Isolation and Characterization of High‐Molecular‐Weight (HMW) Gliadins from Wheat Flour

The aim of this study was to isolate high‐molecular‐weight (HMW) gliadins from wheat flour and to characterize the protein components that contribute to HMW gliadins. Wheat flour Akteur was extracted with a modified Osborne procedure, and the fraction soluble in 60% ethanol (total gliadins) was separated by gel‐permeation HPLC, yielding three fractions, GP1–GP3. GP1 (21.5%) consisted of oligomeric HMW gliadins, GP2 (15.2%) of ω5‐gliadins, and GP3 (63.3%) of ω1,2‐, α‐, and γ‐gliadins. Two‐dimensional SDS‐PAGE of HMW gliadins showed that interchain disulfide bonds were present in HMW gliadins. The molecular mass distribution of HMW gliadins determined by gel‐permeation HPLC was in a range from 66,000 to 680,000 with an average degree of polymerization of 13. Reduced HMW gliadins were further separated by preparative reversed‐phase HPLC into four subfractions (RP1, RP2, RP3, and RP4), which were characterized by SDS‐PAGE and semiquantitative N‐terminal sequencing. HMW gliadins of the wheat flour Akteur contained all types of gluten proteins: 48% low‐molecular‐weight glutenin subunits, 18% γ‐gliadins, 13% α‐gliadins, 9% ω1,2‐gliadins, 8% HMW glutenin subunits, and 4% ω5‐gliadins. We postulate that the existence of HMW gliadins can be explained by the presence of terminators, which interrupt the polymerization of glutenin subunits during biosynthesis and lead to polymers of limited size (oligomers) that are still soluble in aqueous ethanol.     

ATR-FT/IR study on the interactions between gliadins and dextrin and their effects on protein secondary structure

The effects of heat treatment and dextrin addition on the secondary structure of gliadins were investigated by means of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT/IR). Gliadins and gliadin/dextrin mixtures (before and after thermal treatment) were prepared as a dried protein film on the ATR-FT/IR zinc selenide cell plate and equilibrated at a water activity (a(w)) of 0.06. The results show that gliadins undergo conformational changes upon thermal treatment both in the absence and in the presence of dextrin. In particular, in the thermally treated gliadins, the decrease of the band at around 1651 cm(-)(1) and the increase of the bands at around 1628 and 1690 cm(-)(1) suggest a loss of alpha-helix structure and a higher content of protein aggregates. The same trend was observed in the presence of dextrin. Concerning the interactions between gliadins and dextrin, gliadin/dextrin mixtures show variations in the amide I region compared to native gliadins (e.g., an increase of the band at 1645 cm(-)(1) and the absence of the band at around 1668 cm(-)(1)) that might be due to hydrogen bond formation between gliadins and dextrin. It was also found that the spectrum of gliadin/dextrin mixtures was less affected by the hydration state than that of native gliadins, as observed from the differential spectra obtained by subtraction of the spectrum obtained at a(w) = 0.06 (driest condition tested) from the spectrum of the sample equilibrated at a(w) = 0.84. This could be due to the fact that C=O and N-H groups of gliadins are engaged to form hydrogen bonds with the hydroxyl groups of dextrin, and so they are not perturbed by the presence of water molecules. Finally, water activity effects on the secondary structure of gliadins are also discussed.     

Alkylation of Free Thiol Groups During Extraction: Effects on the Osborne Fractions of Wheat Flour

Wheat proteins are classified according to solubility into the so‐called Osborne fractions. Because wheat flour contains both free thiol and disulfide groups, thiol–disulfide interchange reactions are possible during extraction. Osborne fractionation of 12 different wheat flour samples was performed in the presence of N‐ethylmaleinimide (NEMI) to alkylate free thiol groups and without addition of NEMI (control). The addition of NEMI during extraction tended to decrease the content of gliadins (predominantly α‐gliadins) and caused an increase of the content of glutenins in most flour samples. Thus, alkylation of free thiol groups during extraction led to a decline of the gliadin/glutenin ratio from 2 (control) to approximately 1.5 (NEMI). NEMI and control gliadins were separated by gel‐permeation HPLC into an oligomeric subfraction (high‐molecular‐weight [HMW] gliadins) and two monomeric subfractions. In most flours (8 of 12), the addition of NEMI led to a significant increase of the content of HMW gliadins. HMW gliadins from cultivar Akteur wheat were preparatively isolated from NEMI and control gliadins and characterized by HPLC, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and N‐terminal sequencing. HMW gliadin isolated in the presence of NEMI had a significantly higher content of low‐molecular‐weight glutenin subunits and disulfide‐bound cysteine as well as a lower content of α‐gliadins and disulfide‐bound glutathione compared with the control.     

Effect of Microbial Transglutaminase on Dough Proteins of Hard and Soft (Triticum aestivium) and Durum (Triticum durum) Wheat Cultivars

Microbial transglutaminase (MTGase), a protein‐glutamine γ‐glutamyl transferase (E.C. 2.3.2.13), catalyzes acyl transfer reactions by introducing a covalent cross‐link between l ‐lysine and l ‐glutamine residues. The use of this enzyme has been proposed as an improver to increase dough strength. The objective of this study was to assess and compare the effect of MTGase on different fractions of dough proteins found in hard, soft, and durum wheat. Three different concentrations of the MTGase (0, 5, and 10U/g of gluten) were tested. Moisture, protein, and dry gluten contents were determined for each concentration in addition to rheological measurements done with the farinograph. Following each treatment, the dough proteins were extracted and analyzed by SE‐HPLC and RP‐HPLC. Soluble polymeric protein, gliadins, albumins, and globulins were quantified in addition to the gliadin subclasses and glutenin subunit types. The combustion procedure was used to determine the amount of insoluble polymeric protein. Differences were observed in susceptibility to MTGase catalysis among the dough proteins of the cultivars studied: the cultivar Cortazar (soft wheat) was the most susceptible. The proteins of this cultivar had a characteristically higher amount of ω and α+β gliadins when compared with the other cultivars. As reported earlier, solubility of high molecular weight glutenin subunits and ω‐gliadins was reduced because of the MTGase treatment. However, all gliadin subclasses, including the γ and α+β gliadins, also participated in cross‐linking. The proteins of the cultivar Altar (durum wheat) were the least susceptible to the effects of MTGase. Albumins and globulins did not show any reduction in solubility, implying that they did not participate in cross‐linking.     

Identification of microphases in mixed alpha- and omega-gliadin protein films investigated by atomic force microscopy

Pure and mixed films of alpha- and omega-gliadins were studied by tapping mode atomic force microscopy (AFM). The technique was sensitive to the chemistry of the surface properties of the films, allowing imaging of the mixed gliadin phases at different ratios. In addition to the study of the phases at the micrometer level, higher resolution images allowed visualization of the protein films at the molecular level. These studies may have relevance to the formation of phases in developing protein bodies in grain, where gliadins and glutenins are deposited together. It has been assumed that the protein bodies consist of a random network of proteins; these studies indicate that microphases could be present in protein bodies. The technique provides novel methods for studying mixed biopolymer systems.     

Effect of Single Substitution of Glutenin or Gliadin Proteins on Flour Quality of Alpha 16, a Canada Prairie Spring Wheat Breeders' Line

The effect of genetic variation in the glutenin and gliadin protein alleles of Alpha 16, a Canada Prairie Spring (CPS) wheat line, on the dough mixing, bread, and noodle quality properties were evaluated. The presence of a gliadin component (BGGL) and the low molecular weight glutenin subunit (LMW-GS) 45 found in the selection Biggar BSR were associated with significant increases in dough strength characteristics. The results of the study showed that gliadins, LMW-GS, and high molecular weight glutenin subunits (HMW-GS) can influence bread- and noodle-making properties of wheat flour. Genotype-by-environment interactions were not significant for most of the quality parameters studied, indicating that the differences observed in quality characteristics were mainly due to the effect of genotype.     

Studies of Partial Amino Acid Sequences of γ‐40k Secalins of Rye

Though γ‐40k secalins are a major protein type within rye storage proteins, total amino acid sequences are not as well known as the gluten proteins of wheat. Well‐reputed structural features such as amino acid compositions and molecular masses indicated a close relationship between γ‐40k secalins and γ‐gliadins of wheat, but the degree of homology of amino acid sequences and the positions of intramolecular disulfide bonds are unknown. Therefore, two major components of γ‐40k secalins (R1, R2) were analyzed for partial amino acid sequences. The R1 and R2, derivatized with 4‐vinylpyridine, were isolated from the prolamin fraction of rye cultivar Danko by means of a two‐step RP‐HPLC on C₁₈ silica gel. The proteins were digested in parallel with trypsin and thermolysin, and the partial hydrolyzates were separated by RP‐HPLC. Simultaneous measurement of UV absorbance at 210 and 254 nm allowed the detection of all peptides eluted as well as the specific detection of pyridylethylated cysteine peptides. Isolated peptides were characterized by sequence analysis, and in parts by mass spectrometry, and assigned to known sequences of γ‐gliadins. The results demonstrated that the N‐terminal domain of R1 and R2 remained undigested after tryptic hydrolysis; they were in agreement with the N‐terminal domain of γ‐gliadins in their molecular masses and in the absence of cysteine residues. Most of the isolated peptides originated from the C‐terminal domains, they covered 83% (R1) and 77% (R2), respectively, of the C‐terminal domain of a known γ‐gliadin (clone pW1020). Comparison of R1 and R2 revealed differences only in a few sequence positions. The degree of homology between the C‐terminal domains present in γ‐40k secalins and γ‐gliadins was ≈85%. All eight cysteine residues of γ‐gliadins were found in R1 and R2 sequences. Remarkably, sequences close to corresponding cysteine residues were identical for γ‐40k secalins and γ‐gliadins. Therefore, it can be assumed that the positions of intramolecular disulfide links are homologous.     

Immunocytochemical Localization of Gluten Proteins Uncovers Structural Organization of Glutenin Macropolymer

The content and composition of the disulfide‐bonded glutenin macropolymer has been shown to influence dough properties, although its structural organization is poorly characterized. The structure of the glutenin macropolymer in dough was studied using an immunolocalization transmission electron microscopy (TEM) technique by localizing gliadins, low molecular weight glutenin subunits (LMW‐GS), and high molecular weight glutenin subunits (HMW‐GS) in sections of dough using antibody probes selective for each of the three classes of gluten polypeptides. Distinct differences in the distribution patterns of gliadins, LMW‐GS, and HMW‐GS were observed, which suggests that proteins have different roles in the structural organization of the gluten matrix. On the basis of the observed distribution of the proteins in dough, it is speculated that gliadins, which are randomly distributed as individual particles, fill space within the glutenin macropolymer; LMW‐GS, which are present as clusters, are speculated to form aggregated branch structures; and HMW‐GS, which are present as chains, are speculated to form a network from which the LMW‐GS branches are formed. Changes in the distribution of gliadins, LMW‐GS, and HMW‐GS in dough during mixing were also noted. Such an arrangement supports previous biochemical evidence which has established that gliadins, LMW‐GS, and HMW‐GS have specific roles in the structural organization of the glutenin macropolymer in doughs.     

Mechanism and enzymatic contribution to in vitro test method of digestion for maize starches differing in amylose content

To determine the rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) contents in a starch sample, the addition of amyloglucosidase is often used to convert hydrolyzates from α-amylase digestion to glucose. The objectives of this study were to investigate the exact role of amyloglucosidase in determining the digestibility of starch and to understand the mechanism of enzymatic actions on starch granules. Four maize starches differing in amylose content were examined: waxy maize (0.5% amylose), normal maize (≈27% amylose), and two high-amylose starches (≈57 and ≈71% amylose). Notably, without amyloglucosidase addition, the RS content increased from 4.3 to 74.3% for waxy maize starch, 29.7 to 76.5% for normal maize starch, 65.8 to 88.0% for starch with 57% amylose, and 68.2 to 90.4% for the starch with 71% amylose. In the method without α-amylase addition, less RS was produced than without added amyloglucosidase, except in maize at 71% amylose content. Scanning electron microscopy (SEM) revealed the digestive patterns of pinholes with α-amylase and burrowing with amyloglucosidase as well as the degree of digestion between samples. To understand the roles of amyloglucosidase and α-amylase in the in vitro test, multiple analytical techniques including gel permeation chromatography, SEM, synchrotron wide-angle X-ray diffraction, and small-angle X-ray scattering were used to determine the molecular and crystalline structure before and after digestion. Amyloglucosidase has a significant impact on the SDS and RS contents of granular maize starches.     

Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities

Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavins with membranes was explored by simulating the interactions of three theaflavin molecules (theaflavin, theaflavin-3-gallate, and theaflavin-3,3'-digallate) with a mixed bilayer composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). The simulations show that the theaflavins evaluated have an affinity for the lipid bilayer surface via hydrogen bonding. The molecular structure of theaflavins influenced their configuration when binding to the bilayer surface, as well as their ability to form hydrogen bonds with the lipid headgroups. The theaflavin-bilayer interactions studied here help to define structure-function relationships of the theaflavins and provide a better understanding of the role of theaflavins in biological processes. The significance of the results are discussed in the context of black tea composition and bioactivity.     

Immunochemical and Electrophoretic Analysis of the Modification of Wheat Proteins in Extruded Flour Products

Antibodies specific for wheat proteins were used to identify protein fractions modified during extrusion of Hard Red Spring wheat flour (14% protein) under four different combinations of extrusion conditions (18 and 24% feed moisture and 145 and 175°C die temperature). Antibody binding was assessed on immunoblots of proteins extracted from flour and extrudates separated by SDS‐PAGE. Antibodies to high molecular weight glutenin subunits (HMW‐GS) and to B‐group low molecular weight glutenin subunits (LMW‐GS) recognized intact subunits from both flour and extrudates. Antibodies to C‐group LMW‐GS had diminished binding to extruded proteins. Glutenin‐specific antibodies also recognized protein in the extrudates migrating as a smear at molecular weights higher than intact subunits, indicating cross‐linked proteins. Antibodies recognized albumins or globulins in flour but not in extrudates, evidence that these fractions undergo significant modification during extrusion. Acid‐PAGE and antibody reaction of gliadins extracted in 1M urea and in 70% ethanol revealed total loss of cysteine‐containing α, β, γ‐gliadins but no obvious effects on sulfur‐poor ω‐gliadins, suggesting gliadin modification involves replacing intramolecular disulfides with intermolecular disulfide cross‐links. Identifying protein fractions modified during different extrusion conditions may provide new options for tailoring extrusion to achieve specific textural characteristics.     

Effects of High and Low Molecular Weight Glutenin Subunits on Rheological Dough Properties and Breadmaking Quality of Wheat

High molecular weight (HMW) or low molecular weight (LMW) subunits of different chemical state (reduced, reoxidized with KBrO₃, or KIO₃) or gliadins were added in 1% amounts to a base flour of the wheat cultivar Rektor and mixed with water. The corresponding doughs were then characterized by microscale extension tests and by microbaking tests and were compared to doughs from the base flour without additives. The maximum resistance of dough was strongly increased by HMW subunits in a reduced state and by HMW subunits reoxidized with KBrO₃. A moderate increase of resistance was caused by HMW subunits reoxidized with KIO₃ and by LMW subunits reoxidized with KBrO₃ or KIO₃. This resistance was strongly lowered by LMW subunits in a reduced state and by gliadins. The extensibility of dough was significantly increased only by gliadins and reduced HMW subunits; HMW subunits reoxidized with KBrO₃ had no effect, and all other fractions had a decreasing effect. In particular, glutenin subunits reoxidized with KIO₃ induced marked decrease of extensibility, resulting in bell‐shaped curve extensigrams, which are typical for plastic properties. The effect of reoxidized mixtures (2:1) of HMW and LMW subunits on maximum resistance depended on the oxidizing agent and on the conditions (reoxidation separated or together); extensibility was generally decreased. Bread volume was increased by addition of HMW subunits (reduced or reoxidized with KBrO₃) and decreased by LMW subunits (reoxidized with KBrO₃ or KIO₃) and by a HMW‐LMW subunit mixture (reoxidized with KBrO₃). The volume was strongly decreased by addition of reduced LMW subunits. A high bread volume was related to higher values for both resistance and extensibility.     

Effect of solutes and matrix structure on water mobility in glycerol-agar-water gel systems: a nuclear magnetic resonance approach

Nuclear magnetic resonance spectroscopy (NMR) has been widely used to determine water molecular mobility in food systems. This study aimed to examine the effects of matrix structure and solutes on the dynamics of water molecules in model mixed systems, glycerol-agar-water gels, using low- and high-resolution NMR. Simple models to explain water relaxation rates and self-diffusion coefficients in mixed systems were developed using the experimental values obtained for the individual binary systems (glycerol-water solutions and agar-water gels). The spin-lattice relaxation of mixed systems was influenced by interactions of both glycerol and agar with water, while the spin-spin relaxation of mixed systems was dominated by the interaction of agar with water. Water diffusion was influenced by not only molecular interactions between all components but also the gel matrix structure. These models are able to differentiate the effect of solutes from that of matrix structure on water molecular dynamics.     

Effect of Aelia spp. and Eurygaster spp. Damage on Wheat Proteins

The effect of Aelia spp. and Eurygaster spp. wheat bugs on the protein fractions of different wheat cultivars has been studied by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and free‐zone capillary electrophoresis (FZCE). Those methods were used to quantify and characterize the extent of protein modification. A decrease in the amount of alcohol‐insoluble polymeric proteins along with an increase in the alcohol‐soluble polymeric proteins and gliadins were observed in damaged wheat. The high molecular weight (HMW) and low molecular weight (LMW) glutenin fractions were barely detected in the incubated damaged wheat from some cultivars, which indicated hydrolysis of those proteins by the bug proteinases. In damaged wheats, both incubated and unincubated, gliadin electrophoregrams revealed the presence of some new peaks with mobilities similar to the ω gliadins. The overall results suggest that the bug proteinases are potent enzymes that appear to be nonspecific because they hydrolyze all gluten proteins.     

Simple Determination of Gluten Protein Types in Wheat Flour by Turbidimetry

A simple method based on turbidimetry has been developed for the quantitative determination of total gliadins, glutenin subunits, and high and low molecular weight (HMW and LMW) subunits of glutenin. The standard procedure includes the subsequent extraction of wheat flour (100 mg) with a salt solution, with 50% 2‐propanol (gliadins), and with 50% propanol under reducing conditions and increased temperature (glutenin subunits). Aliquots of the gliadin and the glutenin extracts are mixed with 2‐propanol to a final concentration of 83%, and the turbidity of the precipitates is measured photometrically at 450 nm and 20°C after 40 min. Another aliquot of the glutenin extract is mixed with acetone to a final concentration of 40% acetone, and precipitated HMW subunits are determined turbidimetrically after 30 min. The sample is then filtered, and an aliquot of the filtrate is mixed with 2‐propanol to a final concentration of 77% to determine the precipitated LMW subunits. Control analyses with reversed‐phase HPLC on C₈ silica gel indicate that the precipitation of the different protein types is quantitative and specific, and studies of 16 different wheat flours demonstrate the strong correlation between quantification by HPLC and turbidimetry. The turbidimetric measurements are reproducible, linear over a wide absorbance range (0.2–1.7), and sufficiently sensitive to analyze 40 μg of protein or 20 mg of flour. The absolute amounts of protein types in flour can be determined by means of calibration curves with protein standards (gliadins, HMW, and LMW subunits). Altogether, the developed method is simple, accurate, sensitive, and specific for the different protein types. The total procedure takes ≈6 hr for the analysis of six flour samples in parallel or ≈4 hr for three samples in overlapping extraction steps. The chemicals used are inexpensive, scarcely toxic, and easy to dispose.     

Splitting nitrogen applications improves wheat storage protein composition under low N supply

For the baking quality of wheat flours, the composition and concentration of grain protein are crucial. It is common practice to use late nitrogen (N) application to increase grain protein concentration (GPC) and hence, improve baking quality. However, the use of N fertilizer—particularly shortly before harvest—involves environmental risks. With the suitability of GPC as a parameter for baking quality predictions being more and more questioned, there are further investigations needed considering not only the GPC but also the composition of grain protein. Gluten protein composition varies depending on genotype and environmental factors, such as weather conditions and fertilization rate. To examine whether the effect of a split N application varies under different amounts of total N supply, this study investigates the effects of split nitrogen application on grain protein concentration and composition of wheat (Triticum aestivum L. cv. JB Asano) at four different N fertilization levels (0.8; 1.0; 1.2, and 1.4 g N pot^?1) in a pot experiment. The GPC was affected by both, N fertilization level and split N application. In this experiment, the minimum GPC of 13%, which is required for class A wheat varieties, was only achieved when N supply was moderate (at least 1.2 g N per pot). Considering the storage protein composition, the split N application influenced the proportion of α‐/β‐gliadins and γ‐gliadins, the alterations being inconsistent. The ratio of high molecular weight (HMW) to low molecular weight (LMW) glutenin subunits was increased by the split N application only at the lowest N fertilization level. It is concluded that splitting N fertilization into three doses and hence applying one dose of N late in the season can still be a useful approach to improve GPC as well as protein composition – especially when the total N supply is low.     

Proteolysis in model sourdough fermentations

Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker's yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits.     

Compared use of HPLC and FZCE for cluster analysis of Triticum spp and for the identification of T. durum adulteration

Wheat quality criteria continually evolve in response to market pressure and consumer preference. Characterization of cereal cultivars for quality and agronomic properties, have widely shown the importance of the protein content to ensure good quality products. The aim of this work is a comparison of reversed-phase high performance liquid chromatography (RP-HPLC) and free zone capillary electrophoresis (FZCE) in the identification of Italian wheat cultivars and detection of durum wheat flour adulteration. Mainly alcohol soluble (gliadins) and water soluble (albumins) proteins were extracted from 14 common wheat cultivars and from 9 durum wheat cultivars. In RP-HPLC chromatograms, wheat albumins and gliadins eluted between 3 and 9 min and between 10 and 42 min, respectively. Even if the chosen chromatographic conditions (reversed phase) did not permit a complete resolution of hydrophilic proteins such as albumins, a good reproducibility was observed for both albumins and gliadins. In FZCE electropherograms, wheat albumins and gliadins migrated between 8 and 14 min and 16-25 min, respectively. A good reproducibility was found for wheat albumins, while the relatively poor reproducibility of gliadin fractions was a consequence of the selected separation conditions aimed to separate in the same run either hydrophilic (albumins) and alcohol-soluble (gliadins) proteins. The principal component analysis (PCA) of HPLC and FZCE data evidenced that both techniques allowed the univocal identification of the great proportion of investigated wheat cultivars. Three peaks were exclusively detected in RP-HPLC chromatograms of common wheat cultivars, while three unique peaks were found in FZCE electropherograms of common wheat cultivars. These peaks were investigated as a basis for detecting and estimating the adulteration of durum wheat flour with flour from common wheat. The direct relationship between the area of the peaks and adulteration level enabled standard curves to be constructed. The standard curves showed that adulteration may be quantified by either RP-HPLC or FZCE.     

Correlation of Quality Parameters with the Baking Performance of Wheat Flours

The baking performance of a set of flours from 13 wheat cultivars was determined by means of two different microscale baking tests (10 g of flour each). In the micro‐rapid‐mix test the dough was mixed for a fixed time at a high speed, whereas the microbaking test used mixing to optimum dough consistency in a microfarinograph. Quality parameters such as sedimentation value, crude protein content, dough and gluten extension data, and microfarinograph data were also determined. Finally, quality‐related protein fractions (gliadins, glutenins, SDS‐soluble proteins, and glutenin macropolymer) were quantitated by extraction/HPLC methods with reversed‐phase and gel‐permeation columns. All quality parameters were correlated with the bread volumes of both baking tests. The results demonstrated that the microbaking test (adapted mixing time) was much more closely related to the quality parameters than the micro‐rapid‐mix test (fixed mixing time), which hardly showed any correlation. Among the standard quality parameters, only the crude protein content showed a medium correlation with the bread volume of the microbaking test (r = 0.71), whereas the contents of gliadins (r = 0.80), glutenins (r = 0.76), and glutenin macropolymer (r = 0.80) appeared to be suitable parameters to predict the baking performance of wheat flour. All other quality parameters were not or were only weakly correlated and unsuitable for predicting baking performance.     

Effect of ascorbic acid in dough: reaction of oxidized glutathione with reactive thiol groups of wheat glutelin

The reactions of oxidized glutathione generated from endogenous glutathione by the addition of ascorbic acid (AA) prior to dough mixing on free thiol groups of gluten proteins have been investigated. A small amount of (35)S-labeled glutathione was added as a tracer to identify the reaction products of GSSG and free protein thiols by radioactivity measurement. First, gluten was isolated from the dough, then the gliadins were extracted, and residual glutenin was partially hydrolyzed with thermolysin. After preseparation by gel permeation chromatography, the fractions with the highest radioactivity were separated by high-performance liquid chromatography. Radioactive peptides were identified, isolated, sequenced, and assigned to amino acid sequences of gluten protein components. The isolated peptides contained exclusively the cysteine residues C(b) and C(x) of low molecular weight subunits of glutenin, which are supposed to be highly reactive in forming intermolecular disulfide bonds. From these results it can be assumed that the cysteine residues C(b) and C(x) of the low molecular weight subunits of glutenin are at least partly present in the thiol form in flour. During dough mixing they are converted to protein-protein disulfides or glutathione-protein mixed disulfides by thiol/disulfide interchange reactions. Oxidized glutathione necessary for this reaction is generated from glutathione by the action of AA. These results are in accordance with the major hypothesis about the mechanism of action of AA.