Our study was focused on the evaluation of the content of a wider spectrum of eight avenanthramides (AVNs) as unique components of oat grain under the effects of four selected factors (cultivar, locality, cropping system, and year). The weather effects on changes in the AVN content and their relationship to other important parameters of oat grain were further evaluated in more detail. A sensitive UHPLC system coupled with a QExactive Orbitrap mass spectrometer was used for AVN quantification. AVNs confirmed a high variability (RDS = 72.7–113.5%), which was dominantly influenced by the locality and year factors. While most AVN types confirmed mutually high correlations (r = 0.7–0.9), their correlations with the other 10 grain parameters were lower (r ≤ 0.48). Their significant correlations (0.27–0.46) with β-D-glucan could be used in perspective in breeding programs for the synergetic increase of both parameters. PCA analysis and Spearman correlations based on individual cultivars confirmed a significant effect of June and July precipitation on the increase of Σ AVNs. However, the results also indicated that higher precipitation can generate favorable conditions for related factors, such as preharvest sprouting evoking a direct increase of AVNs synthesis in oat grain.

Current clinical studies confirm that the consumption of oats for people suffering from celiac disease is safe. Some studies have confirmed different levels of immunoreactive gluten epitopes of oats in different cultivars, while others explain these differences due to contamination with gluten-rich species or as random cross-reactivity ELISA of homologous oat epitopes with anti-wheat gliadin antibodies. The aim of our two-year study was therefore to map cross-reactive oat epitopes in a set of 132 oat cultivars using a G12-based ELISA kit. The results were focused on the varietal and annual level of cross-reactivity (interference) of avenin epitopes with the G12 antibody on the identification of potential cultivars with significantly different interferences and assessing the degree of risk of possible false-contamination with external gluten. Although repeated evaluations confirmed high year-to-year variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the content of interfering avenin epitopes with G12 did not exceed the considered safe limit (20 mg·kg⁻¹) for celiacs. At the same time, not only annual but, above all, significant cultivar dependences in the interference of avenins to the G12 antibody were demonstrated. Genetic dependence was further confirmed in connection with the proven avenin polymorphism as well as immunoblotting with the identification of interfering peptides with the G12 antibody in the 25 and 30 kDa regions. It was the occurrence of two bands around 30 kDa that predominantly occurred in oat cultivars with a relatively higher content of cross-reactive avenins (12–16 mg·kg⁻¹). Due to the fact that the contents of interfering avenins ranged in several cultivars even over 16 mg·kg⁻¹, the choice of a suitable oat cultivar may be crucial for gluten-free food producers, as it reduces the risk of a possible false-response of the commercial ELISA kits when checking the real-gluten contamination.

There is still an ambiguous opinion on oat regarding its safety for people with celiac disease (CD). Some studies have confirmed different content of oat immunoreactive epitopes in different cultivars while others explain the differences in consequence of cross-contamination with gluten-rich species or as ELISA cross-reactivity of oat homological epitopes with antibodies against wheat gliadin. Our study was based on a two-year mapping of oat immunoreactive epitopes in a set of 132 oat cultivars using the G12-based ELISA kit. Although repeated evaluation confirmed high interannual variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the permitted gluten content (20 mg kg-1) has not been exceeded except for contaminated cultivar Sirene. The polymorphism of purified avenins determined by SDS-PAGE revealed the occurrence of 2 bands around 30 kDa in oat cultivars with relatively high gluten content (12-16 mg kg-1) except for the cultivar Leo while this pattern occurred only in 50% oat cultivars with low gluten content. Quantification of gluten epitopes on purified avenin unit further revealed the three materials (Mojacar, Maris Oberon, SG-K 16370) with the lowest gluten content and presence of one band at 30 kDa. The band pattern at 30 kDa thus represents a promising breeding marker.