Protein Enrichment of Wheat Bread with Microalgae: Microchloropsis gaditana, Tetraselmis chui and Chlorella vulgaris.

Cell wall disrupted and dried Microchloropsis gaditana (Mg), Tetraselmis chui (Tc) and Chlorella vulgaris (Cv) microalgae biomasses, with or without ethanol pre-treatment, were added to wheat bread at a wheat flour substitution level of 12%, to enrich bread protein by 30%. Baking performance, protein quality and basic sensory properties were assessed. Compared to wheat, Mg, Tc and Cv contain higher amounts of essential amino acids and their incorporation markedly improved protein quality in the bread (DIAAS 57-66 vs. 46%). The incorporation of microalgae reduced dough strength and bread volume and increased crumb firmness. This was most pronounced for Cv and Tc but could be improved by ethanol treatment. Mg gave adequate dough strength, bread volume and crumb structure without ethanol treatment. To obtain bread of acceptable smell, appearance, and colour, ethanol treatment was necessary also for Mg as it markedly reduced the unpleasant smell and intense colour of all algae breads. Ethanol treatment reduced the relative content of lysine, but no other essential amino acids. However, it also had a negative impact on in vitro protein digestibility. Our results show that Mg had the largest potential for protein fortification of bread, but further work is needed to optimize pre-processing and assess consumer acceptance.

Ancestral Wheat Types Release Fewer Celiac Disease Related T Cell Epitopes than Common Wheat upon Ex Vivo Human Gastrointestinal Digestion.

Celiac disease (CeD) is an autoimmune enteropathy triggered by immunogenic gluten peptides released during the gastrointestinal digestion of wheat. Our aim was to identify T cell epitope-containing peptides after ex vivo digestion of ancestral (einkorn, spelt and emmer) and common (hexaploid) wheat (Fram, Bastian, Børsum and Mirakel) using human gastrointestinal juices. Wheat porridge was digested using a static ex vivo model. Peptides released after 240 min of digestion were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (HPLC-ESI MS/MS). Ex vivo digestion released fewer T cell epitope-containing peptides from the ancestral wheat varieties (einkorn (n = 38), spelt (n = 45) and emmer (n = 68)) compared to the common wheat varieties (Fram (n = 72), Børsum (n = 99), Bastian (n = 155) and Mirakel (n = 144)). Neither the immunodominant 33mer and 25mer α-gliadin peptides, nor the 26mer γ-gliadin peptide, were found in any of the digested wheat types. In conclusion, human digestive juice was able to digest the 33mer and 25mer α-gliadin, and the 26mer γ-gliadin derived peptides, while their fragments still contained naive T cell reactive epitopes. Although ancestral wheat released fewer immunogenic peptides after human digestion ex vivo, they are still highly toxic to celiac patients. More general use of these ancient wheat variants may, nevertheless, reduce CeD incidence.

Microdochium majus and other fungal pathogens associated with reduced gluten quality in wheat grain.

The bread-making quality of wheat depends on the viscoelastic properties of the dough in which gluten proteins play an important role. The quality of gluten proteins is influenced by the genetics of the different wheat varieties and environmental factors. Occasionally, a near complete loss of gluten strength, measured as the maximum resistance towards stretching (Rmax), is observed in grain lots of Norwegian wheat. It is hypothesized that the loss of gluten quality is caused by degradation of gluten proteins by fungal proteases. To identify fungi associated with loss of gluten strength, samples from a selection of wheat grain lots with weak gluten (n = 10, Rmax < 0.3 N) and strong gluten (n = 10, Rmax ≥ 0.6 N) was analyzed for the abundance of fungal operational taxonomic units (OTUs) using DNA metabarcoding of the nuclear ribosomal Internal Transcribed Spacer (ITS) region ITS1. The DNA quantities for a selection of fungal pathogens of wheat, and the total amount of fungal DNA, were analyzed by quantitative PCR (qPCR). The mean level of total fungal DNA was higher in grain samples with weak gluten compared to grain samples with strong gluten. Heightened quantities of DNA from fungi within the Fusarium Head Blight (FHB) complex, i.e. Fusarium avenaceum, Fusarium graminearum, Microdochium majus, and Microdochium nivale, were observed in grain samples with weak gluten compared to those with strong gluten. Microdochium majus was the dominant fungus in the samples with weak gluten. Stepwise regression modeling based on different wheat quality parameters, qPCR data, and the 35 most common OTUs revealed a significant negative association between gluten strength and three OTUs, of which the OTU identified as M. majus was the most abundant. The same analysis also revealed a significant negative relationship between gluten strength and F. avenaceum detected by qPCR, although the DNA levels of this fungus were low compared to those of M. majus. In vitro growth rate studies of a selection of FHB species showed that all the tested isolates were able to grow with gluten as a sole nitrogen source. In addition, proteins secreted by these fungi in liquid cultures were able to hydrolyze gluten substrate proteins in zymograms, confirming their capacity to secrete gluten-degrading proteases. The identification of fungi with potential to influence gluten quality can enable the development of strategies to minimize future problems with gluten strength in food-grade wheat.

Gluten-Degrading Proteases in Wheat Infected by Fusarium graminearum-Protease Identification and Effects on Gluten and Dough Properties.

Recently, we have observed a relationship between poor breadmaking quality and protease activities related to fungal infection. This study aims to identify potential gluten-degrading proteases secreted by fungi and to analyze effects of these proteases on rheological properties of dough and gluten. Fusarium graminearum-infected grain was used as a model system. Zymography showed that serine-type proteases secreted by F. graminearum degrade gluten proteins. Zymography followed by liquid chromatography-mass spectrometry (MS)/MS analysis predicted one serine carboxypeptidase and seven serine endo-peptidases to be candidate fungal proteases involved in gluten degradation. Effects of fungal proteases on the time-dependent rheological properties of dough and gluten were analyzed by small amplitude oscillatory shear rheology and large deformation extensional rheology. Our results indicate that fungal proteases degrade gluten proteins not only in the grain itself, but also during dough preparation and resting. Our study gives new insights into fungal proteases and their potential role in weakening of gluten.

Rapid seedling establishment and a narrow root stele promotes waterlogging tolerance in spring wheat.

Improving the waterlogging tolerance of wheat varieties could alleviate yield constraints caused by excessive rain and poor soil drainage. In this study, we investigated root and shoot growth as well as anatomical traits of six spring wheat genotypes with contrasting waterlogging tolerance properties. Our aim was to identify root traits that differentiate tolerant from sensitive genotypes. Two experiments were conducted using rhizoboxes and photography for data acquisition. In experiment one, root growth of the genotypes was studied during seedling establishment and a subsequent waterlogging treatment, starting at the 3-leaf stage and maintained for seven days. In the second experiment, root and shoot growth of previously waterlogged plants was compared between the genotypes during seven days of recovery. At harvest of experiment two, root segments were sampled to investigate genotype differences of root cross sectional area, root cortex area, stele area and percentage of aerenchyma. The results show that tolerant, in contrast to sensitive genotypes, developed seminal roots faster in the seedling establishment phase and more nodal roots during the waterlogging treatment. NK93602 and Bjarne were the best performing genotypes. Bjarne in particular had a narrower relative stele size of nodal (13.4%) and seminal roots (11.7%) compared to other genotypes (e.g. 16.3% in nodal roots and 13.9% in seminal roots of sensitive Quarna). The results from this study suggests that early vigor is an important trait for waterlogging tolerance in the field. Anatomical root traits, such as a narrow stele and aerenchyma may contribute to improving waterlogging tolerance furthermore.

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition.

BACKGROUND: The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. RESULTS: The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax ) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. CONCLUSION: Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperatures.

Proteome changes in wheat subjected to different nitrogen and sulfur fertilizations.

Controlling the quality of wheat for breadmaking is a major concern for the milling and baking industry. Wheat flour quality depends on both the genetic background and environmental factors during growth and storage. Amount and timing of application of fertilizer are factors that affect wheat quality. This study investigated the effect of different levels of nitrogen and sulfur on the tris-soluble and glutenin protein fractions by 2D-electrophoresis. Multivariate analysis was performed to study changes in the proteome pattern. In the tris-soluble fraction 20 proteins were changed in abundance due to S fertilization, whereas 16 proteins were changed in the glutenin protein fraction. It was found that induced sulfur deficiency during growth resulted in the most pronounced effect on protein composition. Understanding which proteins are affected by varying levels of fertilizers may help tailor specific traits in various wheat varieties.

Multivariate analysis of 2-DE protein patterns--practical approaches.

Practical approaches to the use of multivariate data analysis of 2-DE protein patterns are demonstrated by three independent strategies for the image analysis and the multivariate analysis on the same set of 2-DE data. Four wheat varieties were selected on the basis of their baking quality. Two of the varieties were of strong baking quality and hard wheat kernel and two were of weak baking quality and soft kernel. Gliadins at different stages of grain development were analyzed by the application of multivariate data analysis on images of 2-DEs. Patterns related to the wheat varieties, harvest times and quality were detected on images of 2-DE protein patterns for all the three strategies. The use of the multivariate methods was evaluated in the alignment and matching procedures of 2-DE gels. All the three strategies were able to discriminate the samples according to quality, harvest time and variety, although different subsets of protein spots were selected. The explorative approach of using multivariate data analysis and variable selection in the analyses of 2-DEs seems to be promising as a fast, reliable and convenient way of screening and transforming many gel images into spot quantities.

Challenges related to analysis of protein spot volumes from two-dimensional gel electrophoresis as revealed by replicate gels.

Assumptions that need to be considered prior to statistical analysis of protein spot volumes from two-dimensional gel electrophoresis (2-DE) data are studied using replicate gels of the same sample. The most important observation is that the data tables of protein spot volumes from 2-DE images contain a large number of missing values, which are not consistent with the presence or absence of the proteins. This implies both loss of information and problems for the subsequent statistical analysis. Challenges with 2-DE protein spot volumes are viewed in light of multiple gel comparisons and multivariate data analysis.

Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for celiac disease.

BACKGROUND AND AIMS: Celiac disease is a prevalent disorder characterized by a chronic intestinal inflammation driven by HLA-DQ2 or -DQ8-restricted T cells specific for ingested wheat gluten peptides. The dominant T-cell responses are to epitopes that cluster within a stable 33mer fragment formed by physiologic digestion of distinct alpha-gliadins. Celiac disease is treated by excluding all gluten proteins from the diet. Conceivably, a diet based on baking-quality gluten from a wheat species that expresses no or few T-cell stimulatory gluten peptides should be equally well tolerated by the celiac patients and, importantly, also be beneficial for disease prevention. METHODS: To identify baking quality, harmless wheat, we followed the evolution of the wheat back to the species that most likely have contributed the AA, BB, and DD genomes to the bread wheat. Gluten were extracted from a large collection of these ancient wheat species and screened for T-cell stimulatory gluten peptides. RESULTS: Distinct differences in the intestinal T-cell responses to the diploid species were identified. Interestingly, we found that the fragments identical or equivalent to the immunodominant 33mer fragment are encoded by alpha-gliadin genes on the wheat chromosome 6D and thus absent from gluten of diploid einkorn (AA) and even certain cultivars of the tetraploid (AABB) pasta wheat. CONCLUSIONS: These findings have implications for celiac disease because they raise the prospect of identifying or producing by breeding wheat species with low or absent levels of harmful gluten proteins.