Simultaneously identify the gluten peptides that are most active in activating the human immune system
The method aims to identify, through the use of specific antibodies, the most toxic peptides instead of, as with the usual methods used today, the total gluten content. Considering that the research is also focused on producing grains with smaller amounts of toxic peptides, as well as using specific pools of enzymes / bacteria / etc in the preparation of products with the same purpose, the possibility of identifying and “weighing” only the presence, in the final product, more active peptides become a very useful tool. The highlighted study traces the history of the methods used to quantify gluten in food and presents advantages and limitations of the new method. All the methods mentioned, including the new method, have been developed to be able to certify whether a food is safe for celiacs or not, but the new methods become even more interesting for non-celiac and / or pre-celiac gluten sensitive subjects. The toxic fraction harmful to celiacs is the same as in people who are sensitive to gluten NOT celiac (1) but has a very different and less harmful impact: the new method is, therefore, very useful also in this case with the advantage of being able to have a greater tolerance in the result.
NEW IMMUNOCHEMICAL DETECTION METHODS
“Techniques that allow for the simultaneous detection of multiple different peptides are avail able, and are making their way into the field of food allergen detection. Another interesting possibility in gluten detection with the use of a multiplex immunoassay is to narrow down the focus even further to the harmful gluten epitopes. If antibodies were raised against the most relevant gluten epitopes, the detection of these specific epitopes could prove more relevant than detecting the total gluten content. A multiplex immunoassay can be updated by adding antibodies against more epitopes, and therefore can keep up with our increasing knowledge on harmful gluten epitopes. Also, by combining antibodies against the most relevant epitopes in a single detection method, the possibility of a false negative result decreases. Van den Broeck et al have investigated the possibilities of breeding a wheat variety with reduced CD-epitopes, based on small varieties in amino acid sequences between different gluten peptides (van den Broeck et al., 2011). If such a wheat variety could be bred, quantifying the total gluten content of food products containing this variety would be less appropriate. However, a detection method that can detect the presence of the harmful epitopes in these products would be very welcome. If the obstacles for developing a multiplex immunoassay can be overcome, this detection method would help providing consumers with more accurate food labels. This would further improve both food safety and the variety of choice in food products for CD patients everywhere”.
(1) – Gluten Immunogenic Peptides as Standard for the Evaluation of Potential Harmful Prolamin Content in Food and Human Specimen.
Ángel Cebolla , María de Lourdes Moreno , Laura Coto and Carolina Sousa
Published: 5 December 2018
Einkorn, emmer and durum wheat: they do not have the “33mer” fraction considered the most active in activating the adverse response of the immune system in celiac subjects. Also for this reason they are the most suitable genotypes for the researches whose aim is to “detoxify” the flours or to intervene with particular enzymes to hydrolyse the “toxic peptides”, however present; they are also more suitable for non-celiac gluten sensitive subjects.
“Quantitation of the immunodominant 33-mer peptide from α-gliadin in wheat flours by liquid chromatography tandem mass spectrometry.
Kathrin Schalk , Christina Lang , Herbert Wieser , Peter Koehler & Katharina Anne Scherf. Scientific Reports volume 7, Article number: 45092 (2017)
Coeliac disease (CD) is triggered by the ingestion of gluten proteins from wheat, rye, and barley. The 33-mer peptide from α2-gliadin has frequently been described as the most important CD-immunogenic sequence within gluten. However, from more than 890 published amino acid sequences of α-gliadins, only 19 sequences contain the 33-mer. In order to make a precise assessment of the importance of the 33-mer, it is necessary to elucidate which wheat species and cultivars contain the peptide and at which concentrations. This paper presents the development of a stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry to quantitate the 33-mer in flours of 23 hexaploid modern and 15 old common (bread) wheat as well as two spelt cultivars. All flours contained the 33-mer peptide at levels ranging from 91–603 μg/g flour. In contrast, the 33-mer was absent (<limit of detection) from tetra- and diploid species (durum wheat, emmer, einkorn), most likely because of the absence of the D-genome, which encodes α2-gliadins. Due to the presence of the 33-mer in all common wheat and spelt flours analysed here, the special focus in the literature on this most immunodominant peptide seems to be justified……Omissis…..
Analysis of durum wheat, emmer and einkorn
The 33-mer peptide was also analysed in two durum wheat and two emmer cultivars (genome AABB) as well as two diploid einkorn cultivars (genome AA) (Table 1). In each of these wheat species, the 33-mer was not detected (<LOD). In comparison to hexaploid common wheat, durum wheat, emmer, and einkorn do not contain the D-genome, which originated from hybridisation of T. turgidum dicoccum (genome AABB) with Aegilops tauschii (genome DD)36. The absence of the 33-mer peptide can be explained by the fact that this peptide is encoded by genes located in the Gli-2 locus on chromosome 6D, which is missing in durum wheat, emmer, and einkorn. Studies by Molberg et al. showed clear variations in intestinal T-cell responses between common wheat and tetra- or diploid species due to different degrees of T-cell immunoreactivity between the gluten proteins encoded on the A-, B-, and D-genome. Einkorn cultivars were only recognized by DQ2.5-glia-α1a-specific T-cell clones, but not by DQ2.5-glia-α1b- and DQ2.5-glia-α2-specific T-cell clones. Emmer and durum wheat cultivars were all recognized by DQ2.5-glia-α1a-specific T-cell clones, but only two out of four emmer cultivars and three out of ten durum wheat cultivars activated DQ2.5-glia-α1b- and DQ2.5-glia-α2-specific T-cell clones37. Consistent with our results, Prandi et al.38 found that the 33-mer was not present in durum wheat. As a consequence, this peptide was used as a marker peptide to identify the presence of common wheat in durum wheat flours. One durum wheat cultivar was also analysed by van den Broeck et al.33 and the 33-mer peptide was not detected either”. https://creativecommons.org/licenses/by/4.0/deed.it
Scientific research has long highlighted the peculiar characteristics of the monococcum wheat reported in (https://glutenlight.eu/2019/03/11/il-grano-monococco/) and can be summarized as follows:
- High digestibility of gluten
- High tolerability in relation to gastro-intestinal disorders (celiac disease excluded)
- High mineral and vitamin content
- High availability of bioactive components
- A different ratio between the components of starch sugar with a prevalence of slow absorption.
Monococcum wheat has, for some time, been the focus of producers-transformers and consumers. The offer, especially of flour, as well as of products is constantly increasing. The products offered, however, do not have, except in rare cases, complete traceability starting from the field to the table. On the flour packages it is rare to find indications regarding the variety of wheat from which it derives; on the final products we find the mandatory ingredients by law but, rarely, the preparation method. The speech, however, applies to all grains both ancient and modern. The greater attention to the monococcum wheat (einkorn) is due to the evocative force of its ancestral origin and to its characteristics of high digestibility, tolerability and healthy contents.
The variety of wheat used and the indicators that inform us about the quantity and “strength” of gluten would be particularly valuable to be able to include more digestible products in our diet when needed. Gluten, as it is formed during water and flour are kneaded, cannot be digested by our intestines, it must first be “broken” by digestive enzymes into very small “fragments”. In this way other digestive enzymes in the intestine will complete the work in order to make the gluten components “amino acids” assimilable. The lower amount of gluten and less strength will sometimes make our task much easier. Products made with monococcoum wheat flour (einkorn) and generally those made with “ancient grains” are advertised as “very digestible” or “highly digestible”. Both terms are very generic since they can present strong differences in terms of gluten quantity and “gluten strength”. Recently I bought two different monococcum wheat flours of which I pointed out the amount of gluten: one has a percentage of dry gluten of 9.6% the other of 17.1%! Same thing with the strength of gluten whose index in one case was 33 in another 71!These indicators are a first valid help that we could have to better balance, with the support of the doctor, our diet. It should also be remembered that the final digestibility of the product made with flours, whatever they may be, is also greatly influenced by the way in which the products are prepared: just think of the considerable contribution to digestibility that we can obtain by using sourdough, but this information is also generally absent or present in an ambiguous way or without specification of which flour was used: “… made with sour dough”. https://glutenlight.eu/2019/05/08/la-fermentazione-della-pasta-acida-ii-parte/.
LC/MS ANALYSIS OF GLUTEN PEPTIDES DERIVED FROM SIMULATED GASTROINTESTINAL DIGESTION OF DIFFERENT WHEAT VARIETIES: QUALITY AND SAFETY IMPLICATIONS. Sforza, Stefano & Prandi, Barbara & Bencivenni, Mariangela & Tedeschi, Tullia & Dossena, Arnaldo & Marchelli, Rosangela & Galaverna, Gianni. (2011):
“Gluten content of wheat is highly variable, depending on the plant genetics and the growing conditions. Beside short peptides, gastrointestinal digestion of gluten also produces longer ones, since the high proline content of gliadins (16-26%) and glutenins (11-13%) makes them very resistant to the degradation by digestive proteases. In the present work, a method for the extraction of the prolamine fraction was applied to different wheat varieties, followed by a simulated gastrointestinal digestion of the gliadin extracted. The peptide mixtures generated were characterized by LC/MS, and most abundant peptides were identified by low- and high-resolution multiple stage MS techniques and through synthesis of authentic standards. These peptides were also semiquantified in the different samples against a suitable internal standard. The peptide mixtures were found to be highly variable, according to the different content and type of gliadins present in wheat varieties, with strong differences among the varieties tested, both qualitatively (the sequences of the peptides generated) and quantitatively (their amount). The greatest difference was found between common and durum wheat varieties. Peptides present only in the former varieties were identified, and used as molecular markers for identifying and quantifying the presence of common wheat when added to durum wheat samples. Most of the peptides identified were also already known to be pathogenic for people affected by celiac disease, an autoimmune enteropathy triggered by gluten proteins, which develops in some genetically susceptible subjects after gluten consumption. Some samples belonging to defined varieties showed a lower amount of celiac-related pathogenic peptides upon digestion, due to a lower gliadin content. Albeit not safe for celiac patients, the use of these varieties in the formulations of baby food could be of great help for lowering the spread of the disease, since the prevalence of celiac disease seems to be promoted by an early exposure to a large amount of gluten peptides”.