No correlation between amylase/trypsin-inhibitor content and amylase inhibitory activity in hexaploid and tetraploid wheat species.

Wheat amylase/trypsin-inhibitors (ATI) are known triggers for wheat-related disorders. The aims of our study were to determine (1) the inhibitory activity against different α-amylases, (2) the content of albumins and globulins (ALGL) and total ATI and (3) to correlate these parameters in wholegrain flour of hexaploid, tetraploid and diploid wheat species. The amount of ATI within the ALGL fraction varied from 0.8% in einkorn to 20% in spelt. ATI contents measured with reversed-phase high-performance liquid chromatography (RP-HPLC) revealed similar contents (1.2-4.2 mg/g) compared to the results determined by LC-MS/MS (0.2-5.2 mg/g) for all wheat species except einkorn. No correlation was found between ALGL content and inhibitory activity. In general, hexaploid cultivars of spelt and common wheat had the highest inhibitory activities, showing values between 897 and 3564 AIU/g against human salivary α-amylase. Tetraploid wheat species durum and emmer had lower activities (170-1461 AIU/g), although a few emmer cultivars showed similar activities at one location. In einkorn, no inhibitory activity was found. No correlation was observed between the ATI content and the inhibitory activity against the used α-amylases, highlighting that it is very important to look at the parameters separately.

Breeding from 1891 to 2010 did not increase the content of amylase/trypsin-inhibitors in wheat (Triticum aestivum).

The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891 to 1950) to modern cultivars (1951-2010). There were also no significant changes in ATI distribution.

Lipidomic insights into the reaction of baking lipases in cakes.

Lipases are promising improvers of cake batter and baking properties. Their suitability for use in various cake formulations cannot be predicted yet, because the reactions that lead to macroscopic effects need to be unravelled. Therefore, the lipidome of three different cake recipes with and without lipase treatment was assessed by ultra high performance liquid chromatography-mass spectrometry before and after baking. By comparing the reaction patterns of seven different lipases in the recipes with known effects on texture, we show that lipase substrate specificity impacts baking quality. Key reactions for the recipes were identified with the help of principal component analysis. In the eggless basic cake, glyceroglycolipids are causal for baking improvement. In pound cake, lysoglycerophospholipids were linked to textural effects. Lipase substrate specificity was shown to be dependent on the recipe. Further research is needed to understand how recipes can be adjusted to achieve optimal lipase substrate specificity for desirable batter and baking properties.

Proteins from Modern and Ancient Wheat Cultivars: Impact on Immune Cells of Healthy Individuals and Patients with NCGS.

In non-celiac gluten sensitivity (NCGS), the elimination of wheat results in a clear symptom improvement, but gluten has still not been proven as (the sole) trigger. Due to the increase in the prevalence of gluten-related diseases, the breeding of high-performance wheat cultivars is discussed as a trigger. To analyze the immune stimulation and signal pathways, the immune cells of healthy subjects and patients with NCGS were stimulated with gliadins from wheat, and the expression and secretion of interleukin 1ß (IL1ß) and interleukin 6 (IL6) were studied. To determine the impact of wheat breeding, the monocyte cell line THP1 and human immune cells were stimulated with gliadin, glutenin, and albumin/globulin fractions of ancient and modern cereals, and expression of inflammatory molecules was checked. Immune cells of patients with NCGS showed an increased expression of IL1ß and IL6 after stimulation with gliadins compared to immune cells of healthy controls. Gliadins caused a strong activation of P-STAT3 in immune cells of healthy controls, and inhibitors of JAK and NFκB pathways considerably reduced this response. In addition to gliadins, we further showed that glutenins and albumin/globulins from all wheat cultivars from the last century, and especially from einkorn and spelt, also markedly induced the expression of inflammatory genes in THP1 and human immune cells. There was no correlation between enhanced immune stimulation and ancient or modern cultivars. This does not support the hypothesis that modern wheat breeding is responsible for the increase in gluten-related diseases. An altered immune situation is suggested in patients with NCGS.

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods.

Do ancient wheats contain less gluten than modern bread wheat, in favour of better health?

Popular media messaging has led to increased public perception that gluten-containing foods are bad for health. In parallel, 'ancient grains' have been promoted with claims that they contain less gluten. There appears to be no clear definition of 'ancient grains' but the term usually includes einkorn, emmer, spelt and Khorasan wheat. Gluten is present in all wheat grains and all can induce coeliac disease (CD) in genetically susceptible individuals. Analyses of 'ancient' and 'modern' wheats show that the protein content of modern bread wheat (Triticum aestivum) has decreased over time while the starch content increased. In addition, it was shown that, compared to bread wheat, ancient wheats contain more protein and gluten and greater contents of many CD-active epitopes. Consequently, no single wheat type can be recommended as better for reducing the risks of or mitigating the severity of CD. An estimated 10% of the population of Western countries suffers from gastrointestinal symptoms that lack a clear organic cause and is often referred to as irritable bowel syndrome (IBS). Many of these patients consider themselves gluten sensitive, but in most cases this is not confirmed when tested in a medical setting. Instead, it may be caused by gas formation due to fermentation of fructans present in wheat or, in some patients, effects of non-gluten proteins. A significant overlap of symptoms with those of CD, IBS and inflammatory bowel disease makes a medical diagnosis a priority. This critical narrative review examines the suggestion that 'ancient' wheat types are preferred for health and better tolerance.

Wheat amylase/trypsin inhibitors (ATIs): occurrence, function and health aspects.

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.

Wheat ATIs: Characteristics and Role in Human Disease.

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.

Comparative quantitative LC-MS/MS analysis of 13 amylase/trypsin inhibitors in ancient and modern Triticum species.

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.

Mass spectrometry of in-gel digests reveals differences in amino acid sequences of high-molecular-weight glutenin subunits in spelt and emmer compared to common wheat.

High-molecular-weight glutenin subunits (HMW-GS) play an important role for the baking quality of wheat. The ancient wheats emmer and spelt differ in their HMW-GS pattern compared to modern common wheat and this might be one reason for their comparatively poor baking quality. The aim of this study was to elucidate similarities and differences in the amino acid sequences of two 1Bx HMW-GS of common wheat, spelt and emmer. First, the sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) system was optimized to separate common wheat, spelt and emmer Bx6 and Bx7 from other HMW-GS (e.g., 1Ax and 1By) in high concentrations. The in-gel digests of the Bx6 and Bx7 bands were analyzed by untargeted LC-MS/MS experiments revealing different UniProtKB accessions in spelt and emmer compared to common wheat. The HMW-GS Bx6 and Bx7, respectively, of emmer and spelt showed differences in the amino acid sequences compared to those of common wheat. The identities of the peptide variations were confirmed by targeted LC-MS/MS. These peptides can be used to differentiate between Bx6 and Bx7 of spelt and emmer and Bx6 and Bx7 of common wheat. The findings should help to increase the reliability and curation status of wheat protein databases and to understand the effects of protein structure on the functional properties. Graphical abstract.

Comparative Study on Gluten Protein Composition of Ancient (Einkorn, Emmer and Spelt) and Modern Wheat Species (Durum and Common Wheat).

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.

Targeted LC-MS/MS Reveals Similar Contents of α-Amylase/Trypsin-Inhibitors as Putative Triggers of Nonceliac Gluten Sensitivity in All Wheat Species except Einkorn.

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.