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Chapter 5: Detection of Gluten and Related Proteins in Foods and Beverages, and Safety Issues Related to Gluten-Free Foods and Beverages — Specific Detection with the R5 Antibody in a Ready-to-Use Test Kit for Industry

Ulrike Immer and Sigrid Haas-Lauterbach, R-Biopharm AG, Landwehrstrasse 54, 64293 Darmstadt, Germany, e-mail info@r-biopharm.de

The Science of Gluten-Free Foods and Beverages
Pages 41-52
DOI: https://doi.org/10.1094/9781891127670.005
ISBN: 978-1-891127-67-0


Celiac disease is becoming one of the major gastrointestinal diseases and is increasingly the focus of scientific discussions. It is a permanent inflammatory disease of the upper small intestine in genetically susceptible individuals induced by ingestion of storage proteins from wheat (gluten), rye, and barley (Marsh, 1992). This disease manifests itself mostly in children with different symptoms. The classical picture is poor growth, weight loss, diarrhoea, and increased fat excretion in the stool. Celiac disease is currently considered to be an autoimmune disease and shows different severe site effects like osteopenia, neurological disorders, anaemia, vitamin deficiency, and others. In the United States, a prevalence of 1 in 133 persons was reported (Fasano et al., 2003). Much higher prevalence, 1 in 100, has been reported for Europe (Catassi et al., 1994). A strict, lifelong, gluten-free diet is the only effective treatment of this disease to achieve restoration of the villous architecture. The diet is based on food that does not contain any toxic storage protein from wheat, rye, and barley. Proteins from wheat, rye, and barley are composed mainly of prolamins (high in proline and glutamine) and glutelins found in the starchy endosperm and are classified as storage proteins of the cereals. The kernels contain about 70% carbohydrates and 8–17% proteins. Eighty percent of the wheat proteins is gluten, which is divided into monomeric prolamins that lack intermolecular disulphide bonds, and polymeric glutenins, which are complexes of disulphide-bonded subunits and are soluble in acid, base, detergent, and some aqueous alcohol solutions under reducing conditions. Generally, the content of prolamin is 50% of the wheat gluten. Prolamins from wheat, rye, and barley are named gliadins, secalins, and hordeins, respectively. The celiac activity of the corresponding oat proteins is currently a matter of debate (Janatuinen et al., 2002; Lundin et al., 2003). Oats have been shown to be non-toxic for celiac patients, with a few exceptional cases (Janatuinen et al., 1995). The risk associated with oat consumption is due to cross-contamination by wheat or barley, because these crops are often grown in close proximity. Consequently, wheat, barley, and rye contamination in oats needs to be confirmed. Traditionally gluten-free cereals or pseudocereals like buckwheat, maize, rice, sorghum, and teff can also be contaminated by toxic cereals and therefore have to be examined to establish their contamination-free status.

Traditionally, cereal proteins have been classified in four types according to their solubility (Osborn, 1907). The prolamin fraction is not soluble in water but is soluble in aqueous ethanol (40–70%). Gliadin, the prolamin fraction from wheat, is classified into α-, β-, γ-, and ω-fractions according to their mobility in the electrical field under acidic conditions. Clinical testing of these gliadin fractions showed that all fractions are toxic (Ciclitira et al., 1984). T-cell toxicity experiments have shown that many gliadin and glutenin sequences are toxic to T-cell populations of celiac patients (Koning, 2003). Particularly, a 33-mer amino acid peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFP), which is resistant to gastric and pancreatic hydrolysis, acts as a strong stimulator to intestinal T-cells (Shan et al., 2002; Arentz-Hansen et al., 2000).

The Codex Alimentarius introduced a threshold of 200 ppm gluten/100 ppm gliadin in 1981. However, this calculation was based on the ratio between nitrogen-gluten in wheat starches and depended on the sensitivity of verification procedures in the past. Today it is known that the ratio of nitrogen-gluten can be used only as an approximation of the gluten content, because most of the gluten-free products consist of a mixture of non-celiac toxic cereals like maize, millet, or others. Additionally, more sensitive analytical methods are available now. There-fore, it is mandatory to measure the prolamin content and not the nitrogen content of a product. The 200 ppm level has been under consideration for many years. The codex distinguishes between gluten-free food and gluten-free food by nature. The lower limit of 20 ppm gluten has been proposed for the declaration of naturally gluten-free food. No consensus could be reached so far as prescriptive limits. Tests with a limit of determination lower than 20 ppm prolamin should be acceptable methods for the determination of prolamins in food. Additionally, the method must be applicable to a wide range of food. There are no reports that gluten toxicity is abolished by heat processing, and consequently a method must be able to measure gluten in food that has been prepared under a wide variety of conditions. Wheat gluten is used mainly to achieve a good texture in bakery products. During kneading, leavening, and baking dough, there is an exchange of disulphide bonds which results in high molecular weight units and an insolubilization of the gliadin monomers. Therefore, a sufficient and complete extraction must be assured. Obviously, it is very difficult to meet these requirements.