‐Performance Liquid Chromatography for Wheat Quality Prediction in Ethiopia

Wheat quality testing facilities in Ethiopia are limited. The aim of this study was to determine whether size‐exclusion high‐performance liquid chromatography (SE‐HPLC) could be used in breeding programs for quality testing. Thirteen Ethiopian and two South African wheat cultivars were evaluated in two diverse environments for milling and dough characteristics. SE‐HPLC was done on the same samples. Across environments, both SDS‐soluble and SDS‐insoluble polymeric proteins significantly influenced important quality characteristics such as SDS‐sedimentation and mixograph development time. The large monomeric proteins, which are mainly gliadins, had a consistently significantly negative effect on quality. The increase of polymeric protein as opposed to monomeric protein led to improvement of quality characteristics. The SDS‐soluble and SDS‐insoluble polymeric proteins were equally important in quality prediction. The amount of polymeric proteins was significantly higher in the high‐protein environment. Despite a large environmental effect on most fractions, a large ratio of polymeric proteins to monomeric proteins (both SDS‐soluble and SDS‐insoluble) can be a good indicator of baking quality. SE‐HPLC is therefore an option to use in breeding programs in Ethiopia for quality evaluation.     

Effect of Microbial Transglutaminase on Dough Proteins of Hard and Soft (Triticum aestivium) and Durum (Triticum durum) Wheat Cultivars

Microbial transglutaminase (MTGase), a protein‐glutamine γ‐glutamyl transferase (E.C. 2.3.2.13), catalyzes acyl transfer reactions by introducing a covalent cross‐link between l ‐lysine and l ‐glutamine residues. The use of this enzyme has been proposed as an improver to increase dough strength. The objective of this study was to assess and compare the effect of MTGase on different fractions of dough proteins found in hard, soft, and durum wheat. Three different concentrations of the MTGase (0, 5, and 10U/g of gluten) were tested. Moisture, protein, and dry gluten contents were determined for each concentration in addition to rheological measurements done with the farinograph. Following each treatment, the dough proteins were extracted and analyzed by SE‐HPLC and RP‐HPLC. Soluble polymeric protein, gliadins, albumins, and globulins were quantified in addition to the gliadin subclasses and glutenin subunit types. The combustion procedure was used to determine the amount of insoluble polymeric protein. Differences were observed in susceptibility to MTGase catalysis among the dough proteins of the cultivars studied: the cultivar Cortazar (soft wheat) was the most susceptible. The proteins of this cultivar had a characteristically higher amount of ω and α+β gliadins when compared with the other cultivars. As reported earlier, solubility of high molecular weight glutenin subunits and ω‐gliadins was reduced because of the MTGase treatment. However, all gliadin subclasses, including the γ and α+β gliadins, also participated in cross‐linking. The proteins of the cultivar Altar (durum wheat) were the least susceptible to the effects of MTGase. Albumins and globulins did not show any reduction in solubility, implying that they did not participate in cross‐linking.     

Amount and Size Distribution of Monomeric and Polymeric Proteins in the Grain of Organically Produced Wheat

In the present study, we evaluated 444 organically grown wheat genotypes for the amount and size distribution of polymeric proteins by size‐exclusion HPLC. The investigated genotypes were divided into six genotype groups—selection, spelt, old cultivar, primitive, landrace, and cultivar—and these were grown in four different locations, namely, Alnarp, Bohuslän, Gotland, and Uppsala in Sweden. The results showed that the percentage of unextractable polymeric proteins in total polymeric proteins (%UPP) and percentage of large unextractable polymeric proteins in total polymeric proteins were higher in the cultivar group as compared with the rest of the investigated genotype groups. The amounts of total extractable polymeric proteins (TOTE) and total unextractable polymeric proteins were low in cultivars and selections, respectively. Spring wheat grain was found to have a significantly higher amount of all protein fractions as compared with winter wheat. The genotype Kenya was found to belong to both groups of the 20 genotypes with the highest TOTE and %UPP. Thus, the genotype Kenya might be of relevance for consumption and future breeding to improve the breadmaking quality of organically produced wheat.     

Influence of nitrogen application rate and timing on grain protein composition and gluten strength in Swedish wheat cultivars

The influence of nitrogen (N) fertilizer application rate (0 vs. 70 vs. 140 kg N ha^–1) and timing (early = at sowing vs. late = at sowing and before heading) on the amount of protein groups, amount and size distribution of mono‐ and polymeric proteins, and gluten strength was investigated in one set of wheat cultivars (Triticum aestivum L.). Due to their genetic background, the cultivars had different protein concentrations and gluten strengths. Despite this, all of them reacted similarly on rate and timing of nitrogen application. The rate of nitrogen fertilizer increased the variation in protein concentration, gluten strength, and also the variation in most of the investigated protein components. Higher nitrogen fertilizer rate increased protein concentration, decreased gluten strength, and increased the total amount of glutenins and gliadins as well as the amounts of most mono‐ and polymeric proteins. Timing of fertilizer did not influence protein concentration. The gluten strength and the relations of proteins were changed by the timing of fertilizer. Early nitrogen feritilizer applications led to higher gluten strength and a higher percentage of total unextractable polymeric protein in the total polymeric protein and large unextractable polymeric protein in the total large polymeric protein, compared to late nitrogen fertilizer applications.     

Effects of Cultivar and Temperature During Grain Filling on Wheat Protein Content,Composition, and Dough Mixing Properties

Three wheat cultivars, Bastian, Polkka, and Tjalve, were grown in growth chambers at 9, 12, 15, 18, and 21°C during grain filling in 1994, 1995, and 1996. The wheat samples were analyzed for protein content and sodium dodecyl sulfate (SDS) sedimentation volume. The mixing properties of sifted flours were determined by mixograph, and the flour protein composition was determined by size-exclusion fast protein liquid chromatography (SE-FPLC). The protein content, sedimentation volume, and mixogram parameters were affected by the temperature during grain filling. The protein content increased as the temperature increased. The sedimentation volumes and the mixograph data showed temperature effects that could not be explained by variation in protein content. The proportion of the polymeric flour proteins increased with increasing temperature. Positive correlations were found between the proportion of polymeric proteins and SDS sedimentation volume and, within each year, between the proportion of polymeric proteins and mixograph peak time. Negative correlations were found between the proportion of low molecular weight flour proteins (proportion of fraction IV) and sedimentation volume. The differences in these quality parameters among cultivars exceeded the effect of temperature during grain filling.     

Fate of Dwarf Bunt Fungus Teliospores During Milling of Wheat into Flour

Wheat contaminated with teliospores of Tilletia controversa Kühn (TCK) was mixed with uncontaminated wheat and processed through the Kansas State University pilot mill. Two 50-bu lots of the contaminated mixture were cleaned, tempered, and milled. Approximately 500 samples of wheat, cleanings, and mill fractions were collected and examined for the presence of intact and broken TCK teliospores. Whole wheat samples (50 g) were washed, sieved through a 60-μm nylon sieve, and pelleted by centrifugation. Contents of the pellet were examined microscopically for the presence of TCK spores. The procedure was modified as needed to accommodate cleanings and mill fractions. Levels of spore contamination in whole wheat samples decreased at each step during the handling process, and large numbers of spores were found in materials that were sieved or aspirated from the grain. Very few spores were found in bran, germ, and shorts; none were detected in red dog or straight-grade flour. The results showed that a high percentage of spores can be removed from wheat by mechanical cleaning but that it is not feasible to remove all of them.     

Distribution of Protein Composition in Bread Wheat Flour Mill Streams and Relationship to Breadmaking Quality

Wheat protein quantity and composition are important parameters for wheat baking quality. The objective of this study was to use fractionation techniques to separate the proteins of flour mill streams into various protein fractions, to examine the distribution of these protein fractions, and to establish a relationship between protein composition and breadmaking quality. Nine break streams, nine reduction streams, and three patent flours obtained from three samples of Nekota (a hard red winter wheat) were used in this study. A solution of 0.3M NaI + 7.5% 1-propanol was used to separate flour protein into monomeric and polymeric proteins. The protein fractions, including gliadin, albumin+globulin, HMW-GS, and LMW-GS, were precipitated with 0.1M NH₄Ac-MeOH or acetone. The fractions were statistically analyzed for their distribution in the mill streams. The quantities of total flour protein and protein fractions in flour were significantly different among mill streams. The ratio of polymeric to monomeric proteins in break streams was significantly greater than in the reduction streams. The relationship between protein composition and breadmaking quality showed that the quantities of total flour protein, albumin+ globulin, HMW-GS, and LMW-GS in flour were significantly and positively correlated with loaf volume. The ratio of HMW-GS to LMW-GS had little association with loaf volume. The gliadin content in total flour protein was negatively and significantly correlated with loaf volume. These results indicated that the quantity and composition of protein among the mill streams was different, and this resulted in differences in breadmaking quality.     

Estimating relative ages from iron-oxide/total-iron ratios of soils in the western Po Valley,Italy

Nineteen samples were collected from deeper layers of as many terraces forming agesequences in five basins. Most samples were collected at depths of 1 m or more, with some below 3 m. Soils at the sample sites were tentatively identified as Alfisols, Entisols or Inceptisols. Total amounts of iron and the amounts extractable by dithionite and by oxalate were determined for the 19 samples. Redness ratings were calculated from sample colors and related to amounts of extractable iron by regression. Mineralogies of the fine sand, silt and clay fractions were also determined.The percentage of the total iron (Fe_t) extracted by dithionite (Fe_d) minus the amount extracted by oxalate (Fe_o) increased gradually with ages of the terraces, which range from early Middle Pleistocene to Upper Pleistocene-Holocene. These percentages are independent of the total amounts of iron in the samples and of the general lithology of a basin. Such ratios thus promise to be useful in estimating relative ages, especially where time-sequences exist. Mineralogies of the samples were consistent with relative ages of terraces as estimated from the ratios of iron in different forms. So were the redness ratings. The regression of extracted iron on redness ratings suggests that colors can be used as preliminary field indicators of relative ages of terraces without time-sequences.     

Selenium-enriched sprouts. A raw material for fortified cereal-based diets

The selenium supply in almost all European countries, including Austria and Germany, is below the recommended daily intake. In these countries, selenium fortification of foods and the use of selenium supplements are quite popular to compensate for low Se intake from diets. In general, wheat (Triticum aestivum) is known to be a good source for bioavailable selenium, and many studies have been performed to enrich selenium in wheat by selenium fertilization of the soil. In the present work, the process of sprouting was investigated as an alternative to enrich selenium in wheat. Sprouting was chosen because it additionally improves the nutritional value of seeds, for example, by a higher vitamin content, a better quality of protein, and some other parameters. Wheat, alfalfa (Medicago sativa), and sunflower (Helianthus annuus) seeds were germinated for 5 and 7 days in solutions containing selenate. The selenium sensitivity of the sprouts was tested by measuring visible germination levels and seedling development. Uptake rates were studied by determination of total selenium using inductively coupled plasma mass spectrometry (ICP-MS). Metabolism of the absorbed selenium was analyzed by determination of selenium species in extracts of the sprouts using anion exchange HPLC coupled to ICP-MS. It was shown that sunflower sprouts were the most resistant and had the highest uptake rates (up to 900 mg/kg), but almost 100% of the selenium was extracted with water and found to be nonmetabolized selenate. Wheat and alfalfa were less resistant and enriched selenium up to concentrations of 100 and 150 mg of Se/kg of dry mass, respectively. The metabolism of the selenate was inversely related to the total uptake rates. At low Se enrichment (approximately 1-2 mg of Se/kg), <20% of the total selenium content within the sprouts remained as inorganic selenium, indicating a high metabolism rate. With increasing uptake the amount of selenate increased to approximately 40-50%. However, with the method used it is possible to produce sprouts containing certain amounts of selenium, which might provide substantial proportions of bioavailable selenium. In combination with the generally high nutritional value of sprouts, they might serve for production of improved cereal-based diets.     

Quantitation of LMW‐GS to HMW‐GS Ratio in Wheat Flours

A comparison was made of methods for measuring the LMW/HMW glutenin subunit (GS) ratio for glutenin. A set of near‐isogenic wheat lines with the number of HMW‐GS varying from 0 to 5 was utilized to provide a wide range of LMW/HMW‐GS. Glutenin preparations were obtained from ground whole meal after solubilization of monomeric proteins by dimethyl sulfoxide (DMSO) or 50% propanol or by fraction collection from a preparative SE‐HPLC column. Analyses were made on the reduced glutenin from each of the three preparations by RP‐HPLC, SE‐HPLC, and SDS‐PAGE. Both solvents, DMSO and 50% propanol, extracted appreciable amounts of polymeric protein, thus casting some doubts on the accuracy of the determinations. This problem was largely avoided when the polymeric fraction was collected from the eluate of a total glutenin extract run on a preparative SE‐HPLC column. Less glutenin was removed by the two solvents for lines with a greater number of HMW‐GS or with strength‐associated HMW‐GS 5+10 coded by the 1D chromosome. Collection of the polymeric protein in SE‐HPLC, followed by separation of the glutenin subunits in RP‐HPLC, was the best method for quantitating the LMW/HMW‐GS ratio. SE‐HPLC gave a clear separation of the two groups of subunits as well as HMW albumins. RP‐HPLC has the potential advantage of being able to quantitate individual subunits.     

Ultrastructure of Developing Hard and Soft Red Winter Wheats After Air- and Freeze-Drying and Its Relationship to Endosperm Texture

A transmission electron microscopic study was conducted on air- and freeze-dried developing wheat to determine the effects of drying on the structure of the starchy endosperm. Field-grown hard red winter wheat (Karl) and soft red winter wheat (Clark) were harvested at 15, 18, 21, 23, 25, 28, and 35 days after flowering (DAF). Wheat was dried by either air-drying in the spike at 28°C or freeze-drying following freezing in liquid nitrogen. Dried wheat was prepared for microscopy. Fresh samples of Karl and Clark were also harvested on the same days and prepared immediately for microscopy. The method of drying greatly affected cellular ultrastructure. The most pronounced change upon air-drying of developing samples was disappearance of individual protein bodies and conversion of the cytoplasm into a matrix-like material similar in appearance to storage protein matrix found in mature wheat endosperm. Freeze-dried wheats maintained nearly natural ultrastructure but exhibited various amounts of freeze damage. Conversion of protein bodies to a matrix was not observed in freeze-dried samples. The results suggest that hardness develops as a result of endosperm senescence rather than accumulation of particular grain components. Senescence may cause changes in the starch granule surface such that surrounding components bind tightly in hard wheats, whereas the binding is weaker in soft wheats. Therefore, the surface of starch granules might be more important than components the starch granules bind to in determining hardness.     

Isolation of extracellular protein from greenhouse soil

Although extracellular proteins may play an important role in the soil environment, these proteins are difficult to isolate because they are immediately degraded by soil microbes, or become associated with clay mineral and humic substances. We developed a method of isolating extracellular proteins from greenhouse soils. Phosphate buffer (pH 6.0) was used to extract protein from soil. A phosphate buffer with higher pH was not recommended because it extracted a large amount of non-proteinaceous organic matter as well as protein and, as a result, the extracted protein was difficult to separate by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After removing cells by filtration, proteins dissolved in the soil extract were recovered by precipitation with 5% trichloroacetic acid (TCA) and isolated by SDS-PAGE. Proteins were detected in 10 of 32 soil samples derived from different greenhouses and the protein bands ranged in apparent molecular mass from 35 to 68 kDa, suggesting that some of soils derived from greenhouse culture contained significant amounts of a specific protein soluble in 67 mM phosphate buffer (pH 6.0). N-terminal amino acid sequence of one of the isolated proteins was found to be a homologue of thermostable cellulase produced by the genus Humicola, a thermophilic fungus.     

Surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients

Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems. In this study, starch surface-associated proteins were extracted from four commercial wheat starches, fractionated by high-performance liquid chromatography and gel electrophoresis, and identified by tandem mass spectrometry analysis. More than 150 proteins were identified, many of which (for example, histones, purothionins, and glutenins) had not been recognized previously as starch-associated. The commercial starches were analyzed by the R-5 enzyme-linked immunosorbent assay method to estimate the amount of harmful gluten protein present. One of these starches had a low gluten content of 7 ppm and actually fell within the range proposed as a new Codex Alimentarius Standard for naturally gluten-free foods (maximum 20 ppm). This low level of gluten indicates that the starch should be especially suitable for use by celiac patients, although wheat starches with levels up to 100 ppm are deemed safe in the proposed Codex standards.     

Genotype and Environment Affect Phytosterol Content and Composition of Wheat

Phytosterols (PS) lower low density lipoprotein (LDL) levels by as much as 10–14% in normal and hypercholesteromic people. High LDL levels in the blood are associated with an increased risk of coronary heart disease. Wheat germ and bran contain significant amounts of PS. The main objective of this study was to evaluate the effect of genotype and environment on PS content and composition in whole wheat grain. Protein and ash contents and mineral composition of the samples were also determined. Grain samples were collected from three cultivars, Jagger, Trego, and Intrada, grown at three locations, Alva, Balko, and Goodwell, OK in 2005. Irrigated and dryland samples were obtained from Goodwell. Total PS content and composition in the grain samples were determined using a gas chromatography system. Whole wheat grain samples varied in PS content from 202 mg/kg to 355 mg/kg. β‐Sitosterol, campesterol, and stigmasterol were the major PS compounds found in all the samples. PS exhibited a significant location‐by‐cultivar random effect (P < 0.048), indicating the presence of genotype‐by‐location interaction. Within each location, a significant cultivar effect was also observed. Protein and ash contents and mineral composition of the samples, except those collected from Goodwell‐dryland, were within the range published in the literature for other wheat cultivars. A fundamental understanding of compositional variation in wheat grain requires multi‐environment testing of genotypes, perhaps over several years. This study is a first step for achieving this goal.     

Nutritional evaluation of ethanol-extracted lentil flours

Lentil flours were extracted with 80% ethanol at 25 and 50 degrees C for 1, 2, or 3 h. The various nitrogen fractions, soluble carbohydrates, three amino acids (Lys, His, and Tyr), available lysine, protein digestibility, and vitamins B(1) and B(2) were analyzed to evaluate the effect of extraction. Extraction resulted in an increase in the total nitrogen content of the extracted flours, with extraction temperature affecting the nature of the nitrogen (protein or nonprotein) content. There was also a large reduction in the oligosaccharides of the raffinose family, although the effect of temperature was appreciable only in the case of stachyose. There was hardly any effect on the concentrations of the amino acids analyzed or on protein digestibility; however, a positive correlation between protein digestibility and the available lysine was recorded in the samples. The vitamin B(1) and B(2) contents underwent variable decreases depending on extraction temperature.     

Compositional investigation of phenolic polymers isolated from Vitis vinifera L. Cv. Pinot Noir during fermentation

Phenolic polymer material extracted during commercial red wine fermentations (Vitis vinifera L. cv. Pinot noir) was isolated and analyzed to characterize its chemical composition. Phenolic polymer isolates were prepared from samples taken throughout fermentation and isolated by adsorption chromatography. Isolates were subjected to phloroglucinolysis to analyze the proanthocyanidin amount as well as the subunit composition. Results of phloroglucinolysis revealed that the proanthocyanidin content of individual phenolic polymer isolates varied from 27 to 54%. Subsequent analyses were done in an attempt to quantify materials other than known proanthocyanidin subunits. Results of all experiments indicate that up to 82% of the phenolic polymer isolates could be accounted for by mass. While this figure accounts for a significant portion of the polymeric phenolic material, further investigation will be needed to qualify the remaining 18%.