Characterization of rye flours and their potential as reference material for gluten analysis

Development of a barley reference material for gluten analysis

“…The barley proteins were extracted according to the modified Osborne fractionation procedure ( Wieser et al, 1998 ). The stepwise extraction (100 mg flour), magnetic stirring, vortexing and centrifugation was done exactly according to Xhaferaj et al (2023) . The extracted solutions of albumins and globulins (ALGL), prolamins and glutelins were diluted to 2 ml with the respective extraction solvents and filtered (0.45 µm Whatman SPARTAN, Cytiva Europe GmbH, Freiburg im Breisgau, Germany).…”

“…In rye or barley these fractions are called secalins and secalinins or hordeins and hordenins, respectively. The terms secalins and hordeins are more common and mainly used for barley and rye gluten protein types, because the separation into prolamins and glutelins according to solubility is inappropriate for rye ( Xhaferaj et al, 2023 ) and barley. Barley gluten can further be classified into the monomeric C- and γ-hordeins and the polymeric B- and d -hordeins ( Schalk et al, 2017 ).…”

“…Other ELISAs such as those utilizing the G12 mAb are also acceptable with similar performance parameters. However, several studies have shown that different ELISA tests often give different results ( Bugyi et al, 2013 , Scherf, 2017 , Amnuaycheewa et al, 2022 , Xhaferaj et al, 2023 ). The main reasons are differences in extraction methods, standards (reference materials (RM)) used for calibration and specificity of the antibodies ( Panda et al, 2017 , Xhaferaj et al, 2020 ).…”

“…However, the use of PWG-gliadin as calibrator in the R5 ELISA kit resulted in an average 5-fold (519%) overestimation of gluten in rye flours ( Xhaferaj et al, 2023 ). One reason is that most ELISA methods mainly target prolamins and calculate the gluten content by multiplying the prolamin content by two.…”

“…Several studies have shown that this conversion factor for determining gluten is inappropriate ( Wieser & Koehler, 2009 ). Especially for rye the conversion factor led to an overestimation of gluten in 32 rye cultivars that had a mean prolamin/glutelin ratio of 4.4 ( Xhaferaj et al, 2023 ). Another reason for the overestimation of gluten is the specificity of the antibodies.…”