Amylase/trypsin-inhibitors (ATIs) are putative triggers of nonceliac gluten sensitivity, but contents of ATIs in different wheat species were not available. Therefore, the predominant ATIs 0.19 + 0.53, 0.28, CM2, CM3, and CM16 in eight cultivars each of common wheat, durum wheat, spelt, emmer, and einkorn grown under the same environmental conditions were quantitated by targeted liquid chromatography-tandem mass spectrometry (LC−MS/MS) and stable isotope dilution assays using specific marker peptides as internal standards. The results were compared to a label-free untargeted LC−MS/MS analysis, in which protein concentrations were determined by intensity based absolute quantitation. Both approaches yielded similar results. Spelt and emmer had higher ATI contents than common wheat, with durum wheat in between. Only three of eight einkorn cultivars contained ATIs in very low concentrations. The distribution of ATI types was characteristic for hexaploid, tetraploid, and diploid wheat species and suitable as species-specific fingerprint. The results point to a better tolerability of einkorn for NCGS patients, because of very low total ATI contents.

The spectrophotometric Bradford assay was adapted for the analysis of gluten protein contents (gliadins and glutenins) of spelt, durum wheat, emmer and einkorn. The assay was applied to a set of 300 samples, including 15 cultivars each of common wheat, spelt, durum wheat, emmer and einkorn cultivated at four locations in Germany in the same year. The total protein content was equally influenced by location and wheat species, however, gliadin, glutenin and gluten contents were influenced more strongly by wheat species than location. Einkorn, emmer and spelt had higher protein and gluten contents than common wheat at all four locations. However, common wheat had higher glutenin contents than einkorn, emmer and spelt resulting in increasing ratios of gliadins to glutenins from common wheat (< 3.8) to spelt, emmer and einkorn (up to 12.1). With the knowledge that glutenin contents are suitable predictors for high baking volume, cultivars of einkorn, emmer and spelt with good predicted baking performance were identified. Finally, spelt, emmer and einkorn were found to have a higher nitrogen partial factor productivity than common and durum wheat making them promising crops for a more sustainable agriculture.

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Amylase/trypsin inhibitors (ATIs) are major wheat allergens and they are also implicated in causing non-celiac gluten sensitivity and worsening other inflammatory conditions. With only few studies on ATI contents in different Triticum species available so far, we developed a targeted liquid chromatography-tandem mass spectrometry (LC–MS/MS) method based on stable isotope dilution assays to quantitate the 13 most important ATIs in a well-defined sample set of eight cultivars of common wheat and durum wheat (modern species), as well as spelt, emmer and einkorn (ancient species) grown at three locations in Germany, respectively. Only few ATIs with low contents were detected in einkorn. In contrast, spelt had the highest total ATI contents. Emmer and common wheat had similar total ATI contents, with durum wheat having lower contents than common wheat. Due to the lack of correlation, it was not possible to estimate ATI contents based on crude protein contents. The wheat species had a higher influence on ATI contents than the growing location and the heritability of this trait was high. Despite comparatively low intra-species variability, some cultivars were identified that may be promising candidates for breeding for naturally low ATI contents.

Amylase/trypsin-inhibitors (ATIs) comprise about 2–4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs.Note This paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020

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Amylase/trypsin inhibitors (ATIs) are widely consumed in cereal-based foods and have been implicated in adverse reactions to wheat exposure, such as respiratory and food allergy, and intestinal responses associated with coeliac disease and non-coeliac wheat sensitivity. ATIs occur in multiple isoforms which differ in the amounts present in different types of wheat (including ancient and modern ones). Measuring ATIs and their isoforms is an analytical challenge as is their isolation for use in studies addressing their potential effects on the human body. ATI isoforms differ in their spectrum of bioactive effects in the human gastrointestinal (GI), which may include enzyme inhibition, inflammation and immune responses and of which much is not known. Similarly, although modifications during food processing (exposure to heat, moisture, salt, acid, fermentation) may affect their structure and activity as shown in vitro, it is important to relate these changes to effects that may present in the GI tract. Finally, much of our knowledge of their potential biological effects is based on studies in vitro and in animal models. Validation by human studies using processed foods as commonly consumed is warranted. We conclude that more detailed understanding of these factors may allow the effects of ATIs on human health to be better understood and when possible, to be ameliorated, for example by innovative food processing. We therefore review in short our current knowledge of these proteins, focusing on features which relate to their biological activity and identifying gaps in our knowledge and research priorities.

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