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.