Barley Prolamins Research Articles

Intelligent films based on highland barley β-glucan/highland barley prolamin incorporated with black rice bran anthocyanins

In this work, the effect of incorporating highland barley prolamin (HBP) on the properties of highland barley β-glucan (HBBG) films was investigated. Besides, the improvement of black rice bran anthocyanins (BRA) on intelligence effect of HBBG/HBP film was studies. The addition of HBP enhanced the barrier properties of HBBG film, indicating that the water vapor permeability and oxygen permeability of HBBG film decreased. In addition, the mechanical, thermal, optical properties and water contact angle of HBBG film were also found to be improved by the addition of HBP. The colors of HBBG/HBP/BRA films changed quickly and could be distinguished by eye in different pH solution and ammonia environment due to the abundant anthocyanins in BRA. Furthermore, the stability of anthocyanins in BRA was enhanced through hydrogen bond among BRA, HBBG and HBP. Therefore, HBBG/HBP/BRA films have broad prospects in intelligent packaging.

Proposal for C-Hordein as Reference Material in Gluten Quantification.

The concentration of residual barley prolamin (hordein) in gluten-free products is overestimated by the R5 ELISA method when calibrated against the wheat gliadin standard. The reason for this may be that the composition of the gliadin standard is different from the composition of hordeins. This study showed that the recognition of whole hordein by R5 antibody mainly came from C-hordein, which is more reactive than the other hordeins. The proportion of C-hordein in total hordein ranged from 16 to 33% of common Finnish barley cultivars used in this study and was always higher than that of ω-gliadin, the homologous protein class in the gliadin standard, which may account for the overestimation. Thus, a hordein standard is needed for barley prolamin quantification instead of the gliadin standard. When gluten-free oat flour was spiked with barley flour, the prolamin concentration was overestimated 1.8-2.5 times with the gliadin standard, whereas estimates in the correct range were obtained when the standard was 40% C-hordein mixed with an inert protein. A preparative-scale method was developed to isolate and purify C-hordein, and C-hordein is proposed as a reference material to calibrate barley prolamin quantification in R5-based assays.

Proteome-Based Analysis of Colloidal Instability Enables the Detection of Haze-Active Proteins in Beer.

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals. Consequently, a treatment for the chemical deglycation with trifluoromethanesulfonic acid was applied, which resulted in the identification of protein Z4, LTP1, CMb, CMe, pUP13, 3a, and Bwiph as constituents of the haze proteome. Because only one hordein was detectable and the proline content in haze hydrolysates was lower than those of barley prolamins, our results suggest that this class of proteins is of minor importance for haze development.

Oxidation of proline decreases immunoreactivity and alters structure of barley prolamin

Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC–MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins.

Caracterización de patrones de hordeínas en variedades mexicanas de cebada maltera

A group of storage proteins highly abundant in cereal seeds are the prolamins, characterized by the frequent presence of proline in their sequence. The barley prolamins are known as hordeins. The aim of this study was to obtain the hordein banding patterns for five different Mexican barley cultivars in mature seeds. In addition, the hordein patterns in processed malt of four mexican and a canadian (Metcalfe) barley cultivars were obtained. Mass Spectrometry (MS), using distinct digestion protocols, identified differential bands in the patterns. Major differences in the seed patterns between cultivars consisted in discrete bands at 100 kDa, 65 kDa and in the range of 37 to 45 kDa. In malt, the patterns were highly contrasting among Mexican cultivars as well as in Metcalfe, suggesting that hordein processing during germination and malt processing is particular to each cultivar. Finally, the MS identification demonstrated that trypsin digestion is appropriate to distinguish B and γ hordeins in malt, whereas sequential digestion with chymotrypsin and trypsin allows the identification of C hordeins in seed.

Hairpin-based DNA electrochemical sensor for selective detection of a repetitive and structured target codifying a gliadin fragment.

High selectivity of genosensors is crucial for certain applications such as those involving species with high genetic variability. This is an unresolved problem when dealing with long target sequences that is furthercomplicated when the target contains repetitive sequence domains. As a model for this situation, the problem of detecting gluten in food with identification of the source is studied. In order to discriminate the specific DNA sequence that encodes the wheat prolamin (gliadin) from rye and barley prolamins, the exquisite selectivity of a rationally designed hairpin capture probe is proposed and compared to a nonstructured capture probe. An electrochemical sandwich assay is proposed, involving capture probes chemisorbed on Au surfaces and biotinylated-signaling probes in combination with streptavidin-peroxidase labeling conjugates. As a result, a genosensor with similar sensitivity to that observed with linear probes but with complete specificity against closely related species was achieved. The surface-attached DNA stem-loop yields a device capable of accurately discriminating wheat DNA from rye and barley with a limit of detection of 1nM.

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 24h 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.

Gluten Fragment Detection with a Competitive ELISA

The second generation of a competitive ELISA for prolamin quantification based on the R5 antibody was studied for method performance and suitability to detect partially hydrolyzed prolamins in food. To be able to convert signal intensities to gluten concentrations, as required by the Codex Alimentarius Standard, a new calibrator consisting of a peptic-tryptic digest of wheat, rye, and barley prolamins was used for the first time. LOD and LOQ of the assay were 1.36 and 5.0 mg prolamin/kg food, respectively. Analysis of beer samples and a hydrolyzed wheat product showed that the assay provided significantly higher prolamin concentrations, compared to the sandwich ELISA based on the same antibody, which is only suitable for the detection of intact prolamins. Spiking experiments with defined concentrations of partially hydrolyzed prolamins gave recoveries ranging from 92 to 136%.

Dissecting the T‐cell response to hordeins in coeliac disease can develop barley with reduced immunotoxicity

Wheat, rye and barley prolamins are toxic to patients with coeliac disease. Barley is diploid with pure inbred cultivars available, and is attractive for genetic approaches to detoxification. To identify barley hordein fractions which activated the interferon-γ (IFN-γ) secreting peripheral blood T-cells from coeliac volunteers, and compare immunotoxicity of hordeins from experimental barley lines. To reactivate a T-cell response to hordein, volunteers underwent a 3-day oral barley challenge. Peripheral blood mononuclear cells (PBMC) were isolated from twenty-one HLA DQ2(+) patients with confirmed coeliac disease. IFN-γ ELISpot assays enumerated T-cells activated by purified prolamins and positive controls. Hordein-specific T-cells were induced by oral barley challenge. All prolamin fractions were immunotoxic, but D- and C-hordeins were most active. Barley lines lacking B- and C-hordeins had a 5-fold reduced hordein-content, and immunotoxicity of hordein extracts were reduced 20-fold compared with wild-type barley. In vivo oral barley challenge offers a convenient and rapid approach to test the immunotoxicity of small amounts of purified hordeins using fresh T-cells from patients in high throughput overnight assays. Barley lines that did not accumulate B- and C-hordeins were viable, yet had substantially reduced immunotoxicity. Creation of hordein-free barley may therefore be possible.

Commercial Assays to Assess Gluten Content of Gluten-Free Foods: Why They Are Not Created Equal

A standardized method of analysis is needed to quantitatively determine the gluten content of food and provide the basis for enforcing regulations regarding use of the term gluten-free in food labeling. People with celiac disease should feel confident that foods labeled “gluten-free” have been assessed for gluten using the same “best available” methodology. The Association of Analytical Communities and the Codex Alimentarius Commission endorse different methods. Both are used by manufacturers in the United States to determine the gluten-free status of food. The sandwich ω-gliadin enzyme-linked immunosorbent assay (ELISA) is the official method of the Association of Analytical Communities. It is able to quantify native and heated gluten. It is unable to accurately detect and quantify barley prolamins, can over- or underestimate gluten content, and cannot accurately quantify hydrolyzed gluten. The sandwich R5 ELISA was endorsed by Codex for gluten determination. It is able to quantify native and heated gluten. One criticism is that it overestimates barley hordein. It also is unable to accurately quantify hydrolyzed gluten. Foods that can be reliably assessed for gluten using a validated commercially available ELISA are those contaminated with native and heated proteins from wheat, barley, and rye. The degree of confidence that can be placed in a manufacturer's assertion that a product is gluten-free is based on the assay used to determine the gluten content and the specific food analyzed.

Making oats safer for patients with coeliac disease

(Table is included in full-text article). Studies suggest that oats can be tolerated in the gluten-free diet by a majority of patients with coeliac disease. Concerns remain, however, about the possibility of contamination of commercially available oat products by wheat, barley and rye. The R5 ELISA allows the identification and quantification of wheat, barley and rye prolamins, and has demonstrated significant contamination in a range of products. If patients with coeliac disease are to take oats, it is important that those consumed are free of contamination. This assay should allow the identification of safe oat products.

Celiac disease in children - modern diagnostic approach

Celiac disease presents a hereditary disorder of gluten tolerance, i.e. of gliadin and related prolamins of wheat, rye and barley. It primarily occurs in Caucasians (1:100-300),while it is considerably or exceptionally rare in colored races. It is particularly frequent in close relatives of the patient, as well as in persons with congenital IgA deficiency and in patients with autoimmune and some chromosomal diseases. The basis of the disease, as well as the key finding in its diagnostics, lies in small bowel inflammation, which withdraws on gluten free diet. Beside clinically manifest or non-manifest enteropathy, changes involving other organs and systems are also frequently seen. The diagnosis of the manifest form of the disease is based on characteristic pathohistological finding detected by the examination of small bowel mucosa in patients on standard diets and their clinical improvement after the introduction of gluten free diet. However, in the diagnosis of the asymptomatic form of the disease, it is necessary to perform enterobiopsy, which confirms the normalization of the appearance of small bowel mucosa in patients on the elimination diet. In children with gluten sensitive enteropathy detected in the first two years of life, as well as in cases in which mucosa samples taken on the first enterobiopsy do not have typical appearance or are inadequate for a reliable interpretation, a definite diagnosis is made based on biopsy finding during the provocation of gluten tolerance. As the quality of permanent teeth can be disturbed, this procedure is not suggested to be done before the completed age of 6 years, and due to adverse effects on the growth and development of the child, it should not be done during puberty. Due to incomplete sensitivity and specificity, the serological indicators of the disease do not have diagnostic value. Therefore, they are primarily used in the disclosure of asymptomatic and atypical forms of celiac disease, as well as in the assessment of the consistency of elimination diet in cases with already verified disease. In addition, the application of these tests makes easier passing the decision to perform pathohistological examination of small bowel mucosa in patients with provoked gluten tolerance, which also gives a more complete understanding into the remission of the disease during the initial phase of treatment.

Stanovení prolaminů ječmene v pivu a pivovarských materiálech.

Prace se zabýva možnostmi imunochemicke detekce hordeinove frakce bilkovin jecmene a připadných stěpných derivatů, respektive charakterizaci monoklonalnich protilatek připravených za ucelem vývoje rychleho, specifickeho a citliveho stanoveni hordeinových bilkovin v potravinařstvi, konkretně v pivu. Byla vyvinuta metoda nepřime kompetitivni ELISA prolaminů jecmene a stanoveny pracovni koncentrace vsech imunoreagencii. Dale byly testovany křižove interakce jednotlivých monoklonalnich protilatek s proteiny ostatnich cerealii. Detekcni limit metody cinil 2 ppm, přicemž linearni rozsah kalibracni křivky se pohybuje v rozmezi 1-100 ppm.

Prolamin Hydrolysis in Wheat Sourdoughs with Differing Proteolytic Activities

The prolamins of wheat, rye, and barley contain structures that are harmful to gluten-sensitive people, and an extensive degradation of these prolamins during food processing might eliminate this problem. Sourdough fermentation is a cereal food process during which some protein degradation occurs. In this study, the prolamin hydrolysis that occurred in a high-proteolytic-activity germinated-wheat sourdough (GWSD) was compared with that of wheat sourdough systems which contained moderate or no proteolytic activities. Virtually all of the wheat prolamins (gliadins and glutenins) were degraded during the GWSD fermentation. Quantification of its prolamin levels confirmed that extensive prolamin hydrolysis had occurred in the GWSD. This hydrolysis was attributed to the cysteine proteinase activities of the germinated wheat. The use of high-proteolytic sourdoughs in baking could make it possible to prepare new low-prolamin cereal-based products for use by gluten-sensitive people, who could then diversify their diets by including these whole-grain containing products into their every-day diets.

Analysis of barley contamination in oats using R5 and ω-gliadin antibodies

Immunological methods based on monoclonal antibodies with varying specifities towards gluten proteins have been used to control the purity of gluten-free products. Commercially available methods have been developed to detect gluten proteins found in wheat. However, difficulties have occurred in quantifying barley proteins with the same accuracy as wheat proteins. Barley is also a common contaminant in oat products. In this study, oat flour samples were deliberately contaminated with known amounts of barley flour and analysed using two commercial enzyme linked immunosorbent assays (ELISA). The methods were based on an ω-gliadin antibody and an R5 antibody. The results obtained with the R5 antibody were overestimates while the ω-gliadin antibody underestimated the higher barley prolamin content. This study showed that inaccuracies in ELISA assays in quantifying barley contaminations can possibly be eliminated by using a hordein standard. However, it is necessary to know the source of contamination. This would prevent overestimation of the hordein content of gluten-free products. Overestimation unnecessarily reduces the variety of gluten-free products and may decrease compliance to a gluten-free diet.

The effects of malting and mashing on barley protein extractability

Proteins in unmalted and malted barley and in brewers’ spent grain (BSG) obtained after mashing were fractionated on the basis of their differential extractability in different media and characterised by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Albumins and globulins were first extracted with 5.0% NaCl and hordeins (barley prolamins) were extracted with 55.0% 1-propanol in the presence, or absence, of 1.0% DTT. Glutelins were then extracted with 2.0% SDS/6.0 M urea/1.0% DTT or with 55.0% 1-propanol/6.0 M urea/1.0% DTT/0.036 M Tris-HCl (pH 8.4). Under non-reducing conditions, monomeric C hordeins and some B hordeins were extracted from unmalted barley, whereas most if not all B, C and D hordeins were extracted under reducing conditions. During malting, disulfide bonds are reduced and B and D hordeins are broken down by proteolysis. No D hordeins were extracted from malt and nearly the same levels of malt B hordeins were extracted both under non-reducing and reducing conditions. B hordeins present in BSG proteins were only extractable under reducing conditions. Whereas most of the C hordeins were extracted from BSG under non-reducing conditions, more C hordeins were extracted under reducing conditions. Mashing probably induced disulfide bond formation resulting in aggregation. Although earlier literature suggested the formation of an aggregate composed of B and D hordein (and glutelin) during mashing, the present work suggests the formation of an aggregate composed of B hordeins in which C hordeins are entrapped.

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.

Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue

Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

The use of extended amino acid motifs for focussing on toxic peptides in coeliac disease.

Cereal prolamins of wheat, rye and barley are the major proteins that have been implicated in toxicity in patients with coeliac disease. The gliadins of wheat are the best characterised with the identification of toxic peptides from rye and barley not as well advanced. This study has employed extended motifs, based on the known toxic motifs are derived from the sequence of A-gliadin, to search protein databases for matches with coeliac-toxic cereals. The results obtained have provided pointers to specific regions in rye and barley prolamins, which have received little attention in in vitro and in vivo studies of toxicity in coeliac disease. The results obtained in this study indicate that the size of the extended motif is critical when searching for coeliac-toxic cereals using protein databases. Extended motifs that are common to all three coeliac-toxic cereals and found in active wheat gliadin peptides are QQPYP, PQQPY and QQQPFP.

Infrared Spectroscopic Study of the Secondary Structure of Wheat, Rye, and Barley Prolamins

The secondary structure of prolamins, the storage proteins of wheat (Triticum aestivum L.), rye (Secale cereale L.), and barley (Hordeum vulgare L.), was studied by infrared (IR) spectroscopy. The secondary structure of wheat gliadin was characterized by a high content of unordered conformations of polypeptide chains (64–67%) and a low content of short α-helical regions (4–5%). Elongated conformations, formed by poly-L-proline II helices (61 and 53%) and β-turns (15 and 19%), predominated in rye secalins and barley hordeins. Prolamin proteins of cereal crops also contained β-sheet structure regions comprising 9–12% of the polypeptide chains.