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.     

Phytate negatively influences wheat dough and bread characteristics by interfering with cross-linking of glutenin molecules

The influence of added phytate on dough properties and bread baking quality was studied to determine the role of phytate in the impaired functional properties of whole grain wheat flour for baking bread. Phytate addition to refined flour at a 1% level substantially increased mixograph mixing time, generally increased mixograph water absorption, and reduced the SDS-unextractable protein content of dough before and after fermentation as well as the loaf volume of bread. The added phytate also shifted unextractable glutenins toward a lower molecular weight form and increased the iron-chelating activity of dough. It appears that phytate negatively affects gluten development and loaf volume by chelating iron and/or binding glutenins, and consequently interfering with the oxidative cross-linking of glutenin molecules during dough mixing. Phytate could be at least partially responsible for the weak gluten network and decreased loaf volume of whole wheat flour bread as compared to refined flour bread.     

Effect of high-nitrogen fertilizer on gliadin and glutenin subproteomes during kernel development in wheat(Triticum aestivum L.)

Nitrogen(N),a macronutrient essential for plant growth and development,is needed for biosynthesis of protein and starch,which affect grain yield and quality.Application of high-N fertilizer increases plant growth,grain yield,and flour quality.In this study,we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and twodimensional difference gel electrophoresis(2D-DIGE).Application of high-N fertilizer led to significant increases in gluten macropolymer content,total gliadin and glutenin content,and the accumulation of individual storage protein components.Of 126 differentially accumulated proteins(DAPs)induced by high-N conditions,24 gliadins,12 high-molecularweight glutenins,and 27 low-molecular-weight glutenins were significantly upregulated.DAPs during five kernel developmental stages displayed multiple patterns of accumulation.In particular,gliadins and glutenins showed respectively five and six accumulation patterns.The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.     

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.     

Application of Polymer Science to Properties of Gluten

Much of the knowledge from polymer science studies can be usefully applied to increase understanding of the properties of gluten and how they are related to composition. Low solubility of gluten proteins compared to many other proteins that have been studied evidently arises from the low entropy of mixing of the largest-sized glutenins and a relatively high value for the Flory-Huggins interaction parameter mainly due to a low concentration of ionisable groups. Grain hardness is a property that appears to depend on the proteins that are concentrated at the starch/matrix interface and how they control adhesion. Dough mixing properties, dough strength and extensibility can be understood in terms of the extension of large glutenin molecules giving rise to rubber elasticity and to the presence of molecular entanglements which contribute strength and extensibility to dough systems.     

Effect of Allelic Variation of Glutenin Subunits and Gliadins on Baking Quality in the Progeny of Two Biotypes of Bread Wheat cv. Ulla

During the determination of the HMW glutenin subunit composition of Finnish varieties, the variety Ulla was observed to contain two biotypes which differed from each other at two loci:Glu-A1andGlu-A3/Gli-A1. One of them, called Ulla 1, contained subunit 2* (Glu-A1b) andGlu-A3o/Gli-A1o, and Ulla 2 contained the null allele (Glu-A1c) andGlu-A3a/Gli-A1c. In order to determine the effect of this allelic variation on quality, the two biotypes were crossed and random lines were produced from the progeny by single seed descent. In total, 95 F6 lines were analysed from four bulked Ulla progeny lines. Significant interaction between the allelic variants of HMW glutenins and LMW gluten proteins affected the SDS-sedimentation volume at the mean flour protein level of 13·1% (dmb); the effect of LMW gluten variants was larger in the lines deficient of a HMW glutenin subunit than in lines having a HMW glutenin subunit (2*). At the higher flour protein levels (mean=15·1%, dmb) the effect on SDS-sedimentation volume was additive; progeny carrying alleles b (subunit 2*) and o/o atGlu-A1andGlu-A3/Gli-A1had significantly greater sedimentation volumes than the progeny carrying alleles c (no subunit) and a/c, respectively. The SDS-sedimentation volumes indicated differences in the quantities of the polymeric glutenins, gel proteins which have been shown to reflect dough strength. In the four bulked Ulla progeny lines, the variation in HMW glutenin subunits affected the dough strength values of the Extensigraph. However, the variation in LMW glutenin subunits did not affect Extensigraph dough strength values, as was predicted by SDS-sedimentation volumes. In the Ulla progeny, adding a HMW glutenin subunit affected Extensigraph dough strength more than adding a LMW glutenin subunit, although both increased the SDS-sedimentation volumes. Moreover, the variation in LMW gluten proteins affected the dough mixing stability in the Farinograph and test baking results of the Ulla progeny.     

Effects of vacuum mixing and mixing time on the processing quality of noodle dough with high oat flour content

The effects of different mixing parameters (vacuum mixing and mixing time) on oat (70% oat flour) and wheat noodle dough were investigated on the basis of textural properties and gluten formation. The results showed that at a vacuum degree of −0.06 MPa and mixing time of 10 min, oat and wheat dough sheets exhibited the highest resistance to extension and glutenin macropolymer (GMP) content, and had the most compact and uniform gluten network. Compared with wheat noodle dough, oat dough had lower resistance to extension, lower tightly bound water content, and higher GMP content. Microstructural examination showed that oat noodle dough had a more aggregated distribution of gluten protein compared with wheat noodle dough under the optimum mixing parameters. Furthermore, the poor binding ability of vital wheat gluten with water molecules caused the indexes of oat noodle dough to be more strongly affected by the changes in mixing parameters than wheat noodle dough.     

Study of the mechanism of improvement due to waxy wheat flour addition on the quality of frozen dough bread

Waxy wheat flour (WWF) was substituted for 10% regular wheat flour (RWF) in frozen doughs and the physicochemical properties of starch and protein isolated from the frozen doughs stored for different time intervals (0, 1, 2, 4 and 8 weeks) were determined to establish the underlying reasons leading to the effects observed in WWF addition on frozen dough quality. Using Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimeter (DSC) and X-ray Diffraction (XRD) among others, the gluten content, water molecular state, glutenin macropolymer content, damaged starch content, starch swelling power, gelatinization properties, starch crystallinity and bread specific volume were measured. Compared to RWF dough at the same frozen storage condition, 10% WWF addition decreased dry gluten and glutenin macropolymer contents and T₂₃ proton density of frozen dough, but increased the wet gluten content, T₂₁ and T₂₂ proton density. 10% WWF addition also decreased damaged starch content, but increased starch swelling power, gelatinization temperature and enthalpy, crystallinity of starch and bread specific volume of frozen dough. Results in the present study showed that the improvement observed due to WWF addition in frozen dough bread quality might be attributed to its inhibition of redistribution of water molecules bound to proteins, increase in damaged starch content and decrease in starch swelling power.     

Understanding the link between GMP and dough: from glutenin particles in flour towards developed dough

Clear correlations exist for glutenin macropolymer (GMP) quantity and rheological properties vs. wheat quality and dough rheological properties, but real insight in understanding these links is still missing. The observation that GMP consists of glutenin particles opens up new possibilities to reveal the underlying mechanism linking glutenin network properties with dough preparation. GMP was isolated from flour of three wheat varieties: Estica, Soissons and Baldus, strongly varying in their mixing requirements (expressed as time-to-peak, TTP). Decrease of GMP quantity and G′ vs. mixing energy was confirmed. More detail was obtained by studying the changes in GMP particles when mixing flour into dough. Mixing leads to a decrease in the average size of the particles. Interestingly, the TTP coincided with the work-input at which all particles just became soluble in SDS. At TTP, the average size of the GMP particles was the same for each variety. During mixing particles lost their globule shapes and appeared ruptured. Particle size analysis confirmed that particles were still present near TTP. Analysis of the change in particle size vs. energy input using physical principles revealed the following: (1) mixing energy is the predominant actuator in decreasing GMP particle size; (2) the initial GMP particle size in flour strongly determines the practical mixing requirements; and (3) the derived mixing energy vs. GMP particle size relationship was shown to be applicable for both Mixograph and Farinograph mixing. Our results demonstrate that, for the flour samples used, glutenin particle size determines TTP and GMP rheology, showing that glutenin particle properties could be a new key to understand the link between GMP and dough properties.     

Enhanced separation and characterization of gluten polymers by asymmetrical flow field-flow fractionation coupled with multiple detectors

Asymmetrical flow field-flow fractionation (AsFlFFF) coupled with refractive index (RI) and multi-angle light scattering (MALS) detectors was used for macromolecular characterization of four different industrial wheat protein preparations (native, enzymatically hydrolyzed, physically separated, and denatured). The fractionation conditions were optimized separately for each protein sample and molar masses were determined from RI and MALS signals. Decaying cross-flow gradient seemed to produce best results for most of the gluten samples in terms of resolution and sample recovery. Sonication of the samples enabled the solubilization of the high-molar mass components with molar mass ranging from 8 × 10⁶ to 3.5 × 10⁸ g/mol. In case of lower-molar mass glutenins (α-gliadins, ω-gliadins, and high molecular weight glutenin subunits), AsFlFFF results were also compared with the results obtained with capillary gel electrophoresis.     

Hydration,polymerization and rheological properties of frozen gluten-water dough as influenced by thermostable ice structuring protein extract from Chinese privet (Ligustrum vulgare) leaves

The effects of thermostable ice structuring proteins (TSISPs) extracted from Chinese privet (Ligustrum vulgare) leaves on water molecular state, dehydration of gluten proteins, secondary structure of proteins, glutenin subunit of glutenin macropolymer (GMP) and rheological properties of gluten doughs during frozen storage were investigated by nuclear magnetic resonance (NMR), attenuated total reflectance-Fourier transform infrared reflectance (ATR-FTIR), reversed phase-high performance liquid chromatography (RP-HPLC) and dynamic rheometry. After frozen storage for 5 weeks, the control sample showed dehydration of gluten proteins and mobility of water molecules in gluten dough increased, significantly indicating ice formation and water redistribution. Secondary structure of gluten proteins changed significantly, α-helix decreased and β-sheet increased. Glutenin subunits depolymerized, indicated by the decrease in high molecular weight glutenins/low molecular weight-glutenins (HMW/LMW) ratio. The decrease in elastic moduli (G′) and viscous moduli (G′') showed the deterioration of rheological properties of gluten dough. The addition of TSISPs inhibited the dehydration of gluten proteins, decrease in α-helix, increase in β-sheet and HMW/LMW ratio, resulting in improved rheological properties of gluten dough.     

Redox Reactions in Wheat Dough as Affected by Ascorbic Acid

Ascorbic acid (AA) is used as bread improver, as its addition to dough causes an increase in loaf volume and an improvement in crumb structure. To explain these effects we review the stereospecificity of the improver action and the properties of ascorbate oxidase and glutathione dehydrogenase and the occurrence of low molecular thiols in flour and their concentration changes during dough mixing in the presence and absence of AA. On the basis of the results the improver action of AA is explained by a reaction sequence leading to a rapid removal of endogenous GSH, which otherwise would cause dough weakening by sulphhydryl/disulphide interchange reactions with gluten proteins. To test this hypothesis the binding sites of endogenous GSH in gluten proteins have been determined by the addition of³⁵S-labelled GSH as a tracer to flour before dough mixing. The distribution of radioactivity in the gliadin and glutenin fractions of gluten obtained from dough indicates that the major portion of GSH is bound to glutenins. The isolation and sequence analysis of radioactive cystine peptides from an enzymatic digest of glutenins demonstrates that GSH is almost exclusively linked to those cysteine residues of LMW subunits that have been proposed to form intermolecular disulphide bonds.     

小麦籽粒发芽对品质的影响

选用来自黑龙江、辽宁和北京的11个冬、春性小麦品种,对其发芽籽粒和耒发芽籽粒进行了11个品质性状的分析,以研究籽粒发芽对小走品质性状的影响。结果表明,小麦籽粒发芽对其蛋白质、淀粉和馒头加工品质均造成负面影响,籽粒发芽与不发芽相比,蛋白质舍量、湿面筋含量、沉淀值、吸水率、形成时间、稳定时间、峰值粘度、稀懈值、降落值、馒头体积和馒头评分等品质性状的差异均达到5％或1％显著水平。籽粒发芽造成蛋白质质量的下降程度大于蛋白质含量的下降程度。籽粒发芽对品质影响程度在品种（系）间也存在差异。发芽处理的小走高分子量走谷蛋白亚基组成和醇溶蛋白电泳图谱均未发生变化。     

Characterisation of an s-type low molecular weight glutenin subunit of wheat and its proline and glutamine-rich repetitive domain

The low molecular weight glutenin subunits (LMW-GS) are major components of the glutenin polymers which determine the elastomeric properties of wheat (Triticum aestivum L.) gluten and dough. They comprise a complex mixture of components and have proved to be difficult to purify for detailed characterisation. The mature LMW subunit proteins comprise two structural domains, with one domain consisting of repeated sequences based on short peptide motifs. DNA sequences encoding this domain and a whole subunit were expressed in Escherichia coli and the recombinant proteins purified. Detailed comparisons by spectroscopy (CD, FT-IR) and dynamic light scattering indicated that the repetitive and non-repetitive domains of the proteins formed different structures with the former having an extended conformation with an equilibrium between poly-L-proline II-like structure and type II' β-turns, and the latter a more compact globular structure rich in α-helix. Although the structures of these two domains appear to form independently, dynamic light scattering of the whole subunit dissolved in trifluoroethanol (TFE) suggested that they interact, leading to a more compact conformation. These observations may have relevance to the role of the LMW-GS in gluten structure and functionality.     

Incorporation of soy proteins into the wheat–gluten matrix during dough mixing

Farinograph methodology was used to evaluate the possible incorporation of soy proteins into a glutenin–soy complex during mixing and to study the contribution of soy proteins to the chemical and physical properties of the dough. To facilitate the interaction of soy and wheat proteins, a redox process was used, which allowed the partial reduction (using dithiothreitol, DTT) and subsequent reoxidation (using potassium iodate) of glutenin without changing its functionality in the dough (a composite of equal parts of wheat and soy flours, 300 g in total). Either raw soy flour (RSF) or physically modified soy flour (PMSF) was used as the soy component. Dough samples were taken at peak mixing time and at break time during mixing, and these were freeze dried for SE-HPLC analysis and capillary electrophoresis (Lab-on-a-chip).     

The final established physicochemical properties of steamed bread made from frozen dough: Study of the combined effects of gluten polymerization,water content and starch crystallinity on bread firmness

The gluten polymerization behavior, water content, starch crystallinity and firmness of Chinese steamed bread made from frozen dough were investigated and their correlations were also established in this study. The decreased degree of gluten polymerization in steamed bread was observed by the enhanced SDS-extractable proteins (SDSEPs) upon frozen storage. Less incorporation of glutenin in the glutenin–gliadin crosslinking of steamed bread mainly contributed to the decreased degree of gluten polymerization. The decreased moisture of steamed bread had a significant negative correlation with the sublimated water in frozen dough (r = −0.8850, P < 0.01). Frozen storage also induced an increase in starch crystallinity and bread firmness. A multiple linear regression model with SDS-extractable proteins, water content and melting enthalpy of starch crystals of steamed bread accounted for 86% of the variance in the natural logarithm of firmness and further revealed that starch crystallinity mainly contributed to bread firmness.     

Surface properties and locations of gluten proteins and lipids revealed using confocal scanning laser microscopy in bread dough

Surface properties of gluten proteins were measured in a dilation test and in compression and expansion tests. The results showed that monomeric gliadin was highly surface active, but polymer glutenin had almost no surface activity. The locations of those proteins in bread dough were investigated using confocal scanning laser microscopy and compared with polar and nonpolar lipids. Added gluten proteins participated in the formation of the film or the matrix, surrounding and separating individual gas cells in bread dough. Gliadin was found in the bulk of dough and gas ‘cell walls’. Glutenin was found only in the bulk dough. Polar lipids were present in the protein matrix and in gas ‘cell walls’, as well as at the surface of some particles, which appeared to be starch granules. However, nonpolar lipid mainly occurred on the surface of particles, which may be starch granules and small lipid droplets. It is suggested that the locations of gluten proteins in bread dough depends on their surface properties. Polar lipid participates the formation of gluten protein matrix and gas ‘cell walls’. Nonpolar lipids may have an effect on the rheological properties by associating with starch granule surfaces and may form lipid droplets.     

Scanning Electron Microscopic Study of Dough and Chapati from Gluten-reconstituted Good and Poor Quality Flour

Hard and soft wheat flours, which were used in the study, resulted in good and poor quality chapatis respectively. Gluten was isolated and interchanged among the two whole wheat flours and studied by scanning electron microscopy for its influence on structural characteristics of dough and its relation to chapati-making quality. Microscopic observations clearly indicated that larger gluten strands covered starch granules in hard wheat flour dough, while gluten was short and starch granules exposed in dough prepared from soft wheat flour. Greater film forming ability of gluten in hard wheat flour dough manifested in long and bulky starch strands interwoven with protein matrix in its chapati crumb. Higher moisture retention and starch gelatinization as a consequence of greater film forming ability of gluten in hard wheat flour resulted in pliable and soft textured chapati.     

Thermal stability of wheat gluten protein: its effect on dough properties and noodle texture

There is a need to develop more sensitive and reliable tests to help breeders select wheat lines of appropriate quality. Gluten thermostability, measured by the viscoelasticity of heated gluten, was assessed for its usefulness in evaluating quality of wheats in breeding programs. Two sets of wheat samples were used: Set I consisting of 20 cultivars and/or breeders' lines (BL), with diverse dough strengths and allelic variations of high M_r glutenin subunits coded at the Glu-A1, Glu-B1 and Glu-D1 loci (N=20) and Set II consisting of 16 near isogenic BL of F7 generation that had been in a quality selection program for three years. Thermostability of the isolated wet gluten was determined by measuring its viscoelastic properties, and was related to noodle texture, flour protein content, protein composition, dough physical properties and other quality predicting tests.Viscoelasticity of heat-treated gluten, isolated with 2% NaCl solution, significantly correlated with most of the tests used to measure dough and/or gluten strength and Chinese white salted noodle texture. The rate of thermal denaturation of proteins depends on M_r and packing density. High ratios of monomeric proteins such as gliadins and low M_r glutenin subunits to high M_r glutenin subunits increase the thermostability of the gluten. The measurement of viscoelasticity of heat-denatured gluten can be a useful test to determine gluten quality. Our study showed that gluten viscoelasticity and most of the tests related to dough and/or gluten strength are independent of allelic variations of the high molecular weight glutenin subunits. This test has been developed for predicting white salted noodle quality.     

河南省小麦品种(系)的品质状况及演变规律

为了解河南省不同时期育成的小麦品种(系)的加工品质状况,选取河南省1980年以来审定或种植的176份小麦品种(系)进行品质性状检测。结果表明,供试材料面粉的蛋白质含量在7.53%~15.34%之间;湿面筋含量在26.70%~46.00%之间;微量SDS沉淀值在6.66~31.05 mL之间;面团稳定时间在1.00~29.90min之间,平均值为4.63min。弱筋材料(稳定时间≤2.5min)为63份;中筋材料(2.5min稳定时间7.0min)为80份;强筋材料(稳定时间≥7.0min)只有33份,占总数的18.75%。1980-2002年审定品种(系)的稳定时间平均值为4.35min,2003-2008年的为4.41 min,而2009-2014年的为5.73min,2015年、2016年和正参加河南省区试的为3.95min,表明近年来河南省审定的小麦品种仍以中、低筋为主,但2002年以后审定的优质品种数目明显增多。对优质品种(系)的系谱分析发现,大多数优质品种为小偃号(小偃6号及其姊妹系)的后代,推测小偃号是河南小麦的主要优质源。此外,发现和面仪参数如峰值时间、峰高、尾宽等原参数及其组成的复合参数与粉质仪的形成时间、稳定时间和粉质质量指数等参数均有很好的线性相关关系,表明可以用和面仪检测育种材料的品质状况。综上所述,随着20世纪末对小麦品质需求的不断提升,河南省优质小麦新品种选育的进程大大加快,小麦品质得到显著改良,小偃6号作为重要的优质源在优质新品种的选育中起着骨干作用。