**The Gluten-Free Diet: Testing Alternative Cereals Tolerated by Celiac Patients**

**Isabel Comino 1 , María de Lourdes Moreno 1 , Ana Real 1 , Alfonso Rodríguez-Herrera 2 , Francisco Barro <sup>3</sup> and Carolina Sousa 1,\*** 


*Received: 29 August 2013; in revised form: 7 October 2013 / Accepted: 15 October 2013 / Published: 23 October 2013* 

**Abstract:** A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease, an autoimmune disorder of the small intestine associated with a permanent intolerance to gluten proteins. The complete elimination of gluten proteins contained in cereals from the diet is the key to celiac disease management. However, this generates numerous social and economic repercussions due to the ubiquity of gluten in foods. The research presented in this review focuses on the current status of alternative cereals and pseudocereals and their derivatives obtained by natural selection, breeding programs and transgenic or enzymatic technology, potential tolerated by celiac people. Finally, we describe several strategies for detoxification of dietary gluten. These included enzymatic cleavage of gliadin fragment by Prolyl endopeptidases (PEPs) from different organisms, degradation of toxic peptides by germinating cereal enzymes and transamidation of cereal flours. This information can be used to search for and develop cereals with the baking and nutritional qualities of toxic cereals, but which do not exacerbate this condition.

**Keywords:** celiac disease; gluten-free diet; cereals; pseudocereals; gluten detoxification

#### **1. Introduction**

Celiac disease is a food intolerant related syndrome that, despite being under-diagnosed, is one of the most frequent chronic gastrointestinal disorders. It develops in genetically predisposed individuals in whom unidentified environmental factors (infections, changes in microbial flora, *etc.*) can trigger intolerance to gluten contained in wheat, barley, rye and oats [1,2]. Gluten is a complex mixture of proteins called prolamins. This protein fraction has specific name: wheat prolamins are termed gliadins and glutenins, barley prolamins are hordeins, rye prolamins are secalin and those from oats are avenins. A common characteristic of these proteins is the presence of multiple proline and glutamine residues, making them resistant to gastrointestinal digestion and more exposed to deamination by tissue transglutaminase.

Several epitopes responsible for the toxicity of gluten have been identified based on their ability to stimulate proliferation of gluten-responsive T cells in celiac patient-derived small intestine biopsies. Considering only wheat, in the Immune Epitope Database (IEDB) [3] can be found 190 T-cell stimulatory epitopes related to celiac disease£# ¤ -gliadin genes, 74 \$-gliadin -gliadin genes, 8 in low molecular weight (LMW) glutenin genes, and 2 in high molecular weight (HMW) genes.

The most accepted model for explaining the immunopathogenesis of celiac disease is the two-signal model, characterized by a first innate immune response and a subsequent secondary adaptive response, which will promote a histological lesion characterized by a massive intraepithelial infiltration of lymphocytes, crypt hyperplasia and villous atrophy [2]. The ingestion of these proteins leads to the inflammation, atrophy, and hyperplasia of the small-intestinal crypts of the celiac patient. However, this disease not only affects the gut, but it is a systemic disease that may cause injury to the skin, liver, joints, brain, heart, and other organs.

Celiac disease goes in remission when the patients are put on a gluten-exclusion diet, and patients relapse when gluten is reintroduced into the diet [1,2]. Complying with a GFD is difficult and affects the patients' quality of life, but a strict diet is critical to reduce morbidity and mortality [4].

Gluten has many special characteristics that favor its use in various food products. Because a large amount of gluten is generated during the manufacture of starch, it has a relatively low price. This may turn out to be problematic for people on a GFD, since gluten proteins may be found in unexpected sources such as meat, fish or milk products. This is the reason why alternative approaches to the GFD are actively sought [5], which include the search for and development of new cereals or gluten with no or low immunogenic content. In this article, we will review the current status of alternative cereals and their derivatives obtained by natural selection, breeding programs and transgenic or enzimatic technology, which may be potential tolerated by patients with celiac disease.

#### **2. Natural Varieties of Cereal and Pseudocereals Suitable for Patients with Celiac Disease**

#### *2.1. Wheat and Barley*

Cultivated wheat is genetically very complex due to its origin from ancestral diploid species through a process of natural hybridization and subsequent polyploidization. The two wheat species of agricultural importance, the pasta wheat, and the bread wheat, are tetraploid (two genomes, AABB) and hexaploid (three genomes, AABBDD), respectively (Figure 1). The tetraploids originated in nature through spontaneous hybridization of two diploid species, each donor genomes A and B, between 0.5 and 2 million years ago. Bread wheat (AABBDD) originated in the fields, about 8000 years ago, through spontaneous hybridization between durum wheat (AABB) and *Aegilops tauschii*, the diploid donor of the D genome (Figure 1).

One species of wheat, *Triticum aestivum*, is predominantly used in the modern industrialized world, due to its increased protein production as well as its hardiness in colder climates. It has also been determined that the proteins that are the most immunogenic for celiac disease reside in the gliadin fraction of *T. aestivum*. However, there are almost 20 other species of wheat that are either not being cultivated by modern societies or are cultivated in select regions of the world [6]. With such a large number of wheat species available, a significant amount of research has been focused on the exploration of different species and cultivars of wheat as an alternative to a strict GFD for celiac patients [7].

**Figure 1.** Diagram showing the evolutionary relationships among species of wheat and related species of *Aegilops*. The dotted line separates the wild and domesticated species of wheat. Ancestral or unknown species are surrounded by a double dashed rectangle. Ploidy level and the number of chromosomes are indicated. Nomenclature according to van Slageren [8].

Attempts have been made to quantify the toxicity of a range of bread wheat and pasta wheat varieties and of species that contain only one of the three genomes of bread wheat [9,10]. Using specific T-cell clones and monoclonal antibodies, the results demonstrated that large quantitative differences exist in the presence of toxic gluten peptides, with some cultivars completely lacking particular harmful peptides [11]. Diploid wheat species are among the suitable candidates for their low capability to activate intestinal T cell responses in celiac patients [11,12]. Compared with tetraploid and hexaploid wheat, commonly used in the making of bread and pasta, the ancient diploid *Triticum monococcum* ssp. monococcum wheat showed a marked reduction, or even a lack, of toxicity *in vitro* cellular assays [13,14]. Gianfrani *et al.* [15] compared the immunological properties of 2 lines of diploid monococcum wheat, Monlis and ID331, with those of *T. aestivum*. They found that both lines activate celiac T cell response. However, ID331 was less effective to activate the innate immune pathways. The reduced ability of some diploid wheat lines to *in vitro* activate the innate immune response in celiac mucosa could render these cultivars less active in inducing celiac disease. However, more analyses are required to explore their potential use as new dietary opportunities for celiac patients.

Some approaches were performed to remove celiac toxic proteins in barley. Double-null hybrid plants, largely devoid of both B- and C-hordeins, were produced by conventional crossing [16]. Barley is a diploid and unlike the situation in bread wheat, the genetics of hordeins are relatively straightforward. There are four protein families of hordeins: B-, C-, D- 
\$-hordeins, with the Band C-hordeins together accounting for over 90% of barley hordeins. Isolation of hordein double-null barley lines from hybrids of Risø 56 and Risø 1508 has produced a line which does not accumulate B- or C-hordein and only has 3% of wild type hordein along with a 20-fold reduction in reactivity in T-cell assays [16,17]. In addition, studies carried out demonstrated that some malting lines (*Hordeum vulgare*) were less immunogenic compared with wild lines (*Hordeum chilense*) [18]. These findings could raise the prospect of breeding barley species with low levels of harmful gluten, and the attractive goal of developing non-toxic barley cultivars with a potential use in the manufacture of beverages as consumed worldwide as are the beers. However, nothing is known about the variability in celiac toxicity of other species or varieties of toxic cereals such as rye.

#### *2.2. Oats*

Cultivated oats are hexaploid cereals belonging to the genus *Avena* L., which is found worldwide in almost all agricultural environments [19]. Recently, oats have been receiving increasing interest as human food, mainly because the cereal could be suitable for consumptions by celiac patients. Several varieties of oats are available. It is a rich source of protein, contains a number of important 
  -glucan, a mixed-linkage polysaccharide, which forms an important part of oat dietary fiber, and also contains various other phytoconstituents like avenanthramides, an indole alkaloid-gramine, flavonoids, flavonolignans, triterpenoid saponins, sterols, and tocols. Traditionally oats have been in use since long and are considered as stimulant, antispasmodic, antitumor, diuretic, and neurotonic. Oat possesses different pharmacological activities like antioxidant, anti-inflammatory, antidiabetic, anticholesterolaemic, *etc.* [20].

The presence of oats in a GFD is still a subject of controversial. Oats differ from other cereals in their prolamin content. The percentage of proline and glutamine (amino acids abundant in toxic regions) in avenin is lower than in other toxic cereals. Some clinical researchers state that patients with celiac disease tolerate oats without signs of intestinal inflammation [21]. According to the Codex Alimentarius for food for special dietary use for persons intolerant to gluten, CODEX STAN118-1979 (revised 2008, [22]), oats can be tolerated by most but not all people who are intolerant to gluten. Moreover, according to the Commission Regulation (EC) No 41/2009 [23] concerning the composition and labeling of foodstuffs suitable for people intolerant to gluten, a

major concern is the contamination of oats with wheat, rye or barley that can occur during grain harvesting, transport, storage and processing. Therefore, the risk of gluten contamination in products containing oats should be taken into consideration with regard to labeling of those products. In contrast, other studies confirmed the toxicity of oats in certain types of patients with celiac disease. Arentz-Hansen *et al.* [24] described the intestinal deterioration suffered by some patients with celiac disease following the consumption of oats while on a GFD. Avenin can trigger an immunological response in these patients similar to the response produced by the gluten of wheat, rye or barley. The monitoring of 19 adult patients with celiac disease who consumed 50 g/day of oats over 12 weeks showed that one of the subjects was sensitive to oats. Therefore, it is critical to clarify either qualitatively or quantitatively the potential immunotoxicity of oats to patients with celiac disease [21,24].

Comparison of the different studies are complicated by the different study designs, the different conditions used in the testing, the number of subjects included in each study and the reporting of the purity control of the oat material used in the clinical trials. Another relevant factor in different designs is the absence of information on the oat variety used. Silano *et al.* [25] investigated the immunogenic effect of avenins from four oat cultivars using peripheral lymphocytes from patients with CD. All the varieties of oats tested (Lampton, Astra, Ava, and Nave) by these investigators were immunogenic with differences in their capacity to induce a response. However, other study confirmed that *Avena genziana* and *Avena potenza* do not display *in vitro* activities related to CD pathogenesis [26].

The utility of the G12 antibody to identify potentially toxic oat varieties for celiac patients has been reported [27]. This finding allowed classification of oat varieties into three groups based in their degree of affinity for the G12 antibody: a highly recognized group, one of moderate recognition, and one with no reactivity [27]. These results were confirmed by MALDI-TOF, SDS-PAGE and western blot by showing that the number, relative intensity of the peaks and protein profile obtained for the nine oat varieties differ from one another. The potentially immunotoxicity of the different types of oats was determined by T cell proliferation and interf \$ 
 The reactivity that T-cells isolated from celiac patients exhibited with three oat varieties (one from each of the classified groups) correlated directly with the moAb G12 reactivity. The diversity observed in the reactivity to the different oat cultivars suggests variations in the avenin composition, and therefore in the amount of immunotoxic epitopes similar to the 33-mer present in these varieties. This gives a rational explanation for why only some oats trigger an immunological response.

In comparison with wheat gliadins, the avenins have been little studied, and the number of full avenin genes present at the moment in the databases is limited and from few genotypes, so that the variability of avenin genes in oats is not well represented. It has recent been known that, like wheat, oat grains have both monomeric and polymeric avenins [28]. A direct correlation between the immunogenicity of the different varieties of oats and the presence of the specific peptides with a higher/lower potential immunotoxicity has been found, that could explain why certain varieties of oats are toxic for celiac patients and other not [28]. The incorporation of some varieties of oats in food products not only may improve the nutritional quality but also may provide a treatment for various illnesses and would be welcomed by patients with celiac disease.
