Data_Sheet_2_Preparation and Characterization of Avenin-Enriched Oat Protein by Chill Precipitation for Feeding Trials in Celiac Disease.xlsx

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.

对于患有乳糜泻的人群而言，燕麦的安全性问题尚无定论。燕麦富含营养特性，能够显著提升限制性低纤维无麸质饮食的品质与适口性，然而，针对其在乳糜泻患者中的安全性进行的严谨喂养研究仍属必要。对燕麦球蛋白蛋白（avenins）进行独立评估，并严格控制麸质污染及其他可能引发非特异性效应和症状的燕麦成分，如纤维，实为关键。此外，avenin应包含所有已知的免疫原性T细胞表位，并以能够使生物反应与临床效应相关联的剂量进行递送。迄今为止，从足量纯净的食品级avenin中提取，以供喂养研究使用尚不可行。本研究报告了一种新的麸质分离技术，在严格的无麸质和食品级条件下，从400公斤无麦燕麦中提取出2公斤的avenin。该提取物中85%为蛋白质，其中96%为avenin。最终avenin制备中淀粉（干重1.8%）、β-葡聚糖（干重0.2%）和自由糖（干重1.8%）的浓度均较低。其他糖类，包括寡糖、小果聚糖和其他复杂糖类，在干重2.8%的含量下也相对较低。对这些制备物中的蛋白质进行液相色谱串联质谱（LC-MS/MS）分析显示，它们仅由燕麦蛋白质组成，且未受到含有麸质的谷物（包括小麦、大麦或黑麦）的污染。与使用其他提取程序获得的样本相比，avenin富集样本的蛋白质组学分析检测到更多avenin亚型和较少的其他蛋白质。所识别的蛋白质代表了五个主要组别，其中四组含有已知的免疫刺激avenin肽。所有五个组别均在50%（体积比）的乙醇提取物中被识别，然而携带表位DQ2.5-ave-1b的组别在代表性上较低。avenin富集的蛋白质组分通过反相高效液相色谱（HPLC）进行定量收集，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行分析。代表约40%蛋白质含量的三个反相HPLC峰富含携带DQ2.5-ave-1a表位的蛋白质。所得高质量avenin将有助于进行受控且确切的喂养研究，以确立患有乳糜泻的人群食用燕麦的安全性。