Designing new materials from wheat protein

We recently discovered that wheat gluten could be formed into a tough, plasticlike substance when thiol-terminated, star-branched molecules are incorporated directly into the protein structure. This discovery offers the exciting possibility of developing biodegradable high-performance engineering plastics and composites from renewable resources that are competitive with their synthetic counterparts. Wheat gluten powder is available at a cost of less than dollars 0.5/lb, so if processing costs can be controlled, an inexpensive alternative to synthetic polymers may be possible. In the present work, we demonstrate the ability to toughen an otherwise brittle protein-based material by increasing the yield stress and strain-to-failure, without compromising stiffness. Water absorption results suggest that the cross-link density of the polymer is increased by the presence of the thiol-terminated, star-branched additive in the protein. Size-exclusion high performance liquid chromatography data of molded tri-thiol-modified gluten are consistent with that of a polymer that has been further cross-linked when compared directly with unmodified gluten, handled under identical conditions. Remarkably, the mechanical properties of our gluten formulations stored in ambient conditions were found to improve with time.     

Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates

The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance.     

Celiac disease: how complicated can it get?

In the small intestine of celiac disease patients, dietary wheat gluten and similar proteins in barley and rye trigger an inflammatory response. While strict adherence to a gluten-free diet induces full recovery in most patients, a small percentage of patients fail to recover. In a subset of these refractory celiac disease patients, an (aberrant) oligoclonal intraepithelial lymphocyte population develops into overt lymphoma. Celiac disease is strongly associated with HLA-DQ2 and/or HLA-DQ8, as both genotypes predispose for disease development. This association can be explained by the fact that gluten peptides can be presented in HLA-DQ2 and HLA-DQ8 molecules on antigen presenting cells. Gluten-specific CD4⁺ T cells in the lamina propria respond to these peptides, and this likely enhances cytotoxicity of intraepithelial lymphocytes against the intestinal epithelium. We propose a threshold model for the development of celiac disease, in which the efficiency of gluten presentation to CD4⁺ T cells determines the likelihood of developing celiac disease and its complications. Key factors that influence the efficiency of gluten presentation include: (1) the level of gluten intake, (2) the enzyme tissue transglutaminase 2 which modifies gluten into high affinity binding peptides for HLA-DQ2 and HLA-DQ8, (3) the HLA-DQ type, as HLA-DQ2 binds a wider range of gluten peptides than HLA-DQ8, (4) the gene dose of HLA-DQ2 and HLA-DQ8, and finally,(5) additional genetic polymorphisms that may influence T cell reactivity. This threshold model might also help to understand the development of refractory celiac disease and lymphoma.     

Characterization of gluten processing streams

Corn gluten meal (CGM) is a major coproduct of corn wet milling; it has value because of high protein. However, variation in composition and high P content reduce market value. Data that characterize gluten streams would be helpful in identifying key processing steps that could be modified to improve the quality of CGM and increase processing efficiency. Few data are published in the literature on the detailed composition of gluten processing streams. The objective was to characterize the gluten process streams in a corn wet milling plant.Samples were obtained from one plant over a six month period and analyzed for dry matter (DM), total N (protein), ash and elements. DM and macroelement content of the streams were increased significantly during processing. Ash, priority pollutant elements and microelement concentrations were low and of little concern. About 38% of the N (protein) in light gluten was not recovered in the CGM; most of this was lost at the gluten thickener step into the gluten thickener overflow. Much of the P also was removed at this step. Modification of the gluten thickener overflow to increase N and reduce P could make CGM a more valuable coproduct and improve processing efficiency.     

Effect of gluten on the digestive tract and fat body of Telodeinopus aoutii (Diplopoda)

Adult specimens or larvae of invertebrates used as food for vertebrates are often maintained close to gluten so they might become vectors for cereal proteins. However, the tissues and internal organs can respond differently in animals with different feeding habits. The midgut epithelium might be a first and sufficient barrier preventing uptake and effects of gluten on the whole body, while the fat body is the main organ that accumulates different xenobiotics. Good models for such research are animals that do not feed on gluten-rich products in their natural environment. The project's goal was to investigate alterations in the midgut epithelium and fat body of the herbivorous millipede Telodeinopus aoutii (Diplopoda) and analyze cell death processes activated by gluten. It enabled us to determine whether changes were intensified or reversed by adaptive mechanisms. Adult specimens were divided into control and experimental animals fed with mushrooms supplemented with gluten and analyzed using transmission electron microscopy, flow cytometry, and confocal microscopy. Two organs were isolated for the qualitative and quantitative analysis: the midgut and the fat body. Our study of the herbivorous T. aoutii which does not naturally feed on gluten containing diet showed that continuous and prolonged gluten feeding activates repair processes that inhibit the processes of cell death (apoptosis and necrosis) and induce an increase in cell viability.     

Isolation of a Spore Germination Inhibitor from a Cellular Slime Mold Dictyostelium discoideum

The functional properties of gluten obtained by treating with chymotrypsin at alkali pH were investigated. The gluten was treated by chymotrypsin at pH 10.0 and 20°C, and was found to be deamidated to a state that was scarcely subject to proteolysis by chymotrypsin. The degree of deamidation of the gluten reached about 25% by this treatment for 2 hr. The functional properties of the gluten thus obtained were investigated in regard to deamidation. The enzymatically deamidated gluten greatly improved such functional properties as solubility and emulsifying ability. In particular, the solubility of the treated gluten was remarkably high in the pH range of 5 to 8, in which native gluten is insoluble. It was apparent that the improvement in functional properties of gluten was mainly due to the deamidation induced by treating with chymotrypsin at pH 10.0 and 20°C.     

A novel enzyme reactor using gluten membrane entrapping cell-associated enzyme.

A membrane enzyme reactor consisting of variable pieces of replaceable cell-immobilized membranes was proposed for the continuous production of bioproducts. To demonstrate the characteristics of the reactor, cell-immobilized membranes were prepared by the entrapment of permeabilized recombinant Escherichia coli cells containing penicillin G acylase within the gluten matrices. A stainless-steel net that was created with a mesh frame was used to support each gluten membrane so that the membranes could be filled into the rectangular-shaped reactor. The reactor equipped with either six or 12 pieces of cell-immobilized gluten membranes containing a biomass concentration of 5%, w/w was effective in catalyzing the production of 6-aminopenicillanic acid from penicillin G. In comparison with intact cells, the cell-immobilized preparation was more stable and the half-life time of the immobilized cell-associate enzyme in gluten membrane was estimated to be 36 days by a long-term operation. As the substrate solution was forced to flow through the reactor equipped with six membranes and in the direction perpendicular to the membranes, the pressure drop was determined to be less than 50 cm H(2)O with a flow-rate up to 50 mL/min. This low pressure due to the porous structure of gluten membrane would lead to a lower operational cost. Increasing either the number of membranes or the area of each cell-immobilized membrane can easily do scaling-up of this membrane reactor.     

Use of xylan,an agricultural by-product,in wheat gluten based biodegradable films: mechanical,solubility and water vapor transfer rate properties

The possibility of using xylan, as an agricultural by-product, for production of composite films in combinations with wheat gluten was investigated. Different levels of xylan (0-40% w/w) were incorporated into wheat gluten to form biodegradable composite films. Films were prepared at pH 4 and 11, and dried at either uncontrolled or controlled conditions. The mechanical properties, solubilities and water vapour transfer rate (WVTR) of the composite films were studied. Films were obtained with added xylan without decreasing film-forming quality. Xylan can be used as an additive, as much as 40% (w/w), in wheat gluten films. Changing pH, wheat gluten/xylan ratio, xylan type and drying conditions affected mechanical and solubility properties, however, WVTR was not affected by xylan additions. Wheat gluten/xylan composite films having different characteristics can be produced depending on xylan type, composition and process conditions.     

Large-scale characterization of natural ligands explains the unique gluten-binding properties of HLA-DQ2

Celiac disease is an enteropathy caused by intolerance to dietary gluten. The disorder is strongly associated with DQA1*0501/DQB1*0201 (HLA-DQ2) as approximately 95% of celiac patients express this molecule. HLA-DQ2 has unique Ag-binding properties that allow it to present a diverse set of gluten peptides to gluten-reactive CD4+ T cells so instigating an inflammatory reaction. Previous work has indicated that the presence of negatively charged amino acids within gluten peptides is required for specific binding. This, however, only partly explains the scale of the interaction. We have now characterized 432 natural ligands of HLA-DQ2 representing length variants of 155 distinct sequences. The sequences were aligned and the binding cores were inferred. Analysis of the amino acid distribution of these cores demonstrated that negatively charged residues in HLA-DQ2-bound peptides are favored at virtually all positions. This contrasts with a more restricted presence of such amino acids in T cell epitopes from gluten. Yet, HLA-DQ2 was also found to display a strong preference for proline at several anchor and nonanchor positions that largely match the position of proline in gluten T cell epitopes. Consequently, the bias for proline at p6 and p8 facilitates the enzymatic conversion of glutamine into glutamic acid in gluten peptides at p4 and p6, two important anchor sites. These observations provide new insights in the unique ability of HLA-DQ2 to bind a large repertoire of glutamine- and proline-rich gluten peptides. This knowledge may be an important asset in the development of future treatment strategies.     

ESR Studies on Lipid-protein Interaction in Gluten

The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4,4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.     

Microfiltration of gluten processing streams from corn wet milling

In corn wet milling, dry matter can be separated from liquids in process streams with centrifuges or vacuum belt filtration (VBF). Because separations usually are not complete, dry matter can be lost in the liquid streams (overflow from the gluten thickener centrifuge and filtrate from VBF). This represents a loss of nutrients, especially protein, to low valued coproducts and reduces quality of water for recycling within the process. The objective was to compare microfiltration of light and heavy gluten process streams to conventional separation methods. Batches of light and heavy gluten were obtained from a wet mill plant and processed by microfiltration. Samples of permeate and concentrate from microfiltration were analyzed and compared to corresponding streams from wet milling. Microfiltration of light gluten resulted in concentrate and permeate streams similar in composition to conventionally processed light gluten using a centrifuge, suggesting that microfiltration is as effective as centrifugation in partitioning solids and water in light gluten. Dewatering of heavy gluten found that conventional VBF caused dry matter concentrations in gluten cake to be higher than concentrate from microfiltration. Permeate from microfiltration of heavy gluten had higher concentrations of ash and lower soluble nitrogen than filtrate from VBF. Microfiltration was able to remove more ash from concentrate, which may improve the value of wet milling coproducts. These data demonstrated microfiltration has potential for separation of light and heavy gluten streams, but more data are needed on effectiveness and practicality.     

Design of azidoproline containing gluten peptides to suppress CD4+ T-cell responses associated with celiac disease

Celiac disease is an intestinal disease caused by intolerance for gluten, a common protein in food. A life-long gluten-free diet is the only available treatment. As it is well established that the interaction between proline-rich gluten derived peptides and the human HLA-DQ2 molecules induces immune responses that lead to disease development, we have now designed a series of gluten peptides in which proline residues were replaced by azidoprolines. These peptides were found to bind to HLA-DQ2 with an affinity similar to that of the natural gluten peptide. Moreover, some of these peptides were found to be non-immunogenic and block gluten induced immune responses. These can thus serve as lead compounds for the development of HLA-DQ2 blocker peptides.     

Gluten screening of several dietary supplements by immunochromatographic assay

Celiac disease (CD) is a chronic intestinal disorder of public health concern caused by gluten ingestion in sensitive individuals. Gluten is a protein found not only in gluten-containing food but also as normal component of drugs and dietary supplements. Detection of gluten in dietary supplements is a very important task required for establishing their gluten status, which is highly important for the safety of products consumed by CD and gluten-sensitive patients. In this paper, we investigated the presence of gluten in twenty one common dietary supplements from the national market using the immunochromatographic assay. This visual assay proved to be an efficient rapid tool for gluten screening as an alternative to the ELISA techniques. The results have shown the presence of gluten in 23.8% of the investigated samples (vitamins, minerals, plant extracts, probiotics supplements, lactoferrin, propolis supplements). The results provide information which may contribute to the completion of the existing lists of gluten-free pharmaceuticals. It is known that for CD patients obtaining accurate information about the gluten content of a particular item is a difficult and time-consuming process.     

A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid

In this study, a novel generic feedstock production strategy based on solid-state fermentation (SSF) has been developed and applied to the fermentative production of succinic acid. Wheat was fractionated into bran, gluten and gluten-free flour by milling and gluten extraction processes. The bran, which would normally be a waste product of the wheat milling industry, was used to produce glucoamylase and protease enzymes via SSF using Aspergillus awamori and Aspergillus oryzae, respectively. The resulting solutions were separately utilised for the hydrolysis of gluten-free flour and gluten to generate a glucose-rich stream of over 140gl(-1) glucose and a nitrogen-rich stream of more than 3.5gl(-1) free amino nitrogen. A microbial feedstock consisting of these two streams contained all the essential nutrients required for succinic acid fermentations using Actinobacillus succinogenes. In a fermentation using only the combined hydrolysate streams, around 22gl(-1) succinic acid was produced. The addition of MgCO(3) into the wheat-derived medium improved the succinic acid production further to more than 64gl(-1). These results demonstrate the SSF-based strategy is a successful approach for the production of a generic feedstock from wheat, and that this feedstock can be efficiently utilised for succinic acid production.     

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA.     

Reactivity of wheat gluten protein during mechanical mixing: radical and nucleophilic reactions for the addition of molecules on sulfur

Mechanical properties of gluten-based biomaterials, such as break stress, were known to be influenced by temperature and shear stresses applied during processing. It is well documented in literature that these processing parameters promoted wheat gluten protein aggregation. Exchange between disulfide bonds and thiol groups oxidation are the postulated mechanisms that lead to gluten protein solubility loss in sodium dodecyl sulfate buffers. Both nucleophilic and radical reactions were postulated to act during gluten aggregation. To graft molecules on gluten, a study was carried out to explore the reactivity of its thiol and disulfide groups during thermomechanical mixing. A range of reactants able to react via radical or nucleophilic pathways with thiol groups were synthesized. Reactivity between gluten and functions was quantified by gluten solubility measurements. This investigation and literature observations allowed proposal of a general gluten aggregation mechanism during mixing.     

Structural bases of T cell antigen receptor recognition in celiac disease

Celiac disease (CeD) is a human leukocyte antigen (HLA)-linked autoimmune-like disorder that is triggered by the ingestion of gluten or related storage proteins. The majority of CeD patients are HLA-DQ2.5⁺, with the remainder being either HLA-DQ8⁺ or HLA-DQ2.2⁺. Structural studies have shown how deamidation of gluten epitopes engenders binding to HLA-DQ2.5/8, which then triggers an aberrant CD4⁺ T cell response. HLA tetramer studies, combined with structural investigations, have demonstrated that repeated patterns of TCR usage underpins the immune response to some HLADQ2.5/8 restricted gluten epitopes, with distinct TCR motifs representing common landing pads atop the HLA–gluten complexes. Structural studies have provided insight into TCR specificity and cross-reactivity towards gluten epitopes, as well as cross-reactivity to bacterial homologues of gluten epitopes, suggesting that environmental factors may directly play a role in CeD pathogenesis. Collectively, structural immunology-based studies in the CeD axis may lead to new therapeutics/diagnostics to treat CeD, and also serve as an exemplar for other T cell mediated autoimmune diseases.     

Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques

Background and Aims

In a previous report, we characterized a condition of gluten sensitivity in juvenile rhesus macaques that is similar in many respects to the human condition of gluten sensitivity, celiac disease. This animal model of gluten sensitivity may therefore be useful toward studying both the pathogenesis and the treatment of celiac disease. Here, we perform two pilot experiments to demonstrate the potential utility of this model for studying intestinal permeability toward an immunotoxic gluten peptide and pharmacological detoxification of gluten in vivo by an oral enzyme drug candidate.

Methods

Intestinal permeability was investigated in age-matched gluten-sensitive and control macaques by using mass spectrometry to detect and quantify an orally dosed, isotope labeled 33-mer gluten peptide delivered across the intestinal epithelium to the plasma. The protective effect of a therapeutically promising oral protease, EP-B2, was evaluated in a gluten-sensitive macaque by administering a daily gluten challenge with or without EP-B2 supplementation. ELISA-based antibody assays and blinded clinical evaluations of this macaque and of an age-matched control were conducted to assess responses to gluten.

Results

Labeled 33-mer peptide was detected in the plasma of a gluten-sensitive macaque, both in remission and during active disease, but not in the plasma of healthy controls. Administration of EP-B2, but not vehicle, prevented clinical relapse in response to a dietary gluten challenge. Unexpectedly, a marked increase in anti-gliadin (IgG and IgA) and anti-transglutaminase (IgG) antibodies was observed during the EP-B2 treatment phase.

Conclusions

Gluten-sensitive rhesus macaques may be an attractive resource for investigating important aspects of celiac disease, including enhanced intestinal permeability and pharmacology of oral enzyme drug candidates. Orally dosed EP-B2 exerts a protective effect against ingested gluten. Limited data suggest that enhanced permeability of short gluten peptides generated by gastrically active glutenases may trigger an elevated antibody response, but that these antibodies are not necessarily causative of clinical illness.     

Effects of High-voltage Electric Field Treatment on the Water Activity of Bread

Treating bread dough by a high-voltage electric field (HVEF) during the first fermentation enabled the bread dough to retain water in the gluten fibers. The microstructure of the gluten fibers was still visible even with an HVEF treatment at 50 kV for 20 min, but could no longer be observed when the gluten was treated for more than 30 min. The crumb temperature of the HVEF treated bread during baking began to rise a few minutes sooner than that of the untreated bread, but the maximum temperature reached was the same in both cases: 99°C. The fact that the water activity of the HVEF-treated bread was 0.987 ± 0.0056, being higher than that of the untreated bread by 0.011, is in good agreement with the growing tests of Rhizopus nigricans. Furthermore, a distinct decrease in the water loss of the baked gluten was observed after the HVEF treatment. These results suggest that the HVEF treatment increased the ability of the gluten fibers, rather than the starch granules, to absorb and retain water; the state of the remaining water is considered to have become immobile, so that migration of moisture to the starch granules in the bread could be minimized.     

DNA synthesis by jejunal mucosa in responsive and non-responsive coeliac disease.

DNA synthesis by jejunal biopsy specimen from patients with coeliac disease and from controls was measured by an organ culture technique. The rate of synthesis in the mucosa of patients with untreated coeliac disease was almost eight times that in normal mucosa. Patients whose jejunal mucosa remained flat despite prolonged gluten withdrawal showed a rate of DNA synthesis significantly lower than that of the untreated patients, while those whose jejunal mucosa had responded to gluten withdrawal showed a rate similar to that of normal subjects. Impaired enterocyte production in nonresponsive coeliac disease may be responsible for the failure to regenerate villi after gluten withdrawal.