Päivi Kanerva

Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods.

The assortment and quality of bakery products designed for celiac patients may be improved by designing whole grain ingredients with low residual prolamin contents. The main objective of this study was to evaluate the extent of prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs (GRSDs). Size-exclusion chromatography analyses, the fate of fluorescent prolamin, and immunological analyses determined the extent of prolamin hydrolysis and pentosan solubilization. Hydrolysis of rye prolamins was extensive in GRSDs, and according to enzyme-linked immunosorbent assay analyses, more than 99.5% of the prolamins were hydrolyzed. Pentosan solubilization occurred in native-rye sourdoughs, whereas in GRSDs, pentosans were partially hydrolyzed to monosaccharides. Test baking showed that the use of GRSD improved the overall quality of oat bread and that an estimated daily gluten intake from 100 g of bread would be less than 10 mg. However, the clinical safety must be assured before making any recommendations for celiac patients to use such products.

Improved extraction of prolamins for gluten detection in processed foods.

A problem in gluten analysis has been inconsistent extractability of prolamins, particularly from processed foods consisting of unknown portions of prolamins from wheat, barley, and rye. This study aimed at improving the extraction of prolamins for immunological analysis, regardless of the cereal species and the production process. The prolamins were extracted with varying concentrations of ethanol, 1-propanol, and 2-propanol. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting were applied to study the protein composition of the extracts and the antibody recognition of the prolamin subgroups. We characterized the affinities of prolamin-specific antibodies that are used in gluten analysis against the prolamin groups that were soluble in 40% 1-propanol. The antibody R5 recognized more abundantly the medium-molecular weight groups, including polymeric proteins, and less the high-molecular weight groups than the anti-ω-gliadin antibody. In the present study, the prolamins were most efficiently extracted by 40% 1-propanol with 1% dithiothreitol at 50 °C . The prolamins were extracted from processed bread samples with efficiency similar to that from untreated meal samples.

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.

OATS—AN OVERVIEW FROM A CELIAC DISEASE POINT OF VIEW

ABSTRACT Oats, belonging to tribe Aveneae, are phylogenetically different from the Triticeae cereals of wheat, rye, and barley. Oats also differ in their protein composition, and a vital difference in the amino acid sequence from the coeliac-toxic Triticeae cereals has been identified. Ever since the introduction of the gluten-free concept more than 50 years ago, there has been uncertainty and controversy about the status of oats. Today, results from more than 15 clinical studies published in the past 15 years have shown that considerable amounts (50 g or more) of oat flakes or oat flour can be safely consumed by most adults and children with coeliac disease, including those with the dermatitis herpetiformis form of the disease. In 2007, the U.S. Food and Drug Administration (FDA) put forward a definition for the term “gluten-free” and proposed that oats should not be classified as prohibited grains. The international standard for foods for persons intolerant to gluten, as formulated by the Codex Alimentarius, recognised in 2008 that oats can be tolerated by most people intolerant to gluten, although oats may not be tolerated by all of them. The EU regulation (2009) declared that oats contained in foodstuffs for people intolerant to gluten must be specially produced and the gluten content must not exceed the new limit of 20 mg/100 g. In spite of the evidence suggesting oats are safe, two areas of concern remain, namely the possible comingling (mixing, “contamination”) of oats with wheat, rye, or barley, and the existence of a possible subgroup among coeliac patients that develop mucosal inflammation and symptoms when eating oats. The comingling problem can be eliminated by suitable agronomic, logistical, and food processing practices. This problem, in particular with barley, might have been overestimated due to problems with the interpretation of results obtained with the newly introduced ELISA R5 method. The number of coeliac patients sensitive to oats is probably very low; so far, only a few oat-sensitive individuals have been reported, whereas there have been hundreds of coeliac patients participating in studies showing oats have no adverse effect. Most of the celiac patients in Finland use oat products, and the authorised labelling of products uses the wording, “contains oats and gluten-free ingredients.” In conclusion, recent evidence from clinical research and practice shows that oat products, in particular when made specifically for coeliac customers, cause no harm to most coeliac disease patients. With oat products included, there is more variation and nutritional value in the coeliac diet, so better compliance and an improved quality of life can be expected. The new regulatory limit, 20 mg/kg in oats, provides an analytical and technological challenge.

Degradation of C-hordein by metal-catalysed oxidation.

C-hordein is a monomeric prolamin protein in barley. The unusual primary structure of C-hordein has highly repetitive sequences and forms a secondary structure of beta-turns. C-hordein structure is similar to that of collagen protein, whose degradation by metal-catalysed oxidation has been intensively studied. No information exists on the metal catalysed oxidation of C-hordein, however. In this study, copper-catalysed hydrogen peroxide induced oxidation of C-hordein caused substantial degradation and formed some insoluble compounds. The use of a gliadin standard in R5 ELISA determinations causes an overestimation of hordeins in a sample. A C-hordein standard was therefore directly used as a standard, thus allowing the C-hordein to be analysed as its oxidised prolamin product. After 48 h of oxidation, the prolamin concentration of oxidised C-hordein decreased to 20% of its original amount for competitive ELISA, and to 3% for sandwich ELISA methods. Carbonyl groups were formed during the oxidation. Backbone fragmentation and side-chain modification suggested structural changes of R5 epitopes in C-hordein. Oxidation is an alternative to enzymatic hydrolysis when degrading and modifying C-hordein.

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.