Detectable Gluten Research Articles

Effects of transamidation vs. fermentation to reduce the gluten content of the Triticum monococcum wheat cultivar Hammurabi: analysis of biochemical, baking and sensory parameters

SummaryThe Triticum monococcum cultivar Hammurabi has been considered a wheat with reduced gluten toxicity for intolerant people. To further reduce gluten toxicity, we tested two biotechnological treatments on Hammurabi flour, fermentation by endogenous microbiota and transamidation by microbial transglutaminase. After 1 day of fermentation, the detectable gluten content dropped to 51.4% ± 6.4% (R5‐ELISA). Interestingly, microbial hydrolysis did not change the content of the water‐insoluble gliadin fraction. Transamidation of Hammurabi caused a greater reduction of the native gluten to 33.5% ± 3.3% (P < 0.01) and changed its physical properties, as it became water soluble. Interestingly, only dough from transamidated Hammurabi flour could leaven, similarly to the untreated dough, while in fermented Hammurabi dough, the rising activity of the added yeast was inhibited. Baking analysis indicated no substantial difference between transamidated and untreated Hammurabi breads. Descriptive sensory analysis showed that darkness appearance parameters and texture parameters were significantly altered in transamidated bread. Importantly, the treatment did not influence wheaty flavour or cereal odour, two mainly expected organoleptic features. Our data may have important implications in the perspective of manufacturing innovative foods for people predisposed to or suffering from gluten intolerance.

Intestinal fatty acid binding protein is a disease biomarker in paediatric coeliac disease and Crohn’s disease

BackgroundThere is a clinical need to develop biomarkers of small bowel damage in coeliac disease and Crohn’s disease. This study evaluated intestinal fatty acid binding protein (iFABP), a potential biomarker of small bowel damage, in children with coeliac disease and Crohn’s disease.MethodsThe concentration iFABP was measured in plasma and urine of children with ulcerative colitis, coeliac disease, and Crohn’s disease at diagnosis and from the latter two groups after treatment with gluten free diet (GFD) or exclusive enteral nutrition (EEN), respectively. Healthy children (Controls) were also recruited.Results138 children were recruited. Plasma but not urinary iFABP was higher in patients with newly diagnosed coeliac disease than Controls (median [Q1, Q3] coeliac disease: 2104 pg/mL 1493, 2457] vs Controls: 938 pg/mL [616, 1140], p = 0.001). Plasma or urinary iFABP did not differ between patients with coeliac on GFD and Controls. Baseline iFABP in plasma decreased by 6 months on GFD (6mo GFD: 1238 pg/mL [952, 1618], p = 0.045). By 12 months this effect was lost, at which point 25% of patients with coeliac disease had detectable gluten in faeces, whilst tissue transglutaminase IgA antibodies (TGA) continued to decrease. At diagnosis, patients with Crohn’s disease had higher plasma iFABP levels than Controls (EEN Start: 1339 pg/mL [895, 1969] vs Controls: 938 pg/mL [616, 1140], p = 0.008). iFABP did not differ according to Crohn’s disease phenotype. Induction treatment with EEN tended to decrease (p = 0.072) iFABP in plasma which was no longer different to Controls (EEN End: 1114 pg/mL [689, 1400] vs Controls: 938 pg/mL [616, 1140], p = 0.164). Plasma or urinary iFABP did not differ in patients with ulcerative colitis from Controls (plasma iFABP, ulcerative colitis: 1309 pg/mL [1005, 1458] vs Controls: 938 pg/mL [616, 1140], p = 0.301; urinary iFABP ulcerative colitis: 38 pg/mg [29, 81] vs Controls: 53 pg/mg [27, 109], p = 0.605).ConclusionsPlasma, but not urinary iFABP is a candidate biomarker with better fidelity in monitoring compliance during GFD than TGA. The role of plasma iFABP in Crohn’s disease is promising but warrants further investigation.Trial registration: Clinical Trials.gov, NCT02341248. Registered on 19/01/2015.

Catcher of the Rye: Detection of Rye, a Gluten-Containing Grain, by LC-MS/MS.

Rye, wheat, and barley contain gluten, proteins that trigger immune-mediated inflammation of the small intestine in people with celiac disease (CD). The only treatment for CD is a lifelong gluten-free diet. To be classified as gluten-free by the World Health Organization the gluten content must be below 20 mg/kg, but Australia has a more rigorous standard of no detectable gluten and not made from wheat, barley, rye, or oats. The purpose of this study was to devise an LC-MS/MS method to detect rye in food. An MS-based assay could overcome some of the limitations of immunoassays, wherein antibodies often show cross-reactivity and lack specificity due to the diversity of gluten proteins in commercial food and the homology between rye and wheat gluten isoforms. Comprehensive proteomic analysis of 20 rye cultivars originating from 12 countries enabled the identification of a panel of candidate rye-specific peptide markers. The peptide markers were assessed in 16 cereal and pseudocereal grains, and in 10 breakfast cereals and 7 snack foods. One of two spelt flours assessed was contaminated with rye at a level of 2%, and trace levels of rye were found in a breakfast cereal that should be gluten-free based on its labeled ingredients.

Detection of gluten in a pilot-scale barley-based beer produced with and without a prolyl endopeptidase enzyme

ABSTRACT Immunochemical and mass spectrometric methods were used to examine the gluten composition of a gluten-reduced beer produced by brewing with barley malt in the presence of prolyl endopeptidase (PEP) and a final filtration treatment with diatomaceous earth and perlite. The competitive ELISA is generally considered appropriate for the analysis of hydrolysed gluten, but it is not considered a scientifically valid method for the quantification of gluten in fermented or hydrolysed foods due to the lack of an appropriate reference standard. As no single analytical method can capture the spectrum of gluten-derived products in beer, a comprehensive approach was employed to analyse the intact and hydrolysed fractions of gluten with complementary methods. The combination of PEP addition and diatomaceous earth/perlite filtration was more effective at reducing the concentration of detectable gluten than each of the treatments alone. However, gluten proteins and/or polypeptides were observed in filtered, PEP-treated beers using sandwich ELISA methods, western blot, and bottom-up mass spectrometry. In addition, mass spectrometry results showed that the number of hydrolysed gluten peptides was almost unaffected by the filtration process. Gluten peptides that contained potentially immunopathogenic sequences were identified in the filtered PEP-containing beers by MS. Variability in gluten composition was observed between three replicate pilot-scale productions, suggesting that the gluten profile in beer could differ from batch to batch. As there is uncertainty in the detection and quantification of gluten in hydrolysed and fermented foods, characterisation of hydrolysed gluten by complementary analytical methodologies is recommended.

Prospective longitudinal study: use of faecal gluten immunogenic peptides to monitor children diagnosed with coeliac disease during transition to a gluten-free diet.

SummaryBackgroundTreatment for coeliac disease is a lifelong strict gluten‐free diet. Although guidelines recommend regular follow‐up with dietary interviews and coeliac serology, these methods may be inaccurate.AimTo evaluate the usefulness of faecal gluten immunogenic peptides to support the diagnosis and to determine the adherence to the gluten‐free diet in coeliac children.MethodsMulticentre prospective observational study including 64 coeliac children. Faecal gluten peptides, and tissue transglutaminase and deamidated gliadin peptide antibodies were analyzed at diagnosis, and 6, 12 and 24 months thereafter. Gluten consumption was estimated from gluten peptide levels.ResultsMost children (97%) had detectable gluten peptides at diagnosis. On a gluten‐free diet, the rate of gluten peptides increased from 13% at 6 months to 25% at 24 months. Mean estimated gluten exposure dropped from 5543 mg/d at diagnosis to 144 mg/d at 6 months, then increased to 606 mg/d by 24 months. In contrast, deamidated gliadin peptide antibodies normalised and only 20% had elevated tissue transglutaminase antibody by 24 months. The elevation of tissue transglutaminase antibody was more prolonged in patients with detectable gluten peptides (P < 0.05). Nevertheless, absolute levels of tissue transglutaminase antibody had low sensitivity to identify patients with detectable gluten peptides (P > 0.1). Dietitian assessment was only moderately correlated with gluten peptide detection (κ = 0.5).ConclusionsFaecal gluten peptides testing may guide treatment of coeliac disease prior to diagnosis and during the assessment diet adherence. Further studies could determine if early identification of gluten exposure reduces the need for expensive/invasive investigations for non‐responsive coeliac disease. ClinicalTrials.gov Number: NCT02711397.

Detection of Gluten in Gluten-Free Labeled Restaurant Food: Analysis of Crowd-Sourced Data.

Adherence to a gluten-free (GF) diet is the mainstay of therapy for celiac disease. Until now, those wishing to avoid gluten in restaurants had to rely on menu labels, word of mouth, intuition, and restaurant workers' advice, with a relative dearth of supporting data. We used crowd-sourced data from users of a portable gluten detection device to estimate rates of, and identify risk factors for, gluten contamination of supposed GF restaurant foods. We analyzed data from a portable gluten detection device (Nima), collected across the United States during an 18-month period by users who opted to share the results of their point-of-care tests. Data were sorted by region, time of day, median household income in the restaurant's vicinity, restaurant genre, and food items. We used the χ test for bivariate analysis and multiple logistic regression for multivariate analysis to identify predictors of gluten detection in restaurant food. There were 5,624 tests, performed by 804 users, in the examined period. Gluten was detected in 32% of GF labeled foods. Rates of gluten detection differed by meal, with 27.2% at breakfast and 34.0% at dinner (P = 0.0008). GF labeled pizza and pasta were most likely to test positive for gluten, with gluten detected in 53.2% of pizza and 50.8% of pasta samples. On multivariate analysis, GF labeled food was less likely to test positive for gluten in the West than in the Northeast United States (odds ratio 0.80; 95% confidence interval 0.67-0.95). This study of crowd-sourced data suggests that a substantial fraction of GF labeled restaurant foods contain detectable gluten. Although the highly sensitive Nima device may detect gluten at levels <20 parts per million (ppm), leading to gluten exposure of unknown clinical significance, our findings raise a potential concern. In addition, our findings of higher rates of gluten detection in pizza and pasta provide practical data when providing dining strategies for patients with celiac disease.

The Validation of the RIDA®QUICK Gliadin for AOAC Research Institute.

RIDA®QUICK Gliadin is an immuno-chromatographic test for the detection of gluten in foods, on surfaces, and in Cleaning-in-Place (CIP) waters. This test kit has been adopted as Final Action AOAC INTERNATIONAL Official Methods of AnalysisSM 2015.16 for gluten in corn products. The assay is based on the monoclonal antibody R5, which recognizes gluten in wheat, barley, and rye. Four different surfaces were contaminated with a gliadin material and analyzed by a direct swabbing of the surface with the dip-stick. The outcome was an LOD95% concentration of the assay between 1.6 and 3.0μg/100cm2 gluten. For CIP waters that contain cleansing reagents, 100% positive results were obtained for minimum gluten concentration between 50 and 100ng/mL. If the CIP water does not contain these reagents, the minimum detectable gluten level is 10ng/mL. The independent validation study consisted of a method comparison study of recovery from a CIP solution and from a stainless-steel surface. The test kit was evaluated at six different concentration levels for both matrices, with 20 or 30 replicates per concentration level. The probability of detection was calculated for each contamination level. Additionally, the LOD95% concentration was estimated for each matrix analyzed.

Gluten Quantitation in Cosmetic Products by Enzyme-Linked Immunosorbent Assay.

The "gluten-free" final rule, published by the U.S. Food and Drug Administration (FDA) in 2013, is intended to provide safe food choices for people suffering from celiac disease. This final rule is not applicable for cosmetic products. As a result, analytical methods for gluten estimation in cosmetics are warranted to aid in the determination of the presence of gluten in these products. A survey of 36 cosmetic products was conducted to measure gluten content by three different ELISAs. None of the nine gluten-free-labeled cosmetics included detectable gluten. The gluten content in six cosmetics was 15.2-1453.5 ppm by one competitive ELISA, whereas three cosmetics contained 11.7-572.3 ppm gluten when quantitated with two sandwich ELISAs. Gluten or hydrolyzed wheat protein was found as an ingredient in five cosmetics with detectable gluten levels. A poor recovery of gluten was observed for most of the cosmetics in the foundation/blush/powder category by ELISA kits. This finding warrants a need for improved analytical methods for gluten estimation in cosmetics.

Investigation of the effects of food processing and matrix components on the analytical results of ELISA using an incurred gliadin reference material candidate

Disorders induced by cereal proteins (e.g. wheat allergy, celiac disease) are widespread in human population. Since their only effective treatment is the avoidance of the problematic proteins, patients have to be familiar with the composition of food products. For checking special foods produced for them, proper analytical methods are necessary. At the moment, in gluten analysis there are no reference methods and reference materials which model real food matrices. During the production and experimental utilisation of our previously developed reference material candidate, numerous questions emerged. As our model product is a real food matrix, interactions can be present between gluten proteins and other macro and micro components. Fat content of the baked cookies is almost 20%, which might affect the results of ELISA measurements. The detectable gluten content is significantly increasing after the defatting procedure, as a pre-treatment of samples. Moreover, baking is a common food processing step that mig...

Nutritional inadequacies of the gluten‐free diet in both recently‐diagnosed and long‐term patients with coeliac disease

Life-long gluten-free diet (GFD) is the only recognised treatment for coeliac disease (CD). The present study aimed to determine the nutritional adequacy of the 'no detectable gluten' diet. Seven-day prospective food intake was assessed in 55 patients who were adherent to a GFD for more than 2years and in 50 newly-diagnosed age- and sex-matched patients (18-71years, 24% male) studied prospectively over 12months on GFD. Historical precoeliac intake was also assessed in the latter group. Intake was compared with Australian Nutritional Recommendations and the Australian population data. Nutritional intake was similar between groups. Of macronutrients, only starch intake fell over 12months (26% to 23%, P=0.04). Fibre intake was inadequate for all except in diet-experienced men. More than one in 10 of both newly-diagnosed and experienced women had inadequate thiamin, folate, vitamin A, magnesium, calcium and iron intakes. More than one in 10 newly-diagnosed men had inadequate thiamin, folate, magnesium, calcium and zinc intakes. Inadequate intake did not relate to nutrient density of the GFD. Inadequacies of folate, calcium, iron and zinc occurred more frequently than in the Australian population. The frequency of inadequacies was similar pre- and post-diagnosis, except for thiamin and vitamin A, where inadequacies were more common after GFD implementation. Dietary intake patterns at 12months on a GFD are similar to longer-term intake. Dietary inadequacies are common and may relate to habitual poor food choices in addition to inherent deficiencies in the GFD. Dietary education should also address the achievement of adequate micronutrient intake. Fortification of GF foods also need to be considered.

Reduced Chemokine Receptor 9 on Intraepithelial Lymphocytes in Celiac Disease Suggests Persistent Epithelial Activation

Celiac disease is caused by an inappropriate immune response to dietary gluten, with increased epithelial lymphocyte infiltration in the duodenum/jejunum as a hallmark. The chemokine receptor 9 (CCR9) is a small intestinal homing receptor normally found on most mucosal T cells in this organ. Because CCR9 expression appears to be activation dependent, we examined CCR9 on duodenal T cells from untreated and treated (gluten-free diet) patients with celiac disease and healthy controls. Duodenal biopsy specimens and blood samples were obtained for histologic analysis and flow-cytometric CCR9 analysis of isolated lymphocytes. CCR9 expression after activation was studied in peripheral blood T cells from healthy volunteers. The median number of CCR9(+) cells among CD3(+) T cells in epithelium and lamina propria, respectively, was 56% and 48% in controls, 11% and 40% in treated patients, and 1% and 8% in untreated patients. Significant differences occurred between controls and treated or untreated patients in the epithelium but only between controls and untreated patients in the lamina propria (P=.008, all comparisons). No such differences were seen in peripheral blood, but stimulation with phorbol myristate acetate and ionomycin and, to a lesser extent, stimulation via NKG2D reduced the CCR9 expression on blood T cells. CCR9 expression is reduced on epithelial and lamina propria T cells in untreated celiac disease. Down-regulation of CCR9 persists in intraepithelial T cells from well-treated patients. This suggests ongoing immune activation preferentially within the epithelium.

Immunochemical analysis of various foods and food ingredients for detectable gluten content: Implications for wheat-allergic and celiac sprue patients

Rationale To reduce the risk for wheat-allergic and celiac patients while allowing them the maximum number of food products in their diet, determining the presence of wheat gluten in non-wheat-containing, “wheat-free”, “gluten-free” foods/ingredients is necessary. Methods A commercially-available wheat gluten immunoassay kit (lower detection limit is 10 ppm gluten) was used to test a variety of foods. Food samples tested included various non-wheat foods at high risk for cross-contamination with wheat, foods labeled “wheat-free” or “gluten-free”, and foods or ingredients commonly avoided by celiac and wheat-allergic patients that originate from wheat or related cereals, but which have questionable gluten content. At least two different lot numbers of each product were tested. Results Most products with questionable wheat content registered below 10 ppm gluten. Food products labeled as wheat or gluten “free” showed levels ranging from 10 ppm to 277 ppm. Malt syrup and extracts registered between 1800 ppm to 2000 ppm gluten. Wheat starches had gluten content ranging from 10 ppm to >5000 ppm. Conclusions Based on the results of this study, distilled products can likely be ingested by celiac patients without problem. Gums are expected to be safe as well. Wheat starches contain varying levels of gluten. It is not recommended that celiac patients consume wheat starches. Malt syrup and extracts had appreciable amounts of gluten and should not be considered safe for celiac patients. Caution must be taken when choosing and consuming food products labeled “wheat-free” or “gluten-free”, as some can contain appreciable amounts of gluten

Immunochemical analysis of various foods and food ingredients for detectable gluten content: Implications for wheat-allergic and celiac sprue patients

Abstract Rationale To reduce the risk for wheat-allergic and celiac patients while allowing them the maximum number of food products in their diet, determining the presence of wheat gluten in non-wheat-containing, “wheat-free”, “gluten-free” foods/ingredients is necessary. Methods A commercially-available wheat gluten immunoassay kit (lower detection limit is 10 ppm gluten) was used to test a variety of foods. Food samples tested included various non-wheat foods at high risk for cross-contamination with wheat, foods labeled “wheat-free” or “gluten-free”, and foods or ingredients commonly avoided by celiac and wheat-allergic patients that originate from wheat or related cereals, but which have questionable gluten content. At least two different lot numbers of each product were tested. Results Most products with questionable wheat content registered below 10 ppm gluten. Food products labeled as wheat or gluten “free” showed levels ranging from 10 ppm to 277 ppm. Malt syrup and extracts registered between 1800 ppm to 2000 ppm gluten. Wheat starches had gluten content ranging from 10 ppm to >5000 ppm. Conclusions Based on the results of this study, distilled products can likely be ingested by celiac patients without problem. Gums are expected to be safe as well. Wheat starches contain varying levels of gluten. It is not recommended that celiac patients consume wheat starches. Malt syrup and extracts had appreciable amounts of gluten and should not be considered safe for celiac patients. Caution must be taken when choosing and consuming food products labeled “wheat-free” or “gluten-free”, as some can contain appreciable amounts of gluten

Persistent Mucosal Abnormalities in Coeliac Disease Are Not Related to the Ingestion of Trace Amounts of Gluten

Background: It is expected that in patients with coeliac disease the small-bowel mucosal mucosa will return to normal if they adhere to a gluten-free diet (GFD). However, in many this is not the case. This study aims to determine whether this persistent villous atrophy (VA) could be due to continued ingestion of the trace amounts of gluten in `gluten-free' foods, as defined by the WHO/FAO Codex Alimentarius. Methods: Duodenal biopsy specimens from 89 adults with long-standing coeliac disease were examined, and the findings correlated with their form of gluten-free diet. Results: In 51 subjects the duodenal specimen was normal, whereas in 38 there was villous atrophy (partial, 28; subtotal, 8; total, 2). There was no relationship between the presence or absence of VA and ingestion of either a GFD as defined by the Codex Alimentarius (Codex-GFD; 39 patients) or a GFD that contained no detectable gluten (NDG diet; 50 patients). Intraepithelial lymphocyte counts were higher, and lactase levels lower, in subjects with an abnormal biopsy specimen than in those in whom it was normal. However, within each of these biopsy groups there was no difference in these variables between patients on a Codex-GFD and those on an NDG-GFD. IgA antigliadin antibody was detected in 4 of 29 patients on a Codex-GFD and in 3 of 13 on a NDG-GFD (NS). Conclusion: The persistent mucosal abnormalities seen in patients with coeliac disease on a GFD are not due to the ingestion of trace amounts of gluten. The consequences of these abnormalities have yet to be determined.

Dietary Analysis in Symptomatic Patients with Coeliac Disease on a Gluten-Free Diet: the Role of Trace Amounts of Gluten and Non-Gluten Food Intolerances

Background: Whereas many people with coeliac disease (CD) are asymptomatic when consuming a gluten-free diet (GFD), a proportion continues to experience symptoms. The reasons for this are unclear. Methods: Thirty-nine adult members of The Coeliac Society of New South Wales, all of whom had persistent gastrointestinal symptoms despite adhering to a GFD, were evaluated. Dietary analysis indicated that 22 (56%) were consuming a GFD as defined by the WHO/FAO Codex Alimentarius (Codex-GFD), in which foods containing up to 0.3% of protein from gluten-containing grains can be labelled as `gluten free'. The remaining 17 were following a no detectable gluten diet (NDG)-GFD, as defined by Food Standards Australia. All subjects were required to follow a NDG-GFD during the study. Those in whom symptoms persisted after changing from a Codex-GFD and those who entered the study already on a NDG-GFD began an elimination diet followed by open and double-blind challenges to identify specific non-gluten food or food chemical intolerances. Results: Of 22 patients who switched to a NDG-GFD symptoms resolved in 5 (23%) and were reduced in 10 others (45%). Thirty-one subjects commenced the elimination diet. Symptomatic improvement was experienced in 24 (77%). Subsequent food or food chemical challenges resulted in a mean of five positive challenges per individual. Diarrhoea was the most commonly provoked symptom, followed by headache, nausea, and flatulence. Symptoms were especially provoked by amine, salicylate and soy. Conclusion: The consumption of trace amounts of gluten, traditionally allowed in a Codex-GFD, may be responsible for the continuing symptoms seen in some patients with CD. Further investigation for non-gluten food intolerances should follow if symptoms persist after adherence to a NDG-GFD.

Effects of dietary gluten elimination in patients with recurrent minor aphthous stomatitis and no detectable gluten enteropathy

Fifty patients with a history of minor recurrent aphthous stomatitis were entered into a 6-month double-blind controlled trial of a gluten-free diet. After hematologic and biochemical investigations and jejunal biopsy, one half of the group was given a gluten-free diet; the other half served as controls and received a gluten-free diet supplemented by gluten given blind (control diet). Twenty-three patients completed the trial, 11 patients on a gluten-free diet, 12 on the control diet. Four of the 11 on the gluten-free diet and 7 of the 12 on the control diet reported significant benefit in terms of minor recurrent aphthous stomatitis, but there was no statistical difference between the responses. The results fail to support studies that report widespread benefit in aphthae of gluten withdrawal and suggest simply a marked placebo response in the condition.