Compliance with Gluten‐Free Labelling Regulation in the Brazilian Food Industry

Gluten is an important protein complex for baking products found in wheat, rye, barley, and some oat varieties. However, some people need to avoid these grains and their products because they result in gluten‐related disorders. The only treatment for these individuals is to engage in a gluten‐free diet. The objective of this work was to verify if the gluten content of several commercial food products sold in Brazil complied with their labeling. The Méndez ELISA R5 sandwich method was used to analyze 437 samples, and of these, 70% were labeled as gluten‐free, 26% as containing gluten, and 4% not labeled in relation to gluten. The results indicated that 89% of the products labeled as gluten‐free were correctly labeled and 11% were not, which represented a risk for celiac people.     

Improved Protocols for ELISA Determination of Gliadin in Glucose Syrups

Simple modifications of existing protocols for high‐sensitivity detection of gluten proteins by immunochemical methods allowed rapid and sensitive determination of residual gluten in highly viscous samples of glucose and maltose syrups obtained from processing wheat starch. Dilution of the original syrup to no less than 15–20% in solids allowed retention of gluten proteins in a soluble form so that ELISA determination of gliadin was possible without an extraction step in aqueous ethanol. An ultrafiltration step may be added to concentrate residual gluten proteins in the diluted syrup samples and allow a further increase in sensitivity. The results are relevant for quality assessment of wheat starch derived syrups as raw materials for use in gluten‐free foods for celiac individuals.     

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.     

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.     

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.     

Influence of Degree of Protein Aggregation on Mass Transport Through Wheat Gluten Membranes and Their Digestibility—An In Vitro Study

The influence of the network structure of wheat gluten on the barrier properties against enzymes was investigated in vitro. The changes in the network structure were introduced by different temperature treatments. The modifications were assessed with solubility studies of wheat gluten proteins in sodium dodecyl sulfate (SDS). The physical barrier properties of wheat gluten membranes were investigated with transport studies examining the transfer of a model protein with no enzymatic activity (BSA) through gluten membranes. The protein network was an effective barrier for BSA, although lightly cross‐linked films were mechanically instable. Membrane breaks occurred in function of the cross‐linking density (percentage of SDS‐insoluble proteins) after only 24 hr for lightly cross‐linked films (≈30% SDS‐insoluble proteins), while highly cross‐linked membranes (≈80% SDS‐insoluble protein) were tight up to more than 33 days. The digestion experiments of the gluten films with pepsin showed that the hydrolysis of wheat gluten films with >72% of SDS‐insoluble protein was significantly retarded. In conclusion, technological treatments to increase the cross‐linking density of gluten have the potential to slow the digestion of cereal‐based foodstuff and to reduce the degradation rate of composite biomaterials.     

Evaluating the Performance of Gluten ELISA Test Kits: The Numbers Do Not Tell the Tale

A wide variety of enzyme‐linked immunosorbent assays (ELISAs) are commercially available for gluten detection in food, including new formats and assays with antibodies against relevant gluten epitopes. Nevertheless, problems persist to accurately determine the gluten content of products. In this study, the performance of a set of 14 ELISA kits for gluten detection, representative of the current ELISA methods available on the market, was evaluated. These tests were used to determine gluten content in a series of relevant food matrices varying in complexity. Our results show that, currently, there is no single ELISA method that can accurately detect and quantify gluten in all different matrices. This includes the current type I method R5 as recommended by Codex Alimentarius. We conclude that further improvements are urgently needed and recommend focusing on competitive formats, improving extraction methods, and the detection of relevant gluten peptides (in order of priority).     

Textural Comparisons of Gluten‐Free and Wheat‐Based Doughs,Batters, and Breads

Studies were conducted with two newly developed gluten‐free bread recipes. One was based on corn starch (relative amount 54), brown rice (25), soya (12.5), and buckwheat flour (8.5), while the other contained brown rice flour (50), skim milk powder (37.5), whole egg (30), potato (25), and corn starch (12.5), and soya flour (12.5). The hydrocolloids used were xanthan gum (1.25) and xanthan (0.9) plus konjac gum (1.5), respectively. Wheat bread and gluten‐free bread made from commercial flour mix were included for comparison. Baking tests showed that wheat and the bread made from the commercial flour mix yielded significantly higher loaf volumes (P < 0.01). All the gluten‐free breads were brittle after two days of storage, detectable by the occurrence of fracture, and the decrease in springiness (P < 0.01), cohesiveness (P < 0.01), and resilience (P < 0.01) derived from texture profile analysis. However, these changes were generally less pronounced for the dairy‐based gluten‐free bread, indicating a better keeping quality. Confocal laser‐scanning microscopy showed that the dairy‐based gluten‐free bread crumb contained network‐like structures resembling the gluten network in wheat bread crumb. It was concluded that the formation of a continuous protein phase is critical for an improved keeping quality of gluten‐free bread.     

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.     

Degradation of HMW Glutenins During Wheat Sourdough Fermentations

Bakeries use sourdoughs to improve bread properties such as flavor and shelf life. The degradation of gluten proteins during fermentation may, however, crucially alter the gluten network formation. We observed changes that occurred in the HMW glutenins during wheat sourdough fermentations. As fermentation starters, we used either rye sourdough or pure cultures of lactobacilli and yeast. In addition, we incubated wheat flour (WF) in the presence of antibiotics under different pH conditions. The proteolytic activities of cereal and sourdough‐derived proteinases were studied with edestin and casein. During sourdough fermentations, most of the highly polymerized HMW glutenins degraded. A new area of alcohol‐soluble proteins (≈30.000 MW) appeared as a result of the proteolytic breakdown of gluten proteins. Very similar changes were observable as WF was incubated in the presence of antibiotics at pH 3.7. Cereal and sourdough‐derived proteinases hydrolyzed edestin at pH 3.5 but showed no activity at pH 5.5. An aspartic proteinase inhibitor (pepstatin A) arrested 88–100% of the activities of sourdough enzymes. According to these results, the most active proteinases in wheat sourdoughs were the cereal aspartic proteinases. Acidic conditions present in sourdoughs create an ideal environment for cereal aspartic proteinases to be active against gluten proteins.     

Formation of Homopolymers and Heteropolymers Between Wheat Flour and Several Protein Sources by Transglutaminase‐Catalyzed Cross‐Linking

The effect of different protein sources (soy flour, lupin flour, egg albumin, gelatin powder, protein‐rich beer yeast flour) on wheat dough functionality was tested by determining gluten index, texture properties, and thermomechanical parameters. Transglutaminase (TG) was also added to improve the dough functionality by forming cross‐links. The presence of protein sources had a significant effect on the gluten index, with the exception of lupin flour. Gelatin and the presence of TG resulted in significant single effects on the texture properties of the wheat‐protein dough. All the protein sources significantly modified the mixing characteristics of the dough or the thermal behavior. Capillary electrophoresis studies of the water‐soluble, salt‐soluble, and glutenin proteins indicated that interactions were mainly within proteins, thus homologous polymers. Scanning electron microscopy studies of the doughs made from blends of wheat and protein sources doughs supported the formation of heterologous structures in the wheat‐lupin blends. The combination of TG and lupin would be a promising method to be used on the treatment of insect‐damaged or weak flours, to increase the gluten strength.     

Accessibility of Amino Groups in Gluten Proteins Studied by a Combination of Chemical Labeling and Immunochemical Detection

The accessibility of primary amino groups to an external probe in durum wheat gluten proteins in the flour itself, and after the extraction and separation of gluten proteins, was studied by labeling the free amino groups with the hemisuccinate of 2‐(2,4‐dichlorophenyl)‐3‐(1H‐1,2,4‐triazol‐1‐yl) propanol (FF18) and evidencing the label immunochemically. Within the flour, the amino groups were less available to the probe than after extraction, and gliadins were less accessible than glutenins, differences that decrease after solubilization and separation of the proteins. Data are discussed in relation to the structural organization of proteins within gluten.     

Wheat Gluten Polymer Structures: The Impact of Genotype,Environment, and Processing on Their Functionality in Various Applications

For a number of applications, gluten protein polymer structures are of the highest importance in determining end‐use properties. The present article focuses on gluten protein structures in the wheat grain, genotype‐ and environment‐related changes, protein structures in various applications, and their impact on quality. Protein structures in mature wheat grain or flour are strongly related to end‐use properties, although influenced by genetic and environment interactions. Nitrogen availability during wheat development and genetically determined plant development rhythm are the most important parameters determining the gluten protein polymer structure, although temperature during plant development interacts with the impact of the mentioned parameters. Glutenin subunits are the main proteins incorporated in the gluten protein polymer in extracted wheat flour. During dough mixing, gliadins are also incorporated through disulfide‐sulfhydryl exchange reactions. Gluten protein polymer size and complexity in the mature grain and changes during dough formation are important for breadmaking quality. When using the gluten proteins to produce plastics, additional proteins are incorporated in the polymer through disulfide‐sulfhydryl exchange, sulfhydryl oxidation, β‐eliminations with lanthionine formation, and isopeptide formation. In promising materials, the protein polymer structure is changed toward β‐sheet structures of both intermolecular and extended type and a hexagonal close‐packed structure is found. Increased understanding of gluten protein polymer structures is extremely important to improve functionality and end‐use quality of wheat‐ and gluten‐based products.     

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.     

Quantitative and Qualitative Role of Added Gluten on White Salted Noodles

A commercial gluten and glutens isolated from four soft and four hard wheat flours were incorporated into a hard and a soft white flour by replacement to directly determine the quantitative and qualitative role of gluten proteins in making noodles. Gluten incorporation (6%) decreased water absorption of noodle dough by 3%, shortened the length of the dough sheet by 15 and 18%, and increased the thickness of the dough sheet by 18 and 20% in soft and hard wheat flour, respectively. Noodles imbibed less water and imbibed water more slowly during cooking with gluten incorporation, which resulted in a 3‐min increase in cooking time for both soft and hard wheat noodles. Despite the extended cooking time of 3 min, noodles incorporated with 6% gluten exhibited decreases in cooking loss by 15% in soft wheat. In hard wheat flour, cooking loss of noodles was lowest with 2% incorporation of gluten. Tensile strength of fresh and cooked noodles, as well as hardness of cooked noodles, increased linearly with increase in gluten incorporation, regardless of cooking time and storage time after cooking. While hardness of cooked noodles either increased or showed no changes during storage for 4 hr, tensile strength of noodles decreased. There were large variations in hardness and tensile strength of cooked noodles incorporated with glutens isolated from eight different flours. Noodles incorporated with soft wheat glutens exhibited greater hardness and tensile strength than noodles with hard wheat glutens. Tensile strength of cooked noodles incorporated with eight different glutens negatively correlated with SDS sedimentation volume of wheat flours from which the glutens were isolated.     

Microbial Transglutaminase as a Tool to Restore the Functionality of Gluten from Insect‐Damaged Wheat

In some wheat‐growing countries, considerable quantities of commercial wheat are rendered unusable in standard baking because of preharvest damage of the grain by protease‐injecting bugs. In the present study, we studied the ability of transglutaminase (TG) treatment of damaged wheat flour to return the functionality of the gluten network. To confirm the TG cross‐linking, the degree of protein hydrolysis, the amount of free thiol groups, and the electrophoresis properties of glutenin subunits were determined. The effectiveness of the TG treatment on insect‐damaged wheat was analyzed by measuring the dough mixing behavior and the gluten quality. A decrease in the degree of hydrolysis (or free amino groups), a reduction in thiol group concentration, and a decrease of extractable high molecular weight glutenin subunits (HMW‐GS) (measured by high‐performance capillary electrophoresis) confirmed the protein cross‐linking catalyzed by TG, the simultaneous formation of disulfide bonds by the proximity of the cross‐linked polypeptide chains, and the formation of aggregates of high molecular weight. The TG treatment of the damaged wheat flour led to a recovery of the consistograph parameters and gluten index value, and the covalent nature of the bonds ensured the stability of the protein changes.     

Observations on the Quality Characteristics of Waxy (Amylose‐Free) Winter Wheats

Previous investigations have suggested waxy (amylose‐free) wheats (Triticum aestivum L.) possess weak gluten properties and may not be suitable for commercial gluten extraction. This limitation could prevent the use of waxy wheat as a source of unique starch, because gluten is a by‐product of the wheat starch purification process. Fifty waxy wheat lines were used to determine the extent to which gluten protein and other grain quality related traits might vary and, consequently, allow the development of waxy wheat with acceptable gluten properties. Among the waxy lines, significant variation was observed for all measured quality traits with the exception of flour protein concentration. No waxy entries statistically equaled the highest ranking nonwaxy entry for grain volume weight, falling number, flour yield, or mixograph mix time. No waxy lines numerically exceeded or equaled the mean of the nonwaxy controls for falling number, flour yield, or mixograph mix time. For grain and flour protein related variables, however, many waxy lines were identified well within the range of acceptability, relative to the nonwaxy controls used in this study. Approximately 50% of the waxy lines did not differ from the highest ranking nonwaxy cultivar for grain and flour protein concentrations. Forty‐three (86%) of the tested waxy lines were not sig‐nificantly different from the nonwaxy line with the highest mixograph mixing tolerance, 22/50 (44%) of the waxy wheat lines did not differ from the highest ranking nonwaxy line in gluten index scores, and 17/50 (34%) did not differ from the highest ranking nonwaxy line in extracted wet gluten. All waxy experimental lines produced gluten via Glutomatic washing. The quality of the gluten, as measured both by mixograph and gluten index, varied widely among the waxy lines tested. These observations suggest that weak gluten is not a natural consequence of the waxy trait, and waxy cultivars with acceptable gluten properties can be developed.     

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.     

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.