Dry Fractionation and Gluten-Free Sourdough Bread Baking from Quinoa and Sorghum.

The roller milling of sorghum and quinoa seeds into flour fractions (coarse, middle, and fine) was investigated, chemically analysed, and applied in the baking of gluten-free sourdough bread. The gap settings were adjusted to 0, 5, 8, and 10 for quinoa, and 3, 5, and 7 for sorghum. The fine fractions reached values of up to about 41% (gap 8) for quinoa and around 20% for sorghum (gap 5). SEM pictographs illustrated the clear separation of each fraction with the chemical analysis showing high contents of protein, TDF (total dietary fibre), and IDF (insoluble dietary fibre) in the coarse fraction. Up to 77% starch content was obtained in the fine fraction with significant amounts of SDF (soluble dietary fibre), which has good health benefits. Increasing the dough moisture up to 90% helped in decreasing the bread crumb firmness, while low Avrami parameters and RVA pasting behaviour indicated a slow bread-staling rate for both sourdough breads.

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.

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

The safety of gluten-free products relies on accurate gluten analysis, most commonly using ELISA. These test kits are calibrated to gliadins or wheat gluten, because there is no reference material (RM) for rye. Our aim was to select representative samples out of 32 rye cultivars for use as RM. All cultivars were characterized by RP-HPLC, gel permeation HPLC and R5 and G12 ELISA. The protein and gluten content ranged from 5.5 to 11.2 g/100 g and 3.0 to 7.8 g/100 g, respectively. The average protein distribution was 40% albumins/globulins, 23% γ-75k-secalins, 17% γ-40k-secalins, 14% ω-secalins and 6% high-molecular-weight-secalins. The mean prolamin/glutelin ratio was 4.4 for rye and this translates to an estimated conversion factor from rye prolamins to gluten of 1.2, instead of the usual factor of 2. Seven cultivars were selected for RM production based on cluster analysis, geographical origin and availability to comprehensively cover the diversity of rye.

Variation in the Content and Composition of Tocols in a Wheat Population.

Wheat is a well-known source of B vitamins but also contains significant amounts of vitamin E and related tocols, which have a number of positive health benefits. However, there are no reports on increasing the tocol content of wheat. A prerequisite for increasing the tocol content is the identification of variation in its amount within wheat and related cereals. We therefore determined the tocol content and composition in the grain of 230 recombinant inbred lines (RILs) of a diverse biparental wheat population (Mv Toborzó/Tommi), showing variation in the total content from 13.69 to 45.18 µg/g d.m. The total content also showed transgressive segregation in the population. The effect of the genotype on the variance components of tocols was studied, and the broad-sense heritability was calculated to be 0.71. The lines were also grouped based on their tocol content and analyzed for their chemical composition and breadmaking quality. The high heritability value and the wide variation found in the total amount indicate that increasing the content of tocols is a possible breeding strategy.

Investigation of Protein and Epitope Characteristics of Oats and Its Implications for Celiac Disease.

The use of pure oats (oats cultivated with special care to avoid gluten contamination from wheat, rye, and barley) in the gluten-free diet (GFD) represents important nutritional benefits for the celiac consumer. However, emerging evidence suggests that some oat cultivars may contain wheat gliadin analog polypeptides. Consequently, it is necessary to screen oats in terms of protein and epitope composition to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variability of oat protein composition directly related to health-related and techno-functional properties. Elements of an oat sample population representing 162 cultivated varieties from 20 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by size exclusion-high performance liquid chromatography (SE-HPLC) while the 70% ethanol-extracted proteins were analyzed by RP-HPLC. Protein extracts separated into three main groups of fractions on the SE-HPLC column: polymeric proteins, avenins (both containing three subgroups based on their size), and soluble proteins, representing respectively 68.79-86.60, 8.86-27.72, and 2.89-11.85% of the total protein content. The ratio of polymeric to monomeric proteins varied between 1.37 and 3.73. Seventy-six reversed phase-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. Their distribution among the cultivars varied significantly, 6-23 peaks per cultivar. The number of appearances of peaks also showed large variation: one peak has been found in 107 samples, while 15 peaks have been identified, which appeared in less than five cultivars. An estimation method for ranking the avenin-epitope content of the samples has been developed by using MS spectrometric data of collected RP-HPLC peaks and bioinformatics methods. Using ELISA methodology with the R5 antibody, a high number of the investigated samples were found to be contaminated with wheat, barley, or rye.

Are current analytical methods suitable to verify VITAL® 2.0/3.0 allergen reference doses for EU allergens in foods?

Food allergy affects up to 6% of Europeans. Allergen identification is important for the risk assessment and management of the inadvertent presence of allergens in foods. The VITAL® initiative for voluntary incidental trace allergen labeling suggests protein reference doses, based on clinical reactivity in food challenge studies, at or below which voluntary labelling is unnecessary. Here, we investigated if current analytical methodology could verify the published VITAL® 2.0 doses, that were available during this analysis, in serving sizes between 5 and 500 g. Available data on published and commercial ELISA, PCR and mass spectrometry methods, especially for the detection of peanuts, soy, hazelnut, wheat, cow's milk and hen's egg were reviewed in detail. Limit of detection, quantitative capability, matrix compatibility, and specificity were assessed. Implications by the recently published VITAL® 3.0 doses were also considered. We conclude that available analytical methods are capable of reasonably robust detection of peanut, soy, hazelnut and wheat allergens for levels at or below the VITAL® 2.0 and also 3.0 doses, with some methods even capable of achieving this in a large 500 g serving size. Cow's milk and hen's egg are more problematic, largely due to matrix/processing incompatibility. An unmet need remains for harmonized reporting units, available reference materials, and method ring-trials to enable validation and the provision of comparable measurement results.

Further Steps Toward the Development of Gluten Reference Materials - Wheat Flours or Protein Isolates?

Celiac disease is a gluten-induced hypersensitivity reaction that requires a lifelong gluten-free diet. Gluten-free foods must not contain more than 20 mg/kg gluten as laid down by Codex Alimentarius. Measuring the presence of gluten with routine immunoanalytical methods in food is a serious challenge as many factors affect accurate determination. Comparability of the results obtained with different methods and method validation are hindered by the lack of a widely accepted reference material (RM). The core questions of RM development from wheat are the number of cultivars to be included and the format of gluten (i.e., flour, gluten, or gliadin isolates) to be applied. Therefore, the aim of our work was to produce an appropriate gluten RM from wheat. For this, five previously selected wheat cultivars and their blend were used to produce flours, gluten and gliadin isolates under laboratory conditions. Protein content, protein composition and responses to different ELISA methods were compared and widely evaluated in our study. The protein contents of the flours were 12.1-18.7%, those of the gluten isolates 93.8-97.4% and those of the gliadin isolates 72.7-101.9%. The gluten and gliadin isolates had similar protein profiles as the source flours. By comparing the different wheat cultivars and their protein isolates, we found that the isolation had a smaller effect on protein composition than genetic variability. The choice of a blend would be more suitable for the production of a RM in case of flours and also isolates. The immunoanalytical results showed that the isolation had an effect on the analytical results, but its extent depended on the ELISA method. The use of flour would be more applicable in this regard, but handling of the material and long-term stability should also be considered in the final decision of gluten RM production.

A novel approach to the characterization of old wheat (Triticum aestivum L.) varieties by complex rheological analysis.

BACKGROUND: Lines of the internationally recognized old Hungarian Bánkúti 1201 variety are important genetic resources for breeding programmes. Their protein composition and gluten dependent technological traits have been comprehensively studied, however, little information is available about their carbohydrate dependent viscous properties. The aim of this work was to obtain comprehensive rheological characterization of all sublines of Bánkúti 1201 maintained at Martonvásár and to investigate their variability if the carbohydrate dependent viscous behaviour was also included in the analyses. RESULTS: The majority of the lines reflected the famously good mixing quality of Bánkúti, however, much higher diversity of pasting behaviour was detected. Cluster analysis of the Mixolab data was performed resulting in four sample groups. Since several lines of similar mixing properties had significantly different pasting characteristics, it was assumed that classification was mainly based on the viscous properties. From each cluster two to three representative samples were selected for wider examination using conventional testing methods. These results also supported the higher variability of pasting behaviour of the lines, which can be critical for end product quality. The members of the second cluster can be highlighted due to their waxy wheat like behaviour. CONCLUSIONS: Possible reasons for the great variability of pasting behaviour could be the compositional and structural differences of starch and other carbohydrates (e.g. arabinoxylans). Complex rheological characterization and study of molecular background can provide information about important traits from the point of view of technology and product development, which are unknown in the case of old wheat varieties and landraces. © 2020 Society of Chemical Industry.

Stability analysis of wheat lines with increased level of arabinoxylan.

Plant breeders have long sought to develop lines that combine outstanding performance with high and stable quality in different environments. The high-arabinoxylan (AX) Chinese variety Yumai-34 was crossed with three Central European wheat varieties (Lupus, Mv-Mambo, Ukrainka) and 31 selected high-AX lines were compared for physical (hectolitre weight, thousand grain weight, flour yield), compositional (protein content, gluten content, pentosan) and processing quality traits (gluten index, Zeleny sedimentation, Farinograph parameters) in a three-year experiment (2013-2015) in the F7-F9 generations. The stability and heritability of different traits, including the relative effects of the genotype (G) and environment (E), were determined focusing on grain composition. The contents of total and water-soluble pentosans were significantly affected by G, E and G × E interactions, but the heritability of total (TOT)-pentosan was significantly lower (0.341) than that of water-extractable (WE)-pentosan (0.825). The main component of the pentosans, the amount and composition (arabinose:xylose ratio) of the arabinoxylan (AX), was primarily determined by the environment and, accordingly, the broader heritability of these parameters were 0.516 and 0.772. However, genotype significantly affected the amount of water-soluble arabinoxylan and its composition and thus the heritability of these traits was also significant (0.840 and 0.721). The genotypes exhibiting higher stability of content of TOT-pentosan also showed more stable contents of WE-pentosan. There was a positive correlation between the stability of contents of WE-pentosan and WE-AX, while the stability of the WE-AX content and AX composition were also strongly correlated. Water absorption was strongly genetically determined with a heritability of 0.829 with the genotype determining 38.67% of the total variance. Many lines were grouped in the GGE biplot, indicating that they did not significantly differ stability.

Characterisation and comparison of selected wheat (Triticum aestivum L.) cultivars and their blends to develop a gluten reference material.

The reliability and comparability of gluten analytical results in gluten-free foods is hampered by the lack of reference materials (RM). This is partly caused by the yet incomplete knowledge of the effect of genetic and environmental variability of wheat proteins on immunochemical analyses, which affects the choice of gluten source to be applied for RM production. We investigated the genetic variability and the effect of harvest year on the protein composition of five previously selected wheat cultivars. We also compared the magnitude of these effects on ELISA results to get closer to the question of choosing individual cultivar or a mixture as an RM. Our results proved that the application of a blend for this purpose is advantageous. The candidates were also produced on pilot scale to investigate the feasibility of upscaling. The results of comparison studies showed that the pilot scale blended flour can also be suitable for RM.

Variation in protein composition among wheat (Triticum aestivum L.) cultivars to identify cultivars suitable as reference material for wheat gluten analysis.

Gluten proteins of certain cereals (wheat, rye and barley) can trigger hypersensitivity reactions. In special dietary products for people intolerant to gluten, their amount must not exceed the regulatory threshold levels. The source of gluten can influence gluten quantitation due to variability in protein profile of grain cultivars and species. A proper reference material is crucial for accurate measurement of gluten and evaluating assay performance. It should be as representative of the commodity as possible. In this study, protein content and composition of a set of 23 common wheat cultivars grown around the world were determined. According to qualitative and quantitative selection criteria, cultivars that possessed a typical gluten composition were identified. Five cultivars were selected for subsequent experiments to confirm their suitability as a basis for reference material production.

Optimization of Arabinoxylan Isolation from Rye Bran by Adapting Extraction Solvent and Use of Enzymes.

Physicochemical and functional properties of arabinoxylans (AXs) can be significantly influenced by their isolation method. Finding balanced process conditions that allow optimal extraction yields while preserving AXs functionality is a challenge. The aim of this study was to determine the effect of different chemical solvents with neutral and alkaline pH on the intrinsic properties and extraction yield of AXs isolated from rye bran. Additionally, the application of xylanases and other cell wall degrading enzymes (Pentopan Mono BG, Deltazym XL-VR, Viscoflow BG) to solubilize bound AXs was investigated. Results show that the use of Ca(OH)2 for isolation was superior to water and Na2 CO3 , as it selectively solubilized AXs and delivered isolates with a purity of up to 43.92% AX and a moderate ferulic acid (FA) content (209.35 ± 16.79 mg FA/100 g AX). Application of xylanases was further able to duplicate these achieved AX yields (7.50 to 9.85g AX/100 g bran). Additionally, isolates displayed highest ferulic acid contents (445.18 to 616.71 mg FA/100 g AX) and lowest impurities in comparison to chemical extracted AXs. Rheological characterization of the isolates showed a pronounced shear thinning behavior which fitted well to the power-law model (R2 > 0.989). Differences in pseudoplasticity of the isolates suggested that structural and chemical properties might have been responsible for this behavior.

Protein interactions during flour mixing using wheat flour with altered starch.

Wheat grain proteins responses to mixing and thermal treatment were investigated using Mixolab-dough analysis systems with flour from two cultivars, Ventura-26 (normal amylose content) and Ventura-19 (reduced amylose content). Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis revealed that, stress associated and metabolic proteins largely interacted with dough matrix of Ventura-26 after 26min (56°C); gliadins, avenin-like b proteins, LMW-GSs, and partial globulins showed stronger interactions within the dough matrix of Ventura-26 at 32min/C3 (80°C), thereafter, however, stronger protein interactions were observed within the dough matrix of Ventura-19 at 38min/C4 (85°C) and 43min (80°C). Thirty-seven proteins associated with changes in dough matrix due to reduced amylose content were identified by mass spectrometry and mainly annotated to the chromosome group 1, 4, and 6. The findings provide new entry points for modifying final product attributes.

Protein-transitions in and out of the dough matrix in wheat flour mixing.

Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour.

Rheological and stability aspects of dry and hydrothermally heat treated aleurone-rich wheat milling fraction.

Novel aleurone-rich wheat milling fraction developed and produced on industry scale is investigated. The special composition of the novel flour with high protein, dietary fiber and fat content results in a unique combination of the mixing and viscosity properties. Due to the high lipid concentration, the fraction is exposed to fast rancidity. Dry heat (100°C for 12min) and hydrothermal treatment processes (96°C for 6min with 0-20 L/h steam) were applied on the aleurone-rich flour to modify the technological properties. The chemical, structural changes; the extractability of protein, carbohydrate and phenolic components and the rheological characteristics of the flours were evaluated. The dry treated flour decreased protein and carbohydrate extractability, shortened dough development time, reduced gel strength and enhanced the gelling ability. Hydrothermal treatment caused changes in the phenolic content improved the dough stability and -resistance. Heat treatment processes were able to extend the stability of the flour.

Hydroxyl radical oxidation of feruloylated arabinoxylan.

Feruloylated arabinoxylan (AX) has a unique capacity to form covalent gels in the presence of certain oxidizing agents. The present study demonstrates that hydroxyl radical oxidation does not provoke ferulic acid dimerization and thus oxidative gelation. We studied the hydroxyl radical mediated oxidation of an alkali-extracted AX preparation (purity: 92g/100g dry matter) that showed gel-forming capability upon peroxidase/hydrogen peroxide treatment. Hydroxyl radicals were produced with ascorbate-driven Fenton reaction and the radical formation was monitored with electron paramagnetic resonance, using a POBN/EtOH spin trapping system. Oxidation was carried out at different catalytic concentrations of iron (50 and 100µM) and at different temperatures (20°C, 50°C, and 80°C). It was demonstrated that hydroxyl radical oxidation does not provoke gel formation, but viscosity decrease in AX solution, which suggests polymer degradation. Furthermore, it was demonstrated that hydroxyl radical formation in AX solution can be initiated merely by increasing temperature.

Comparison of the effects of different heat treatment processes on rheological properties of cake and bread wheat flours.

Dry and hydrothermal heat treatments are efficient for modifying the technological-functional and shelf-life properties of wheat milling products. Dry heat treatment process is commonly used to enhance the volume of cakes. Hydrothermal heat treatment makes wheat flours suitable as thickener agents. In this study, cake and bread wheat flours that differed in baking properties were exposed to dry (100 °C, 12 min) and hydrothermal (95 °C, 5 min, 5-20 l/h water) heat treatments. Rheological differences caused by the treatments were investigated in a diluted slurry and in a dough matrix. Dry heat treatment resulted in enhanced dough stability. This effect was significantly higher in the cake flour than the bread flour. Altered viscosity properties of the bread flour in the slurry matrix were also observed. The characteristics of hydrothermally treated samples showed matrix dependency: their viscosity increases in the slurry and decreases in the dough matrix. These results can support us to produce flour products with specific techno-functional properties.

Wheat storage proteins in transgenic rice endosperm.

Transgenic rice seed expressing wheat HMW glutenin subunit was characterized to study the effects of the wheat prolamin on the protein expression pattern and protein size distribution in the endosperm and the functional and rheological properties of the rice flour and dough. Significant differences were found in the protein expression pattern between the transgenic and wild type samples. Comparing the protein expression profiles of transgenic and nontransgenic plants, combined with proteomic-based studies, indicated increased protein disulfide isomerase (PDI) levels in the transgenic rice lines. The accurate molecular size of HMW-GS in rice endosperm was identified by MALDI-TOF-MS analysis. The expressed wheat HMW (subunit 1Dx5) GS showed a positive effect on the functional properties of rice dough by significantly increasing the size distribution of the polymeric protein fraction and modifying the dough mixing parameters.

Development of incurred reference material for improving conditions of gluten quantification.

Celiac disease and wheat allergy are the most common adverse reactions triggered by cereal proteins, mainly gluten, which is one of the 14 allergenic food ingredients that must be labeled on food products in the European Union (EU). To meet the requirements of this regulation, reliable analytical methodology for proper quantification of gluten is necessary. However, validation of presently used methods (ELISA and lateral flow device) is limited partly due to the lack of reference methods and incurred reference materials. To solve this problem, the goal of our work was to develop an incurred reference material for the quantification of gluten under the auspices of EU-FP6 funded Network of Excellence MoniQA. During this work, we produced a processed model product (cookie) containing gliadin (major allergenic fraction of gluten) in a defined amount. This paper addresses the development process of this material together with the associated problems (insufficient homogeneity and low recovery) and their solutions. As a result, an incurred food matrix was produced on a laboratory-scale with a potential use as a reference material. The model product was tested by an ELISA method followed by a comparative study of commercially available ELISA kits to investigate the applicability of the product. Preliminary results of this study are also presented.

Comparative study of the effect of incorporated individual wheat storage proteins on mixing properties of rice and wheat doughs.

The aim of this work was to compare the effects of incorporated wheat storage proteins on the functional properties of rice and wheat flours. The advantage of rice as a base flour compared to wheat is that it does not contain any wheat flour components and, therefore, has no interactive effect between wheat glutenin proteins. The incorporation of individual HMW glutenin subunit proteins (Bx6, Bx7, and By8) in different ratios had significant positive effects on the mixing requirements of both rice and wheat doughs. Reconstitution experiments using two x+y type HMW-GS pairs together with a bacterially expressed LMW-GS have been also carried out in this study. The largest effects of polymer formation and mixing properties of rice flour dough were observed when Bx and By subunits were used in a 1:1 ratio and HMW and LMW glutenin subunits in a 1:3 ratio. However, using the same subunit ratios in wheat as the base flour, these synergistic effects were not observed.