6 research outputs found
Development of a barley reference material for gluten analysis
Celiac disease (CD) can be triggered in susceptible individuals by the consumption of gluten, a complex storage protein mixture present in wheat, rye and barley. There is no specific reference material (RM) available for barley and this leads to inaccurate quantitation of barley gluten in supposedly gluten-free foods. Therefore, the aim was to select representative barley cultivars to establish a new barley RM. 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. The mean gluten and protein content was 7.2 g/100 g and 11.2 g/100 g, respectively. The prolamin/glutelin ratio (1:1) commonly used in ELISAs to calculate the gluten content was found to be inappropriate for barley (1.6 ± 0.6). Eight cultivars suitable as potential RMs were selected to ensure a typical barley protein composition and improve food safety for CD patients
Classics in a new perspective: gluten as a special food safety and analytical challenge
In the last couple of decades, the nutritional role and perception of gluten became controversial. In one hand, gluten proteins play a central role in determining the baking quality of wheat and other cereals. On the other hand, hypersensitivity reactions triggered by gluten in susceptible individuals have become subjects of growing interest. Of these gluten-related disorders, with an estimated global prevalence of 1%, the most important one is celiac disease (CD), which is an autoimmune disorder accompanied by villous atrophy. CD can manifest in a wide range of symptoms, its only treatment option is a lifelong gluten-free (GF) diet. To support compliance to this diet, current EU legislation maximizes the gluten-content of products sold with a GF label in 20 mg/kg. It necessitates accurate quantification of gluten in this low concentration range. The method-of-choice for this purpose is the immunoanalytical-based ELISA (enzyme-linked immunosorbent assay). However, validation of different ELISA methods and the comparability of their results and, consequently, the reliability of the data they provide is problematic. The major goal of this paper is to introduce the analytical and protein chemistry issues behind this problem and the efforts to improve the conditions of the methodology. We are also including the special role of oats in the GF diet in an attempt to provide the widest possible overview of the food safety and analytical challenges represented by gluten
Klasszikus tĂ©mák Ăşj megvilágĂtásban: a glutĂ©n mint speciális Ă©lelmiszerbiztonsági Ă©s analitikai kihĂvás
A glutĂ©n, vagy sikĂ©r fehĂ©rjĂ©k táplálkozás-Ă©lettani szerepe Ă©s megĂtĂ©lĂ©se az utĂłbbi Ă©vtizedekben kettĹ‘ssĂ© vált. A glutĂ©n fehĂ©rjĂ©k egyrĂ©szt központi szerepet töltenek be a bĂşza Ă©s más gabonák sĂĽtĹ‘ipari minĹ‘sĂ©gĂ©nek kialakĂtásában. MásrĂ©szt azonban egyre inkább elĹ‘tĂ©rbe kerĂĽlnek olyan tĂşlĂ©rzĂ©kenysĂ©gi reakciĂłk, melyeket szintĂ©n a glutĂ©n fehĂ©rjĂ©k váltanak ki az arra Ă©rzĂ©keny populáciĂłban. A glutĂ©n által okozott rendellenessĂ©gek közĂĽl 1% körĂĽli globális elĹ‘fordulásával az egyik legjelentĹ‘sebb a lisztĂ©rzĂ©kenysĂ©g, vagy más nĂ©ven cöliákia, mely a vĂ©konybĂ©l bolyhainak sorvadásával járĂł autoimmun betegsĂ©g. A tĂĽnetek szĂ©les skáláját okozhatja, jelenleg egyetlen ismert kezelĂ©si mĂłdja az Ă©lethosszig tartĂł glutĂ©nmentes diĂ©ta. A diĂ©ta betartásának elĹ‘segĂtĂ©sĂ©re a jelenleg Ă©rvĂ©nyes EU szabályozás 20 mg/kg-ban maximalizálja a glutĂ©nmenteskĂ©nt Ă©rtĂ©kesĂthetĹ‘ termĂ©kek glutĂ©ntartalmát. Ez pedig szĂĽksĂ©gessĂ© teszi a glutĂ©n mennyisĂ©gĂ©nek minĂ©l pontosabb meghatározását ebben az alacsony koncentráciĂł-tartományban. A meghatározás rutinmĂłdszere az immunanalitikai elven működĹ‘ ELISA (enzyme-linked immunosorbent assay). A kĂĽlönbözĹ‘ ELISA mĂłdszerek validálása Ă©s eredmĂ©nyeik összehasonlĂthatĂłsága, vagyis az általuk szolgáltatott adatok megbĂzhatĂłsága azonban problĂ©mát jelent. CikkĂĽnk fĹ‘ cĂ©lkitűzĂ©se az ennek hátterĂ©ben állĂł analitikai Ă©s fehĂ©rjekĂ©miai kĂ©rdĂ©sek, valamint a mĂłdszertan feltĂ©telrendszerĂ©nek javĂtását cĂ©lzĂł törekvĂ©sek bemutatása. Emellett kitĂ©rĂĽnk a zab glutĂ©nmentes diĂ©tában betöltött kĂĽlönleges szerepĂ©re is, Ăgy kĂsĂ©relve meg minĂ©l szĂ©lesebb körben rálátást nyĂşjtani a glutĂ©n által kĂ©pviselt Ă©lelmiszerbiztonsági Ă©s analitikai kihĂvásokra
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.
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% Îł-75k-secalins, 17% Îł-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/Îł-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
Klasszikus tĂ©mák Ăşj megvilágĂtásban: a glutĂ©n mint speciális Ă©lelmiszerbiztonsági Ă©s analitikai kihĂvás
A glutĂ©n, vagy sikĂ©r fehĂ©rjĂ©k táplálkozás-Ă©lettani szerepe Ă©s megĂtĂ©lĂ©se az utĂłbbi Ă©vtizedekben kettĹ‘ssĂ© vált. A glutĂ©n fehĂ©rjĂ©k egyrĂ©szt központi szerepet töltenek be a bĂşza Ă©s más gabonák sĂĽtĹ‘ipari minĹ‘sĂ©gĂ©nek kialakĂtásában. MásrĂ©szt azonban egyre inkább elĹ‘tĂ©rbe kerĂĽlnek olyan tĂşlĂ©rzĂ©kenysĂ©gi reakciĂłk, melyeket szintĂ©n a glutĂ©n fehĂ©rjĂ©k váltanak ki az arra Ă©rzĂ©keny populáciĂłban. A glutĂ©n által okozott rendellenessĂ©gek közĂĽl 1% körĂĽli globális elĹ‘fordulásával az egyik legjelentĹ‘sebb a lisztĂ©rzĂ©kenysĂ©g, vagy más nĂ©ven cöliákia, mely a vĂ©konybĂ©l bolyhainak sorvadásával járĂł autoimmun betegsĂ©g. A tĂĽnetek szĂ©les skáláját okozhatja, jelenleg egyetlen ismert kezelĂ©si mĂłdja az Ă©lethosszig tartĂł glutĂ©nmentes diĂ©ta. A diĂ©ta betartásának elĹ‘segĂtĂ©sĂ©re a jelenleg Ă©rvĂ©nyes EU szabályozás 20 mg/kg-ban maximalizálja a glutĂ©nmenteskĂ©nt Ă©rtĂ©kesĂthetĹ‘ termĂ©kek glutĂ©ntartalmát. Ez pedig szĂĽksĂ©gessĂ© teszi a glutĂ©n mennyisĂ©gĂ©nek minĂ©l pontosabb meghatározását ebben az alacsony koncentráciĂł-tartományban. A meghatározás rutinmĂłdszere az immunanalitikai elven működĹ‘ ELISA (enzyme-linked immunosorbent assay). A kĂĽlönbözĹ‘ ELISA mĂłdszerek validálása Ă©s eredmĂ©nyeik összehasonlĂthatĂłsága, vagyis az általuk szolgáltatott adatok megbĂzhatĂłsága azonban problĂ©mát jelent. CikkĂĽnk fĹ‘ cĂ©lkitűzĂ©se az ennek hátterĂ©ben állĂł analitikai Ă©s fehĂ©rjekĂ©miai kĂ©rdĂ©sek, valamint a mĂłdszertan feltĂ©telrendszerĂ©nek javĂtását cĂ©lzĂł törekvĂ©sek bemutatása. Emellett kitĂ©rĂĽnk a zab glutĂ©nmentes diĂ©tában betöltött kĂĽlönleges szerepĂ©re is, Ăgy kĂsĂ©relve meg minĂ©l szĂ©lesebb körben rálátást nyĂşjtani a glutĂ©n által kĂ©pviselt Ă©lelmiszerbiztonsági Ă©s analitikai kihĂvásokra
Classics in a new perspective: gluten as a special food safety and analytical challenge
In the last couple of decades, the nutritional role and perception of gluten became controversial. In one hand, gluten proteins play a central role in determining the baking quality of wheat and other cereals. On the other hand, hypersensitivity reactions triggered by gluten in susceptible individuals have become subjects of growing interest. Of these gluten-related disorders, with an estimated global prevalence of 1%, the most important one is celiac disease (CD), which is an autoimmune disorder accompanied by villous atrophy. CD can manifest in a wide range of symptoms, its only treatment option is a lifelong gluten-free (GF) diet. To support compliance to this diet, current EU legislation maximizes the gluten-content of products sold with a GF label in 20 mg/kg.It necessitates accurate quantification of gluten in this low concentration range. The method-of-choice for this purpose is the immunoanalytical-based ELISA (enzyme-linked immunosorbent assay). However, validation of different ELISA methods and the comparability of their results and, consequently, the reliability of the data they provide is problematic. The major goal of this paper is to introduce the analytical and protein chemistry issues behind this problem and the efforts to improve the conditions of the methodology. We are also including the special role of oats in the GF diet in an attempt to provide the widest possible overview of the food safety and analytical challenges represented by gluten