Investigations on the extractability of gluten proteins from wheat bread in comparison with flour

 Immunochemical methods are recommended for the quantitation of small amounts of gluten in food produced for those with coeliac disease. A major problem, however, is the reduced extractability of gliadin, the toxic factor of gluten, with aqueous alcohol, when foods have been heat-processed. A combined extraction/HPLC procedure was used to study the extractability of all gluten protein types from wheat flour and bread under both non-reducing and reducing conditions. Gliadin isolated from wheat flour was used as a reference protein for quantitation. The results indicate that the extractability of gliadin from bread with 60% ethanol under non-reducing conditions is strongly reduced. α- and γ-gliadins are much more affected than ω-gliadins, and less gliadin was extracted from the crust than from the crumb. For a complete extraction of gliadins from bread, reducing conditions and increased temperature are required. However, glutenin subunits are coextracted with the gliadins. This extract can be used for the quantitation of total gluten proteins by RP-HPLC. The recovery of gliadin added to flour before dough-mixing and bread-making is 98%. Received: 16 February 1998     

Studies on the protein composition and baking quality of einkorn lines

White flours from 23 einkorn breeding lines (assortment 1) and wholemeal flours from 24 einkorn lines (assortment 2) were investigated for their qualitative and quantitative protein compositions by means of a combined extraction/HPLC procedure. The HPLC patterns of the gliadin fractions enabled the differentiation of most einkorn samples. The absence of a group of γ-gliadins at the beginning of the γ-gliadin elution region was unique for einkorn compared to all other wheat species. Differences in the patterns of γ-gliadins allowed the classification of einkorns into four groups; a further subdivision of these groups was possible by the number of ω5-gliadins and the different patterns of α-gliadins and low-molecular-weight glutenin subunits. The total gluten protein (gliadins + glutenins) contents of einkorn flours were similar to or even higher than those of common wheat and spelt. Typical for einkorn flours was the extreme excess of gliadins over glutenins with ω5-gliadins being most abundant and high-molecular-weight glutenin subunits being extremely rare. Micro-tests on the mixing properties and baking performance of assortment 2 flours revealed remarkable differences. Dough development time was negatively correlated with the ratio of gliadins to glutenins and positively with the content of glutenins; bread volume was mainly dependent on the content of glutenins. In conclusion, the determination of the quantitative gluten protein compositions offers a reliable indication of the expected baking quality during the early stages of breeding.     

Comparative investigations of gluten proteins from different wheat species I. Qualitative and quantitative composition of gluten protein types

In contrast to the hexaploid common (bread) wheat, little information is available on the qualitative and quantitative compositions of gluten proteins from other cultivated wheat species. Therefore, representatives of hexaploid spelt, tetraploid durum wheat and emmer, and diploid einkorn were compared with three classes of common wheat (winter wheat, spring wheat, wheat rye hybrid). The flours were extracted to yield total endosperm proteins and the gluten protein fractions (gliadins and glutenin subunits). The extracts were characterised using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase HPLC; both methods revealed that gluten protein groups and types known from common wheat (ω-, α-, γ-gliadins, HMW and LMW subunits of glutenin) were present in all species. The HPLC platterns of gliadins and glutenin subunits from species with the same genome composition (common wheat/spelt or durum wheat/emmer) were related, and those of einkorn quite different. According to the quantities determined by reversed-phase HPLC, α-gliadins were predominant in most cases, followed by γ-gliadins and LMW subunits; ω-gliadins and HMW subunits were generally minor components. Common wheats were characterised by the highest proportions of total glutenins and HMW subunits, which are known to be important for breadmaking quality. Moreover, the lower ratio of gliadins to glutenins was typical. Emmer had the lowest proportions of total glutenins and of HMW and LMW subunits, together with einkorn the highest proportion of α-gliadins, and, by far, the highest ratio of gliadins to glutenins. The values for spelt and durum wheat were mostly in a medium range between common wheats, emmer, and einkorn, respectively. Amongst common wheats, spring wheat was characterised by more balanced quantities of α- and γ-gliadins, and wheat rye hybrid by the highest proportions of ω-gliadins. Received: 26 November 1999     

Biochemical and Functional Properties of Wheat Gliadins: A Review

Gliadins account for 40–50% of the total storage proteins of wheat and are classified into four subcategories, α-, β-, γ-, and ω-gliadins. They have also been classified as ω5-, ω1, 2-, α/β-, and γ-gliadins on the basis of their primary structure and molecular weight. Cysteine residues of gliadins mainly form intramolecular disulfide bonds, although α-gliadins with odd numbers of cysteine residues have also been reported. Gliadins are generally regarded to possess globular protein structure, though recent studies report that the α/β-gliadins have compact globular structures and γ- and ω-gliadins have extended rod-like structures. Newer techniques such as Mass Spectrometry with the development of matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight mass spectrometry (TOFMS) have been employed to determine the molecular weight of purified ω- gliadins and to carry out the direct analysis of bread and durum wheat gliadins. Few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b in bread wheat cultivars, γ-45 in pasta, γ-gliadins in cookies, lower gliadin content for chapatti and alteration in Gli 2 loci in tortillas have been reported to improve the product quality, respectively. Further studies are needed in order to elucidate the precise role of gliadin subgroups in dough strength and product quality.     

The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour

The flours of 13 wheat varieties grown at different levels of nitrogen fertilisation were characterised by the quantitative determination of flour protein groups and gluten protein types using a combined extraction/HPLC procedure. The results demonstrate that the quantities of albumins and globulins were scarcely influenced by different nitrogen fertilisation, whereas those of gluten proteins (gliadins, glutenins) were strongly influenced. The effect on gliadins was more pronounced than on glutenins, as well as the effect on major protein types (α-gliadins, γ-gliadins, LMW subunits of glutenin) in comparison with minor types (ω-gliadins, HMW subunits of glutenin). The proportions of hydrophilic proteins (ω-gliadins, HMW subunits of glutenin) were increased by high levels of nitrogen and those of hydrophobic proteins (γ-gliadin, LMW subunits of glutenin) were decreased. The degree of the effects on both quantities and proportions of flour protein groups and gluten protein types was strongly dependent on the variety. © 1998 SCI.     

The polypeptide composition,structural properties and antioxidant capacity of gluten proteins of diverse bread and durum wheat varieties,and their relationship to the rheological performance of dough

The aim of this study was to compare five bread and five durum wheat genotypes for gliadins and glutenins profiles, the concentration of free sulphhydryl groups and disulphide bonds, antioxidant capacity of gluten proteins and their bread‐making performance. On average, bread wheat had significantly higher concentration of total sulphur‐rich (S‐rich) and sulphur‐poor (S‐poor) subunits of gliadins, as well as total low molecular weight (LMW) and high molecular weight (HMW) subunits of glutenins than durum wheat. However, durum wheat had higher concentration of S‐rich γ‐gliadins and S‐poor D‐LMW‐glutenins, but did not possess S‐poor ω‐gliadins. The concentration of disulphide bonds and total cysteine was higher in the durum gluten than that in the bread gluten, as well as antioxidant capacity (on average 90.6 vs. 85.9 mmol Trolox Eq kg^?1, respectively). In contrast to the bread wheat, the concentration of HMW‐glutenins was negatively associated with extensibility, as well as resistance to extension in durum wheat flour dough.     

Detection and quantification of adulteration of durum wheat flour by flour from common wheat using reverse phase HPLC

When common wheat (Triticum aestivum L) gliadins were separated by RP-HPLC, a major doublet peak eluted at 47.20 and 47.94 min. This peak was consistently found to be absent in Durum wheat (Triticum durum Desf) gliadins separated under identical conditions. In Durum wheat gliadins a characteristic small peak eluted at 48.30 min followed at 50.47, 510.37, 52.80 min by larger peaks. The peak area ratio of the peaks eluting at 50.47 and 51.37 min was found to be 2.18 (±0.14). This ratio was found to decrease proportionally on contamination of Durum wheat flour with flour from some common wheat varieties. This ratio alone was not enough to detect and quantify adulteration by all varieties of common wheat. An alternative method was found whereby the peak emerging between 47 and 49 min in the Durum wheat gliadin elution profile was expressed as a ratio of the total protein applied. This ratio was shown to increase when Durum wheat flour was adulterated with flour from common wheat thus enabling quantitative estimation of the level of adulteration. A third method of detecting adulteration of Durum wheat flour is also proposed in which the peak emerging between 47 and 49 min is collected and the protein separated by PAGE. The presence of more than one band of γ/β-gliadins is indicative of adulteration.     

The influence of gamma irradiation on the immunoreactivity of gliadin and wheat flour

The gliadin fraction of wheat flour gluten is potentially responsible for an allergic reaction in individuals with a genetic predisposition to allergy. In order to study the influence of ionising radiation on the immunogenicity of gliadin, samples of commercial gliadin powder and wheat flour were irradiated with doses between 2.2 and 12.8 kGy. Ethanolic extracts (40% ethanol/water, v/v) of treated samples were analysed by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against ω-gliadin or polyclonal anti-gliadin antibodies and by immunoblotting after polyacrylamide gel electrophoresis. Irradiated gliadin samples show the increase in allergenicity measured by ELISA. A linear relationship between the immune response induced by irradiated gliadin and the applied radiation dose was observed. The increase in immunoreactivity was confirmed by immunoblot assays with sera containing anti-gliadin antibodies. Immunoreactivity of gliadin extracted from irradiated wheat flour was higher than the immune response of pure gliadin irradiated with the same dose. RP-HPLC was applied for the analysis of gliadin constituents after irradiation. The content of ω-gliadins in irradiated samples decreased with increasing radiation dose.     

不同面筋蛋白组分对面包品质的影响

为探究麦谷蛋白、麦醇溶蛋白以及两者的不同配比对面包品质的影响,将谷朊粉、提取的麦谷蛋白、麦醇溶蛋白分别添加到面包中,以面包比容为评价指标,研究其对面包品质的影响。结果表明,三者均能提高面包比容,且在质量分数1.5%（占面粉基）的添加量下,麦谷蛋白与谷朊粉的改善效果相当。再将麦谷蛋白与麦醇溶蛋白重新配比添加到面包中,当麦谷蛋白/麦醇溶蛋白质量比为3∶1时,在质量分数1.1%（占面粉基）的添加量下,与空白组相比,面包比容提高了11.4%,面包硬度降低。贮藏5 d后,面包硬度增量低于空白组13.1%,延缓了面包的老化,且效果优于质量分数1.5%（占面粉基）谷朊粉添加量的效果。     

Hearth bread baking quality of durum wheat varying in protein composition and physical dough properties

Thirty durum wheat genotypes from ten countries of origin were grown in field plots for two consecutive years. Three of the genotypes were γ‐gliadin 42 types and the remainder were γ‐gliadin 45 types. Among the γ‐gliadin 45 types, six high‐molecular‐weight glutenin subunit (HMW‐GS) patterns were identified: 6 + 8, 7 + 8, 7 + 16, 14 + 15, 20 and 2*, 20. All the γ‐gliadin 42 genotypes contained low amounts of unextractable polymeric protein (UPP) and exhibited low gluten index values and weak gluten properties. The γ‐gliadin 45 genotypes exhibited a wide range of UPP, gluten index and dough strength. HMW‐GS 20 genotypes were generally weak, whereas HMW‐GS 6 + 8 and 7 + 8 genotypes were generally strong. When baked by a lean formulation, long‐fermentation straight‐dough hearth bread process, the durum wheat genotypes exhibited a wide range of baking quality. Loaf volume and bread attributes were strongly correlated with UPP and gluten index. Some of the genotypes exhibited bread attributes and loaf volume equal or slightly superior to those of a high‐quality bread wheat flour. However, even the strongest durum wheat genotypes exhibited inferior fermentation tolerance to the bread wheat flour, as seen by a requirement for lower baking absorption during dough handling and more fragile dough properties when entering the oven. Among the HMW‐GS groups, HMW‐GS 7 + 8 and 6 + 8 exhibited the best and HMW‐GS 20 the poorest baking quality. Farinograph, alveograph and small‐scale extensigraph properties demonstrated that a combination of dough elasticity and extensibility was needed for superior durum wheat baking performance. Copyright © 2007 Society of Chemical Industry     

Comparative investigations of gluten proteins from different wheat species II. Characterization of ω-gliadins

 Flours of different wheat species (common wheats including winter wheat, spring wheat, and wheat rye hybrid, spelt, durum wheat, emmer, and einkorn) were successively extracted with a salt solution and 60% (v/v) aqueous ethanol. The alcohol extracts (gliadins) were separated by reversed-phase HPLC. Six to nine different ω-gliadins were obtained for each wheat sample and were characterized by their relative amounts, the amino acid compositions, the N-terminal amino acid sequences, and the molecular masses. The wheats investigated showed typical differences in the qualitative and quantitative HPLC patterns. The amino acid compositions of all ω-type gliadins revealed significantly higher proportions of glutamine, proline, and phenylalanine compared with other gluten proteins. These three amino acids accounted for 70 to 86% of the total composition. Typical differences in amino acid compositions, N-terminal sequences, and molecular masses allowed a clear differentiation of the proteins into ω5- and ω1,2-type gliadins; the gliadin fractions of emmer and einkorn contained only the ω5-type, but not the ω1,2-type. ω5-Gliadins were characterized by extremely high proportions of glutamine (52–57 mol %) and relatively high proportions of proline (18–21 mol %) and phenylalanine (9–10 mol %). ω1,2-Gliadins had less glutamine (39–45 mol %) and phenylalanine (6–8 mol %), but much more proline (22–31 mol %). ω5-Gliadins of all wheats except emmer could be assigned to the N-terminal sequence variants SRQLSP or SRLLSP; the typical sequence of emmer ω5-gliadins was SMELQT. The N-terminal sequences of ω1,2-gliadins were characterized by two basic variants beginning with KELQSP or ARQLNP. The wheat rye hybrid had additionally components with the sequence RQLNPS known from ω-secalins of rye. The determination of molecular masses by MALDI-TOF mass spectrometry revealed a range of 44,000–55,000 for the ω5-type and a range of 36,000–44,000 for the ω1,2-type. Thus, the actual masses were by far lower than the values derived from SDS-PAGE mobility. Received: 25 May 2000     

Effects of different <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Enterococcus</Emphasis> strains and chemical acidification regarding degradation of gluten proteins during sourdough fermentation

Dough quality and baking performance of wheat dough are significantly affected by the qualitative and quantitative composition of the gluten. Therefore, the degradation was studied of specific fractions of gluten proteins in sourdough as affected by starter cultures. Doughs were fermented for 0, 5, and 24 h at 30 °C after addition of Lactobacillus sakei, L. plantarum, L. sanfranciscensis or Enterococcus faecalis. Chemically acidified doughs were used as controls. All doughs were analyzed quantitatively for their content of albumins, globulins, gliadins, glutenins, and glutenin macropolymer by means of a combined extraction/HPLC procedure. Protein degradation during sourdough fermentation was primarily due to acidic proteases present in flour. While L. sakei, L. plantarum and L. sanfranciscensis were mostly non-proteolytic, E. faecalis clearly contributed to gluten proteolysis. Single gluten protein types were clearly different in their resistance to proteolytic activities of the dough system and E. faecalis, and, in contrast to total glutenins, the amounts of gluten macropolymer were significantly reduced already after 5 h of incubation. When longer fermentation times were applied, gluten was substantially degraded. The strongest decrease was found for the glutenin fraction leading to an increase of alcohol soluble oligomeric proteins in the gliadin fraction. The extent of the decrease of monomeric gliadins was strongest for the γ-type followed by the α- and the ω-types. This indicates that dough properties residing in specific types of gluten fractions can be influenced by the duration of fermentation and the application of proteolytic strains.     

Comparative investigations of gluten proteins from different wheat species

The only commercially available immunoassay for gliadin determination in gluten-free food which has been ring-tested and in use for many years, is a test kit based on monoclonal antibodies against -gliadins. Various studies of the literature have shown that different gliadin standards resulted in different calibration curves, and it has been proposed that the affinity of -gliadins to the monoclonal antibodies varied among wheat varieties. To clarify this fundamental problem, total gliadins and the -gliadins from a winter wheat ("Rektor"), a spring wheat ("CWRS"), a wheat rye hybrid ("Herzog") and varieties of spelt, durum wheat, emmer and einkorn, were isolated and analyzed by means of an enzyme-linked immunoabsorbent assay (ELISA) kit based on antibodies against -gliadins. Additionally, single - and -gliadins of Rektor wheat were studied. The results demonstrated that the calibration curves derived for total gliadins differed, in parts, strongly from that of the kit gliadin standard; only the curves for the durum wheat and spelt gliadins were in congruence with the kit gliadin. Single -gliadins revealed strong differences between and within wheat species and ELISA equivalents had a range from 10 to 220% according to kit gliadin. The affinity of -gliadins was not correlated with the calibration curves of total gliadins. Some of the -gliadins of Rektor wheat showed ELISA equivalents similar to those of -gliadins. Because the proportions of -gliadins in total gliadins were significantly higher than those of -gliadins, the unspecific binding of -gliadins contributed much more to the total affinity of gliadins than the specific binding of -gliadins. -Gliadins, however, did not show any detectable affinity.     

Dinkel und Z:oliakie

Spelt wheat (Triticum spelta L.) has not been investigated for the toxicity on coeliac disease patients until now. Because clinical studies are out of considerations for ethical reasons, spelt wheat and coeliac-active bread wheat (Triticum aestivum L.) were compared by the analysis of N-terminal sequences of α-gliadins, which have been proposed to be responsible for the toxic effect. The gliadin fractions of the spelt wheats ‘Roquin’ and ‘Schwabenkorn’ and of the bread wheat ‘Rektor’ were preparatively separated by RP-HPLC and major α-gliadin components were then compared by N-terminal sequence analysis. The results did not reveal any significant difference between spelt and bread wheats within the first 25 positions. For the determination of sequences further from the N-terminus, the gliadin fractions of the spelt wheats were hydrolyzed with pepsin and trypsin. The resulting peptides were successively separated by gel permeation chromatography and RP-HPLC. Those peptides derived from the N-terminal part of α-gliadins were identified by reference peptides isolated previously from bread wheat [this journal 194: 229 (1992)]. Retention times upon RP-HPLC and amino acid compositions of corresponding peptides confirmed the identity of spelt and bread wheat concerning the N-terminal sequences of α-gliadins from position 3 to 56. For these reasons, it can be concluded that spelt wheat is a coeliac-toxic cereal and has to be avoided by coeliac patients.     

Physicochemical and bread-making properties of air flow pulverized wheat and corn flours

The air flow pulverized grain flours were prepared from American wheat (AWF), Korean wheat (KWF), and corn (CRF) at 3 different grinding speeds, 9,000, 10,000, and 11,000 rpm. The effects of grinding speed on the physicochemical and bread making characteristics of resulting flours were investigated. Pulverization at low grinding speed produced the flour similar to those from conventional milling in particle size (80–100 μm). The higher speed grinding at 11,000 rpm produced grain flours with smaller particle sizes (20–30 μm) and higher amount of damaged starches. Accordingly, grinding speed increased water holding capacity, swelling power, and water solubility of the resulting grain flour, and also increased pasting properties. Gluten addition was needed for KWF (15% gluten) and CRF (30% gluten) to facilitate optimum bread making. The flour pulverized at high speed produced the KWF and CRF bread with the highest loaf volume and the lowest firmness.     

X-ray micro-computed tomography evaluation of bubble structure of freeze-dried dough and foam properties of bread produced from roasted wheat flour

This study evaluated the bubble structure of freeze-dried dough and foam properties of bread produced from the roasted wheat flour by means of X-ray micro-computed tomography (μCT). Wheat was roasted at 90 °C and 86 Hz, of which the conditions was determined by means of a central composite design, and the flour used to produce dough proofed for 20- and 40 min as well as bread. The use of 10 g dough and bread samples enabled scanning at a much higher resolution. Roasting resulted in a decreased strut thickness of the bread, suggesting a finer crumb structure and softer texture, compared to the control. Porosity increases were observed for the roasted wheat samples. This suggested a slightly weaker gluten strength as was observed with the lower mixograph peak height. Roasting wheat, at the conditions determined by the central composite design, did not negatively affect the foam properties of the breads produced from the roasted wheat flour.     

Protein Characteristics that Affect the Quality of Vital Wheat Gluten to be Used in Baking: A Review

The use of vital wheat gluten in the baking industry and wheat flour mills aims to improve the rheological characteristics of flour considered unsuitable to obtain products such as sliced bread, French bread, high‐fiber breads, and other products that require strong flours. To improve characteristics such as flour strength, dough mixing tolerance, and bread volume, vital wheat gluten is added to flour at levels that can vary from 2% to 10% (flour basis), with 5% being a commonly used dosage. However, the vital wheat gluten commercialized in the market has few quality specifications, especially related to the characteristics of the proteins that constitute it and are responsible for the formation of the viscoelastic gluten network. Information on protein quality is important, because variations are observed in the technological quality of vital wheat gluten obtained from different sources, which could be associated to damage caused to proteins during the obtainment process. Several tests, either physical–chemical analyses, or rheological tests, are carried out to establish gluten quality; however, they are sometimes time‐consuming and costly. Although these tests give good answers to specify gluten quality, flour mills, and the baking industries require fast and simple tests to evaluate the uses and/or dosage of vital gluten addition to wheat flour. This review covers the concepts, uses, obtainment processes, and quality analysis of vital wheat gluten, as well as simple tests to help identify details about protein quality of commercial vital wheat gluten.     

Multivariate optimisation of a capillary electrophoretic method for the separation of glutenins. Application to quantitative analysis of the endosperm storage proteins in wheat

A capillary electrophoretic method has been designed to allow separation of glutenins with high resolution. Several factors, such as buffer composition, running voltage and capillary temperature were optimised using factorial design and response surface methodology. On the other hand, quantification of content of glutenins and gliadins of different wheat varieties were achieved for the first time using a glutenin extract of wheat gluten and a gliadin extract as external standards, respectively, and using the lys-tyr-lys tripeptide as internal standard. The optimised method and an early reported method for the gliadin separation were validated by evaluating linearity, sensitivity, detection and quantitation limits, repeatability and precision.     

小麦粉自由巯基(-SH)测定方法的对比研究

小麦粉自由巯基的含量直接影响小麦粉的加工品质,从而影响小麦粉的用途。对比研究了3种自由巯基检测方法在巯基的提取、显色反应等方面的优劣,并分别以低筋粉、中筋粉和高筋粉面粉加工厂原粉及市售饺子粉(低筋粉)、标准粉(中筋粉)和面包粉(高筋粉)为原料,并分析了几种方法对不同原料的检测适用性。结果表明:从加标回收率的角度看,方法一、方法二均可用于市售低筋粉和中筋粉的自由巯基含量的检测,几种方法均不适于测定市售高筋粉的巯基含量;3种方法均可用于小麦原粉巯基含量的检测。总体来讲,方法一是适用性和方便性最佳的检测小麦粉自由巯基的方法。     

Quality characteristics improvement of low‐phenylalanine toast bread

This study was designed to prepare and evaluate low‐phenylalanine toast bread made from gliadin‐free wheat flour and hydrocolloids. Wheat protein fraction (gliadin) rich in phenylalanine was extracted using aqueous alcohol solution for the production of low‐phenylalanine wheat flour. Pectin, gum arabic and carboxymethylcellulose (CMC) were used separately to improve the quality of bread at levels of 1, 2 and 3%. Chemical, rheological, organoleptic, baking, staling and microstructure of bread were studied. Phenylalanine content of gliadin‐free bread samples reduced by 43.2% compared with control. Separation of gliadin negatively affected the rheological properties of dough and baking quality of bread, while rheological properties, baking quality and staling were improved upon hydrocolloids addition. Microscopic examination of crumb structure revealed remarkable differences in control and treated breads. It was found that acceptable bread could be produced using gliadin‐free wheat flour with the addition of pectin or CMC up to 2 and 3%, respectively.