Gluten contamination of cereal foods in Canada

Persons suffering from celiac disease (CD) must avoid foods containing gluten or those contaminated with wheat, barley, or rye. This study was designed to estimate gluten contamination of cereal-based foods available in Canada, whether labelled gluten-free or not. About half of the 148 foods sampled were labelled as gluten-free. According to R5-enzyme-linked immunosorbent assay (ELISA), twenty-three cereal-based foods (or 15%) contained more than 20 mg of gluten per kg, including sixteen regular and seven gluten-free foods, the latter being the least contaminated. When used in combination with R5-ELISA, AOAC-ELISA (not detecting barley) was a simple and efficient tool to roughly estimate the nature of the gluten contamination, which was later confirmed by real-time polymerase chain reaction for barley, and wheat/barley/rice. Rice-, corn- or quinoa-based foods were the safest for celiac patients. In addition to misleading food labelling for both gluten-rich and gluten-free foods, critical issues for persons with CD included foods made with oats or buckwheat (contaminated with wheat and barley gluten) in addition to those, such as breakfast cereals, specifically enriched with barley malt ingredients.     

Determination of Prolamins in Beers by ELISA and SDS‐PAGE

Coeliac disease is triggered by exposure to the prolamin protein fraction of wheat, barley, or rye. The prolamin content of five lager beers and one wheat beer were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting and seven lager beers and three wheat beers were analyzed by enzyme‐linked immunosorbent assay (ELISA). Most of the lager beers were made from barley and some had varying amounts of rice or corn as adjuncts. One of the beers was “gluten‐free”, having been produced from corn and buckwheat without barley. The lager beer samples were gel‐filtered before ELISA or SDS‐PAGE analysis. Prolamin proteins were found in all but one beer which was made of corn, rice and barley and which was not the “gluten‐free” beer. ELISA analysis was done using a commercially available gluten assay kit. For lager beers, a barley prolamin standard for ELISA was propanol‐extracted from barley malt instead of using the prolamin standard of the gluten assay kit. As expected, the wheat beers contained much higher amounts of prolamins than the lager beers. The samples were studied by SDS‐PAGE to identify different prolamin fractions. Proteins having a relative molecular mass in the range of 8000–17,000 and 38,000 and above were detected in immunoblotting by the prolamin sensitive antibody in the lager beers.     

Is the calculation of the gluten content by multiplying the prolamin content by a factor of 2 valid?

Presently, the only essential therapy of celiac disease (CD) is the permanent strict withdrawal of gluten from the diet. With respect to gluten containing foods from wheat, rye, barley, and oats, CD patients have to consume surrogates that must be gluten-free according to the “Codex Alimentarius Standard for Gluten-Free Foods”. The recent “Draft Revised Standard” proposes a gluten threshold of 20 mg per kg gluten-free product. For gluten quantitation, the alcohol-soluble prolamins should be extracted and analyzed by an immunochemical method; the amount of gluten is calculated by multiplying the prolamin content by the factor of 2. To investigate, whether this calculation is valid in any case of contamination of gluten-free products by wheat, rye, barley, and oats, wholemeal or white flours from common wheat, spelt, durum wheat, kamut, emmer, einkorn, rye, barley and oats were analyzed for the ratio of prolamins to glutenins (PROL/GLUT) by a combination of extraction and reversed-phase HPLC procedures. Additionally, different industrial wheat starches were analyzed for their prolamin and total gluten content using different extraction and concentration steps followed by gel permeation HPLC. The results for the cereal flours revealed that the ratio PROL/GLUT was generally higher than 1.0 as proposed by the “Draft Revised Standard” and strongly influenced by cereal species and variety. Common wheat showed the lowest ratio (1.5–3.1), followed by oats and spelt (1.7–3.3), barley (1.4–5.0), durum wheat and kamut (3.1–3.4), emmer (3.5–7.6), rye (6.3–8.2), and einkorn (4.0–13.9). In any case, the gluten content of gluten-free products contaminated with CD activating cereals was generally overestimated, when the prolamin content was multiplied by the factor of 2. In extreme cases, e.g., contamination with rye, the overestimation amounted to 72–79%. Completely different PROL/GLUT ratios were found in ten commercial wheat starches ranging from 0.2 to 4.9. Obviously, the quality of wheat cultivars used for starch production and/or different process parameters, e.g., washing steps, influenced the composition of gluten proteins adherent to starch granules. For wheat starch, the calculation of the gluten content by 2 × PROL may either lead to underestimation (−71% at most) or overestimation (+66% at most). In conclusion, this calculation is invalid; therefore, a future task will be the development of immunoassays with antibodies against all types of storage proteins from wheat, rye, barley, and oats.     

Variation in gluten protein and peptide concentrations in Belgian barley malt beers

Gluten is the main family of storage proteins found in barley. During malting and brewing, some of the barley malt's proteinaceous material is hydrolysed into peptides or to amino acids. Most of the gluten proteins are removed with the spent grains and with hot‐ and cold‐breaks. However, some gluten proteins and especially gluten‐derived peptides can remain throughout the brewing process and will hamper the gluten‐free (≤20 ppm) status of the beer. In this work, three production batches (a, b and c) of 51 Belgian barley malt beers from 24 breweries were analysed with the sandwich (R7001) and competitive (R7021) Ridascreen gliadin R5‐ELISA to quantify gluten proteins and peptides. Although the majority of the beers contained low‐gluten protein concentrations of ≤20 ppm (a/45, b/47, c/48), only a minority were truly gluten‐free with ≤20 ppm gluten peptides (a/18, b/17, c/15). The grain bill had no influence on the measured gluten concentration, but the use of (combined) clarification techniques and presence of wheat malt in the grist was respectively a positive and negative influence. Ten beers, from four breweries, were gluten free in all analysed samples. These included two wheat beers, reflecting the importance of effective clarification in the management of gluten. These results explore the feasibility of the production of gluten‐free barley malt beers. Copyright © 2018 The Institute of Brewing & Distilling     

Analysis of “gluten-free” foods for trace amounts of gluten using gel electrophoresis

The presence of gluten in speciality foods for individuals with coeliac condition can be estimated quantitatively in the range of 0.1–1.0 mg gluten nitrogen per gram sample by a specific extraction procedure followed by electrophoresis and densitometry. After preliminary lipid removal by extraction with petroleum spirit (containing 1% formic acid), gliadin is extracted with chloroform–methanol (equal volume mixture). The extract is evaporated to dryness and the residue, suspended in acidic urea solution, is used for starch gel electrophoresis at pH 3.1. The amount of β, γ and ω gliadins is estimated by densitometry. The procedure detects (and distinguishes) gluten-type protein from wheat, rye, triticale, barley and oats. Proteins from maize, soy, egg and milk do not interfere. The method is primarily designed for testing unbaked foods, and is less satisfactory with baked goods.     

Pasting properties of starch and protein in selected cereals and quality of their food products

In an attempt to improve intake of dietary fibre and antioxidants and quality of whole grain products, whole grain meals from barley, millet, rye and sorghum were evaluated individually and in blends with wheat flour in terms of starch pasting properties and protein heat damage, during cycles of heating and cooling in RVA tests. The whole grain meals were blended with either hard or soft wheat flour and processed into bread, cake, cookie or snack products. The products were then evaluated with regard to physical properties and acceptability. Significant differences were observed between cereals in starch peak, breakdown and setback viscosities as well as in protein peak viscosity. The results showed that RVA could be used to help formulate cereal blends with certain pasting properties. Substitution of wheat flour, with 15% of barley, rye, millet or sorghum whole grain, did not have significant detrimental effects on physical properties or acceptability of pita bread. Additionally, replacement of wheat flour with up to 30% of barley, rye, millet or sorghum whole grain meal had no significant effects on quality of cakes or cookies. A multigrain snack-like food was also developed as a healthy product and was highly acceptable in a sensory test. The developed product would help enhance consumption of whole grain foods, resulting in improved intake of fibre and health-enhancing components.     

Gluten weight in ancient and modern wheat and the reactivity of epitopes towards R5 and G12 monoclonal antibodies

Differences in the level of coeliac‐active gluten epitopes in wheat might have some significance for individuals reporting noncoeliac gluten sensitivity. The aim of this study was to compare the reactivity of epitopes towards ELISA R5 and G12 monoclonal antibodies in ancient (emmer; Khorasan wheat; spelt) and modern wheat (common bread wheat; durum), and to check whether the bread‐making process leads to the degradation of epitopes. Data from ELISA R5 and G12 did not match gluten dry weight in wheat. Bread dough fermentation and extensive baking did not change the reactivity of coeliac‐active epitopes towards monoclonal antibodies. Compared to hexaploid bread‐type wheat (spelt; common bread wheat), ancient and modern pasta‐type tetraploid wheat (emmer; Khorasan; durum) had less epitopes reactive towards ELISA R5 and G12 and might be preferable for wheat‐sensitive individuals looking for food with reduced coeliac‐active epitopes.     

A study on malt modification,used as a tool to reduce levels of beer hordeins

Storage proteins from barley, wheat and rye are toxic to gluten sensitive consumers. These consumers include those suffering from coeliac disease, which account for up to 1% of the global population, and non‐coeliac gluten sensitivity that may affect even greater numbers of the population. Codex Alimentarius has published guidelines and limits of gluten in gluten‐free foods, which are applied in Europe, and similar guidelines apply in the rest of the world. The storage proteins present in barley are hordeins. These proteins are broken down and used by the plant as a source of amino acids during germination and growth of the barley embryo. The objective of this study was to extend the germination stage of the malting process and look at the effect on beer hordeins. Standard MEBAK methods were used to develop an extended malting process and produce three different malts, germinated for 3, 5 or 7 days. The quality of malt was assessed and model beers were produced from each malt to test the effect of modification on levels of beer hordeins. Malt germinated for 7 days produced beer 18 mg/kg hordeins corresponding to a reduction of 44% compared with the beer made from malt germinated for 3 days characterized by a hordein content equal to 32 mg/kg. The malting loss was increased during the 7 days of germination but otherwise all malts were of high quality. The results showed that malting conditions have a significant impact on beer hordeins. Copyright © 2018 The Institute of Brewing & Distilling     

高粱食品:一种古老作物带来新的健康

高粱是一种古老的作物初次被驯化于非洲,并在公元前3000年左右推广到了世界各地.高粱是一种抗旱的谷类作物,生长在其它作物无法生存的半干旱气候条件下,在2002年世界高粱产量达到5450万公吨.美国高粱年产量可达1300～1500万公吨左右,其中30%～50%用于出口,在西方国家中用高粱喂养动物已非常普遍,大约世界高粱产量的40%被用于作为非洲和印度等国家人民的食物.在美国对于患有腹乳糜泻疾病无法适应面筋蛋白的人已经用白色食品级的杂交作物生产出运用于无小麦产品作为食用,不能食用小麦或其它谷类作物比如黑麦,大麦,他们提供了些刺激性小的面粉已成为用于制造无小麦食品的原料.当高粱缺乏面筋时,高粱粉便不能生产有黏性和弹性的生面团,因此蛋奶糊配方被用于制造高粱产品,包括面包威化饼干、面条和比萨饼皮,这些原来都是由面粉制成.研究表明,高粱系生产高质量食物,因此种植高梁将被用于改善提高食物质量,最近一些研究表明由于这种古老作物包括含有二十烷醇(policosanols)的多酚复合物和高粱蜡的高抗氧化水平,可能对健康有独特的益处,对于保护心血管的健康至关重要.尤其在把小麦作为主要食物的西方世界,对于给患有腹乳糜泻疾病的人们提供了健康稳定的主食是一个挑战.因此高粱这种古老的作物作为21世纪的主要谷类作物引来了新的兴趣和前途,这是由于(1)它对于患有腹乳糜泻疾病的人是一种富有潜力的主食.(2)保持食物的营养和健康的高抗氧化水平和蜡含量.(3)由于它的抗干旱性和有限供水量的高生产量,2030年预期人口指数增长明显.     

Real Time PCR for the detection and discrimination of cereal contamination in gluten free foods

Real-time PCR methods, using melting curve analysis for product identification, were established for the specific discrimination of wheat, rye, barley and oats in food samples. Specific primers targeting cereal prolamin genes were chosen for the amplification. The lengths of the amplicons varied between 104 and 181 base pairs and the melting point between 81.2 and 85.0 °C. The specificity of the wheat PCR was shown for spring and autumn wheat but also for durum wheat, spelt wheat and kamut. The methods were applied for final food products and for detection of toxic cereal contamination in oat samples. The results of the analysis were compared with those obtained with an established enzyme immuno assay for gluten analysis. The PCR methods give a good correlation with the protein assay and are rapid and sensitive. The PCR methods can thus be used as confirmatory methods in food analysis of gluten-free and naturally gluten-free foods.     

The nutritive value for ruminants of thin stillage and distillers' grains derived from wheat,rye, triticale and barley

A study was conducted to determine nutrient degradabilities of thin stillages and distillers' grains derived from wheat‐, rye‐, triticale‐ and barley‐based ethanol production. In vitro protein degradabilities of wheat, rye, triticale and barley thin stillages were determined using a protease enzyme assay. One ruminally fistulated cow was used to determine ruminal nutrient degradabilities for wheat, rye, triticale and barley distillers' grains. Results of the in vitro study showed that the soluble protein fraction was highest for rye thin stillage and lowest for barley thin stillage. The degradation rate of the slowly degradable protein fraction was higher for wheat and triticale thin stillage than rye thin stillage and was higher for rye than barley thin stillage. Effective degradability of crude protein followed the order rye (659 g kg⁻¹) > triticale (632 g kg⁻¹) > wheat (608 g kg⁻¹) > barley (482 g kg⁻¹) thin stillage. Ruminal degradability of dry matter was highest for rye and lowest for barley distillers' grains. Ruminal degradability of dry matter was also higher for wheat than triticale distillers' grains. Crude protein from barley distillers' grains had a lower ruminal degradability relative to crude protein from wheat and rye distillers' grains. Ruminal degradability of neutral detergent fibre was highest for rye distillers' grains (470 g kg⁻¹), intermediate for wheat and triticale distillers' grains (average 445 g kg⁻¹) and lowest for barley distillers' grains (342 g kg⁻¹). It was concluded that thin stillage and distillers' grains derived from barley had a lower nutritive value for ruminants compared with those derived from wheat, rye and triticale. © 2000 Society of Chemical Industry     

Gluten contamination in oat products and products naturally free from gluten

Some countries now allow celiac patients to include oats in their gluten-free diet. Studies have shown that commercially available oats can be contaminated with gluten. The aim of the study was to analyse oat products and products naturally free from gluten to determine the degree of contamination. A total of 88 oat products and 22 products based on maize, rice, millet or buckwheat were analysed, using a commercially available enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody to gliadin. The ELISA is quantitative with a detection limit of 20 mg/kg gluten. Some of the positive samples were also analysed for the presence of DNA from wheat, barley or rye. Thirteen percent of the oat products had gluten content over 200 mg/kg. Of the products naturally free from gluten 14% had a gluten content over 200 mg/kg. There was a tendency for higher levels of contamination with increased processing. The risk of gluten contamination in oat products as well as in non-gluten cereals must be considered. The level of contamination was in most cases low. The results regarding oats can be compared to those of such cereals as maize, rice, buckwheat and millet.     

Kernel‐based gluten contamination of gluten‐free oatmeal complicates gluten assessment as it causes binary‐like test outcomes

Gluten‐free (GF) foods, whose claim compliance is controlled at the ‘serving level’, hold better chances of protecting gluten‐intolerant consumers. This is particularly true for GF oatmeal, as oats are easily contaminated with gluten‐rich kernels of wheat, rye and barley, which remain intact to the spoon as pill‐like flakes. A single contaminant kernel in otherwise pure oats results in GF labelling noncompliance, thereby posing a risk to patients with coeliac disease. Our in‐market survey of 965 GF oatmeal servings uncovered that one in fifty‐seven servings exceeded the GF labelling maximum of 20 mg kg^?1 (i.e. 20 ppm). The noncompliance pattern was ‘binary‐like’, with kernel‐based contamination the suspected pass/fail driver. We have highlighted probabilities of misassessment for various sample sizes in light of oat's natural propensity for kernel‐based contamination and proposed use of attribute‐based sampling for compliance assessment, thereby providing a way to assess/manage/control ‘rates of servings containing a contaminant kernel’ within acceptable limits with high confidence.     

Effects of edible plant polyphenols on gluten protein functionality and potential applications of polyphenol–gluten interactions

Expanding plant‐based protein applications is increasingly popular. Polyphenol interactions with wheat gluten proteins can be exploited to create novel functional foods and food ingredients. Polyphenols are antioxidants, thus generally decrease gluten strength by reducing disulfide cross‐linking. Monomeric polyphenols can be used to reduce dough mix time and improve flexibility of the gluten network, including to plasticize gluten films. However, high‐molecular‐weight polyphenols (tannins) cross‐link gluten proteins, thereby increasing protein network density and strength. Tannin–gluten interactions can greatly increase gluten tensile strength in dough matrices, as well as batter viscosity and stability. This could be leveraged to reduce detrimental effects of healthful inclusions, like bran and fiber, to loaf breads and other wheat‐based products. Further, the dual functions of tannins as an antioxidant and gluten cross‐linker could help restructure gluten proteins and improve the texture of plant‐based meat alternatives. Tannin–gluten interactions may also be used to reduce inflammatory effects of gluten experienced by those with gluten allergies and celiac disease. Other potential applications of tannin–gluten interactions include formation of food matrices to reduce starch digestibility; creation of novel biomaterials for edible films or medical second skin type bandages; or targeted distribution of micronutrients in the digestive tract. This review focuses on the effects of polyphenols on wheat gluten functionality and discusses emerging opportunities to employ polyphenol–gluten interactions.     

Immunochemical determination of gluten in malts and beers.

The gluten content in different varieties of barley and malts, and in different types of beers, was determined by a 'sandwich' enzyme immunoassay (RIDASCREEN Gliadin kit). The gluten levels in barley wheat, rye and spelt malts ranged 18.8-45.0, 44.0-68.0, 41.6 and 21.2 g kg-1, respectively. When various types of beer were compared, the gluten concentration increased as follows: alcohol-free beer (<3.0), lager beers (<3.0-8.7 mg l-1), stouts (9.0-15.2 mg l-1) and wheat beers (10.6-41.2 mg l-1). When 10 Czech lager beers were analysed, using both sandwich and competitive ELISA, the results showed that the latter method provided values several times higher than the former. Gluten balance was carried out during the brewing process, starting from the raw materials and terminating at the final beer. Gluten levels decreased due to precipitation during the mashing process, primary and secondary fermentation and, lastly, as a result of adsorption during beer stabilization. The gluten content in beer is, thus, approximately three orders of magnitude lower than in the raw malt.     

Identification of Bioactive Peptides from Cereal Storage Proteins and Their Potential Role in Prevention of Chronic Diseases

Cereal grains, such as wheat, barley, rice, rye, oat, millet, sorghum, and corn, have been staples in human diets since ancient times. At present, there is a significant body of scientific evidence showing the health benefits of consuming whole grains in chronic disease prevention, particularly in regards to diabetes, cardiovascular disease, and cancer. The objective was to determine bioactive peptides in cereal grains that may prevent cardiovascular disease, cancer, inflammation, and diabetes. Bioactive peptides that may be obtained from cereal grains, particularly wheat, oat, barley, and rice, were identified. Bioactive peptides that play a role in chronic disease prevention have been found primarily in legumes and dairy products; although research connecting cereal grains with potential bioactive peptide activity is limited. In this review, 4 cereal grains, wheat, oat, barley, and rice, were evaluated for bioactive peptide potential using the BIOPEP database. In addition, research information was compiled for each grain regarding evidence about the effect of their proteins in prevention of chronic diseases. All 4 grains showed high occurrence frequencies of angiotensin‐converting enzyme‐inhibitor peptides (A = 0.239 to 0.511), as well as of dipeptidyl peptidase‐inhibitor and antithrombotic, antioxidant, hypotensive, and opioid activity. Wheat and rice proteins had anticancer sequences present. Wheat and barley showed the greatest diversity and abundance of potential biological activity among the cereal proteins. Further research needs to be conducted to learn how these biologically active peptide sequences are released from cereal grains. This study supports the notion that cereal grains are a nutritious part of a healthy diet by preventing chronic diseases.     

Comparative immunological and chromatographic study of some plant β-Amylases

A comparative study has been made of the β-amylases of barley, wheat, rye, oats and sweet-potato by means of exclusion chromatography and immunochemical analysis. The reactivity of barley malt and wheat β-amylase was compared with different anti-barley and anti-wheat sera. In exclusion chromatography on Sephadex G100, barley β-amylase yielded four, and both wheat and rye, two active components, whereas oat and sweet-potato had only one active component. During the storage of barley, wheat and rye β-amylases the large-molecule components were split into smaller ones; no changes occurred in oat and sweet-potato β-amylases. On analysis against a specific barley β-amylase antiserum, wheat and rye β-amylase gave a reaction which indicated that they were immunologically partly identical with barley β-amylase, and identical with each other. This serum induced no reaction in β-amylases of sweet-potato and oats. The rye β-amylase precipitation line did not display enzymic activity after reaction with this antiserum. Analyses with different antisera of barley and wheat confirmed the partial immunological identity of barley malt and wheat β-amylase. With some barley antisera, partial inhibition of wheat β-amylase activity was observed. A similar phenomenon was apparent when barley malt β-amylase was precipitated with some wheat antisera.     

Phytic Acid Degradation in Complementary Foods Using Phytase Naturally Occurring in Whole Grain Cereals

Complementary foods based on cereals and legumes often contain high amounts of phytic acid, a potent inhibitor of mineral and trace element absorption. The possibility to degrade phytic acid during the production of complementary foods by using whole grain cereals as the phytase source was investigated. Whole grain rye, wheat, or buckwheat (10%) were added to cereal‐legume‐based complementary food mixtures, and phytic acid was shown to be completely degraded in a relatively short time (1.5 to 3 h) when incubated at optimal conditions for cereal phytase. The potential usefulness of the method for industrial production was demonstrated with a complementary food based on wheat and soybean.     

Immunocrossreactivity of antisera against grain prolamines of wheat, rye, barley and oats

Prolamines extracted from wheat (gliadin), rye (secalin), barley (hordein) and oats (avenin) were used to raise antibodies in rabbits. The four prolamines were separated by SDS-PAGE and blotted onto nitrocellulose. The immunocrossreactivity of the separated prolamines with the four antisera was studied. The close botanical relationship between wheat and rye, and to a lesser extent barley, is clearly established. The crossreactivity of gliadin, secalin and hordein with anti-avenin serum was found to be weak. In contrast, avenin shows a strong crossreactivity with anti-gliadin serum.     

Oxidative modification of a proline-rich gliadin peptide

Prolamins are proline-rich proteins occurring in cereal grains. Prolamins of wheat, barley and rye, or gluten protein, can cause coeliac disease in individuals not tolerating gluten. Degrading harmful prolamins can reduce their toxicity. A model peptide sequenced in α-gliadin, 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), was chosen for our study. The metal-catalysed oxidation of 33-mer was studied, instead of enzymatic hydrolysis. Peptide 33-mer was treated in several oxidative systems. Iron-catalysed hydrogen peroxide-induced oxidation showed the greatest modification of 33-mer. Carbonyl groups and dityrosine cross-links were readily formed. At best, the immunological activity of 33-mer was reduced to 18% of its initial level after 24 h of oxidation. Oxidative treatment can be further applied for the modification of cereal prolamin proteins, since it appears to be a potential alternative for reduction of coeliac immunological activities in gluten proteins.