Search Results (18)

During fermentation, bacterial and fungal species synthesize substrate-specific enzymes to obtain nutrients. During this process, potential allergenic products, including immunologically important gluten peptides, can be created. Current protocols for assessing the levels of these peptides often overlook the specific gluten source. In this study, wheat sources provided by commercial enzyme suppliers underwent gluten extraction before being pooled into a Complete Gluten Mix, which then underwent variations of hydrolysis utilizing the digestive enzymes, pepsin and trypsin complexes. The resulting gluten peptide profiles were examined using the Wes automated Western blot system to confirm the presence of small, immunologically relevant gluten peptides. These hydrolysates were further tested for suitability as a relevant calibrant against commercially available ELISA standards. The PT3 calibrant, a hydrolyzed version of the Complete Gluten Mix, was found to be the most suitable, as it contained <50 kDa gluten peptides and gave similar absorbance readings to the majority of ELISA kit standards tested, and overlaid the GlutenTox^® Competitive G12 antibody calibration curve, which was designed against the 33-mer immunogenic peptide from wheat. Additionally, no gluten bands were observed on the Wes for the enzymes of interest, which was confirmed through ELISA analysis. Full article

A lifelong gluten-free diet (GFD) is the only treatment for celiac disease and other gluten-related disorders. Nevertheless, strict adherence to the GFD is often challenging due to concerns about social isolation, risk of gluten contaminations, high cost, poor quality and the taste of gluten-free products. Moreover, although the GFD is effective in achieving mucosal healing, it may lead to dietary imbalances due to nutrient deficiencies over a long period of time. To overcome these issues, several gluten-free wheat flours have been developed to create products that closely resemble their gluten-containing counterparts. Furthermore, given the critical importance of adhering to the GFD, it becomes essential to promote adherence and monitor possible voluntary or involuntary transgressions. Various methods, including clinical assessment, questionnaires, serology for celiac disease, duodenal biopsies and the detection of Gluten Immunogenic Peptides (GIPs) are employed for this purpose, but none are considered entirely satisfactory. Since adherence to the GFD poses challenges, alternative therapies should be implemented in the coming years to improve treatment efficacy and the quality of life of patients with celiac disease. The aim of this narrative review is to explore current knowledge of the GFD and investigate its future perspectives, focusing on technology advancements, follow-up strategies and insights into a rapidly changing future. Full article

Immunogenic peptides from wheat gluten can be produced during digestion, which are difficult to digest by gastrointestinal proteases and negatively affect immune responses in humans. Gluten intolerance is a problem in countries where wheat is a staple food, and a gluten-free diet is commonly recommended for its treatment and prevention. Enzyme approaches for degradation of the peptides can be considered as a strategy for its prevention. Here, we isolated a gluten-degrading bacterium, Bacillus amyloliquefaciens subsp. plantarum, from wheat grains. The culture conditions for enzyme production or microbial use were considered based on gluten decomposition patterns. Additionally, the pH range for the activity of the crude enzyme was investigated. The bacterium production of gluten-degrading enzymes was temperature-dependent within 25 °C to 45 °C, and the production time decreased with increasing culture temperature. However, it was markedly decreased with increasing biofilm formation. The bacterium decomposed high-molecular-weight glutenin proteins first, followed by gliadin proteins, regardless of the culture temperature. Western blotting with an anti-gliadin antibody revealed that the bacterium decomposed immunogenic proteins related to α/β-gliadins. The crude enzyme was active in the pH ranges of 5 to 8, and enzyme production was increased by adding gliadin into the culture medium. In this study, the potential of the B. amyloliquefaciens subsp. plantarum for gluten-degrading enzyme production was demonstrated. If further studies for purification of the enzyme specific to the immunogenic peptides and its characteristics are conducted, it may contribute as a strategy for prevention of gluten intolerance. Full article

Roughly 1% of the global population is susceptible to celiac disease (CD)—inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum—cathepsin L—hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2–4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L—loss of the catalytically competent rotameric state of one of the active site residues, His 275. To “fix” the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people. Full article

Gliadins proteins make up around 30% of total wheat flour proteins. They are involved in many immune disorders affecting an increasing number of people who eat foods made with wheat flour. The triggering factor is the accumulation in the gut of immunogenic peptides derived from incomplete degradation of gliadins by gastric proteases. Previous research has revealed the effectiveness of sourdough-fermentation technology or related lactic acid bacteria in reducing wheat flour allergenic proteins. However, there are no single yeast cultures for producing reduced allergenicity wheat products. This study evaluated sourdough-related yeast Wickerhamomyces anomalus strains for their ability to hydrolyze gliadin proteins. All yeast strains were able to degrade gliadins and use them as carbon and nitrogen sources. The proliferation of the yeast strains depended on the gliadin addition; complete hydrolysis was observed after 24 h. The strain showing higher proteolytic activity fermented, acceptably wheat flour dough. The gliadin content of the leavened dough was reduced by 50%. Bread made from the W. anomalus-fermented dough showed a 78% reduction in immunogenic α-gliadins. 50% of the decrease was attributed to the proteolytic activity of the yeast cells, and the other 35% to the baking process. These results show the potential of the yeast W. anomalus as a starter for reducing immunogenicity wheat products. Full article

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance. Full article

Gluten proteins are responsible for the wheat breadmaking quality. However, gluten is also related to human pathologies for which the only treatment is a gluten-free diet (GFD). GFD has gained popularity among individuals who want to reduce their gluten intake. Tritordeum is a cereal species that originated after crossing durum wheat with wild barley and differs from bread wheat in its gluten composition. In this work, we have characterized the immunogenic epitopes of tritordeum bread and results from a four-phase study with healthy adults for preferences of bread and alterations in the gut microbiota after consuming wheat bread, gluten-free bread, and tritordeum bread are reported. Tritordeum presented fewer peptides related to gluten proteins, CD-epitopes, and IgE binding sites than bread wheat. Participants rated tritordeum bread higher than gluten-free bread. Gut microbiota analysis revealed that the adherence to a strict GFD involves some minor changes, especially altering the species producing short-chain fatty acids. However, the short-term consumption of tritordeum bread does not induce significant changes in the diversity or community composition of the intestinal microbiota in healthy individuals. Therefore, tritordeum bread could be an alternative for healthy individuals without wheat-related pathologies who want to reduce their gluten consumption without harming their gut health. Full article

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD. Full article

Celiac disease (CeD) is an autoimmune enteropathy triggered by immunogenic gluten peptides released during the gastrointestinal digestion of wheat. Our aim was to identify T cell epitope-containing peptides after ex vivo digestion of ancestral (einkorn, spelt and emmer) and common (hexaploid) wheat (Fram, Bastian, Børsum and Mirakel) using human gastrointestinal juices. Wheat porridge was digested using a static ex vivo model. Peptides released after 240 min of digestion were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (HPLC-ESI MS/MS). Ex vivo digestion released fewer T cell epitope-containing peptides from the ancestral wheat varieties (einkorn (n = 38), spelt (n = 45) and emmer (n = 68)) compared to the common wheat varieties (Fram (n = 72), Børsum (n = 99), Bastian (n = 155) and Mirakel (n = 144)). Neither the immunodominant 33mer and 25mer α-gliadin peptides, nor the 26mer γ-gliadin peptide, were found in any of the digested wheat types. In conclusion, human digestive juice was able to digest the 33mer and 25mer α-gliadin, and the 26mer γ-gliadin derived peptides, while their fragments still contained naive T cell reactive epitopes. Although ancestral wheat released fewer immunogenic peptides after human digestion ex vivo, they are still highly toxic to celiac patients. More general use of these ancient wheat variants may, nevertheless, reduce CeD incidence. Full article

While durum wheat is a major food source in Mediterranean countries, storage (i.e., gluten) proteins are however responsible for celiac disease (CD), a serious autoimmune disease that occurs in genetically predisposed subjects. Different gluten epitopes—defined as “immunogenic” (IP) and “toxic” (TP) peptides—are involved in the pathology and their content in wheat grain depends on environmental and genetic factors. Detection of IP and TP is not trivial, and no work has been conducted so far to identify the genomic regions associated with their accumulation in wheat. In the present study, a genome-wide association study was performed on a durum wheat collection to identify marker–trait associations (MTAs) between 5730 high quality SNPs and the accumulation of CD-related peptides and gluten protein composition measured in two consecutive cropping seasons (2015/2016 and 2016/2017). High-molecular-weight glutenin subunits (HMW-GS) were more stable between the two years, and differences in total gluten proteins were mainly due to low-molecular-weight glutenin subunits (LMW-GS) and accumulation of gliadins. In the first instance, association tests were conducted on yellow pigment content (YP), a highly inheritable trait with a well-known genetic basis, and several significant MTAs were found corresponding to loci already known for being related to YP. These findings showed that MTAs found for the rest of the measured traits were reliable. In total, 28 significant MTAs were found for gluten composition, while 14 were found to be associated with IP and TP. Noteworthy, neither significant (−log10p > 4.7) nor suggestive (−log10p > 3.3) MTAs for the accumulation of CD-triggering epitopes were found on Gli-A1/Glu-A3 and Gli-B1/Glu-B3 loci, thus suggesting regulatory rather than structural gene effect. A PBF transcription factor on chromosome 5B, known to be involved in the regulation of the expression of CD-related peptides, was identified among the positional candidate genes in the LD-decay range around significant SNPs. Results obtained in the present study provide useful insights and resources for the long-term objective of selecting low-toxic durum wheat varieties while maintaining satisfactory gluten quality. Full article

Microbial transglutaminase (mTG) is a survival factor for microbes, but yeasts, fungi, and plants also produce transglutaminase. mTG is a cross-linker that is heavily consumed as a protein glue in multiple processed food industries. According to the manufacturers’ claims, microbial transglutaminase and its cross-linked products are safe, i.e., nonallergenic, nonimmunogenic, and nonpathogenic. The regulatory authorities declare it as “generally recognized as safe” for public users. However, scientific observations are accumulating concerning its undesirable effects on human health. Functionally, mTG imitates its family member, tissue transglutaminase, which is the autoantigen of celiac disease. Both these transglutaminases mediate cross-linked complexes, which are immunogenic in celiac patients. The enzyme enhances intestinal permeability, suppresses mechanical (mucus) and immunological (anti phagocytic) enteric protective barriers, stimulates luminal bacterial growth, and augments the uptake of gliadin peptide. mTG and gliadin molecules are cotranscytosed through the enterocytes and deposited subepithelially. Moreover, mucosal dendritic cell surface transglutaminase induces gliadin endocytosis, and the enzyme-treated wheat products are immunoreactive in CD patients. The present review summarizes and updates the potentially detrimental effects of mTG, aiming to stimulate scientific and regulatory debates on its safety, to protect the public from the enzyme’s unwanted effects. Full article

Celiac disease, wheat sensitivity, and allergy represent three different reactions, which may occur in genetically predisposed individuals on the ingestion of wheat and derived products with various manifestations. Improvements in the disease diagnostics and understanding of disease etiology unveiled that these disorders are widespread around the globe affecting about 7% of the population. The only known treatment so far is a life-long gluten-free diet, which is almost impossible to follow because of the contamination of allegedly “gluten-free” products. Accidental contamination of inherently gluten-free products could take place at any level from field to shelf because of the ubiquity of these proteins/grains. Gluten contamination of allegedly “gluten-free” products is a constant threat to celiac patients and a major health concern. Several detection procedures have been proposed to determine the level of contamination in products for celiac patients. The present article aims to review the advantages and disadvantages of different gluten detection methods, with emphasis on the recent technology that allows identification of the immunogenic-gluten peptides without the use of antibodies. The possibility to detect gluten contamination by different approaches with similar or better detection efficiency in different raw and processed foods will guarantee the safety of the foods for celiac patients. Full article

Although ancient, heritage, and modern wheat varieties appear rather similar from a nutritional point of view, having a similar gluten content and a comparable toxicity linked to their undigested gluten peptide, whenever the role of ancient end heritage wheat grains has been investigated in animal studies or in clinical trials, more anti-inflammatory effects have been associated with the older wheat varieties. This review provides a critical overview of existing data on the differential physiological responses that could be elicited in the human body by ancient and heritage grains compared to modern ones. The methodology used was that of analyzing the results of relevant studies conducted from 2010 through PubMed search, by using as keywords “ancient or heritage wheat”, “immune wheat” (protein or peptides), and immune gluten (protein or peptides). Our conclusion is that, even if we do not know exactly which molecular mechanisms are involved, ancient and heritage wheat varieties have different anti-inflammatory and antioxidant proprieties with respect to modern cultivars. It is, therefore, reasonable to assume that the health proprieties attributed to older cultivars could be related to wheat components which have positive roles in the modulation of intestinal inflammation and/or permeability. Full article

Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten that is associated with gastrointestinal issues, including diarrhea, abdominal pain, and malabsorption. Gluten is a general name for a class of cereal storage proteins of wheat, barley, and rye that are notably resistant to gastrointestinal digestion. After ingestion, immunogenic peptides are subsequently recognized by T cells in the gastrointestinal tract. The only treatment for CD is a life-long gluten-free diet. As such, it is critical to detect gluten in diverse food types, including those where one would not expect to find gluten. The utility of liquid chromatography-mass spectrometry (LC-MS) using cereal-specific peptide markers to detect gluten in heavily processed food types was assessed. A range of breakfast products, including breakfast cereals, breakfast bars, milk-based breakfast drinks, powdered drinks, and a savory spread, were tested. No gluten was detected by LC-MS in the food products labeled gluten-free, yet enzyme-linked immunosorbent assay (ELISA) measurement revealed inconsistencies in barley-containing products. In products containing wheat, rye, barley, and oats as labeled ingredients, gluten proteins were readily detected using discovery proteomics. Panels comprising ten cereal-specific peptide markers were analyzed by targeted proteomics, providing evidence that LC-MS could detect and differentiate gluten in complex matrices, including baked goods and milk-based products. Full article

The wheat varietal selection undertaken by breeders in recent decades has been tailored mainly to improve technological and productivity-related traits; however, the latter has resulted in a considerable impoverishment of the genetic diversity of wheat-based products available on the market. This pitfall has encouraged researchers to revalue the natural diversity of cultivated and non-cultivated wheat genotypes in light of their different toxic/immunogenic potential for celiac disease and wheat-allergic patients. In the present investigation, an advanced proteomic approach was designed for the global characterization of the protein profile of selected tetraploid wheat genotypes (Triticum turgidum). The approach combined proteins/peptides sequence information retrieved by specific enzymatic digestions (single and dual proteolytic enzymes) with protein digestibility information disclosed by means of in-vitro simulated human gastroduodenal digestion experiments. In both cases, the peptide pools were characterized by discovery analysis with liquid chromatography high-resolution tandem mass spectrometry, and specific amino acid sequences were identified via commercial software. The peptide list was screened for in silico toxicity/immunogenicity risk assessment, with the aid of various open-source bioinformatics tools for epitopes matching. Given the global information provided by the designed proteomic approach, the in silico risk assessment not only tackled toxicity implication for celiac disease patients, but also scouted for immunogenic sequences relevant for wheat allergic patients, achieving a comprehensive characterization of the protein profile of the selected genotypes. These latter were assessed to encrypt a variable number of toxic/immunogenic epitopes for celiac disease and wheat allergy, and as such they could represent convenient bases for breeding practices and for the development of new detoxification strategies. Full article