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According to legislation, foods bearing a gluten-free label must not contain more than 20 mg of gluten per kilogram, which is crucial to ensure food safety for celiac disease patients. Gluten is detected by immunological, genomic, chromatographic and/or mass spectrometric methods, but the complexity of gluten poses various analytical challenges.
Gluten is the main storage protein present in the starchy endosperm of wheat, rye, and barley grains. The complex mixture of more than one hundred individual gluten proteins is a combination of the polymeric glutelin fraction and the mostly monomeric alcohol-soluble fraction named prolamin. The unique rheological properties of wheat gluten make dough elastic and stretchable. Such properties are essential for products such as bread, noodles and pastries. However, gluten has received much attention because of its ability to trigger hypersensitivity reactions in genetically predisposed individuals, including celiac disease, wheat allergy, diarrhea-predominant irritable bowel syndrome and non-celiac gluten sensitivity [1].
In most western populations the prevalence of celiac disease is about 1%, making it one of the most common food intolerances. In affected patients the ingestion of certain gluten peptides (protein building blocks) leads to a hypersensitivity reaction, which causes a chronic inflammation and the destruction of the small intestinal mucosa [2]. This leads to serious health consequences such as anemia, mineral and vitamin deficiencies or reduced bone mineralization due to decreased nutrient absorption. Therefore, a strict and lifelong gluten-free diet is the only treatment that improves gastrointestinal symptoms. This means that all types of gluten-containing cereals including wheat, barley, rye, and in some cases oats, should be avoided in the diet. This includes all potentially contaminated foodstuffs such as convenience foods. A gluten intake for patients with celiac disease below 20 mg per day prevents intestinal damage and is regarded as safe [3]. According to Regulation (EU) No 1169/2011 and the Codex Alimentarius Standard 118-1979 (2015), foods bearing a gluten-free label must not exceed 20 mg/kg of gluten in the final product [4]. Therefore, an accurate quantitation of gluten traces is necessary in order to ensure food safety for celiac disease patients.
The requirements for the analytical quantitation of gluten are regulated in the Codex Alimentarius Standard 118-1979 (2015). To ensure the appropriate quantitation of gluten, an immunological method or any other method equivalent in terms of sensitivity and specificity is required. The antibodies used must react with the fractions of gluten proteins that are toxic to individuals with gluten-dependent diseases. Furthermore, they must not show any cross-reactivity with other ingredients, and a certified reference material should be used for method validation, ensuring a detection limit of 10 mg gluten/kg or lower [4].
Fig. 1 The principles behind enzyme-linked immunosorbent assays (ELISAs). A) sandwich ELISA. B) competitive ELISA. For explanation of the subsequent steps, please refer to the text.
The most common method that can be easily applied meets the requirement of the Codex for fast gluten quantitation: the enzyme-linked immunosorbent assay (ELISA). The quantitation is based on the antibody-antigen reaction where the antigen binds to the antibody resulting in a measurable signal which is either directly (sandwich ELISA) or indirectly (competitive ELISA) proportional to the gluten concentration in the sample. In sandwich ELISAs, the plate is coated with a known quantity of the capture antibody. Adding the antigen from the sample results in antigen/antibody binding (step a). After washing, the enzyme-labelled detection antibody is added to bind to a second site of the antigen (step b). Excess antibody is removed and the addition of the enzymatic substrate results in a color reaction (step c, Fig. 1A). In competitive ELISAs, a known quantity of antigen is immobilized on the surface of the plate. The sample containing the antigen is added simultaneously with a limited, constant quantity of enzyme-labelled antibody, so that immobilized and free antigens compete for the antibody binding sites (step a). After washing and addition of the enzymatic substrate, a colored product is formed (step b, Fig. 1B). The sandwich ELISA according to Méndez et al. using the R5 antibody is particularly recommended for intact gluten proteins [5]. The competitive R5 ELISA is suitable for partially hydrolyzed gluten proteins that are found, for example, in sourdough products, starch syrup, malt extract and beer [6].
However, due to the complex structure and special properties of gluten, the correct quantitation in food matrices using immunological methods faces numerous analytical challenges. Different ELISA test systems do not always achieve the same result, due to differences in materials used for calibration and in the specificity of the antibodies. Moreover, most analytical methods target only the alcohol-soluble prolamin fraction, which is assumed to be about 50 % of the total gluten content. However, prolamin and glutelin contents in cereals vary depending on the species, the cultivar as well as on the environmental conditions during plant growth [7]. Furthermore, the lack of standardized reference materials and harmonized analytical methods makes quantitative results difficult to compare.
Fig. 2 A typical workflow for proteomics experiments that combine untargeted and targeted liquid chromatography tandem mass spectrometry (LC-MS/MS).
A promising and powerful alternative to ELISA is the detection of gluten using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method is based on the prior enzymatic digestion of samples containing gluten. This results in specific peptides, which can be quantitated using different relative (label-free quantitation, intensity-based absolute quantitation) or absolute (stable isotope dilution assay) methods. To identify suitable marker peptides, untargeted LC-MS/MS experiments need to be conducted to generate a proteomic profile of the digested samples. Marker peptides are then quantitated with the help of databases and bioinformatics tools as well as by adding labelled internal standards (Fig. 2) [7,8].
Compared to ELISAs, however, the quantitation with LC-MS/MS requires expensive instrumentation and expertise. In addition, the conversion of the peptide content to the original gluten content is especially challenging, because gluten may be partially modified during food processing, leading to differences in gluten composition. Nonetheless, LC-MS/MS methods offer great potential for the quantitation of gluten in food, due to their selectivity, sensitivity, flexibility and applicability. Therefore, MS techniques are recommended as a complementary method to ELISA. A further advantage is the possibility of developing multi-methods for the simultaneous detection of gluten and other allergens.
Genome-based methods such as polymerase chain reaction (PCR) can be used as a sensitive screening method for the presence of wheat, rye, barley or oats in food. The method is based on the amplification and detection of specific DNA or RNA fragments. Compared to ELISAs, quantitative real-time PCR (rt-qPCR) shows higher sensitivity and specificity, especially for raw materials. This method allows low nucleotide concentrations of 20 pg DNA/mg to be determined [9]. However, it is not suitable for the quantitation of highly hydrolyzed and processed food samples, due to DNA degradation. Therefore, it does not allow conclusions to be drawn about the protein content and the gluten proteins responsible for the disease. Nevertheless, it is used to confirm the presence of certain cereals and can be used complementary to other analytical methods [7].
For people diagnosed with celiac disease, gluten consumption can cause serious health issues, because it triggers hypersensitivity reactions. The only remedy is a lifelong gluten-free diet. For this reason, the accurate quantitation of gluten traces found in foods declared as gluten-free is necessary. According to EU regulations gluten-free labelled foods must not exceed 20 mg/kg of gluten in the final product. Gluten in food can be quantified by methods such as ELISA and LC-MS/MS. Nevertheless, quantitation still poses challenges due to conflicting results, lack of standardized reference materials and inadequate comparability of quantitative results. Consequently, there is still a great need for research to improve the detection and quantitation of gluten in order to ensure food safety for celiac disease patients.
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Category: Food Safety | Gluten
Literature:
[1] Scherf, K.A., Catassi, C., Chirdo, F., Ciclitira, P.J. et al. (2020) Recent progress and recommendations on celiac disease from the Working Group on Prolamin Analysis and Toxicity, Front Nutr., 7, 29, DOI: 10.3389/fnut.2020.00029
[2] Jericho, H., Guandalini, S. (2018) Celiac Disease, Curr Pediatr Rep., 6, 40–49, DOI: 10.1007/s40124-018-0154-y
[3] Catassi, C., Fabiani, E., Iacono, G., D'Agate, C. et al. (2007) A prospective, double-blind, placebo-controlled trial to establish a safe gluten threshold for patients with celiac disease, Am J Clin Nutr., 85, 160–166, DOI: 10.1093/ajcn/85.1.160
[4] Codex Alimentarius Commission, Codex Standard 118-1979. Codex Standard for Foods for Special Dietary Use for Persons Intolerant to Gluten.
[5] Méndez, E., Vela, C., Immer, U., Janssen, F.W. (2005) Report of a collaborative trial to investigate the performance of the R5 enzyme linked immunoassay to determine gliadin in gluten-free food, Eur J Gastroenterol Hepatol., 17, 1053–1063. DOI: 10.1097/00042737-200510000-00008
[6] Gessendorfer, B., Koehler, P., Wieser, H. (2009) Preparation and characterization of enzymatically hydrolyzed prolamins from wheat, rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten, Anal Bioanal Chem., 395, 1721–1728, DOI: 10.1007/s00216-009-3080-6
[7] Scherf, K.A., Poms, R.E. (2016). Recent developments in analytical methods for tracing gluten, J Cereal Sci., 67, 112–122, DOI: 10.1016/j.jcs.2015.08.006
[8] Colgrave, M.L., Goswami, H., Byrne, K., Blundell, M., et al. (2015) Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination, J Proteome Res., 14, 2659–2668, DOI: 10.1021/acs.jproteome.5b00187
[9] Mujico, J.R., Lombardía, M., Mena, M.C., Méndez, E., Albar, J.P. (2011) A highly sensitive real-time PCR system for quantification of wheat contamination in gluten-free food for celiac patients, Food Chem., 128, 795–801, DOI: 10.1016/j.foodchem.2011.03.061
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