Gluten and amylase/trypsin-inhibitors (ATIs) are known triggers for wheat-related disorders. Ancient wheat species like einkorn, emmer, Khorasan wheat, Polish wheat and spelt are suggested to have fewer of these immunoreactive proteins compared to modern common wheat, because breeding for higher yields and increased resistance to plant diseases might have resulted in changes in the protein composition. Currently, there are only very few studies on the protein composition of ancient wheat species. This comparative study investigated the bread volume, protein content as well as gluten and ATI content and composition, along with the inhibitory activity in ten ancient wheat varieties cultivated under organic conditions. The varieties had different ploidy levels and were harvested in three consecutive years. The various analyses consistently concluded that differences in protein composition were associated with the different ploidy levels. This means that the results were similar for the diploid species (einkorn), the tetraploid species (emmer, durum wheat, Khorasan wheat and Polish wheat) and for the hexaploid species (spelt and common wheat), respectively. These results suggest that breeding has likely not increased the immunoreactive potential of wheat and ancient wheats are unlikely to provide improved tolerability for individuals with wheat-related disorders.

Flour (50 mg) was extracted twice with Abic solution (0.5 mL, 50 mmol/L, pH 7.8) for 30 min at 22 °C using a magnetic stirrer. After every extraction step, the suspensions were centrifuged (25 min, 22°C, 3750 rcf) and the supernatants were combined. Using a rotational vacuum concentrator (Christ, Osterode, Germany), the extracts were then evaporated to dryness. The residue was dissolved in 350 µL of Tris–HCl (0.5 mol/L, pH 8.5) and 350 µL of 1 propanol. Then 50µL of standard solution, containing IS1-22, was added. The concentration of each IS in this solution was adjusted to the expected peptide content in the samples. After that, 50 µL of TCEP were added (0.05 mol/L TCEP in 0.5 mol/L Tris–HCl, pH 8.5) and incubated for 30 min at 60°C in a thermal shaker (Hettich Lab Technology, Tuttlingen, Germany) to perform reduction of the disulfide bonds. Alkylation was performed in the same device by adding 100 µL of CAA (0.5 mol/L CAA in 0.5 mol/L Tris–HCl, pH 8.5) and incubating for 45 min at 37°C in the dark. The solvent was again removed by evaporation to dryness. Last, 0.5 mL of trypsin solution (enzyme-to-substrate ratio 1:30, 0.04 mol/L urea in 0.1 mol/L Tris–HCl, pH 7.8) was added for tryptic digestion for 18 h overnight at 37°C in the dark. After that, the reaction was stopped with 5 µL of TFA. The solution was removed by evaporation and the residue was then dissolved in 1 mL of water containing 2% acetonitrile and 0.1% formic acid. For calibration, two solutions (25 – 100 µg/mL of each peptide) were prepared from the stock solutions, solution 1 with P1-P22 and solution 2 with IS1-IS22. An aliquot of each solution 1 and 2 was reduced and alkylated similar to the samples and as described in Geisslitz et al. 2020. Alkylated solutions 1 and 2 (0.3 – 1.3 µg/mL of each peptide) were mixed in molar ratios n(P)/n(IS) between 9.1 and 0.1 (9+1, 7+1, 5+1, 3+1, 1+1, 1+3, 1+5, 1+7 and 1+9) and used for quantitation. Limits of detection and quantitation were also determined according to Geisslitz et al. 2020

Ten German varieties of different wheat species were cultivated at the Bavarian State Research Center for Agriculture LfL (Bayerische Landesanstalt für Landwirtschaft, Ruhstorf an der Rott, Germany) and harvested in three consecutive years (2021–2023). Two spelt (Babenhausener Zuchtvesen (BZV) and Muellers Gaiberger (MGA)), two emmer (Weihenstephan Emmer 1 (WEM1) and Weihenstephan Emmer 2 (WEM2)), one durum wheat (Winterdurum Sambadur (WDS)), one Khorasan wheat (KSW), one Polish wheat (POW) and three einkorn varieties (wild einkorn (WEK), Enkidu (ENK) and Terzino (TER)) were included. The two einkorn varieties ENK and TER were added after the first year of the trial. The cultivation was carried out under organic growing conditions without fertilization or any other treatment. The experimental layout with plot sizes of 2 × 4 m was a randomized complete block design (Latin rectangle) with three replications, which were harvested and milled together. The kernels were milled with a Quadrumat Junior (Brabender, Duisburg, Germany) to obtain type 550 flours (ash content of 0.51 %–0.63 % based on dry matter) according to the German flour classification system.