Rye and barley reference materials for the analysis of gluten

Gluten-free foods are subject to the Codex Alimentarius as well as the Commission Implementing
Regulation (EU) No. 828/2014. It is stated that gluten-free labelled foods must
not exceed 20 mg/kg of gluten in the final product. To ensure food safety for celiac disease
patients, accurate quantification of gluten in gluten-containing and potentially contaminated
foods is crucial. Immunological methods such as the R5 ELISA are the main methods for
gluten quantification that are used by food producers, as they are certified. However, there
are many disadvantages of the method, due to differences in materials used for calibration
and specificity of antibodies. The differences in the protein structure of different grain
species (wheat, rye and barley) leads to under- or overestimation of the gluten content of
rye- or barley-contaminated foods. The reason for this is the use of wheat-based calibration
standards. There is a lack of standardized gluten reference materials and harmonized
analytical methods in gluten analysis. Moreover, there is only little research on rye and barley
proteins in general but as well as gluten reference materials from rye and barley.
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The objective of this study is to establish representative rye- and barley-based reference materials
for gluten analysis. For this purpose, suitable cultivars were selected and various protein
isolates were produced from their flour mixtures. By characterizing 32 different rye and 35 different
barley cultivars using RP-HPLC, GP-HPLC and two commercially available ELISA kits
(R5 and G12), we were able to identify representative cultivars for the lab-scale production
of new reference materials. In terms of protein distribution, rye cultivars showed an average
composition of 40% albumins/globulins, 23% γ-75k-secalins, 17% γ-40k-secalins, 14%
ω-secalins and 6% high-molecular-weight-secalins. The relative protein composition of the 35
barley cultivars averaged 25% albumins and globulins, 11% D-hordeins, 19% C-hordeins and
45% B/γ-hordeins. Moreover, we discovered that the commonly used prolamin/glutelin ratio
of 1:1 for calculating gluten content was unsuitable for both rye and barley. For rye, a ratio
of 4.4:1 was determined, while for barley, a ratio of 1.6:1 was observed, indicating a higher
proportion of prolamins compared to glutelins. The gluten content in the majority of samples
was overestimated when using both ELISA kits. Additionally, we discovered that separating rye
and barley gluten protein types into prolamins and glutelins using the modified Osborne
fractionation was not straightforward. Of the 32 rye and 35 barley varieties, seven and eight
varieties suitable for the production of the reference materials were selected using statistical
tools such as hierachical cluster analysis, respectively. The gluten composition of the chosen
cultivars were compared in two different harvest years. Moreover, four different protein isolates
were produced using a mixture of the selected cultivars: prolamin, glutelin, total gluten
and an acetonitrile water extractable protein (AWEP). The isolates were characterized using
LC-MS/MS and their reactivity towards the R5 monoclonal antibody in the sandwich ELISA
system was tested. For rye isolates, the reactivity order towards the R5 monoclonal antibody
was as follows: prolamins>AWEP >gluten>glutelins. For barley isolates, the reactivity was
highest for AWEP and prolamins, followed by gluten and glutelins. The results show that
the use of the wheat-based PWG-gliadin standard in ELISA test systems does not lead to an
optimal determination of the gluten content in rye and barley flours. Rye and barley reference
materials are necessary to optimize gluten analysis in rye- and barley-contaminated foods. The
isolates produced in this study represent one possibility for such reference materials to improve
gluten quantification of barley- and rye-contaminated foods and to ensure food safety for celiac
disease patients.