Preparation and Characterization of Avenin-Enriched Oat Protein by Chill Precipitation for Feeding Trials in Celiac Disease

Abstract

The safety of oats for people with celiac disease remains unresolved. While oats have attractive nutritional properties that can improve the quality and palatability of the restrictive, low fiber gluten-free diet, rigorous feeding studies to address their safety in celiac disease are needed. Assessing the oat prolamin proteins (avenins) in isolation and controlling for gluten contamination and other oat components such as fiber that can cause non-specific effects and symptoms is crucial. Further, the avenin should contain all reported immunogenic T cell epitopes, and be deliverable at a dose that enables biological responses to be correlated with clinical effects. To date, isolation of a purified food-grade avenin in sufficient quantities for feeding studies has not been feasible. Here, we report a new gluten isolation technique that enabled 2 kg of avenin to be extracted from 400 kg of wheat-free oats under rigorous gluten-free and food grade conditions. The extract consisted of 85% protein of which 96% of the protein was avenin. The concentration of starch (1.8% dry weight), β-glucan (0.2% dry weight), and free sugars (1.8% dry weight) were all low in the final avenin preparation. Other sugars including oligosaccharides, small fructans, and other complex sugars were also low at 2.8% dry weight. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the proteins in these preparations showed they consisted only of oat proteins and were uncontaminated by gluten containing cereals including wheat, barley or rye. Proteomic analysis of the avenin enriched samples detected more avenin subtypes and fewer other proteins compared to samples obtained using other extraction procedures. The identified proteins represented five main groups, four containing known immune-stimulatory avenin peptides. All five groups were identified in the 50% (v/v) ethanol extract however the group harboring the epitope DQ2.5-ave-1b was less represented. The avenin-enriched protein fractions were quantitatively collected by reversed phase HPLC and analyzed by MALDI-TOF mass spectrometry. Three reverse phase HPLC peaks, representing ~40% of the protein content, were enriched in proteins containing DQ2.5-ave-1a epitope. The resultant high quality avenin will facilitate controlled and definitive feeding studies to establish the safety of oat consumption by people with celiac disease.

Identification of Perspective Oat Cultivars with a Minimum Content of Gluten Homologous Peptides

“…4A and 4B. Consistent with the results of Tanner et al [16], the significant 5 -7 bands (peptides) in the range of 20-30 kDa were detected on electrophoretic gels. At 30 kDa where immunoreactive peptides were detected with G12 monoclonal antibody by Comino et al [7], an important difference between two groups of oat genotypes associated with the presence of either a single band or double bands on the gels was detected.…”

“…Based on these findings, it should be more correct to speak only about detection of aveninderived epitopes homologous to wheat gliadin 33-mer for whose detection the G12 monoclonal antibody was raised. Moreover, the avenin-derived epitopes determined in the present study do not correspond with avenin-derived epitopes reported to stimulate immune response in CD patients as reported by Tanner et al [16]. It can thus be expected that noncontaminated oat samples with gluten-like immunoreactive peptide levels lower than 20 mg kg -1 will be safer for celiacs than analogous wheat-derived products with the same gluten contents.…”

“…Chilly precipitation of avenins was carried out according to Tanner et al [16] with some modifications. 0.5 g oat flour was extracted in 1,5ml of 50% (v/v) ethanol, vortexing regularly over 1 h, and centrifuging at 3000 g for 1 h. The 50% ethanol supernatants were chilled at 4 ºC overnight, centrifuge as above and pellets redissolved in 0,3 ml 60% (v/v) ethanol during 2 h incubation at room temperature.…”

The Gluten Gene: Unlocking the Understanding of Gluten Sensitivity and Intolerance

“…28 Nevertheless, numerous studies, based on findings showing that avenins alone do not induce immune responses in most CD patients and symptomatically tolerated by them, point to the safety of adding oats to their diet. [29][30][31][32] In this regard, however, Hardy et al 33 in their in vivo study on 73 biopsy-confirmed HLA-DQ2.5+ CD patients showed that the ingestion of oats (100 g/day) for 3 days mobilizes polyclonal avenin-specific T-cells in blood in fewer than 10% of studied patients. Half of the patients had at least one digestive symptom during this challenge, which was due to a high daily intake of oats (100 g) and a high amount of fiber in them.…”

Perennial Ryegrass Contains Gluten-Like Proteins That Could Contaminate Cereal Crops

“…In wheat, these proteins are gliadins; in rye, they are secalins; in barley, hordeins, and oats, they are avenins. The phylogenetic relationship between prolamin proteins from different species has been demonstrated with homology comparisons between avenin sequences and α- and γ-gliadins from wheat, B-hordeins from barley, and γ-secalins from rye ( 28 – 30 ). Herein, we showed that ryegrass has gliadin-like peptides that share a certain level of similarity to avenins and γ-gliadins.…”