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.     

Effects of cell wall components on the functionality of wheat gluten

Normal white wheat flours and especially whole meal flour contain solids from the inner endosperm cell walls, from germ, aleurone layer and the outer layers of cereal grains. These solids can prevent either gluten formation or gas cell structure. The addition of small amounts of pericarp layers (1–2%) to wheat flour had a marked detrimental effect on loaf volume. Microstructural studies indicated that in particular the epicarp hairs appeared to disturb the gas cell structure. The detrimental effects of insoluble cell walls can be prevented by using endoxylanases. It has been shown that some oxidative enzymes, naturally present in flour or added to the dough, will oxidise water-extractable arabinoxylans via ferulic acid bridges, and the resulting arabinoxylan gel will hinder gluten formation. The negative effects of water-unextractable arabinoxylans on gluten yield and rheological properties can be compensated by the addition of ferulic acid. Free ferulic acid can probably prevent arabinoxylan cross-linking via ferulic acid.     

土壤紧实度变化对小麦籽粒产量和品质的影响

以济南17(强筋品种)、烟农15(中筋品种)、鲁麦22(弱筋品种)为供试品种,设置人为碾压和不碾压2种处理,研究了土壤紧实度(以土壤容重表示)变化对不同类型小麦品种的籽粒产量和加工品质的影响。结果表明,随着土壤紧实度提高,3个品种的分蘖成穗率均显著降低,从而导致单位面积穗数和籽粒产量降低。3个品种相比较分蘖成穗率低的鲁麦22籽粒产量降幅最大。相关品质指标测定结果显示,提高土壤紧实度,对3个品种的蛋白质含量、湿面筋含量、沉淀值和吸水率均无显著影响,但济南17的面筋指数明显降低,面团断裂时间和面团稳定时间显著缩短,单位重量面粉烘焙所得的面包体积变小,而烟农15和鲁麦22受影响较小。其原因可能与土壤紧实度提高条件下济南17籽粒中谷蛋白／醇溶蛋白比例和谷蛋白大聚体含量降低有关。将济南17面团流变学特性年际间变化幅度与紧实度变化的处理效应相比较发现,土壤紧实度是影响强筋小麦品种品质性状稳定性的重要因素之一。     

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.     

根部喷施FeSO_4对春小麦籽粒组分和面粉品质的影响

以4个春小麦品种为材料,在温室条件下研究了根部喷施不同量FeSO4对小麦籽粒硫含量、铁含量、蛋白质含量、淀粉组成、SDS沉淀值、面筋品质和面团流变学特性的影响.结果表明:喷施FeSO4后4个品种的硫含量、铁含量、蛋白含量、SDS沉淀值、湿面筋含量和面筋指数均显著高于对照,但各处理间干面筋含量无显著变化,且不同品种、不同品质参数达到最高值的FeSO4喷施量并不一致;2个品种的直链淀粉含量、直支比和总淀粉含量比对照显著降低,另2个品种直链淀粉含量和直支比却无显著变化;各品种的面团形成时间、稳定时间和断裂时间随着FeSO4喷施量的增加呈现出先升高后降低的趋势,并均在15 g/m2或22.5 g/m2 FeSO4喷施量水平达到最佳,但均与FeSO4喷施量无显著相关性.研究发现,根部喷施适宜浓度的FeSO4可在一定程度上改善春小麦籽粒组分和面粉品质,但改善的效果因品种和品质参数而异.     

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.     

基因型与生态环境对小麦籽粒品质与蛋白质组分的影响

通过2年3点试验,研究了40个小麦(Triticum aestivum)品种(品系)籽粒品质性状和蛋白质组分含量的变异。结果表明,籽粒品质和蛋白质组分在基因型间存在较大的差异;根据小麦籽粒品质的综合性状,可将40个小麦品种(品系)分为6组不同的品质类型,在本试验点的生态环境条件下。基本以中筋及弱筋小麦为主;生态环境对小麦籽粒的容重、沉降值、湿面筋含量、蛋白质含量、赖氨酸含量与蛋白质组分含量均有极显著的影响,而面筋指数、淀粉含量和直链淀粉含量对环境反应不敏感,适宜的生态环境条件有利于形成合理的谷蛋白／醇溶蛋白比。面粉面筋质量好,基因型与生态环境的互作对小麦籽粒品质。谷蛋白与醇溶蛋白及两者的比值有显著影响,对球蛋白影响不大,而面粉蛋白质含量、面筋含量、沉降值及千粒重主要受基因的表达和环境的独立影响,蛋白质组分含量在基因型间和环境间的变化与小麦籽粒烘烤品质密切相关。     

Alpha gliadin antibody levels: a serological test for coeliac disease.

The diagnostic value in coeliac disease of circulating antibodies to casein, crude gliadin, and alpha gliadin was assessed using an adaption of the enzyme linked immunosorbent assay system. alpha Gliadin was the only antigen which consistently separated 26 patients with untreated coeliac disease from 26 normal controls and 13 patients with chronic inflammatory bowel disease. The mean assay index for the 26 patients was 3.1 (SD 1.2) compared with 1.05 (0.5) for the normal controls and 1.1 (0.6) for patients with chronic inflammatory bowel disease. The alpha gliadin antibody levels of six patients with coeliac disease who had maintained a gluten free diet for at least two years were not significantly higher than normal (1.0 (0.4)). The validity of the test was determined in 90 consecutive patients who were being investigated for the presence of coeliac disease. Levels of alpha gliadin antibody were raised in 36 out of 44 patients found to have histologically proved coeliac disease and in six out of 46 subjects whose jejunal mucosa was normal. Serial alpha gliadin concentrations were measured in 12 patients with coeliac disease who had repeat jejunal biopsies performed six months after starting a gluten free diet. The levels of antibody fell in seven of the eight patients whose jejunal mucosa improved on maintaining the diet. They remained raised in four patients who did not adhere to the diet and whose mucosa did not improve. Although a test measuring alpha gliadin antibodies is unlikely to replace jejunal biopsy in the diagnosis of coeliac disease it may be useful in screening for the disease among outpatients.     

The Isolation of Bacterial Polysaccharides with Anti-inflammatory Activity

It was found in the previous paper that wheat gluten polypeptides gave higher molecular weights in SDS-PAGE than in sedimentation equilibrium. To clear the cause of the abnormality of gluten polypeptides in SDS-PAGE, behaviors of the SDS complex of gliadin IV were investigated in comparison with those of the standard proteins. The amount of bound SDS for reduced and alkylated gliadin IV was not different from the value obtained with usual proteins. In accordance with the results of Reynolds et al., the plot of log [η] against log M gave a slope of 1.1, supporting a rodlike structure of the complex. The intrinsic viscosity of the SDS complex of gliadin IV gave a higher value of 0.28 dl/g in comparison with the corresponding value of 0.15 dl/g for the standard proteins. The sedimentation constant was lower in gliadin IV (1.61 S) than in the standard (1.77 S). These facts indicate that the gliadin IV complex has a higher frictional coefficient for its molecular weight of protein, suggesting a more elongated structure than usual. The helix content of the complex of gliadin IV was extremely low (12%). It was suggested that the high proline content of gliadin gives an elongated structure to the SDS complex and this structure causes a low electrophoretic migration mobility and overestimation of molecular weight in SDS-PAGE.     

Analysis of dough functionality of flours from transgenic wheat

The rheological properties of flours from five different lines of transgenic wheat that either express or over-express subunits 1Ax1 and 1Dx5 were analyzed by mixograph assays and SDS sedimentation tests. In one case, the over-expression of subunit 1Dx5 resulted in a ca. 2-fold increase in mixing time, associated with a significant improvement in dough strength, and a lower resistance breakdown, suggesting an important increase in dough stability. However, the flour failed to develop properly without mixing with control flour because the rate of mixing was insufficient to develop the dough, i.e., the flour was overstrong. In two wheat transgenic lines, the expression of 1Ax1 and 1Dx5 transgenes, associated with silencing of all the endogenous high-molecular-weight glutenin subunits, resulted in flours with lower mixing time, peak resistance and sedimentation volumes, suggesting a lower gluten strength.     

Protein markers of plant traits in breeding spring wheat for grain yield and quality

A comparative study of the composition of gliadin proteins in grains of different-quality wheat cultivars Rollo and Drott and four forms of the hybrids F₉ and F₁₀ obtained by crossing these cultivars has been performed. The grain yield of ears, quality of flour, dough, and gluten were compared. Groups of genetically linked gliadin components, controlled by chromosomes 1 and 6 of homologous groups, have been identified as protein markers of selectively valuable plant traits.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 121–126.Original Russian Text Copyright © 2005 by Berezovskaya, Trufanov, Mitrofanova, Kazmiruk.     

Protein markers of traits of plants in breeding spring wheat for grain productivity and quality

A comparative study of the composition of gliadin proteins contained in grains of different-quality wheat cultivars Rollo and Drott and four forms of hybrids F9 and F10, obtained by crossing these cultivars, has been performed. The traits of grain productivity of ears, quality of flour, dough, and gluten were analyzed. Groups of genetically linked gliadin components, controlled by chromosomes 1 and 6 of homoeologous groups, have been identified as protein markers of selectively valuable traits of plants.     

收获期对BNS杂交小麦面粉和馒头品质的影响

以BNS低温敏感雄性不育系衍生的3个杂交种为材料,设置5月27日、5月31日和6月4日3个收获期,分析了不同收获时期对小麦面筋含量、面团流变学特性、淀粉糊化特性以及馒头加工品质的影响.结果表明: 收获时期对BNS杂交小麦的面粉白度、蛋白质和干湿面筋含量均有一定影响.最佳收获期因品种而异,百杂1号和百杂2号的最佳收获期为5月27日,百杂3号的最佳收获期为6月4日,此时期收获的小麦面粉品质最好,馒头综合评分和口感也均最好.其中百杂2号于5月27日收获的小麦同时适合馒头和面条的加工.     

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten‐free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α‐, γ‐, ω‐gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP‐HPLC and mass spectrometry (LC‐MS/MS), whereas gluten immunogenicity was assayed by an anti‐gliadin 33‐mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ‐ and α‐gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω‐gliadins and γ‐gliadins, and three of these also silenced α‐gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC‐MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α‐ and ω‐gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.     

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour

Background

Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity.

Methods

SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC–ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour.

Results

Gliadin and glutenin yields decreased to 7.6 ± 0.5% and 7.5 ± 0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC–ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31–49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31–49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics.

Conclusions

The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties.

General significance

Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.     

Humoral response to wheat protein in patients with coeliac disease and enteropathy associated T cell lymphoma.

Features that might distinguish uncomplicated coeliac disease from enteropathy associated T cell lymphoma were investigated. Of 76 patients with coeliac disease, 71 (93%) had raised levels of alpha gliadin antibody and all responded clinically and histologically to treatment with a gluten free diet. In contrast, none of 16 patients with enteropathy associated T cell lymphoma had raised levels of alpha gliadin antibody, and treatment with a gluten free diet resulted in histological improvement in one and transient clinical improvement in six patients. The ratio of women to men was 2.2:1 in the group with coeliac disease and 1:1.6 in the patients with enteropathy associated T cell lymphoma. Thus patients with enteropathy associated T cell lymphoma do not display a humoral immune response to wheat protein (alpha gliadin), rarely respond to a gluten free diet, and are often men. Patients with uncomplicated coeliac disease usually have raised levels of alpha gliadin antibody, always respond to a gluten free diet, and are frequently women. These findings suggest the presence of two separate forms of enteropathy: one is benign and sensitive to wheat protein whereas the other runs a malignant course.     

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.     

Mechanical properties and network structure of wheat gluten foams

This Article reports the influence of the protein network structure on the mechanical properties of foams produced from commercial wheat gluten using freeze-drying. Foams were produced from alkaline aqueous solutions at various gluten concentrations with or without glycerol, modified with bacterial cellulose nanosized fibers, or both. The results showed that 20 wt % glycerol was sufficient for plasticization, yielding foams with low modulus and high strain recovery. It was found that when fibers were mixed into the foams, a small but insignificant increase in elastic modulus was achieved, and the foam structure became more homogeneous. SEM indicated that the compatibility between the fibers and the matrix was good, with fibers acting as bridges in the cell walls. IR spectroscopy and SE-HPLC revealed a relatively low degree of aggregation, which was highest in the presence of glycerol. Confocal laser scanning microscopy revealed distinct differences in HMW-glutenin subunits and gliadin distributions for all of the different samples.     

Identification and analysis of multivalent proteolytically resistant peptides from gluten: implications for celiac sprue

Dietary gluten proteins from wheat, rye, and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably alpha- and gamma-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from alpha-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of alpha-2 gliadin confirmed that the DQ2 restricted T cell response to the alpha-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative gamma-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.     

HLA-DQ determines the response to exogenous wheat proteins: a model of gluten sensitivity in transgenic knockout mice

We have investigated the genetic basis of the immune response to dietary gluten in HCD4/DQ8 and HCD4/DQ6 double transgenic mice. Mice were immunized with gluten i.p. or individual peptides s.c. and spleen or draining lymph node T cells were challenged in vitro. Strong proliferative responses to gluten were seen in the HCD4/DQ8 mice, whereas the HCD4/DQ6 mice responded to gluten poorly. A series of overlapping peptides spanning gliadin were synthesized. The HCD4/DQ8 mice reacted to many of the individual peptides of gliadin, while the HCD4/DQ6 mice were relatively unresponsive. T cells isolated from HCD4/DQ8 mice also responded well to modified (deamidated) versions of the gliadin peptides, whereas HCD4DQ6 mice did not. The T cell response to gluten was CD4 dependent and DQ restricted and led to the production of cytokines IL-6, TGF-beta, and IL-10. Finally, intestinal lymphocytes isolated from gluten-fed HCD4/DQ8 mice displayed an activated phenotype. These data suggest that this HLA class II transgenic murine model of gluten sensitivity may provide insight into the initiation of the MHC class II-restricted gluten sensitivity in celiac disease.