Keywords

2.1 Introduction

Gluten is a complex mixture of proteins found in cereal kernels from the Triticeae family. This group includes wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and rye (Secale cereale L.), which are evolution-related and contain homologous peptide groups. Although oats belong to the same sub-family, they come from the Aveneae family (Fig. 2.1). There is an ongoing debate concerning the toxicity of oat prolamins, which present some characteristics that are different than those from Triticeae [1].

Fig. 2.1
figure 1

Taxonomic relationship of cereals (Modified from Belitz et al. [2])

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In 1907 Osborne [3] divided cereal proteins into four groups according to their solubility: water-soluble albumins; salt-soluble globulins (0.4 M); 60–70% aqueous ethanol-soluble prolamins; and glutelins, which are soluble in dilute acids or bases, detergents, and reducing agents. Each fraction takes different names, depending on the cereal from which it comes, and the content varies from one cereal to another (Table 2.1). Rye has the highest albumin content, and the prolamin content is highest in corn and wheat, whereas oat and rice have the lowest content, and glutelins are the major property in rice and oats.

Table 2.1 Protein distribution (%) and designations of fractions separated by Osborne
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Albumins and globulins are derived from the original cytoplasm of the cell and from other sub-cellular fractions, and they have metabolic and structural functions because of the presence of enzymes. Prolamins and glutelins are both proteins whose function is storing nitrogen, carbon, and sulfur in the endosperm of the kernel [2, 4]. The term “prolamin” is used due to its high content in the amino acid proline (Pro), and “glutelin” is used because of the content of glutamine (Gln). Proline has the ability to make β-turns, which has been shown to be more efficient than α-turns in packing proteins into a small space. This characteristic is very useful for storing amino acids, but the tight structure that is created hinders the hydrolyzation of the prolamin [5].

Gluten is a complex protein formed by a mixture of prolamins and glutelins at a ratio of 1:1 that represents around 80% of the total proteins of most cereals. These proteins can be found in cereals either as monomers or as oligomers and polymers linked by disulphide bonds – something that causes different physicochemical properties.

Wheat prolamin and glutelin are responsible for the rheological characteristics of dough; while gliadin brings viscosity, glutenin provides elasticity and strength for the dough. Gliadins form monomers with intramolecular disulphide bonds, and molecular weights ranging from 30 to 60 kDa that can be divided into α-, β-, γ-, and ω-gliadins (Fig. 2.2). Gliadins are also grouped by their N-terminal sequence: S-rich α/β-, γ-, and S-poor ω-gliadins that contain no cysteine residue [6]. On the other hand, glutenins are found as polymers through interchain disulfide bonds as well as intramolecular bonds, with molecular weights ranging from 80 kDa to several million kilodaltons. These proteins are divided into two groups: low–molecular weight glutenins and high–molecular weight glutenins, as a consequence of their molecular weight range [7, 8]. In other grains, like rye, gluten fractions are sub-grouped into ω-secalins, γ-secalins, and HMW glutelins. In barley, this heterogeneous protein is divided into B-, C-, and D-hordeins.

Fig. 2.2
figure 2

Wheat protein classification

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2.2 Toxicity Factors in Cereal Proteins

A great heterogeneity of peptides seems to be involved in the pathogenesis of celiac disease, although the characterization of all the relevant epitopes has not yet been achieved [9, 10]. Furthermore, the chemical diversity resulting from the various amino acid compositions makes the quantification of immunogenic peptide sequences difficult.

In order to develop an ideal antibody for gluten analysis in foods, it is important to take into account the fact that besides being able to determine various cereal prolamins, it should also recognize the specific intramolecular regions responsible for toxicity in CD [1]. The toxicity of prolamins depends on their amino acid sequences and molecular properties. Celiac-harmful proteins are rich in glutamine and proline [11,12,13] and these aminoacids are located in the protein repetitive domains. Alfa- and ω-gliadin-derived peptides are involved in immunological responses in adults, whereas other peptides, such as low–molecular weight glutenins and γ-gliadins, have been responsible for toxicity in children and occasionally in adults [14, 15]. In this context, many protein regions had been proposed by their immunogenic properties, and even today some of them remain unidentified.

Accordingly, several approaches have been developed to identify the gluten peptides that can be recognized by T cells from the celiac population. The proline-rich repetitive region of gliadins is responsible for carrying epitopes for a respective lymphocyte receptor and is connected to CD. One of the most popular is 33-mer from α-gliadin, which contains three overlapping glutamine (Gln)-Pro rich epitopes (12,52,95): PFPQPQLPY, PQPQLPYPQ (3 copies), and PYPQPQLPY (2 copies) [16].

A 33-amino acid peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) from α/β-gliadin has been shown to be resistant to gastric and pancreatic hydrolysis and to exhibit celiac disease toxicity in vivo and in vitro. Furthermore, this 33-mer peptide acts as a strong stimulator to intestinal T-cells [16]. Nevertheless, other potential immunogenic peptides had been described (Table 2.2).

Table 2.2 Some immunogenic peptides found in wheat prolamins (modified from Kanerva [5])
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2.3 The Gliadin Standard

It is crucial for celiac patients to know the gluten content of the foods they eat, but detecting the toxic fraction of these molecules is not easy. Currently, immunochemistry is the most-used technology for food analysis, but some technical aspects remain to be solved. A critical point in this assay is the availability of a suitable reference standard in order to assess the results. The Working Group on Prolamin Analysis and Toxicity prepared the European Gliadin Standard (called PWG standard or PWG gliadin) from the 28 most common wheat cultivars grown in Europe [27]. These prolamins (gliadins) were separated from albumins and globulins using 0.4 M NaCl and then extracted with 60% ethanol. The gliadin extracts were finally concentrated, desalted by ultrafiltration, freeze-dried, and homogenized. This standard has been characterized by polyacrylamide gel electrophoresis, capillary electrophoresis, RP-HPLC, MALDI-TOF MS, and immunoassays, and its solubility and stability have been also evaluated.

However, the number of proteins in cereals is greater than those present in the PWG gliadin standard. Moreover, a great number of foodstuffs (produced using processes of fermentation and hydrolysis, like sourdough products, starch syrup, malt extracts, or beer) can suffer a partial or total hydrolysis of proteins. In these cases, using a hydrolyzed standard and a competitive immunoassay to determine gluten in foods has been suggested. This hydrolyzed standard has been prepared with prolamin fractions from wheat (gliadin), barley (hordein), and rye (secalin) [28]. Although the results obtained with the PWG gliadin standard are comparable to those obtained using the partially enzymatically digested prolamin standard in a competitive assay, it is more difficult to prepare a reproducible hydrolyzed standard [29]. Other immunotoxic peptides of prolamin are being proposed as standard with a high degree of repeatability, reproducibility, and stability [30, 31], but they are still not widely used in routine analysis.

2.4 International Food Laws for Gluten-Free Products

Even if the promoter of CD was discovered in the twentieth century, with diet as the only treatment possible for these patients, food safety and food laws were not completely revised to meet their necessities until the twenty-first century. This is due, fundamentally, to the fact that CD and food allergies are becoming more prevalent with time. In the case of CD, it is probably because nowadays the diagnosis is more efficient, even if there is still a high percentage of under-diagnosed people; moreover, many potential celiac sufferers start diet treatment on their own, with no official diagnosis and without the supervision of doctors.

Food allergen management and control (including gluten) has become a food safety issue in recent years. In the case of gluten, it has been brought to light that even if naturally gluten-free ingredients are used, the final product could be cross-contaminated, and products apparently free of gluten could be made from ingredients that in the end bring a non-admissible gluten level not always reflected on the label. This situation leads to diet transgressions that ultimately affect the health of celiac sufferers and give little chance to reach nutritional balance with a true gluten-free diet. Many products labelled gluten-free have been launched on the market recently, and such a claim has been attempted to be regulated. Laws have been passed all over the world trying to make adequate labelling of gluten-free products mandatory in order to increase the variety of food these patients can use, thereby ensuring their safety.

The Codex Alimentarius (an international organization founded by the Food and Agriculture Organization and the World Health Organization) was established to develop coordinated international food standards. In 1979 this institution adopted a standard for foods for special dietary use for persons intolerant to gluten; the standard was revised and corrected both in 2008 and 2015 [32]. The document set the definition of gluten-free foods and foods specially processed to reduce gluten content as follows:

  1. 1.

    Gluten-free foods are dietary foods

    1. (a)

      Consisting of or made only from one or more ingredients that do not contain wheat (i.e., all Triticum species, such as durum wheat, spelt, and khorasan wheat, which is also marketed under different trademarks such as Kamut), rye, barley, oats, or their crossbred varieties, and the gluten level does not exceed 20 mg/kg in total, based on the food as sold or distributed to the consumer, and/or

    2. (b)

      Consisting of one or more ingredients from wheat (i.e., all Triticum species, such as durum wheat, spelt, and khorasan wheat, which is also marketed under various trademarks such as Kamut), rye, barley, oats, or their crossbred varieties, which have been specially processed to remove gluten, and the gluten level does not exceed 20 mg/kg in total, based on the food as sold or distributed to the consumer.

  2. 2.

    Foods specially processed to reduce gluten content to a level from above 20 to 100 mg/kg

    • are foods that consist of one or more ingredients from wheat (i.e., all Triticum species, such as durum wheat, spelt, and khorasan wheat, which is also marketed under different trademarks such as Kamut), rye, barley, oats or their crossbred varieties, which have been specially processed to reduce the gluten content to a level from above 20 to 100 mg/kg in total, based on the food as sold or distributed to the consumer.

Oats, or at least some varieties [33, 34] of oats, can be tolerated by most but not all people who are intolerant to gluten. Taking this into consideration, the allowance of oats that are not contaminated with wheat, rye, or barley in foods covered by this standard may be determined at the national level. According to the document, nations are responsible for the decisions on the marketing of products, and the decision on whether or not to use the term “gluten-free” on the label lies with each manufacturer, so the product is only subject to the respective regulatory framework if a voluntary gluten-free claim is made.

Most nations have based their laws on this standard. Nevertheless, there are some differences between nations at the level of labelling.

In the European Union (EU), before the revision of the Codex Standard in 2008, the Council Directive 89/398/CEE of May 3, 1989, included gluten-free foods as food for particular uses [35]. This directive forced the approval of other directives that would regulate any of these foods. In the case of gluten-free foods, Directive 2000/13/EC included by amendment a list of allergens in its annex IIIa [36], in which appeared cereals containing gluten (i.e., wheat, rye, barley, oats, spelt, Kamut, or their hybridized strains) and products thereof. The food containing any of the allergens listed in that annex had to be clearly labelled in the list of ingredients or by an indication that will include the word “contains” followed by the names of the ingredient(s) concerned. This list of allergens was recently modified in other directives in order to give details of the non-allergenic derivatives of the ingredients for which there were exemptions from the labelling requirement in force at that moment, such as wheat-based maltodextrins or glucose syrups based on barley.

Lately, and considering the fact that gluten is not introduced into an infant’s diet until several months of age, Commission Directive 2006/141/EC of December 22, 2006, on infant formulas and formulas for infants when appropriate complementary feeding is introduced, prohibited the use of gluten-containing ingredients in such foods [37]. This prohibition extended to the use of the terms “gluten-free” or “low content of gluten” in these products. Even if the directive will be repealed in 2020, the prohibition on using gluten-containing ingredients in these foods will remain in the new regulation (Commission Delegated Regulation (EU) 2016/127 of September 25, 2015 [38]).

After the revision of the Codex Standard, and based on it, the European Union adopted Commission Regulation (EC) No. 41/2009 of January 20, 2009 [39]. This regulation was recently repealed and its framework is now under the Regulation (EU) No. 1169/2011, of October 25, 2011, on the provision of food information to consumers [40], and under the Commission Implementing Regulation (EU) No. 828/2014, which lays out the specific requirements for the provision of information to consumers on the absence or reduced presence of gluten in food [41]. These regulations considered that gluten levels defined in the Codex were scientifically set and they regulated the indications related to gluten content that will be made in labelling in the EU, as seen before. They allowed the use of “very low gluten” and “gluten-free” wordings that enable celiac patients to find on the market a variety of foods suitable to their needs and to their level of sensitivity to gluten (Table 2.3). Moreover, according to this regulation, it is permitted to distinguish between foods that are naturally free of gluten and products that are specially formulated for celiac patients.

Table 2.3 Statements on the absence or reduced presence of gluten in foods that are allowed to be made and conditions thereof by Regulation (EU) No 828/2014
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Outside the EU, before 2013 there were no standards for the food industry to use in labelling products as “gluten-free” in the U.S. That year, the Food and Drug Administration (FDA) established, among other criteria, a gluten limit of less than 20 ppm for foods that carry the label “gluten-free,” “no gluten,” “free of gluten,” or “without gluten” [42]. The rule implements part of the Food Allergen Labelling and Consumer Protection Act of 2004 (FALCPA) [43]. According to FDA opinion, messages such as “low gluten” or “very low gluten” are misleading, and if used, they will be studied case by case (Guidance for Industry, FDA, 2014). The rule excludes those foods whose labelling is regulated by the U.S. Department of Agriculture (USDA) and the Alcohol and Tobacco Tax and Trade Bureau (TTB), but includes supplements. Generally, the USDA regulates the labelling of meats, poultry, and certain egg products, while the TTB regulates the labelling of most alcoholic beverages, including all distilled spirits, wines that contain 7% or more alcohol by volume, and malted beverages that are made with both malted barley and hops.

The U.S.’s neighbor, Canada, for example, does not allow expressions such as “low gluten” or “reduced gluten” for the same reason, as seen in Section B.24.018 of the Canadian Food and Drug Regulations [44]. The U.S., however, does not allow food that has less than 20 ppm gluten but contains a gluten-containing grain to use the claim “gluten-free,” while Canada does. This claim will always be made if the manufacturer includes additional processing steps that are shown to be effective in removing gluten [45].

Furthermore, and as an example of the permanent evolution of the regulation, the FDA is at the moment proposing a rule to establish requirements concerning “gluten-free” labelling for foods that are fermented or hydrolyzed or that contain fermented or hydrolyzed ingredients [46]. The FDA proposes evaluating the compliance of such fermented and hydrolyzed foods that bear a “gluten-free” claim with the gluten-free labelling rule based on records that are made and kept by the manufacturer of the food with the “gluten-free” claim and made available to the agency for inspection. This is undoubtedly good news, as there is confusion in interpreting the results of current gluten test methods for fermented and hydrolyzed foods, as mentioned in Chap. 3. This could also be a good guideline for international legislators to broaden the variety of food that celiac patients could consume safely.

According to this proposed rule, the records would need to provide adequate assurance that the food is “gluten-free” in compliance with the gluten-free food-labelling final rule before fermentation or hydrolysis. In addition, the proposed rule would require the manufacturer of fermented or hydrolyzed foods bearing the “gluten-free” claim to document that it has adequately evaluated the potential for gluten cross-contact and, if identified, that the manufacturer has implemented measures to prevent the introduction of gluten into the food during the manufacturing process. Likewise, manufacturers of foods that contain fermented or hydrolyzed ingredients and bear the “gluten-free” claim would be required to make and keep records that adequately show that the fermented or hydrolyzed ingredients are “gluten-free” according to their regulations. Finally, the proposed rule would state that the FDA would evaluate the compliance of distilled foods by verifying the absence of protein using scientifically valid analytical methods that can reliably detect the presence of protein or protein fragments in the distilled food.

Other countries that are culturally close, such as Australia and New Zealand, regulate gluten-related labelling in their Food Standards Codes. This regulation states that while gluten-free mentions can only be made for products with no gluten-containing ingredients (even if they have been malted or hydrolyzed − including oats), “low gluten” mentions could be made for products with less than 200 ppm of gluten, which doubles the limit of the Codex Standard [47].

Other countries, such as Argentina, are stricter than the Codex, and set the limit for gluten-free products to 10 ppm. Moreover, Argentina defines gluten-free products as those that are naturally free of gluten, and manufacturers must apply GMP (Good Manufacturing Practices) in order to assure the absence of cross-contamination as set by Article 1383 of Chapter XVII of the Argentinian Food Code [48]. In Japan, seven items are mandatory for labelling: shrimp/prawns, crab, wheat, buckwheat, eggs, milk, and peanuts, and another 20 are recommended for labelling. They are aware of allergies, but they do not regulate gluten content-related expressions regarding celiac disease. In fact, manufacturers must declare the presence of these allergens when their content is above 10 ppm, and precautionary expressions such as “may contain” are prohibited [49]. Nevertheless, changes are rapidly being made all over the world.

Other countries have no regulations yet for gluten-free labelling or labelling for allergies and intolerances. Even if legislators have been working hard over the last few years, there is still much work to be done. Hydrolyzed and/or fermented products are an example, as previously mentioned, but industrial processes are complex and not always totally controlled to avoid cross-contaminations; this promotes the use of precautionary labelling (terms such as “may contain,” “made in a factory that also uses...”, or “not suitable for celiac patients”), prohibited in some countries for any allergen. This precautionary labelling is not properly regulated in most countries. It is confusing and turns over to the patients the responsibility of having a potential risk if they consume the product, or it narrows down the availability of products for this group even if, in some cases, the products are probably safe for them. Moreover, the inconsistent use of these messages leads to mistrust in the label and they are often ignored [50, 51].

For oats, the regulation is slightly different. The aforementioned Codex standard pointed out that even if most people with intolerance to gluten can include oats in their diets without any adverse effect on their health, the allowance of oats that are not contaminated with wheat, rye, or barley in foods may be determined at the national level. In the EU, Regulation (EU) No. 828/2014 includes additional requirements for products containing oats [41]. These cereals, if used in products labelled as gluten-free or low-gluten content, must have been specially produced, prepared, and/or processed in a way to avoid contamination by wheat, rye, barley, or their crossbred varieties, and the gluten content of such oats cannot exceed 20 mg/kg. In the U.S., oats are not considered to be a “gluten-containing grain” [41], so oats can be used as an ingredient in foods labelled “gluten-free” as long as the oats contain <20 ppm gluten. Nevertheless, the rule encourages “manufacturers of foods labelled ‘gluten-free’ that use an oat-derived ingredient where the word ‘oat’ does not appear in the ingredient list to indicate in their labelling that an oat-derived ingredient is present.”

It is necessary to label food products adequately, and it is obvious that important efforts have been made in recent years all over the world. Nevertheless, the “homogenization” of the rules worldwide will greatly enhance the quality of life for celiac sufferers. Globalization allows travelling and working in various parts of the globe, and it is difficult for celiac sufferers to follow a safe and varied diet if labelling rules remain different in each country. Nevertheless, it is very important to highlight that GMP in the food industry is fundamental for the production of food without any risk of cross-contamination. It is not only labelling, but also a new inclusive perspective of food-production that takes into account the reality for celiac and allergy sufferers, an ever-expanding group. For this new perspective, new technologies for gluten detection play a crucial role in the industry. Rapid, cost-effective, and reliable methodologies may allow manufacturers to detect gluten in any of the steps of the processes, without any inconvenient time lapses in their production.