Effects of Temperature on Sorption of Water by Wheat Gluten Determined Using Deuterium Nuclear Magnetic Resonance

The effects of lipids and residual starch components of wheat flour gluten on gluten hydration properties were investigated using nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) techniques. Whole or native, lipid-free, starch-free, and lipid- and starch-free gluten samples were prepared from wheat (Triticum aestivum) cv. Mercia. ²H NMR relaxation on gluten samples hydrated with deuterium oxide (D₂O) was measured over a 278–363 K temperature range. FTIR spectra were recorded in dry and fully hydrated material. Transverse relaxation (T₂) results indicated that all four gluten samples were hydrophilic in nature. There was little difference in relaxation behavior of whole and lipid-free gluten samples. T₂ values and populations of the relaxation components were very similar in each. The FTIR spectra of both samples showed an increase in extended β-sheet secondary structures on hydration. These results suggest that lipid binding in gluten, if it occurs, has little effect on wheat gluten properties. Adding starch to the gluten matrix results in an increase in water sorption on heating that may be attributed to the effects of starch gelation. However, the whole water uptake of the gluten cannot be accounted for by the contribution of the residual starch, as estimated by the effects of added starch. Extraction of residual starch required solubilization of the protein, including breaking of the disulfide bonds. This process altered the gluten structure and properties. Light microscope investigation showed that glutens with residual starch extracted were unable to form fibrillar strands on hydration. NMR and FTIR results showed greater water sorption in both samples with extracted starch than in the unextracted samples.     

Impact of Puroindolines on Semisweet Biscuit Quality: A Fractionation–Reconstitution Approach

Puroindoline (PIN) proteins are a factor determining wheat kernel endosperm texture. Biscuits are preferably made from flour from soft wheat (Triticum aestivum L.). Such wheat contains relatively high levels of wild‐type PINs, the impact of which on biscuit quality is unclear. We here studied the impact of PINs on biscuit texture using model flour samples reconstituted from starch and gluten fractions with varying PIN levels. These were obtained by fractionating flour from soft or durum wheat containing either wild‐type or no PINs, respectively. This approach allowed largely retaining the interaction between PINs and either starch or gluten, such as it exists in flour. High PIN levels enhanced air incorporation during dough preparation, increased dough (lateral) expansion, and yielded larger biscuits with higher porosity, which was mainly because of the larger pores. Biscuit fracture stress negatively correlated with PIN level. Porosity contributed to biscuit mechanical properties, but PINs also affected biscuit matrix strength, which in turn affected fracture stress. PINs seem to exert their softening effect when present above a threshold level, and then they have a stronger impact on biscuit fracture stress than the wheat endogenous lipids.     

Surface Lipids Play a Role in the Interaction of Puroindolines with Wheat Starch and Kernel Hardness

Kernel hardness is an important quality characteristic of common wheat. In this study, we investigated the role of starch surface lipids on the interaction of puroindoline proteins and starch granules through in vitro starch–protein binding experiments and flour reconstitution. SDS‐PAGE showed that there were no puroindoline proteins on the starch granule surface when surface lipids were removed or when defatted starch was incubated with puroindoline proteins. However, the puroindoline protein bands were present when defatted starch was incubated with lipids followed by purified puroindoline proteins, which indicated that starch surface lipids play a role in the binding of puroindolines to starch granules. The hardness of flour tablets and dough sheets made from reconstituted flour, which combined defatted starch incubated with lipids and puroindolines with gluten, was lower than for the control reconstituted flour, which was made from defatted starch and gluten. The results of scanning electron microscopy also showed that starch granules were embedded in the gluten in the gluten + defatted starch + lipids + puroindolines treatment. These results confirmed that starch surface lipids are involved in the interaction of puroindolines with wheat starch and kernel hardness.     

Influence of Starch and Gluten Characteristics on Rheological Properties of Wheat Flour Gel at Small and Large Deformation

Starch and gluten were isolated from 10 wheat cultivars or lines with varied amylose content. The rheological properties of 30% wheat flour gel, starch gel, and the gel of isolated gluten mixed with common starch were determined in dynamic mechanical testing under shear deformation, creep‐recovery, and compression tests under uniaxial compression. Variation of wheat samples measured as storage shear modulus (G′), loss shear modulus (G″), and loss tangent (tan δ = G″/G′) was similar between flour and starch gels and correlated significantly between flour and starch gel. The proportion of acetic acid soluble glutenin exhibited a significant relationship with tan δ of gluten‐starch mixture gel. The small difference in amylose content strongly affected the rheological parameters of flour gels in creep‐recovery measurement. Wheat flour gel with lower amylose content showed higher creep and recovery compliance that corresponded to the trend in starch gel. Compressive force of flour gel at 50 and 95% strain correlated significantly with that of starch gel. Gel mixed with the isolated gluten from waxy wheat lines appeared to have a weaker gel structure in dynamic viscoelasticity, creep‐recovery, and compression tests. Starch properties of were primarily responsible for rheological changes in wheat flour gel.     

Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine

Recently, wheat gluten has been proposed as technological adjuvant in order to clarify wines. However, the possibility that residual gluten proteins remain in treated wines cannot be excluded, representing a hazard for wheat allergic or celiac disease patients. In this work, commercial wheat glutens, in both partially hydrolyzed (GBS-P51) and nonhydrolyzed (Gluvital 21000) forms, were used as fining agents in red wine at different concentrations. Beside immunoenzymatic analyses using anti-gliadin, anti-prolamin antibodies and pooled sera of wheat allergic patients, a method based on liquid chromatography coupled to mass spectrometry has been proposed to detect residues of gluten proteins. Residual gluten proteins were detected by anti-prolamin antibodies, anti-gliadin antibodies and sera-IgE only in the wine treated with GBS-P51 at concentration 50, 150, and 300 g/hL, respectively, whereas no residual proteins were detected by these systems in the wine treated with Gluvital 21000. In contrast liquid chromatography-mass spectrometry analyses allowed the detection of proteins in red wines fined down to 1 g/hL of Gluvital 21000 and GBS-P51. Our results indicate that MS methods are superior to immunochemical methods in detecting gluten proteins in wines and that adverse reactions against gluten treated wines cannot be excluded.     

Fractionation and reconstitution experiments provide insight into the role of gluten and starch interactions in pasta quality

Commercial durum wheat (Triticum durum desf.) semolina was fractionated into starch, gluten, and water extractables. Starch surface proteins and surface lipids were removed, and two starches with manipulated granule size distributions were produced to influence starch properties, affecting its interaction with other semolina components. Reconstituted spaghetti was made with untreated (control) or treated starches. The pasta made from the starting semolina material had lower cooking time and was of lower quality than the samples made from reconstituted material. This was not due to changes in gluten properties as a result of the first step of the fractionation process. For the reconstituted samples, starch interaction behavior was not changed after surface protein or surface lipid removal. Starch surface properties thus do not influence the starch interaction behavior, indicating that starch-gluten interaction in raw (uncooked) pasta is mainly due to physical inclusion. All reconstituted pasta samples also had generally the same cooking quality. It was concluded that the small changes in starch gelatinization behavior, caused by the above-mentioned starch modifications, are of little importance for pasta quality.     

A New Method to Study Simple Shear Processing of Wheat Gluten‐Starch Mixtures

This article introduces a new method that uses a shearing device to study the effect of simple shear on the overall properties of pasta‐like products made from commercial wheat gluten‐starch (GS) blends. The shear‐processed GS samples had a lower cooking loss (CL) and a higher swelling index (SI) than unprocessed materials, suggesting the presence of a gluten phase surrounding starch granules. Pictures of dough micro‐structure by confocal scanning laser microscopy (CSLM) showed the distribution of proteins in the shear‐processed samples. This study revealed that simple shear processing could result in a product with relevant cooking properties as compared with those of commercial pasta. Increasing gluten content in GS mixtures led to a decrease in CL and an increase in maximum cutting stress of processed samples, whereas no clear correlation was found for SI values of sheared products. It was concluded that the new shearing device is unique in its capability to study the effect of pure shear deformation on dough development and properties at mechanical energy and shear stress levels relevant to industrial processing techniques like pasta extrusion.     

A new methodology based on GC-MS to detect honey adulteration with commercial syrups

Honey adulterations can be carried out by addition of inexpensive sugar syrups, such as high fructose corn syrup (HFCS) and inverted syrup (IS). Carbohydrate composition of 20 honey samples (16 nectar and 4 honeydew honeys) and 6 syrups has been studied by GC and GC-MS in order to detect differences between both sample groups. The presence of difructose anhydrides (DFAs) in these syrups is described for the first time in this paper; their proportions were dependent on the syrup type considered. As these compounds were not detected in any of the 20 honey samples analyzed, their presence in honey is proposed as a marker of adulteration. Detection of honey adulteration with HFCS and IS requires a previous enrichment step to remove major sugars (monosaccharides) and to preconcentrate DFAs. A new methodology based on yeast (Saccharomyces cerevisiae) treatment has been developed to allow the detection of DFAs in adulterated honeys in concentrations as low as 5% (w/w).     

High pressure liquid chromatographic determination of antihistamine-adrenergic combination products: collaborative study

A reverse phase high pressure liquid chromatographic method in which ion-pairing is used for the determination of combinations of pseudoephedrine hydrochloride with triprolidine hydrochloride or chlorpheniramine maleate in syrups and tablets was collaboratively studied by 8 laboratories. Collaborators were supplied with 12 samples including synthetic and commercial syrup formulations and commercial tablet composites. Mean recoveries of pseudoephedrine hydrochloride and triprolidine hydrochloride from synthetic syrup formulations were 100.5 and 99.6%, respectively. Mean recoveries of pseudoephedrine hydrochloride and chlorpheniramine maleate from synthetic syrups were 98.8 and 100.5%, respectively. Mean coefficients of variation for syrups and tablets ranged from 1.68 to 3.07% for pseudoephedrine hydrochloride, from 2.92 to 3.85% for triprolidine hydrochloride, and from 1.34 to 2.15% for chlorpheniramine maleate. The method has been adopted official first action.     

Globulin Expression in Grain of Australian Hard Wheat Cultivars Is Affected by Growth Environment

Our aim was to study changes in wheat proteomes across different growth locations as the first step in linking protein composition with functional changes in grains produced with commercial production systems. Soluble and insoluble proteins were extracted sequentially from grain of three commercial wheat cultivars grown at four locations in New South Wales, Australia, during a single season. Bands were separated with SDS‐PAGE and identified by peptide mass fingerprinting. Quantitative changes in the electrophoretic patterns were observed mainly in the insoluble polypeptides of molecular mass 40,000–70,000 for all three cultivars grown at two of the four locations. These proteins were identified as mainly globulin and serpin isoforms, as well as triticin. Other proteins with changed expression included disease‐resistance proteins, class III peroxidase, starch branching enzyme I, β‐amylase, and storage proteins. Two‐dimensional electrophoretic analysis was performed on two of the same wheat cultivars grown at one of the locations during two consecutive seasons. Protein spots that varied between seasons consisted of globulin and serpin isoforms, triticin, HMW glutenin, γ‐gliadin, starch branching enzyme IIb, and α‐amylase. The implications of the upregulation of globulin and triticin on whole meal flour quality, through their participation in polymerization of the gluten network, are considered.     

Relationship Between Solvent Retention Capacity and Protein Molecular Weight Distribution,Quality Characteristics,and Breadmaking Functionality of Hard Red Spring Wheat Flour

This research aims to investigate the relationship between the solvent retention capacity (SRC) test and quality assessment of hard red spring (HRS) wheat flour samples obtained from 10 HRS cultivars grown at six locations in North Dakota. The SRC values were significantly (P < 0.05) correlated with flour chemical components (protein, gluten, starch, and damaged starch contents, except arabinoxylan); with farinograph parameters (stability [FST], water absorption, peak time [FPT], and quality number); and with breadmaking parameters (baking water absorption [BWA], bread loaf volume [BLV], and symmetry). Differences in locations and cultivars contributed significantly to variation in quality parameters and SRC values. Suitability of SRC parameters for discriminatory analysis of HRS wheat flour is greatly influenced by molecular weight distribution (MWD) of SDS‐unextractable proteins. SRC parameters, except for sucrose SRC, showed significant (P < 0.01) and positive correlations with high‐molecular‐weight (HMW) polymeric proteins in SDS‐unextractable fractions, whereas only lactic acid SRC exhibited significant (P < 0.01) correlations with low‐molecular‐weight polymeric proteins. HMW polymeric proteins also exhibited positive associations with FPT, FST, BWA, and BLV. The discrepant variation in association of SRC parameters with respect to MWD of SDS‐unextractable proteins could improve segregation of HRS wheat flour samples for quality.     

Ingredient Characterization and Hardening of High‐Protein Food Bars: an NMR State Diagram Approach

A second‐degree simplex lattice mixture design was used to study the effects of soy, dairy, and soy‐dairy blends of powdered proteins in three high‐protein food bar models (sugar syrup, polyol syrup, and reduced‐sugar syrup). Overall protein performance was evaluated based on textural changes during accelerated storage, bar integrity, and dough stickiness and was a strong function of the syrup model used (R² = 92.33%). Nuclear magnetic resonance (NMR) relaxometry was used to measure relaxation times (T₂, T₂*, and T₁) at 20°C and to create state diagrams (temperature, T₂* curves) for the individual powdered proteins and syrups over a temperature range of –35 to 50°C. Increases in relaxation times for powdered protein samples were indicative of better overall protein performance, whereas increases in relaxation times for syrup samples were associated with increases in moisture content and concentration of polyols. Increases in water activity (a_w) of the bars during accelerated storage suggested an elevated rate of hardening for polyol‐containing bars that was caused by a decrease in the amount of water capable of acting as a plasticizer in the product. Proteins were separated into four types (A, B, C, and D) based on the shape of the state diagram curve. Predicted to be the most stable, type D proteins (SUPRO 313 and SUPRO 430) offered the most versatility and, when blended with other proteins, often induced synergistic softening effects in the nutrition bars which led to an extended product shelf life. The NMR state diagram technique appears to be a valuable tool for predicting overall performance of powdered proteins in sugar‐, polyol‐, and reduced‐sugar syrup based food bars.     

Effect of Physical State of Nonpolar Lipids on Rheology and Microstructure of Gluten‐Starch and Wheat Flour Doughs

To clarify the effects of solid fat and liquid oil on dough in more detail in a simpler system, gluten‐starch doughs with different gluten contents were investigated. The results from rheological measurements indicate that dough with a higher starch content has less resistance to strain and dough with a lower starch content has a rubber‐like structure. The effects of the physical state of nonpolar lipids such as fat and oil on gluten‐starch doughs and wheat flour doughs were investigated using rheological measurements and scanning electron microscopy. Fat‐containing dough had more gas cells and a very smooth gluten gel surface with few holes, which may provide higher tolerance to strain. Moreover, the fat seemed to uniformly distribute the gluten gel between the starch granules in the dough, which reduced the friction between starch granules and led to a lower storage modulus. A mechanism governing the effect of fats on loaf volume is proposed based on the phenomena observed in the fat‐containing dough.     

The Biochemical Basis of Celiac Disease

Celiac disease (CD) is an inflammatory disorder of the upper small intestine triggered by the ingestion of wheat, rye, barley, and possibly oat products. The clinical feature of CD is characterized by a flat intestinal mucosa with the absence of normal villi, resulting in a generalized malabsorption of nutrients. The prevalence of CD among Caucasians is now thought to be in a range of 1:100–300. There is a strong genetic association with human leukocyte antigens (HLA‐)DQ2 and DQ8 and currently unknown non‐HLA genes. During the last decade, intense biochemical studies have contributed to substantial progress in understanding the general principles that determine the pathogenesis of CD. The precipitating factors of toxic cereals are the storage proteins, termed gluten in the field of CD (gliadins and glutenins of wheat, secalins of rye, and hordeins of barley). There is still disagreement about the toxicity of oat avenins. The structural features unique to all CD toxic proteins are sequence domains rich in Gln and Pro. The high Pro content renders these proteins resistant to complete proteolytic digestion by gastrointestinal enzymes. Consequently, large Pro‐ and Gln‐rich peptides are cumulated in the small intestine and reach the subepithelial lymphatic tissue. Depending on the amino acid sequences, these peptides can induce two different immune responses. The rapid innate response is characterized by the secretion of the cytokine interleukin‐15 and the massive increase of intraepithelial lymphocytes. The slower adaptive response includes the binding of gluten peptides (native or partially deamidated by tissue transglutaminase) to HLA‐DQ2 or ‐DQ8 of antigen presenting cells and the subsequent stimulation of T‐cells accompanied by the release of proinflammatory cytokines such as interferon‐γ and the activation of matrix metalloproteinases. Both immune responses result in mucosal destruction and epithelial apoptosis. Additionally, stimulated T‐cells activate B‐cells that produce serum IgA and IgG antibodies against gluten proteins (antigen) and tissue transglutaminase (autoantigen). These antibodies can be used for noninvasive screening tests to diagnose CD. The current essential therapy of CD is a strict lifelong adherence to gluten‐free diet. Dietetic gluten‐free foods produced for CD patients underlie the regulations of the Codex Alimentarius Standard for Gluten‐Free Foods. The “Draft Revised Codex Standard” edited in March 2006 proposes a maximum level of 20 mg of gluten/kg for naturally gluten‐free foods (e.g., based on rice or corn flour) and 200 mg/kg for foods rendered gluten‐free (e.g., wheat starch). Numerous analytical methods for gluten determination have been developed, mostly based on immunochemical assays, mass spectrometry, or polymerase chain reaction. So far, only two enzyme‐linked immunosorbent assays have been successfully ring‐tested and are commercially available. During the last decade, future strategies for prevention and treatment of CD have been proposed. They are based on the removal of toxic epitopes by enzymatic degradation or gene engineering and on blocking parts of the immune system. However, any alternative treatment should have a safety profile competitive with gluten‐free diet.     

Surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients

Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems. In this study, starch surface-associated proteins were extracted from four commercial wheat starches, fractionated by high-performance liquid chromatography and gel electrophoresis, and identified by tandem mass spectrometry analysis. More than 150 proteins were identified, many of which (for example, histones, purothionins, and glutenins) had not been recognized previously as starch-associated. The commercial starches were analyzed by the R-5 enzyme-linked immunosorbent assay method to estimate the amount of harmful gluten protein present. One of these starches had a low gluten content of 7 ppm and actually fell within the range proposed as a new Codex Alimentarius Standard for naturally gluten-free foods (maximum 20 ppm). This low level of gluten indicates that the starch should be especially suitable for use by celiac patients, although wheat starches with levels up to 100 ppm are deemed safe in the proposed Codex standards.     

Farinograph Responses for Wheat Flour Dough Fortified with Wheat Gluten Produced by Cold‐Ethanol or Water Displacement of Starch

The objective of this research was to identify and define mixing characteristics of gluten‐fortified flours attributable to differences in the method for producing the gluten. In these studies, a wheat gluten concentrate (W‐gluten) was produced using a conventional process model. This model applied physical water displacement of starch (dispersion and screening steps), freeze‐drying, and milling. W‐gluten was the reference or “vital” gluten in this report. An experimental W‐concentrate was produced using a new process model. The new model applied coldethanol (CE) displacement of starch (dispersion and screening steps), freeze‐drying, and milling. Freeze‐drying was used to eliminate thermal denaturation and thereby focus on functional changes due only to the separation method. The dry gluten concentrates were blended with a weak, low‐protein (9.2%), soft wheat flour and developed with water in a microfarinograph. We found that both water and cold‐ethanol processed gluten successfully increased the stability (St) and improved mixing tolerance index (MTI) to create in the blended flour the appearance of a breadbaking flour. Notably, in the tested range of 9–15% protein, the St for CE‐gluten was always higher then the St for W‐gluten. Furthermore, the marginal increase in St (slope of the linear St vs. protein concentration) for the CE‐gluten was ≈57% greater than that for the W‐gluten. The slope of the MTI vs. protein data was lower for the CE‐gluten by 24%. Flour fortified with CE‐gluten exhibited higher water absorption (up to 1.8% units at 13.5% P) than flour fortified with W‐gluten.     

Incubation of Isolated Wheat Starch with Proteolytic or Lipolytic Enzymes and Different Extraction Media Reveals a Tight Interaction Between Puroindolines and Lipids at Its Granule Surface

Differences in hardness of wheat cultivars have been related to differences in interactions between the starch granule surface and the gluten protein matrix that are mediated by the proteins puroindoline (PIN) A and B. We examined whether or not PINs and (polar) lipids are associated at the starch granule surface, and, if so, how they interact with the starch granule surface itself. Starch was isolated from a soft wheat cultivar containing both wild‐type PINs and incubated with peptidases or lipases, or in extraction media (typically used for defatting). Protein, PIN, and lipid levels revealed that PINs and lipids are tightly associated together at the starch granule surface. Our results imply that PINs need lipids for binding to the granule surface but not vice versa.     

Mixograph Responses of Gluten and Gluten‐Fortified Flour for Gluten Produced by Cold‐Ethanol or Water Displacement of Starch from Wheat Flour

The total protein of gluten obtained by the cold‐ethanol displacement of starch from developed wheat flour dough matches that made by water displacement, but functional properties revealed by mixing are altered. This report characterizes mixing properties in a 10‐g mixograph for cold‐ethanol‐processed wheat gluten concentrates (CE‐gluten) and those for the water‐process concentrates (W‐gluten). Gluten concentrates were produced at a laboratory scale using batter‐like technology: development with water as a batter, dispersion with the displacement fluid, and screening. The displacing fluid was water for W‐gluten and cold ethanol (≥70% vol, ‐12°C) for CE‐gluten. Both gluten types were freeze‐dried at ‐10°C and then milled. Mixograms were obtained for 1) straight gluten concentrates hydrated to absorptions of 123–234%, or 2) gluten blended with a low protein (9.2% protein) soft wheat flour to obtain up to 16.2% total protein. The mixograms for gluten or gluten‐fortified flour were qualitatively and quantitatively distinguishable. We found differences in the mixogram parameters that would lead to the conclusion of greater stability and strength for CE‐gluten than for W‐Gluten. Differences between the mixograms for these gluten types could be markedly exaggerated by increasing the amount of water to the 167–234% range. Mixograms for evaluation of gluten have not been previously reported in this hydration range. Mixograms for fortification suggest that less CE‐gluten than W‐gluten would be required for the same effect.     

Effects of Transglutaminase Enzyme on Fundamental Rheological Properties of Sound and Bug‐Damaged Wheat Flour Doughs

Transglutaminase (TG) catalyzes the formation of nondisulfide covalent crosslinks between peptide‐bound glutaminyl residues and ∊‐amino groups of lysine residues in proteins. Crosslinks among wheat gluten proteins by TG are of particular interest because of their high glutamine content. Depolymerization of wheat gluten proteins by proteolytic enzymes associated with bug damage causes rapid deterioration of dough properties and bread quality. The aim of the present study was to investigate the possibility of using TG to regain gluten strength adversely affected by wheat bug proteases. A heavily bug‐damaged (Eurygaster spp.) wheat flour was blended with sound cv. Augusta or cv. Sharpshooter flours. Dynamic rheological measurements, involving a frequency sweep at a fixed shear stress, were performed after 0, 30, and 60 min of incubation on doughs made from sound or blended flour samples. The complex moduli (G* values) of Augusta and Sharpshooter doughs blended with 10% bug‐damaged flour decreased significantly after 30 min of incubation. These dough samples were extremely soft and sticky and impossible to handle for testing purposes after 60 min of incubation. To test the possibility of using TG to counteract the hydrolyzing effect of bug proteases on gluten proteins, TG was added to the flour blends. The G* values of TG‐treated sound Augusta or Sharpshooter doughs increased significantly after 60 min of incubation. The G* values of the Augusta or Sharpshooter doughs blended with bug‐damaged flour increased significantly rather than decreased after 30 and 60 min of incubation when TG was included in the dough formulation. This indicates that the TG enzyme substantially rebuilds structure of dough hydrolyzed by wheat bug protease enzymes.