Reactivity of gluten detecting monoclonal antibodies to a gliadin reference material

People affected by coeliac disease need to adhere to a life-long gluten-free diet to avoid symptoms. ELISA-tests are seen as the mainstay for the detection of gluten in gluten-free food because of their sensitivity. They can, however, yield different gluten amounts depending on the antibody and reference material used. We compared the reactivity of three prominent mouse anti-gliadin-antibodies to a reference gliadin isolated from 28 common bred European wheat varieties. The reference material proteins were labelled with fluorescent dye Cy3. They were then separated by 2DE and transferred by Western blot onto low fluorescent PVDF-membranes, followed by incubation with the three primary anti-gliadin antibodies one by one. Detection of the reacting proteins used anti-mouse antibody which was labelled with fluorescent dye Cy5. The use of this technique made it possible to co-detect the 2DE-image of the reference material proteins (Cy3) and proteins reacting with the respective antibody (Cy5). The three investigated antibodies had dissimilar reactivities with different proteins of the reference gliadin. Antibodies R5 and PN3 reacted mainly with gliadin fractions, antibody 401.21 mainly with high molecular weight glutenins. The results confirm the individual specificity of these antibodies and demonstrate the importance of validating immunochemical methods for gluten detection.     

The Role of Gluten Proteins in the Baking of Arabic Bread

Fractionation and reconstitution/fortification techniques were utilised to study the role of gluten in Arabic bread. Glutens from two wheat cultivars of contrasting breadmaking quality were fractionated by dilute HCl into gliadin and glutenin. Gluten, gliadin and glutenin doughs from the good quality flour had higher G ′ and lower tan δ values than those from the poor quality flour at all the frequencies examined. Interchanging the gliadin and glutenin fractions between the reconstituted flours showed that the glutenin fraction is largely responsible for differences in the breadmaking performance. Fortification of an average quality flour with the gliadin and glutenin fractions from the poor and good quality flours, at the levels of 1% and 2% (protein to flour mass), induced marked differences in the mechanical properties of bread. The resilience of the loaves was not adversely affected by the addition of gliadins and increased, with a concomitant significant (p<0·05) improvement in quality, at the 2% level of fortification with gliadins from the good quality flour. Addition of glutenin resulted in loaves with leather-like properties that became particularly apparent at the higher level of fortification; the observed deterioration in quality paralleled the increase in the elastic character of the doughs. It is suggested that highly-elastic doughs are not compatible with the rapid expansion of gases at the high-temperature short-time conditions employed in the baking of Arabic bread and that there exists a threshold in dough elasticity beyond which a rapid decline in quality takes place.     

Rheological characteristics of native and steamed wheat flour suspensions

Several Indian snack foods consist of an outer coating made with a wheat flour batter and a sweet or savoury filling. In order to study the possibilities of improving the rheological characteristics of batters used in the batter-coated products, wheat flour was steamed for varying periods of time (5, 15 and 30 min). The studies indicated that SDS-sedimentation values decreased from 35 to 24·5 mL, gluten forming protein was completely denatured, gel mobility increased and solubility of gliadin in the β-region decreased with an increase in the steaming period.The steamed wheat flour was used to make batters having 30, 33 and 36% solids suspended in water. The apparent viscosities of the batter increased from 9·6 to 19·2 Pa·s; the yield stress increased from 5·3 to 7·15 Pa; the consistency index increased from 27·86 to 78·31 Pa·sⁿ. The maximum values of all three parameters were observed in the batter which had a solids concentration of 36%, and which had been made with a flour steamed for 30 min. On the other hand, the flow behaviour index decreased slightly with duration of steaming and with increasing solid concentrations in the batter.     

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.     

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.     

Effect of ultraviolet irradiation on wheat (Triticum aestivum) flour: Study on protein modification and changes in quality attributes

Wheat is one of the most widely consumed cereals, and it witnesses huge variation in its physicochemical properties due to pre- and post-harvest operations, hence, it undergoes many physicochemical treatments to achieve optimum baking characteristics. In the current study, ultraviolet (UV) radiation is proposed as a green technology to modify the wheat flour characteristics. Irradiation of Wheat flour with UV-C radiation (254 nm) of different radiation power and exposure time resulted a significant change in the physicochemical properties (water-binding capacity, amylose content, reducing sugar, sulfhydryl (SH), disulfide (SS) groups level). Further, decrease in gluten content, increase in total volatile basic nitrogen (TVBN) content, and photo-induced thiol-disulfide bridge exchange was observed upon irradiation. FTIR analysis of flour, gluten and gliadin protein confirmed the modification in protein conformations upon irradiation. The β-sheet had the highest contribution in protein conformation for wheat flour, while for gluten protein powder, random coils showed the maximum contribution. The first two principal components could explain 92.55% of total variance during principal component analysis, and the gluten content was correlated positively with SS content but negatively with SH and TVBN content. This study will help researchers and industrialist to understand the interactions of UV radiation with biological material.     

Use of dry-moist heat effects to improve the functionality,immunogenicity of whole wheat flour and its application in bread making

Whole wheat flour samples having protein content of 8.9% and 10.6% were subjected to dry and moist heat conditions to improve the functionality. Dry heat treated flours (DHTF) had higher values of falling number and SDS sedimentation values when compared to moist heat treated flour (MHTF). MHTF showed decrease in water absorption from 75.4 to 56.7%, increase in dough development time from 3.3 to 11.9 min, increase in peak viscosity and cold paste viscosity from 467 to 778 BU and 678 to 1017 BU respectively when compared to untreated flour. MHTF lost its elasticity, SDS-page gel electrophoresis indicated the change especially in the region of gliadin and ELISA indicated 41% reduction in immunogenicity against gliadin. The specific volume of breads prepared from MHTF was significantly lower whereas the crumb firmness value was higher than breads from untreated flours. Breads from treated flours also showed reduction in immunogenicity against gliadin.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten II. Studies on the influence of additives

Cysteine, N-ethylmaleinimide, radical scavengers, various salts or urea were added to wheat gluten. After treatment at increasing pressure (0.1–800 MPa) and temperature (30–80 °C) the resulting material was analysed by micro-extension tests and an extraction/HPLC method to measure protein solubility. Furthermore, cysteine was added to isolated gliadin and glutenin prior to high-pressure treatment and protein solubility was determined. The resistance to extension of gluten strongly increased and the solubility of gliadin in aqueous ethanol decreased with increasing pressure and temperature. As compared to experiments without additive the observed effects were much stronger. Isolated gliadin turned largely insoluble in aqueous ethanol when cysteine was added prior to high-pressure treatment. The S-rich α- and γ-gliadins were much more strongly affected than the S-poor ω-gliadins pointing to a disulphide related mechanism. Monomeric gliadin components were completely recovered after reduction of the aggregates with dithioerythritol. In contrast, samples without free thiol groups such as isolated gliadins or with SH groups, which had been blocked by N-ethylmaleinimide, were hardly affected by high-pressure treatment. The addition of radical scavengers to gluten showed no effect in comparison to the control experiment, indicating that a radical mechanism of the high-pressure effect can be excluded. The observed effects can be explained by thiol-/disulphide interchange reactions, which require the presence of free thiol groups in the sample. The addition of salts and urea showed that unfolding of the protein due to weakening of interprotein hydrogen bonds is strongest for ions with a high radius (e.g. thiocyanate). This leads to weakening of gluten at ambient pressure but it facilitates high pressure induced reactions, e.g. of disulphide bonds.     

Functional Properties of Wheat Gliadins. I. Effects on Mixing Characteristics and Bread Making Quality

The effects of addition of total gliadin and gliadin subfractions on the 2 g Mixograph parameters and loaf volume data of cv. Hereward base flour were studied. The addition of increasing levels of total gliadin and gliadin subgroups to cv. Hereward base flour decreased the overall dough strength, as evidenced by decreases in the values for mixing time (MT), mixing stability (MS) and work input (WI). The decreasing order of these parameters for different gliadins was: ω1- >, γ-, > β-, > α-gliadins. The mixing tolerance, as measured by resistance breakdown (RBD) and bandwidth breakdown (BWBD), decreased as a result of addition of different fractions. However, Peak dough resistance (PDR) values increased with addition of individual groups of gliadins and gluten to the base flour. A linear relationship was found between the PDR and loaf volume when individual groups of gliadin were added to the base flour. The ω-gliadins produced the least positive effects on PDR. Addition of total gliadin and its subgroups substantially improved loaf volumes of pan breads. The ω-gliadins again resulted in a smaller increase in loaf volume.     

Impact of ethanol,succinic acid,and the combination thereof at levels produced during sponge fermentation on hard wheat,soft wheat,and durum wheat farinograph rheology

The sponge and dough mixing process is one of the most common in the world, yet the mechanistic understanding of this process has yet to be sufficiently explored. In this study, aqueous solutions of ethanol, succinic acid, and their combination were prepared at concentrations intended to replicate fermentation times of 3, 4 and 6 h. These solutions were added to a farinograph mixer to make dough using hard wheat, soft wheat, and durum wheat flour. The results indicate that these yeast metabolites (ethanol, succinic acid) impact the mixing resistance, peak mixing value, and dough mixing stability in each of the flour types, likely primarily affected by the ratios of gliadin to glutenin and LMW glutenin in each flour type. Results suggest a stabilizing non-covalent interaction imparted by gliadin at peak mixing time, a stabilizing effect of HMW glutenin during break down, and synergistic effects of ethanol and succinic acid that leads to a faster rate of breakdown in later stages of mixing. It also suggests an increase in mixing resistance when acidulants are added to durum wheat dough. Taken together, this study adds new insights on the sponge and dough mixing process in a way that has not previously been conducted.     

Emulsifying and surface properties of citric acid deamidated wheat gliadin

The properties of citric acid deamidated wheat gliadin (d-gliadin) and d-gliadin emulsions at concentrations of 2% and 0.5% at pH 7 and 3 were investigated. d-gliadin exhibited high solubility at neutral pH and had a different molecular weight distribution compared with control gliadin. Stability of d-gliadin emulsion increased after heat treatment. Heat treatment led to a red shift of the fluorescence emission maxima of emulsions, indicating the rearrangement of d-gliadin molecules and increase of the protein structure flexibility at the oil–water interface. The emulsion was sensitive to NaCl up to 150 mM due to electrostatic screening effects. d-gliadin concentration influenced the droplet size and the saturation surface load of emulsion. d-gliadin slowly adsorbed on the oil–water interface during the adsorption process. Higher d-gliadin concentration exhibited higher surface pressure. Surface adsorption properties confirmed that d-gliadin was a good emulsifier in oil–water emulsions.     

关中平原小麦粉鲜面条制作适宜性研究

针对餐饮业连锁经营和向食品制造转化的背景下,食品生产企业对小麦品种质量特性、原料质量稳定性,特别是面条制作适宜性提出新的要求,对关中平原大面积种植的小麦品种籽粒质量性状进行分析,并通过训练的专业感官评价人员对以其为原料、由面点师实验室制作的鲜面条的烹饪特性进行评价,同时统计分析小麦籽粒性状、面粉理化特性和面条感官评价结果之间的关系。结果表明,关中平原小麦千粒重、容重和硬度较高,面粉湿面筋含量高,面团延展性能良好,优质强筋小麦品种的比例明显提高。面粉的黏度参数,特别是峰值黏度,对面条的感官评价总分,面条质量要素中的色泽、硬度和黏性有较大影响,是评价鲜面条专用粉的重要参考指标。面条感官评价总分与峰值黏度呈显著正相关,与籽粒硬度、破损淀粉含量和面粉吸水率呈显著或极显著负相关。小麦品种（面粉）百农207、F50、F20、中麦578、西农583和郑麦366适宜制作优质鲜面条。     

Interfacial Behaviour of Secalin and Rye Flour-milling Streams in Comparison with Gliadin

Secalin was extracted from rye flour and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-gel pattern showed that some proteins were composed of disulphide linked polypeptide chains. However, a gliadin sample contained more polymeric protein. The behaviour of gliadin and secalin at the air–water interface was compared using the surface balance technique. It was found that secalin was more surface active than gliadin, spreading faster and to a higher surface pressure. The influence of pH on the interfacial behaviour was also studied. The surface pressure after 45 min equilibrium of both gliadin and secalin decreased with decreasing pH. The effect was independent of the acid (hydrochloric acid, lactic acid or ascorbic acid) when compared at the same pH. The behaviour at the gas–liquid interface of five different rye flour-milling streams together with the whole (straight run) flour was also investigated. The fraction with highest protein content spread fastest and reached the highest surface pressure value. When spread on ascorbic acid at pH 3·7 the surface pressure of the flour stream with lowest protein content decreased to the greatest extent, whereas the fraction with the highest protein content was not affected. It was thus found that, although secalin showed an interfacial behaviour similar to gliadin, this behaviour was not necessarily shown by the total protein mixture in a rye flour.     

小麦品种面粉白度的变异及其影响因素分析

为提高小麦品种的面粉白度,以来自不同种植区的216个小麦品种(系)为材料,研究了小麦面粉白度的分布规律及与其他品质指标的相关性,并初步探讨了储藏时间对面粉白度的影响。结果表明,面粉白度在小麦品种(系)间存在极显著差异(P0.01),年份间极显著正相关;参试材料的面粉白度平均为72.9,变幅为54.1~80.1,高于80的品种较少,大多数品种(系)的白度值在70~75之间。来源于西南冬麦区的小麦品种(系)的面粉白度值最高,显著高于黄淮冬麦区和北方冬麦区(P0.05),长江中下游冬麦区的白度值也较高;国外引进品种的白度值普遍较低,平均值为69.53;软麦的白度值显著高于硬麦(P0.05)。面粉白度与出粉率、籽粒硬度、蛋白含量和粉质参数等指标均呈负相关,尤其与硬度的负相关性最强(r=-0.798)。储藏一段时间可略微增加面粉白度,但增幅不明显。同时,筛选出一批优质高白度材料,可作为培育强筋高白度小麦品种的优良亲本。     

Comparison of different rice flour- and wheat flour-based butter cookies for acrylamide formation

Acrylamide (AcA) contents of different rice flour- and wheat flour-based butter cookies baked at 130 °C for 20, 55, or 90 min were investigated. AcA contents of different flour-based cookies increased with baking time. Color parameters in terms of CIE L*, b*, C*, and ΔE values showed significant opposite correlation to the AcA formation in each of the raw flour cookie. The cookies based on white rice flour had the lowest AcA contents ranging from not detectable (ND) to 204 μg/kg, followed by cookies based on brown rice flour (ND to 450 μg/kg), white wheat flour (155 μg/kg to 661 μg/kg), and whole wheat flour (306 μg/kg to 982 μg/kg). Considerably lower AcA levels were found in the rice flour-based cookies than in the wheat flour-based cookies, as well as in the milled flour-based cookies than in the whole-grain cookies. Although the flour source was considered to play a primary role in determining the AcA content, AcA content was apparently not dependent on the quantities of reducing sugars and free asparagine in the starting raw flour and cookies during baking. In summary, given its reducing potential for AcA formation, rice flour could be used in the production of cookies safe from heat-induced contaminants.     

黑粒小麦醇溶蛋白指纹图谱及其遗传分析

黑粒小麦因其具有特殊的营养价值而倍受人们青睐。我国近年来已育成和引入了一批黑粒小麦品种 (系 )和资源 ,它们的黑粒基因来源和色素分布部位不同。本研究利用 ISTA颁布的 A- PAGE标准程序分析了来源不同的 9份黑粒小麦亲本及其 1 3份后代材料的醇溶蛋白指纹图谱。结果表明 ,9份黑粒小麦亲本材料分离出 40条相对迁移率不同的谱带 ,其中 8条为共同带 ,每个亲本均有各自独特的谱带组合。采用“0～ 1”系统记录谱带 ,计算亲本间遗传距离 ,平均值为 0 .34,范围为 0 .1 0～ 0 .48。来自加拿大的京 1 1 70 5和 MY2 4 33的遗传距离为 0 .1 0 ,具有最大的遗传相似性。我国育成的漯珍 1号、乌麦 5 2 6和黑小麦 76号三者间的遗传距离在 0 .33以上。利用非加全成对算术平均法 ( UPGMA)作聚类分析 ,在 0 .34水平上可聚为 3类 ,其中漯珍 1号自成一类。黑粒小麦和白粒小麦杂种后代个体的醇溶蛋白呈共显性遗传 ,但白粒亲本北农 6号的一条带在 F1没有出现。醇溶蛋白指纹图谱的相似程度能反映出品种间的亲缘关系 ,不失为黑粒小麦育种亲本选配的一个重要依据。     

Distribution and function of LMW glutenins,HMW glutenins,and gliadins in wheat doughs analyzed with ‘in situ’ detection and quantitative imaging techniques

The different gluten subunits, gliadins, LMW glutenins, and HMW glutenins have been reported to play different key roles in different type of wheat products. This paper studied the interaction between gliadin, LMW and HMW glutenins in soft, hard and durum semolina flour doughs during different stages of mixing. In order to see how do the gluten subunits (gliadin, LMW glutenin and HMW glutenin) redistribute during mixing, dough samples were taken at maximum strength and 10 min after maximum strength. The doughs have been mixed with the same level of added water (55%), therefore they all have different strengths values due to their changes in proteins content. Oscillatory rheological measurements were performed on the doughs. It has been found that HMW glutenins are relatively immobile because of their less molecular mobility and do no redistribute themselves especially at high strength for doughs such as hard wheat flour. LMW glutenins and gliadins on the other hand redistribute themselves at even at high dough strengths forming a more stable network. In weaker doughs such as soft wheat, the breakdown of the three proteins subunits is responsible for the decay in dough strength. We have also visualized how the greater amount of LMW glutenins in semolina is in constant interaction with HMW glutenins and gliadins allowing the dough to maintain a stable strength for an extended mixing time. Finally, we have found the ‘in situ’ detection and quantitative analysis techniques to be more sensitive to the changes occurring in the gluten network of the dough than the oscillatory rheological analysis.     

Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale

Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum.     

Gluten and non-gluten proteins of wheat as target antigens in autism,Crohn’s and celiac disease

Studies show that patients with celiac disease react not only with gluten wheat proteins but also with non-gluten wheat components. Our goal was to measure IgG or IgA antibodies against wheat proteins or peptides that would provide the most sensitive method for the detection of wheat immune reaction in children with autism spectrum disorder, and patients with Crohn’s and celiac disease (CD). Using ELISA, we measured these antibodies against various gluten and non-gluten wheat proteins. Compared to controls in all three conditions, the strongest reaction was against CXCR3-binding gliadin peptide, followed by a wheat protein mixture, and α-gliadin 33-mer peptide. We determined that a sample that strongly reacted against non-gluten proteins also reacted strongly against gluten proteins. We also found that IgA antibodies against CXCR3-binding gliadin peptide were strongly reactive in a subgroup of 33% in the autism group, 42% in the Crohn’s group, and all tested samples with CD. The results indicate that measuring IgG and IgA antibodies against non-gluten proteins adds nothing to the pathologic relevance of these antibodies. Further research is needed on CXCR3-binding gliadin peptide antibodies as a possible biomarker and as a guide for dietary elimination in CD, Crohn’s disease and a subgroup of children with ASD.     

Rheological Behaviour of Wheat Glutens at Small and Large Deformations. Effect of Gluten Composition

Glutens derived from two wheat cultivars with a known difference in bread making quality, i.e. cv. Katepwa (good) and cv. Obelisk (poor), were fractionated into gliadin and glutenin. Cultivar Katepwa gluten contained more glutenin than cv. Obelisk gluten. Reconstituted glutens were prepared by mixing, in different ratios, gliadin and glutenin fractions that originated from one gluten type or from both glutens. The rheological properties of these mixtures, when hydrated, were studied at small deformations in shear and at large deformations in biaxial extension. The reconstitution of gluten in its original glutenin/gliadin ratio produced a composite that had a somewhat higher resistance to deformation and was more elastic than the unfractionated gluten. This was true for both gluten types. However, the difference between the rheological behaviour of both reconstituted gluten types was comparable with that found between the native glutens. From measurements with glutens reconstituted at various glutenin/gliadin ratios, it appeared that the main factor determining the rheological behaviour of hydrated gluten is the glutenin/gliadin ratio. By interchanging the gliadin and glutenin fractions of the two glutens, it was shown that the source from which the fractions originated, particularly that of the glutenin fraction, was also important.