Most abundant immunogenic and toxic peptides identified in the digested prolamin extracts (known immunogenic and toxic sequences are underlined), together with an indication of the protein of origin and of their relative abundance in the different types of wheat (durum wheat:
Gluten proteins are characterized by the high glutamine and proline content; thus, during gluten digestion, several resistant peptides are produced. Some of them contain sequences that, in celiac patients, are able to trigger an immunological reaction. The prolamin fraction of different wheat samples was submitted to in vitro digestion, and the peptides generated were analysed using liquid chromatography coupled to mass spectrometry techniques. Several wheat varieties were analysed, showing large differences in the production of immunotoxic peptides on digestion. After simulated gastrointestinal digestion of wheat, emerged that peptides containing sequences known to elicit the adaptive immune response derived mainly from γ‐gliadin, whereas peptides containing sequences involved in the innate immune response were distributed among α‐gliadin and γ‐gliadin and low‐molecular‐weight glutenins. From the results, no major differences due to the different cultivation places were observed. On the other hand, statistically significant differences are present among the genotypes tested, especially for the immunogenic peptides. The possible development would be the selection of wheat genotypes with reduced amount of immunogenic sequences, to reduce the exposure of people and decrease the risk of new cases of disease.
- In vitro digestion
- gluten peptides
- celiac disease
- wheat protein
Approximately 8% of children and 1–2% of adults suffer from food allergy worldwide, and the perceived prevalence is even much higher, up to 22% of the population, constituting a fast growing health problem [1, 2]. The prevalence of food allergies is continuously increasing in the last decades, especially in the developed countries. The Big‐8 of food allergens, namely the foods that are mainly involved in these immunological reactions, are milk, eggs, fish, crustaceans, peanuts, tree nuts, soybeans and wheat. These foodstuffs can be eaten by most of the population without problems, but they can give a strong immunological reaction with topic and systemic consequences in sensitive people [3, 4]. Thus, the only therapy available for patients suffering from food allergy is the strict avoidance of the offending food. This means that allergic consumers must absolutely avoid eating foods that could provoke potentially life‐threatening reactions. Successful avoidance depends on having complete and accurate information on food labels. Thus, huge efforts are made by regulatory agencies, with the collaboration of food industry, to protect allergic consumers, to ensure that all food allergens present in the food are declared on the label and that effective controls are used to prevent the presence of unintended allergens . In the case of children, dietary elimination of nutrient‐dense foods may result in inadequate nutrient intake and impaired growth: children with multiple food allergies have a higher risk of impaired growth and may have a higher risk of inadequate nutrient intake than children without food allergies. In addition to this, the social lifestyle of individuals with food allergy and of their families can be severely disadvantaged, since they need to constantly avoid the allergenic ingredient . This task becomes more difficult to manage when people do not eat at home but in restaurants, canteens and other food chains, even if a list of the ingredients of all the dishes must be provided. Moreover, the repercussions of food allergy are not only limited to individuals or households: the food industry must also sustain a lot of extra costs due to food allergy. Primarily, legislative changes, such as the new EU‐legislation on food labelling (EU Directive 2003/89/EC amending Directive 2000/13/EC), force the industry to adapt productive processes, food labelling and monitoring to improve allergic consumer protection. The burden of responsibility falls to the food manufacturer, who is required to manage production processes to ensure allergenic ingredients are labelled . Up to now, the potential social impact and economic costs of food allergy on the individual, families, health‐related services and food industry are relevant.
Wheat is in the list of the eight main allergenic foods, because the gluten contained in it is the main external trigger of celiac disease. Celiac patients eat several types of gluten‐free products, some of them are naturally gluten‐free foods (fruits, vegetables and unprocessed meat, fish and poultry) but some others are gluten‐free substitute foods (pasta, bread, cereals, crackers and snack foods) where wheat flour is replaced by gluten‐free flours. Gluten‐free products can be purchased at general and specialty food stores as well as via Internet. Several studies demonstrated that gluten‐free food is not always readily available, and it is considerably more expensive than regular, gluten‐containing foods . The increasing incidence of celiac disease in the population has negative effects not only on the quality of life but also on the health care system: it has been estimated that the average annual health care costs per‐patient in primary care significantly increased by 91% for CD patients after they had been diagnosed with the disease . The impact is also evident for the agricultural and food sectors: wheat is one of the first three cereals for diffusion and cultivation for human nutrition. Gluten, the main trigger of celiac disease, is at the basis of rheological properties of wheat‐based products. In fact, the formation of a gluten network in the dough is of outmost importance for air bubbles and starch retention (respectively for leavened products and pasta). A low gluten content of the flour leads to loss of product shape in the case of leavened products and to soft and mushy pasta. The consequence is that wheat breeding has been, during the last decades, oriented toward increasing yield and the amounts of amylopectin, gluten and protein .
At the moment, no therapies are available for people that are already celiac, so the only treatment is the gluten‐free diet. But, on the other hand, efforts can be made in the direction of decreasing celiac disease incidence. Different hypotheses have been made on the reasons of the increased incidence of celiac disease. Since celiac disease affects the gastrointestinal tract, the gut microflora can play a key role in the loss of the immunological tolerance. For example, rod‐shaped bacteria in the upper small bowel are present in one‐third of the children with CD but in less than 2% of the controls ; another study showed that the species
Thus, trying to decrease these risk factors could help to stop the rising of celiac disease incidence. It is known for a long time that breast feeding has a protective effect against the development of celiac disease, especially when it is still ongoing during gluten introduction in the diet. Also the improvement of infant milk formula, decreasing protein content and osmolarity, has helped to reduce celiac disease incidence . Obviously, the easiest way to reduce the amount of gluten ingestion is the reduction of wheat‐derived products consumption, but this would mean a kind of “preventive gluten‐free diet”, with all the problems and limitations previously described (first of all the decrease in life quality). An alternative way could be the reduction of gluten content in wheat (in contrast with what has been done in the last decades), but this would mean a dramatic decrease in the texture quality of baked products and pasta. Since gluten proteins have a reserve role (nitrogen stock), they underwent to a limited evolutionary pressure, thus showing a high‐sequence variability with a lot of different isoforms. This lays the groundwork for a possible varietal selection aimed to have the same total gluten amount (maintaining the same rheological properties) but expressing protein isoforms with a reduced content of sequences involved in celiac disease. In this way, the exposure of the population to immunotoxic sequences will be reduced and, possibly, also the incidence of the disease.
2. Characterization of the peptides deriving from gluten digestion
Differences in gluten coding genes have been extensively characterized, besides for their technological and functional implication in baked products, also for evaluating how much the wheat genetic characteristics can impact on the final immunotoxicity of gluten. One of the most studied immunogenic peptides, the 33‐mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, has been demonstrated to be encoded by the 6D chromosome, thus being absent in diploid and tetraploid
This extensive characterization (Table 1) gives useful information for a better understanding of the peptides that presumably come in contact with the intestinal mucosa, triggering the immunological response in celiac patients. Immunogenic peptides are those containing sequences known in literature to elicit the adaptive immune response, through recognition by the HLA‐DQ2 (or DQ8) of the antigen presenting cells (APC), leading to stimulation of T cell response. As it is shown in Table 1, the identified immunogenic peptides derive mainly from α‐gliadin, in particular from the region between the 56th to the 88th amino acid. Toxic peptides are those containing sequences known in literature to elicit the innate immune response: their interaction with epithelial cells, macrophages and dendritic cells leads to the up regulation of different cell mediators, the most important one being interleukin‐15. Also in this case, most of the peptides were identified as deriving from α‐gliadin, more specifically from the N‐terminal region. Using the isotopically labelled internal standard method, peptides containing sequences involved in celiac disease can be quantified: these data can be very helpful for interpretation of the results of immunological assays, since the different response can be due both to different epitopes generation in terms of amino acid sequence and to a different relative amount of pathogenic peptides.
|Gliadin type||Relative amount (durum)||Relative amount (common)|
3. Wheat screening through
digestion and LC‐MS analysis in vitro
3.1. Influence of the cultivation region
To investigate the role of soil and climatic conditions on the total amount of toxic and immunogenic sequences, durum wheat samples harvested in three different Italian regions were submitted to prolamin extraction and
The total amount off peptides containing immunogenic and toxic sequences is reported in Figure 1, mediated for each harvesting area. Statistically significant differences were determined with analysis of variance (two ways ANOVA), with
3.2. Influence of the genotype
The accurate molecular characterization of the
As shown in Figures 2 and 3, there are great differences among the different samples. More specifically, the peptides that are more affected by genetic features are those eliciting the adaptive immune system (immunogenic peptides). This relies on the fact that toxic peptides derive from the N‐term region of gliadins, which is much more conserved than the region that originates immunogenic peptides. In the latter case, the difference is surprisingly high: there is a 6‐fold difference between the highest and the lowest scoring sample (600 µg/g vs 100 µg/g). These data confirm the huge variability in gluten‐coding genes, since also among accessions of the same genetic group, there are noticeable differences, for example, in the first group. Recent studies demonstrated that number of subjects that lost the immunological tolerance to gluten in their adulthood is increasing and among the possible causes there is also the amount and the quality of ingested gluten . This means that the use of less immunogenic wheat varieties (especially in the preparation of baby foods) can reduce the exposure to gluten, possibly decreasing the incidence of the disease. And, moreover, it would be possible to operate a varietal selection aimed to have the same gluten content (thus comparable rheological properties), but expressing different gliadin isoforms, with a reduced content of immunogenic and toxic peptides, to reduce the exposure of genetically predisposed subjects, and possibly to reduce the risk of celiac disease development. These data take in consideration the molecular point of view, so it would be really interesting to cross the data with immunological tests (such as T cell proliferation assays or K562 cells agglutination) on the samples to verify the quality of the correlation between pathogenic peptides content and immune response.
4. Wheat digestion: comparison between two different models
To perform immunological assays on gluten peptides, it is necessary to simulate the human gastrointestinal digestion on gliadin/gluten/wheat samples. In literature,
A qualitative comparison of the peptides generated with the two models is reported in Table 2, together with the protein of origin of the peptide and the retention time. Results clearly showed that the peptide composition obtained is completely different. While with the simplified digestion model, quite all the peptides derive from α‐gliadins; using the physiological digestion model, they are equally distributed among α‐gliadins, γ‐gliadin and low‐molecular‐weight glutenins. This fact can be ascribed to the different solubilisation power of the two methods. Ethanol extraction of the prolamin fraction probably leads to a better extractability of α‐gliadins; on the opposite, in the physiological digestion model, the presence of additional enzymes other than proteases (such as α‐amylases and lipases), together with the bile salts, contributes to matrix degradation, improving the extractability and digestibility of higher molecular weight proteins such as γ‐gliadins and low‐molecular‐weight glutenins. Another important difference among the two models is the presence of specific cleavage sites for the enzymes used. In the simplified digestion model, all the peptides show specific cleavage sites for the three enzymes used (pepsin, chymotrypsin and trypsin), for example, tyrosine, phenylalanine and leucine. In the physiological model indeed, in most cases, there are no specific cleavage sites, due to the action of the exoproteases present in pancreatin. Thus, changing the
|Simplified digestion||Physiological digestion|
|Adaptive immune response||Prot||Rt||Adaptive immune response||Prot||Rt|
A good correlation was found using the Pearson test (
5. Gluten peptides’ fate in the pasta production chain
The quantification of gluten peptides reported in Table 2 was carried out for six steps of pasta production (involving only durum wheat), to verify if some technological treatment have an influence on protein extractability/digestibility. Three different varieties were analyzed to exclude variations exclusively due to the genotype. Results are shown in Figure 5 for whole wheat, flour, dough, extruded pasta, dried pasta and cooked pasta.
As observed in Figure 5, after the physiological digestion method, the amount of toxic peptides is approximately a half than with the simplified method, whereas immunogenic peptides are approximately twice, due to the different type of peptides generated. This should be taken into account when biological tests are done to assess immunological responses. It can be noted that the amount of toxic and immunogenic peptides remains largely constant along the pasta production chain, so none of the processing steps of pasta is at the moment able to decrease wheat immunogenic potential for celiac patients. In other words, if a varietal screening has to be performed, there is no need to use the end product; it is sufficient to test the basic wheat variety. The difference between the two digestion methods becomes more evident after pasta cooking; in fact, heat causes polymerization of gliadins through intermolecular disulphide bridge formation and to a lesser extent for dehydroalanine formation . Thus, the heat treatment leads to the loss of gliadin extractability, which is the reason of the high underestimation of peptides generated after digestion of the ethanolic extract. It is interesting to note that independently from the method adopted, the differences between varieties maintain the same trend at all the steps of processing. This is an ulterior confirmation that traditional pasta processing leaves gluten immunogenic and toxic peptides unaffected.
Several digestion methods applied to gluten proteins are reported in literature. Generally, these models are very simple involving only the use of the main gastric and pancreatic proteases (pepsin, trypsin and chymotrypsin). A buffering agent is also used, to keep the correct pH value at each phase. However, a physiological digestion procedure was previously used in literature to assess the release of mycotoxins and heavy metals from food matrices. This method involves the use of digestive juices whose chemical composition strictly reflects the physiological one. These two methods were compared to assess gluten peptides generated. In both cases (simple and complex model), the peptides generated from the digestion were characterized using liquid chromatography coupled with mass spectrometry. In these
Thus, in the case, a subsequent immunological experiments or biological trials have to be performed, the more physiological method is more suitable than the simplified one, because the peptides generated are really different and the complex method is more similar to what really happens in the human gastrointestinal tract.
The peptides containing immunotoxic sequences were quantified for both the
For what concerns genotype influence, since the cultivar selection operated by breeders in the last years to achieve the desired rheological properties has led to a decrease in the genetic biodiversity of durum wheat varieties present nowadays on the market, 25 durum wheat accessions were selected from a durum wheat panel in order to maximize the genetic biodiversity of the samples (and thus eventual differences in immunotoxic peptides production upon digestion). Results obtained from every single accession were mediated in five groups on the basis of phylogenetic affinity on dendrogram.
For toxic peptides, no significant differences were present while strong variability emerged for immunogenic peptides, with accessions of the second groups (International Center for Agricultural Research in the Dry Areas (ICARDA) accessions for temperate areas) showing a significantly lower content of peptides eliciting adaptive immune response.
The higher variability of immunogenic peptides compared to toxic peptides can be explained on the basis of gliadins sequence variability; in fact, toxic peptides usually derive from the N‐term region of the protein, which is the most conserved. On the contrary, immunogenic peptides derive from a region of the protein showing a much higher variability. So, different wheat genotypes can express different gliadins isoforms thus showing a different final content of immunogenic sequences.
Then, it is possible to select wheat varieties with good gluten content (and good rheological properties) but with a reduced amount of immunogenic sequences in order to reduce the exposure of people to a possible trigger for celiac disease.