Gluten Degradation Research Articles

Screening of gluten hydrolyzing strains for food applications

Wheat contains 8–20% proteins which include albumins, globulins, gliadins and glutenins. Glutenins are the composite of gluten and glutelins that are stored together with starch in the endosperm. Gluten is found in wheat, barley, rye, oat and related species and is known for its viscoelastic properties. They trigger food-sensitive enteropathies in humans: Celiac Disease (CD), Irritable Bowel Syndrome (IBS), wheat allergy, non-celiac gluten sensitivity (NCGS), and so forth, due to inability to digest gluten. Presently, the only effective treatment is strict gluten-free diet, life-long. However, probiotics are used in varying degrees for treating such disorders. The present study highlights the use of protease producing probiotic strains to degrade gluten proteins thus decreasing its toxicity. From the 25 isolates screened, six (Ich1, Ich3, Ic1, Ic3, Ic4, and Lactobacillus plantarum) showed significant gluten and standard gliadin degradation and Ich1 showed the highest gluten and standard gliadin degradation by 85.32 and 70.0% respectively. This could be attributed to their high protease activity, and these isolates clearly demand further studies on their potentiality as novel dietary supplement for therapeutic application. Novelty Impact Statement In the present investigation, highest gluten degradation of 85.32 and 82.12% were observed with ICh1 and IC4 respectively, which was significant than that of Lactobacillus plantarum. It was found from the present investigation that protease producing probiotic isolates were capable of degrading wheat gluten which was directly proportional to the amount of enzyme.

Gluten Degradation, Pharmacokinetics, Safety, and Tolerability of TAK-062, an Engineered Enzyme to Treat Celiac Disease

Celiac disease (CeD) is an immune-mediated disorder triggered by the ingestion of gluten. Despite adhering to a gluten-free diet (the only management option available to patients with CeD), many patients continue to experience symptoms and intestinal injury. Degradation of immunogenic fractions of gluten peptides in the stomach has been proposed as an approach to reduce toxicity of ingested gluten; however, no enzymes evaluated to date have demonstrated sufficient gluten degradation in complex meals. TAK-062 is a novel, computationally designed endopeptidase under development for the treatment of patients with CeD. Pharmacokinetics, safety, and tolerability of TAK-062 100-900 mg were evaluated in a phase I dose escalation study in healthy participants and patients with CeD. Gluten degradation by TAK-062 was evaluated under simulated gastric conditions invitro and in healthy participants in the phase I study, with and without pretreatment with a proton pump inhibitor. Residual gluten (collected through gastric aspiration in the phase I study) was quantified using R5 and G12 monoclonal antibody enzyme-linked immunosorbent assays. Invitro, TAK-062 degraded more than 99% of gluten (3 g and 9 g) within 10 minutes. In the phase I study, administration of TAK-062 was well tolerated and resulted in a median gluten degradation ranging from 97% to more than 99% in complex meals containing 1-6 g gluten at 20-65 minutes postdose. TAK-062 is well tolerated and rapidly and effectively degrades large amounts of gluten, supporting the development of this novel enzyme as an oral therapeutic for patients with CeD. (ClinicalTrials.gov: NCT03701555, https://clinicaltrials.gov/ct2/show/NCT03701555.).

Prototype Gluten-Free Breads from Processed Durum Wheat: Use of Monovarietal Flours and Implications for Gluten Detoxification Strategies.

In this investigation, we reported the production of prototype breads from the processed flours of three specific Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp. durum genome. The differences of the proteins’ primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.

Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme.

Celiac disease is characterized by a chronic immune-mediated inflammation of the small intestine, triggered by gluten contained in wheat, barley, and rye. Rothia aeria, a gram-positive natural colonizer of the oral cavity and the upper digestive tract is able to degrade and detoxify gluten in vitro. The objective of this study was to assess gluten-degrading activity of live and dead R. aeria bacteria in vitro, and to isolate the R. aeria gluten-degrading enzyme. Methods: After an overnight fast, Balb/c mouse were fed a 1 g pellet of standard chow containing 50% wheat (and 4% gliadin) with or without 1.6 × 107 live R. aeria bacteria. After 2 h, in vivo gluten degradation was assessed in gastric contents by SDS-PAGE and immunoblotting, and immunogenic epitope neutralization was assessed with the R5 gliadin ELISA assay. R. aeria enzyme isolation and identification was accomplished by separating proteins in the bacterial cell homogenate by C18 chromatography followed by gliadin zymography and mass spectrometric analysis of excised bands. Results: In mice fed with R. aeria, gliadins and immunogenic epitopes were reduced by 20% and 33%, respectively, as compared to gluten digested in control mice. Killing of R. aeria bacteria in ethanol did not abolish enzyme activity associated with the bacteria. The gluten degrading enzyme was identified as BAV86562.1, here identified as a member of the subtilisin family. Conclusion: This study shows the potential of R. aeria to be used as a first probiotic for gluten digestion in vivo, either as live or dead bacteria, or, alternatively, for using the purified R. aeria enzyme, to benefit the gluten-intolerant patient population.

Effect of sourdough starter and fungal proteases on gluten content and sensory properties of tarhana

SummaryIn this study, gluten degradation during prolonged tarhana fermentation was monitored in a traditional dough as well as in dough containing sourdough starter, peptidase additives and combination of both. Gluten content, determined using the sandwich ELISA assay method, was reduced significantly in the first 5 days of fermentation for all formulations. After 30 days into fermentation, approximately 15% and 20% reduction of gluten was observed in the traditional dough and sourdough starter added dough, respectively. In enzyme supplemented formulations, the mean gluten levels were below 20 ppm by 20 days of fermentation. Results showed that proteolytic enzyme addition to the dough mix can potentially render long fermented tarhana a food which conforms to regulations set forth for gluten‐free foods, even though wheat flour is one of the main production ingredients. Sensory evaluation of the 30 day fermented tarhana samples revealed differences in sourness and odour properties of the products.

Bacterial-Based Strategies to Hydrolyze Gluten Peptides and Protect Intestinal Mucosa

Gluten is a mixture of proteins highly resistant to hydrolysis, resulting in the emergence of toxic peptides responsible for gluten-related disorders. Currently, a gluten-free diet (GFD) is the unique proven therapy for celiac disease (CD). Several research groups and pharmaceutical companies are developing new nondietetic therapeutic strategies for CD. Probiotics are viable microorganisms thought to have a healthy effect on the host. The proteolytic mechanism of lactic acid bacteria comprises an extracellular serine protease, di- and oligopeptide-specific transport systems, and several intracellular peptidases that might affect gluten degradation. Therefore, probiotic supplementation is an attractive therapy because of its possible anti-inflammatory and immunomodulatory properties. Several studies have been performed to assess the effectiveness of various specific probiotic strains, showing positive effects on immune-modulation (inhibition of pro-inflammatory cytokine TNF-α) restoring gut microbiota and decrease of immunogenic peptides. The present review aims to summarize the current knowledge on the ability of probiotic strain (single or mixtures) to digest gliadin peptides in vitro and to modulate the inflammatory response in the gut.

Shift in bacterial diversity in acidogenesis of gelatin and gluten seeded with various anaerobic digester inocula

The aim of this study was to investigate divergence of bacteria degrading model proteins of food-processing wastewater. Gelatin and gluten were used as substrate to represent animal and plant proteins from food wastes, respectively. The inocula were obtained from eight full-scale anaerobic digestion reactors. Food-to-microorganism ratio was 3 g chemical oxygen demand equivalent of substrate per 1 g volatile suspended solids of inoculum. A first-order reaction model revealed reaction constants ranged 1.34 ≤ k ≤ 2.30 d−1 for gelatin and 0.63 ≤ k ≤ 1.69 d−1 for gluten. Metagenomic analysis of 16s rRNA sequences showed that dominant bacteria after gelatin degradation batch were different for each inocula. Klebsiella aerogenes, Hathewaya, Peptoclostridium, or Clostridium collagenovorans were most abundant. Klebsiella aerogenes was the most abundant species after gluten degradation for all inocula.

Gluten-Degrading Proteases in Wheat Infected by Fusarium graminearum-Protease Identification and Effects on Gluten and Dough Properties.

Recently, we have observed a relationship between poor breadmaking quality and protease activities related to fungal infection. This study aims to identify potential gluten-degrading proteases secreted by fungi and to analyze effects of these proteases on rheological properties of dough and gluten. Fusarium graminearum-infected grain was used as a model system. Zymography showed that serine-type proteases secreted by F. graminearum degrade gluten proteins. Zymography followed by liquid chromatography-mass spectrometry (MS)/MS analysis predicted one serine carboxypeptidase and seven serine endo-peptidases to be candidate fungal proteases involved in gluten degradation. Effects of fungal proteases on the time-dependent rheological properties of dough and gluten were analyzed by small amplitude oscillatory shear rheology and large deformation extensional rheology. Our results indicate that fungal proteases degrade gluten proteins not only in the grain itself, but also during dough preparation and resting. Our study gives new insights into fungal proteases and their potential role in weakening of gluten.

Plant Proteases: From Key Enzymes in Germination to Allies for Fighting Human Gluten-Related Disorders.

Plant proteases play a crucial role in many different biological processes along the plant life cycle. One of the most determinant stages in which proteases are key protagonists is the plant germination through the hydrolysis and mobilization of other proteins accumulated in seeds and cereal grains. The most represented proteases in charge of this are the cysteine proteases group, including the C1A family known as papain-like and the C13 family also called legumains. In cereal species such as wheat, oat or rye, gluten is a very complex mixture of grain storage proteins, which may affect the health of sensitive consumers like celiac patients. Since gluten proteins are suitable targets for plant proteases, the knowledge of the proteases involved in storage protein mobilization could be employed to manipulate the amount of gluten in the grain. Some proteases have been previously found to exhibit promising properties for their application in the degradation of known toxic peptides from gluten. To explore the variability in gluten-degrading capacities, we have now analyzed the degradation of gluten from different wheat cultivars using several cysteine proteases from barley. The wide variability showed highlights the possibility to select the protease with the highest potential to alter grain composition reducing the gluten content. Consequently, new avenues could be explored combining genetic manipulation of proteolytic processes with silencing techniques to be used as biotechnological tools against gluten-related disorders.

Comparison of Small Gut and Whole Gut Microbiota of First-Degree Relatives With Adult Celiac Disease Patients and Controls.

Recent studies on celiac disease (CeD) have reported alterations in the gut microbiome. Whether this alteration in the microbial community is the cause or effect of the disease is not well understood, especially in adult onset of disease. The first-degree relatives (FDRs) of CeD patients may provide an opportunity to study gut microbiome in pre-disease state as FDRs are genetically susceptible to CeD. By using 16S rRNA gene sequencing, we observed that ecosystem level diversity measures were not significantly different between the disease condition (CeD), pre-disease (FDR) and control subjects. However, differences were observed at the level of amplicon sequence variant (ASV), suggesting alterations in specific ASVs between pre-disease and diseased condition. Duodenal biopsies showed higher differences in ASVs compared to fecal samples indicating larger disruption of the microbiota at the disease site. The duodenal microbiota of FDR was characterized by significant abundance of ASVs belonging to Parvimonas, Granulicatella, Gemella, Bifidobacterium, Anaerostipes, and Actinomyces genera. The duodenal microbiota of CeD was characterized by higher abundance of ASVs from genera Megasphaera and Helicobacter compared to the FDR microbiota. The CeD and FDR fecal microbiota had reduced abundance of ASVs classified as Akkermansia and Dorea when compared to control group microbiota. In addition, predicted functional metagenome showed reduced ability of gluten degradation by CeD fecal microbiota in comparison to FDRs and controls. The findings of the present study demonstrate differences in ASVs and predicts reduced ability of CeD fecal microbiota to degrade gluten compared to the FDR fecal microbiota. Further research is required to investigate the strain level and active functional profiles of FDR and CeD microbiota to better understand the role of gut microbiome in pathophysiology of CeD.

Construction of Chimera Prolyl Endopeptidases to Determine Role of Domains in Substrate Size Specificity

Eurygaster integriceps Puton (commonly known as the Sunn pest) is a member of the family Scutelleridae. It is a major pest of wheat and is commonly found in Middle East, northern Africa, Russia, and Eastern Europe (Critchley, 1998). The Sunn pest secretes a proteolytic enzyme, a prolyl endopeptidase (spPEP) into the wheat grains with subsequent degradation of gluten (Darkoh et al., 2010), the major protein in wheat. The spPEP is a unique prolyl endopeptidase in the family of S9 serine proteases because it recognizes large proteins (gluten) as substrates ranging from 30kDa to greater than 140kDa. All other prolyl endopeptidases are purported to bind only peptides shorter than 30 amino acids in length. PEPs consist of two conserved domains, a hydrolase domain which includes the catalytic triad (Ser554, Asp641, and His680) and a seven bladed β‐propeller domain (Juhász et al., 2006). To understand the role with which the two domains have on substrate specificity, chimeras were constructed in which domains were swapped between the sunn pest prolyl endopeptidase (spPEP), mammalian prolyl endopeptidase (hPEP), and the bacteria prolyl endopeptidase (bPEP). Forward and reverse primers identical within the hinge region of either spPEP, hPEP, or bPEP connecting the two domains were used in polymerase chain reaction (PCR) to amplify the vector and the respective domain of each PEP. Ligation independent cloning (LIC) was used to clone the insert DNA into the LIC vector (pNYCOMPS‐LIC‐ccdB‐FH10T+(N‐term) expression vector). The product chimeras include spPEP hydrolase/hPEP β‐propeller, spPEP hydrolase/bPEP β‐propeller, hPEP hydrolase/spPEP β‐propeller, hPEP hydrolase/bPEP β‐propeller, bPEP hydrolase/spPEP β‐propeller, and bPEP hydrolase/hPEP β‐propeller. Each of the chimeras were expressed in BL21 (DE3) pTFe. The cell lysates were used to determine the enzyme activity for each of the chimera using Gly‐Pro‐p‐nitroanalide (GPpNA) as substrate.Support or Funding InformationOffice of Research and Sponsored Programs, Stephen F. Austin State University, and Ed &amp; Gowen Cole, Nacogdoches. Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Randomized clinical trial: Effective gluten degradation by Aspergillus niger-derived enzyme in a complex meal setting

The Aspergillus niger-derived prolyl endoprotease (AN-PEP) has previously been shown to degrade gluten in healthy subjects when added to an intragastrically infused meal. The current study investigated the efficacy of AN-PEP in a physiological meal setting. In this randomized placebo-controlled crossover study, 18 gluten-sensitive subjects consumed a porridge containing 0.5 g gluten together with two tablets either containing a high or low dose of AN-PEP, or placebo. Gastric and duodenal content was sampled over 180 minutes, and areas under the curve of gluten concentrations were calculated. The primary outcome, i.e. success rate of high dose AN-PEP defined as at least 50% gluten degradation compared to placebo in the duodenum, was achieved in 10 of 13 comparisons. In the stomach, gluten levels were reduced from 176.9 (median, interquartile range 73.5–357.8) to 22.0 (10.6–50.8, p = 0.001) in the high dose and to 25.4 μg × min/ml (16.4–43.7, p = 0.001) in the low dose. In the duodenum, gluten levels were reduced from 14.1 (8.3–124.7) in the placebo to 6.3 (3.5–19.8, p = 0.019) in the high dose and to 7.4 μg × min/ml in the low dose (3.8–12.0, p = 0.015). Thus even in a physiological meal setting, AN-PEP significantly degraded most gluten in the stomach before it entered the duodenum.

UEG Week 2017 Oral Presentations

Effective gluten degradation in non-coeliac gluten-sensitive subjects by Aspergillus Niger derived enzyme: A placebo-controlled randomized clinical trial

Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase was estimated to be 77kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co2+ and inhibited by Mg2+, K+ and Ca2+ followed by Zn2+, Na+, Mn2+, Al3+, and Cu2+. The Km and kcat values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides (PQPQLPYPQPQLPY (a-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein)) was also confirmed by enzymatic assays and mass spectrometric analysis of cleavage fragments. Addition of the enzyme during small scale mashing of barley malt reduced the gluten content. The findings here demonstrate the potential of enzyme use during mashing to produce gluten free beer, and provide new insights into the effects of proline specific proteases on gluten degradation.

The human digestive tract has proteases capable of gluten hydrolysis

ObjectiveTo identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD). MethodsSixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides. ResultsThe gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171–466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD. ConclusionThe digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.

Salivary Gluten Degradation and Oral Microbial Profiles in Healthy Individuals and Celiac Disease Patients.

Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission (n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints (n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the α-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli (P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine.IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis.

Novel approaches for enzymatic gluten degradation to create high-quality gluten-free products

Celiac disease (CD), a chronic enteropathy of the small intestine caused by ingestion of gluten, is one of the most prevalent food hypersensitivities worldwide. The essential treatment is a strict lifelong gluten-free diet based on the avoidance of gluten-containing products from wheat, rye, barley and, in rare cases, oats. Products made from naturally gluten-free raw materials often have inferior nutritional, textural and sensory properties compared to the corresponding gluten-containing products. Therefore, the incorporation of wheat, rye and barley flours after efficient removal of the harmful component gluten into gluten-free products would be beneficial. Gluten modification resulting in decreased CD-immunoreactivity may be achieved via the formation of crosslinks using microbial transglutaminase. To effectively eliminate CD-immunoreactivity, plant, fungal, bacterial, animal or engineered peptidases are capable of degrading gluten proteins and peptides into harmless fragments. The application of peptidases from germinated cereal grains, fungal peptidases and/or lactic acid bacteria during food processing yielded high-quality sourdough wheat breads, pasta, wheat starch and bran, rye products and beer, all with gluten contents below the Codex Alimentarius threshold of 20mg/kg for gluten-free products. As with all gluten-free products, the legislative compliance of such treated materials needs to be monitored closely. Provided that all safety requirements are met, gluten-containing raw materials treated in an adequate way to remove CD-active gluten fragments may be used together with naturally gluten-free ingredients to create an extended choice of high-quality gluten-free products.

Gluten degradation in wheat flour with Aspergillus niger prolyl-endopeptidase to prepare a gluten-reduced bread supplemented with an amaranth blend

Celiac disease and other-gluten related disorders are characterized by an inadequate immune reaction against dietary gluten. Some strategies have been developed for gluten modification, to reduce or avoid the immune response. The aim of this study was to modify gluten proteins in wheat flour using the Aspergillus niger prolyl-endopeptidase (AnPEP) to elaborate bread supplemented with amaranth blends, evaluating its immunoreactivity and technological quality. The reaction conditions: enzyme (dilutions 1:50, 1:100 and 1:500), temperature (35 or 40 °C), substrate (WF) (10% or 20%, w/v), time (1–8 h), gluten content by R5-ELISA, and the protein composition by SE-HPLC were analyzed. For bread-making, 60% of modified WF and 40% of amaranth blends (20:80, 30:70, 40:60; raw: popped, respectively), and two fermentation times (52 and 90 min) were tested. Specific volume and gluten content were evaluated. The best reaction conditions were (1:50) AnPEP, 20% WF, and 8 h incubation at 35 °C. The bread supplemented with the 20:80 amaranth blend showed a higher specific volume and after a 52 min fermentation had 99% less immunogenic gluten than the wheat bread. The use of the amaranth blend in combination with WF modified by AnPEP is an effective way to obtain gluten-reduced breads with acceptable quality.

Gluten-specific peptidase activity of barley as affected by germination and its impact on gluten degradation

Endogenous cereal peptidases have gained interest concerning the degradation of gluten in raw materials for the production of gluten-free beer. This study demonstrates that enzymes from barley malt are able to degrade celiac-active peptides PQPQLPYPQPQLPY (P1) and SQQQFPQPQQPFPQQP (P2). The gluten-specific peptidase activity can be significantly increased by systematic variation of the germination parameters moisture, temperature, and time. Statistical analysis using normalized, linear regression including non-linear terms showed different maxima between the substrates. Maximum peptidase activity for P1 (25.6 U/kg malt) was obtained at 48% water content, 18 °C germination temperature, and 6.6 d germination time and for P2 (8.3 U/kg) at 48%/12 °C/8d or 42%/18 °C/8d, respectively, with low correlations between the substrates (r = 0.54, α < 0.05). The degradation of hemoglobin (4567 U/kg; 45%/15 °C/4–8d), which has often been used for determination of hydrolytic activity, showed no correlation with the two peptide substrates. The degree of gluten degradation as affected by the germination time correlated significantly with both peptides (r = −0.78–0.86, α < 0.005) and to a lesser extend with hemoglobin (r = −0.67, α < 0.01). The results might help implementing gluten-specific peptidase activity of barley malt as quality parameter in the field of brewing.

Transport of wheat gluten exorphins A5 and C5 through an in vitro model of intestinal epithelium

The gluten exorphins (GEs) A5 and C5 are peptides with a potential opioid-like activity derived from enzymatic degradation of wheat gluten. In this study, a 70% Caco2/30% HT-29 co-culture layer was used as an in vitro model of human small intestine to investigate the transport of the two GEs and their resistance to brush border peptidases. Thirty, 150 or 600μg of the peptides, GE A5 and GE C5, were added to the apical chamber of the transwells. We previously demonstrated these amounts to be released during in vitro digestion of wheat-derived bread and pasta samples. The solutions in apical and basolateral chambers were analyzed for intact GEs or related fragments by UPLC/HRMS. The two peptides behaved differently since after 120min higher amount of intact GE A5 (0.55–24.60%) compared to GE C5 (0.20–1.00%) was recovered in the basolateral chamber, depending on the initial dose that was added to the transwell apical compartment. The GE C5 was strongly hydrolyzed by cellular peptidases. The fluxes of two GEs through the in vitro intestinal epithelium were concentration- and time-dependent. The transport inhibitors Wortmannin and Glycyl-sarcosine did not significantly affect the apical-to-basolateral fluxes of the two GEs. Differently, Cytochalasin D slightly increased the transepithelial transport of both GEs, and decreased the TEER values of the co-culture cells. These results suggest the paracellular route as a possible transport mechanism of both GEs. Overall, this study indicates the possibility that food-derived GEs can cross the human intestinal epithelium as intact peptides, and improves the knowledge for exploiting the opioid-like activity potentially associated with the ingestion of wheat-derived foods.