R.J. Hamer

Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes

BackgroundBread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the α-gliadins contain several peptides that are associated to the disease.ResultsWe obtained 230 distinct α-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All α-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, α-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific.ConclusionOur analysis shows that α-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.

Interaction of water unextractable solids with gluten protein: effect on dough properties and gluten quality

In a previous study, we have shown that water unextractable solids (WUS) interfere with gluten formation and affect the quality of the resulting gluten. In this study we aim to explain how WUS can affect the process of gluten formation. To this end, WUS were modified with NaOH, xylanase, horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Effects of modified WUS on gluten yield, dough properties, and gluten and glutenin macropolymer (GMP) composition and properties were studied. The results showed that addition of WUS to wheat flour led to a lower gluten yield and gluten starch yield, a higher Rmax and a lower E at Rmax of gluten and a more concentrated and elastic GMP gel. Pretreatment of WUS by NaOH, xylanase, HRP and H2O2 cannot correct its negative effect on gluten yield, but addition of xylanase or free ferulic acid (FA) during gluten separation can remove or prevent the negative effect of WUS on gluten yield. Compared to addition of only WUS, addition of WUS and FA together to wheat flour resulted in a higher gluten yield, a higher E at Rmax of gluten, and a less concentrated and elastic GMP gel. Similar to water extractable pentosans (WEP), FA bound WUS plays a key role in the effect of WUS on gluten yield and properties. It appears that there is a common mechanism regarding the effect of WUS and WEP that the oxidative cross-linking during gluten formation could be prevented by FA addition. The difference between both is that WUS have a higher water binding capacity, which is reflected in a higher Rmax of dough and gluten in the presence of WUS.

Number of thiol groups rather than the size of the aggregates determines the hardness of cold set whey protein gels

Variation of protein concentration during heating resulted in the formation of protein aggregates with clearly different structural and chemical characteristics. Heating conditions were chosen such that differences in the degree of aggregation were excluded. Acid induced gelation of dispersions of these aggregates resulted in gels with clearly different hardness. Although gel hardness seemed to correlate with the different structural aggregate features as reported before in literature, the differences in hardness could for the most part be cancelled by blocking of the thiol groups. Application of thiol-blocked protein aggregates enabled us to make a distinction between the effect of structural- and chemical-properties of the aggregates. Formation of larger disulfide cross-linked protein structures paralleled the increase in gel hardness and dominated the effect of structural characteristics on mechanical properties of cold-set gels. In addition, the effect of the presence of native non-aggregated protein on the final gel properties can be excluded, since in our gel-experiments most protein (>95%) participated in the formation of a protein network. Therefore, we can conclude that the hardness of cold set whey protein gels is determined by the number of thiol groups rather than by the size of the aggregates or other structural features.

Understanding the link between GMP and dough: From glutenin particles in flour towards developed dough

Clear correlations exist for glutenin macropolymer (GMP) quantity and rheological properties vs. wheat quality and dough rheological properties, but real insight in understanding these links is still missing. The observation that GMP consists of glutenin particles opens up new possibilities to reveal the underlying mechanism linking glutenin network properties with dough preparation. GMP was isolated from flour of three wheat varieties: Estica, Soissons and Baldus, strongly varying in their mixing requirements (expressed as time-to-peak, TTP). Decrease of GMP quantity and G′ vs. mixing energy was confirmed. More detail was obtained by studying the changes in GMP particles when mixing flour into dough. Mixing leads to a decrease in the average size of the particles. Interestingly, the TTP coincided with the work-input at which all particles just became soluble in SDS. At TTP, the average size of the GMP particles was the same for each variety. During mixing particles lost their globule shapes and appeared ruptured. Particle size analysis confirmed that particles were still present near TTP. Analysis of the change in particle size vs. energy input using physical principles revealed the following: (1) mixing energy is the predominant actuator in decreasing GMP particle size; (2) the initial GMP particle size in flour strongly determines the practical mixing requirements; and (3) the derived mixing energy vs. GMP particle size relationship was shown to be applicable for both Mixograph and Farinograph mixing. Our results demonstrate that, for the flour samples used, glutenin particle size determines TTP and GMP rheology, showing that glutenin particle properties could be a new key to understand the link between GMP and dough properties.

Effects of soaking, germination and fermentation on phytic acid, total and in vitro soluble zinc in brown rice

Rice is an important staple food in Asian countries. In rural areas it is also a major source of micronutrients. Unfortunately, the bioavailability of minerals, e.g. zinc from rice, is low because it is present as an insoluble complex with food components such as phytic acid. We investigated the effects of soaking, germination and fermentation with an aim to reduce the content of phytic acid, while maintaining sufficient levels of zinc, in the expectation of increasing its bioavailability. Fermentation treatments were most effective in decreasing phytic acid (56-96% removal), followed by soaking at 10°C after preheating (42-59%). Steeping of intact kernels for 24h at 25°C had the least effect on phytic acid removal (<20%). With increased germination periods at 30°C, phytic acid removal progressed from 4% to 60%. Most wet processing procedures, except soaking after wet preheating, caused a loss of dry mass and zinc (1-20%). In vitro solubility, as a percentage of total zinc in soaked rice, was significantly higher than in untreated brown rice while, in steeped brown rice, it was lower (p<0.05). Fermentation and germination did not have significant effects on the solubility of zinc. The expected improvement due to lower phytic acid levels was not confirmed by increasing levels of in vitro soluble zinc. This may result from zinc complexation to other food components.

Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease

Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of ‘low CD toxic’ as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

BackgroundGluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).ResultsThe effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties.ConclusionThe consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.

A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat.

The detection, analysis, and quantification of individual celiac disease (CD) immune responsive gluten proteins in wheat and related cereals (barley, rye) require an adequate and reliable extraction protocol. Because different types of gluten proteins behave differently in terms of solubility, currently different extraction protocols exist. The performance of various documented gluten extraction protocols is evaluated for specificity and completeness by gel electrophoresis (SDS-PAGE), immunoblotting and RIDASCREEN Gliadin competitive ELISA. Based on these results, an optimized, two-step extraction protocol has been developed.

Influence of lysophosphatidylcholine on the gelation of diluted wheat starch suspensions

Starch is an omnipresent constituent which is used for its nutritional and structuring properties. Recently concerns have been raised since starch is a source of readily available glucose which is tightly correlated with diabetes type II and obesity. For this reason, the possibilities for modulating the digestibility of starch while preserving its functional properties were investigated; therefore the focus of this paper is on starch gelatinization and the effect of lysophosphatidylcholine (LPC) on the structuring properties of wheat starch. The effect of LPC on thermal properties and viscosity behavior of starch suspensions was studied using DSC and RVA, respectively. The influence on granular structure was observed by light microscopy. The RVA profile demonstrated no viscosity increase at high LPC concentrations which proves intact granular structure after gelatinization. LPC in intermediate concentrations resulted in a notable delay of pasting; however the peak and end viscosities were influenced as well. Lower LPC concentrations demonstrated a higher peak viscosity as compared with pure starch suspensions. DSC results imply that inclusion complexes of amylose-LPC might be formed during pasting time. Since the viscosity profiles are changed by LPC addition, swelling power and solubility of starch granules are influenced as well. LPC hinders swelling power and solubility of starch granules which are stimulated by heating.

Functional properties of low Mr wheat proteins. II: Effects on dough properties

The effects of non-gluten low Mr wheat proteins on bread-making quality and dough properties were investigated. Low Mr proteins retarded the re-polymerisation of SDS-unextractable glutenin (glutenin macropolymer-GMP; gel protein) during resting of doughs from the cultivars Obelisk, Camp Remy, Castan and Rektor. Polymerisation in the overly-strong cultivar, Fresco, was not affected substantially. Additions of 0·2% to 0·4% by weight of low Mr proteins to flour increased the relaxation half-times of doughs from cvs Camp Remy and Rektor as measured in a Bohlin VOR Rheometer. Furthermore, the phase angle, δ (ratio of loss modulus to storage modulus), decreased at these levels of low Mr protein addition, indicating the formation of a dough with greater elastic character. At 0·8% (w/w) addition, the relaxation time decreased and the phase angle increased. The effects on the dough of cv. Obelisk were relatively slight. The complex dynamic moduli of doughs from cvs Camp Remy and Rektor were relatively unaffected by the addition of low Mr proteins, but the moduli decreased when they were added to cv. Obelisk flour. The rate of GMP repolymerisation during resting was related negatively to the phase angle, whereas the complex modulus increased to a plateau value as the rate of GMP reassembly increased. In micro-baking tests with 10-g and 100-g pan loaves, increases in loaf volume of 11% to 12% were observed with up to 0·8% (w/w) addition of low Mr proteins to cv. Camp Remy dough. For the weaker cultivar, Obelisk, addition of low Mr proteins caused either no increase in loaf volume or a decrease of 13%, depending on the level of addition. The results indicate that, although low Mr wheat proteins have inconsistent effects on loaf volume, they have profound effects on both the biochemical and rheological properties of dough.