Structure and dynamics of flour by solid state NMR: effects of hydration and wheat aging

The effects of accelerated aging of wheat seeds on structural and dynamic properties of dry and hydrated (ca 10 wt % H(2)O) flour at a molecular level were investigated by several high and low resolution solid-state NMR techniques. Identification and characterization of domains with different mobility was performed by (13)C direct excitation (DE) and cross-polarization (CP) magic angle spinning (MAS), as well as by (1)H static and MAS experiments. (1)H spin-lattice relaxation times (T(1) and T(1)(rho)) measurements were carried out to investigate molecular motions in different frequency ranges. Experimental data show that the main components of flour (starch and gluten proteins) are in a glassy phase, whereas the mobile fraction is constituted by lipids and, in hydrated samples, absorbed water. A lower proportion of rigid domains, as well as an increased dynamics of all flour components are observed after both seeds aging and flour hydration. Linear average dimensions between 20 and 200 A are estimated for water domains in hydrated samples.     

ESR Studies on Lipid-protein Interaction in Gluten

The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4,4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.     

Hydrophobicity of stored (15, 35 °C), or dry-heated (120 °C) rice flour and deteriorated breadmaking properties baked with these treated rice flour/fresh gluten flour

Rice flour was stored at 15 °C/9 months, at 35 °C/14 days, or dry-heated at 120 °C/20 min. The breadmaking properties baked with this rice flour/fresh gluten flour deteriorated. In addition, the rice flour was mixed with oil in water vigorously, and oil-binding ability was measured. Every rice flour subjected to storage or dry-heated at 120 °C showed higher hydrophobicity, owing to changes in proteins. Then, proteins in the stored rice flour were excluded with NaOH solution, and bread baked with the deproteinized rice flour showed the same breadmaking properties as unstored rice flour/fresh gluten flour. The viscoelasticity of wheat glutenin fraction decreased after the addition of dry-heated rice flour in a mixograph profile. DDD staining increased Lab in color meter, which suggested an increase in SH groups in rice protein. The increase in SH groups caused a reduction in wheat gluten protein resulting in a deterioration of rice bread quality.     

Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer.     

Physicochemical properties of proteins from wheat grown under high-contrast climatic conditions

Studies using electrophoresis, gel chromatography, viscometry, and calorimetry revealed an interrelation of several physicochemical properties of proteins of soft wheat grown under conditions of cool and wet weather with rheological characteristics of gluten and dough and bread quality. The ratio of gliadin and albumin-globulin polypeptides in flour with short-tearing gluten was much lower compared to that in flour with normal gluten. Proteins from flour with short-tearing gluten, including the water-soluble and salt-soluble fraction, had a loose spatial structure. Gluten fractions of this gluten (gliadin and glutenin) were characterized by a more compact and elongated structure compared to normal gluten. As distinct from normal gluten, the conformation of protein particles in short-tearing gluten depended little on hydrophobic interactions. The results suggest that the main components of grain determine the rheological properties of short-tearing gluten.     

Physicochemical properties of proteins from wheat grown under high-contrast climatic conditions

Studies using electrophoresis, gel chromatography, viscometry, and calorimetry revealed an interrelation of several physicochemical properties of proteins of soft wheat grown under conditions of cool and wet weather with rheological characteristics of gluten and dough and bread quality. The ratio of gliadin and albumin-globulin polypeptides in flour with short-tearing gluten was much lower compared to that in flour with normal gluten. Proteins from flour with short-tearing gluten, including the water-soluble and salt-soluble fraction, had a loose spatial structure. Gluten fractions of this gluten (gliadin and glutenin) were characterized by a more compact and elongated structure compared to normal gluten. As distinct from normal gluten, the conformation of protein particles in short-tearing gluten depended little on hydrophobic interactions. The results suggest that the main components of grain determine the rheological properties of short-tearing gluten.     

NMR local range investigations in amorphous starchy substrates: II-Dynamical heterogeneity probed by (1)H/(13)C magnetization transfer and 2D WISE solid state NMR

In the preceding paper, we have investigated the structural heterogeneous character of a series of amorphous samples prepared from various starchy substrates (native potato starch, amylopectin and amylose) following different techniques of preparation (casting, freeze drying and solvent exchange). Spectral decompositions of the C1 resonances of the (13)C CP-MAS (Cross Polarization and Magic Angle Spinning) spectra under (1)H decoupling have shown the existence of five main types of alpha(1-4) linkages. In this part, 2D solid state NMR WISE experiments and the (13)C/(1)H magnetization transfer in CP as a local probe for both structures and dynamics were used. The (13)C CP magnetization curves versus contact time of each C1 component in each recorded spectrum were fitted with an analytic function taking into account two (1)H reservoirs. Interpretation of the characteristic times derived from fitting yields some improvements on the knowledge of the heterogeneity of the samples and on the water molecules distribution.     

Use of (1)H cross-relaxation nuclear magnetic resonance spectroscopy to probe the changes in bread and its components during aging

(1)H nuclear magnetic cross-relaxation spectroscopy was used to probe the molecular mobility/rigidity in bread and its components during storage. The Z-spectra lineshapes, attributed to the solid-like polymer fractions of the samples, differed for the bread, gelatinized waxy starch (GX), gelatinized wheat starch (GW), heated flour (HF), and heated gluten (HG). Upon storage, no change was observed in the Z-spectrum of the bread sample, while the Z-spectra for GX, GW, and HG increased in the width at half height of the decomposed broad component (increased rigidity). These trends in the Z-spectra detected by NMR were contradictory to the DSC results that showed an increase in amylopectin retrogradation enthalpy for all samples containing starch, including bread. These trends in the Z-spectra detected by NMR were not reflected by the DSC results that showed an increase in amylopectin retrogradation enthalpy for all samples, including bread. The differences in molecular mobility could not be therefore, due to recrystallized amylopectin and may be attributed to the role of gluten and/or redistribution of water in the amorphous regions of the samples.     

Herbicidal activity of hydrolyzed corn gluten meal on three grass species under controlled environments

US Patent No. 5,030,268 discloses that corn gluten meal, the protein fraction of corn (Zea mays L.) grain, can be used as a natural preemergence herbicide. However, corn gluten meal is insoluble in water, and this characteristic renders it difficult to apply as a herbicide. To seek a watersoluble material with more potent herbicidal activity, the phytotoxicity of various samples derived from corn gluten meal and other related crop materials were evaluated by using three different grass species under controlled environments. Greenhouse and growth chamber bioassays showed that the sample of enzymatically hydrolyzed corn gluten meal was more herbicidally active than the corn gluten itself and was highly water soluble. Gluten hydrolysate prepared with bacterial source proteinase had the greatest inhibitory activity to the root growth of germinating seeds. This water-soluble material derived from corn gluten meal had a growth-regulating effect on the root system and can be used as a natural herbicide.Journal Paper No. J-15609 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149.     

A Spin-Label Study on Lipids in Dough Formed at Various Concentrations of Oxygen

The effect of oxygen on wheat flour lipids during dough mixing was investigated by analysis of the lipid composition and by an ESR technique with a fatty acid spin-label (4,4’-dimethyl-oxazolidine-N-oxyl derivative of 5-ketostearic acid). Dough was prepared in the presence of the spin-label under an atmosphere of air, nitrogen, 95% nitrogen—5% oxygen or oxygen, and the gluten was obtained by washing out the starch. ESR spectra of the spin-label incorporated into the gluten showed decreases in the order parameter, rotational correlation time and activation energy for rotational viscosity with increasing atmospheric oxygen concentration. During dough mixing in oxygen, oxidation of lipids proceeded and bound lipids slightly decreased. These data indicate that modification of lipids by incorporated oxygen leads to an increase in their fluidity and to a decrease in their hydrophobic interaction with protein in dough.     

Aggregation Behaviors of Glutens,Glutenins and Gliadins from Various Wheats

The aggregation behavior was investigated by the method presented in the previous paper, with the glutens separated from ten kinds of wheat flours different in the mixing property. It was shown that the rate constant of aggregation, k, increased in the order of the weak, medium and strong flour. This is an additional evidence for the correlation between the aggregation behavior of the separated gluten and the mixing property of the flour, which was suggested in the previous paper with four kinds of flours. The aggregation behaviors of glutenin and gliadin were also investigated after fractionation of gluten. A good correlation was observed between the τ₁₀/C values of gluten and glutenin separated from it, showing that the difference of the aggregation behavior of gluten is mainly due to the nature of glutenin.     

Precipitins to Dietary Proteins in Serum and Upper Intestinal Secretions of Coeliac Children

We have used precipitin tests to detect antibodies to 10 dietary proteins in the serum (71 cases) and intestinal secretions (51 cases) of a group of children. Thirty-three of the patients had untreated coeliac disease. Our aims were to find out if, in coeliac patients, there was intestinal secretion of antibodies to wheat proteins only or if, as in coeliac serum, antibodies to many food proteins were present; and to confirm that secretion of antibodies to wheat or gluten was specific for coeliac disease.Precipitins to one or more dietary antigens were detected in the intestinal secretions of 26 out of 30 coeliacs and of 11 out of 21 children who did not have coeliac disease. Most of the positive reactions were with the antigens wheat flour, gluten, oatmeal, and egg. Though precipitins to wheat flour or gluten were present in the intestinal secretions of 22 out of 30 coeliacs this was not specific for coeliac disease for these precipitins were also present in 8 out of 21 non-coeliac children.Serum precipitins were detected in 27 out of 33 coeliacs (to the antigens wheat flour, gluten, oatmeal, rice flour, milk, bovine calf serum, sheep serum, and egg) and in 5 out of 33 non-coeliacs (mainly to milk and calf serum, but two infants aged 3 and 5 months had precipitins to several antigens).     

Use of xylan,an agricultural by-product,in wheat gluten based biodegradable films: mechanical,solubility and water vapor transfer rate properties

The possibility of using xylan, as an agricultural by-product, for production of composite films in combinations with wheat gluten was investigated. Different levels of xylan (0-40% w/w) were incorporated into wheat gluten to form biodegradable composite films. Films were prepared at pH 4 and 11, and dried at either uncontrolled or controlled conditions. The mechanical properties, solubilities and water vapour transfer rate (WVTR) of the composite films were studied. Films were obtained with added xylan without decreasing film-forming quality. Xylan can be used as an additive, as much as 40% (w/w), in wheat gluten films. Changing pH, wheat gluten/xylan ratio, xylan type and drying conditions affected mechanical and solubility properties, however, WVTR was not affected by xylan additions. Wheat gluten/xylan composite films having different characteristics can be produced depending on xylan type, composition and process conditions.     

MYCOMON-monitoring project of toxigenic fungi in food in years 1999 – 2001

An occurrence of the toxigenic fungi producing aflatoxins and ochratoxin A in food was investigated in the study (“MYCOMON”) in years 1999–2001. Twenty five commodities were purchased from retail in twelve collection places in the Czech Republic (300 food samples together). The presence of potentially toxigenic fungiAspergillus flavus was observed in 28% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 1999, in 17% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 2000 and in 18% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 2001.Aspergillus tamarii (aflatoxins producer) was found in black pepper samples (25%) in year 1999, in black pepper samples and black tea (25%) in year 2000 and in black pepper samples and black tea (21%) in year 2001.Aspergillus parasiticus andAspergillus nomius were not isolated.Aspergillus sectionNigri (potential producer of ochratoxin A) was detected in some food (black pepper, black tea, caraway seeds, fine flour, fruit tea, raisins, sweet red pepper).Penicillium verrucosum and Aspergillus ochraceus were not isolated from the tested food.     

13C and 1H solid state NMR investigation of hydration effects on gluten dynamics

The effect of hydration on the molecular dynamics of soft wheat gluten was investigated by solid state NMR. For this purpose, we recorded static and MAS ¹H spectra and SPE, CP, and other selective ¹³C spectra under MAS and dipolar decoupling conditions on samples of dry and H₂O and D₂O hydrated gluten. Measurements of carbon-proton CP times and several relaxation times (proton T₁, T_1ρ and T₂, and carbon T₁) were also performed. The combination of these techniques allowed both site-specific and domain-averaged motional information to be obtained in different characteristic frequency ranges. Domains with different structural and dynamic behaviour were identified and the changes induced by hydration on the dynamics of different domains could be monitored. The proton spin diffusion process was exploited to get information on the degree of mixing among different gluten domains. The results are consistent with the “loop and train” model proposed for hydrated gluten.     

Consumption of hypoallergenic flour prevents gluten-induced airway inflammation in Brown Norway rats

Brown Norway rats were immunized with gluten, and then fed a diet containing hypoallergenic flour or an amino acid mixture. The rats were then made to inhale a solubilized gluten to induce gluten-specific bronchial asthma. The antibody levels in the serum of rats were measured by ELISA, and cell counts were done on cytospin preparations of bronchoalveolar lavage fluid. Body weight was decreased after allergen challenge in rats fed the amino acid mixture but not in rats fed the hypoallergenic flour. Antibody levels in the serum were significantly lower in rats fed hypoallergenic flour than in those fed the amino acid mixture. Differential cell counts in the bronchoalveolar lavage fluid showed that the numbers of eosinophils, lymphocytes, and neutrophils were significantly lower in rats fed the hypoallergenic flour than in those fed the amino acid mixture. These results suggest that hypoallergenic flour actively suppresses the allergic reactions, probably by inducing oral tolerance.     

Immunoblotting detection of lectins in gluten and white rice flour

The gluten lectin was isolated by affinity chromatography, separated by sodium dodecyl sulphate-gel electrophoresis together with purified wheat germ agglutinin (WGA) and electrotransferred to nitrocellulose filters. The binding pattern of anti-WGA to the blotted filters confirmed the presence of WGA in gluten. A lectin from rice bran and white rice flour, respectively, was isolated by affinity chromatography. Both lectins reacted with anti-WA in immunoblotting. As patients with coeliac disease are known to tolerate rice flour, the finding of a WGA-like lectin questioned the suggestion that WGA in gluten is involved in the pathogenesis of coeliac disease. A second lectin was also isolated from rice flour which reacted only with antibodies against soybean lectin on immunoblots. This may indicate a contamination of soybean proteins in rice flour.     

Selective Staining of Protein and Starch in Wheat Flour and its Products

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H₂SO₄ per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable to flour smears, cryostat and wax sections.     

花后灌水次数对强筋小麦籽粒产量和品质的影响

在防雨池栽培条件下,以2个优质强筋小麦品种（济麦20和藁城8901）为试验材料,研究了花后不同水分供应状况对籽粒产量、籽粒品质（粉质仪参数和面包体积）及其蛋白质组分的影响.结果表明：2个品种的籽粒产量、面粉的面团形成时间、面团稳定时间和制成面包体积均随花后灌水次数的增加呈先升高后降低的趋势;其中,花后灌1水（花后7 d）时藁城8901的籽粒产量最高,花后灌2水（花后7 d+花后14 d）时济麦20的籽粒产量最高;2个品种面粉的面团形成时间、面团稳定时间和制成面包体积均以花后灌1水时最优.单体蛋白含量、不溶性谷蛋白含量、谷蛋白总含量、蛋白质含量以及湿面筋含量也呈现类似的变化趋势.逐步回归分析表明,花后不同水分供应状况下,不溶性谷蛋白含量是影响面团稳定时间的关键因素,谷蛋白总含量与面包体积的变化密切相关.因此,为了保持优质强筋小麦品质的稳定性,水分管理应以改善籽粒蛋白质特别是谷蛋白组分的构成为目标.     

Compositional Difference of Wheat Flour Glutens in Relation to Their Aggregation Behaviors

Comparison of the aggregation behavior of gluten was made by following the turbidity change of gluten suspension with four kinds of glutens prepared from flours different in flour quality. The four kinds of glutens showed different behaviors suggesting a relation to the flour quality. From the observation of the aggregation behavior of separated glutensis and gliadins and also of reconstituted glutens, it was concluded that the aggregation behavior of gluten is determined mainly by the nature of glutenin it contains. To investigate the difference of compositions of glutenins, component polypeptides of glutenin were fractionated by gel filtration into three fractions FI, FII and FIII after reduction and cyanoethylation of glutenin The glutenin from a strong flour was rich in FII, while the glutenin from a weak flour in FI. The observation of the aggregation behaviors of FI, FII and FIII suggested that the aggregation behavior of glutenin depends on the contents of these fractions.