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.

BACKGROUND: Gluten 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). RESULTS: The 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 alpha-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 omega-gliadin, gamma-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. CONCLUSION: The 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.

Net charge affects morphology and visual properties of ovalbumin aggregates.

The effect of ovalbumin net charge on aggregate morphology and visual properties was investigated using chromatography, electrophoresis, electron microscopy, and turbidity measurements. A range of differently charged ovalbumin variants (net charge ranging from -1 to -26 at pH 7) was produced using chemical engineering. With increasing net charge, the degree of branching and flexibility of the aggregates decreased. The turbidity of the solutions reflected the aggregate morphology that was observed with transmission electron microscopy. Increasing the stiffness of the aggregates transformed the solutions from turbid to transparent. Artificially shielding the introduced net charge by introducing salt in the solution resulted in an aggregate morphology that was similar to that for low-net-charge variants. The morphology of heat-induced aggregates and the visual appearance of the solutions were significantly affected by net charge. We also found that the morphology of ovalbumin aggregates can be rapidly probed by high-throughput turbidity experiments.

Nutridynamics--studying the dynamics of food components in products and in the consumer.

The concentrations and biological effects of nutrients, antinutrients and bioactive compounds, including microbes and their constituents, are affected by production and processing steps, the food matrix in which they reside, the way they are digested and metabolized in the human body, and whether or not and in what form they subsequently reach their target site. A new scientific concept, denoted here as 'nutridynamics', aims to unravel the dynamics of these processes by using a systematic approach to study how a food component is affected by the food matrix itself and what it does in the body. This holistic concept has potential synergy with the areas of food technology and nutrigenomics, and provides a link between food production and the mechanistic effects of bioactive ingredients.

Oscillatory water sorption test for determining water uptake behavior in bread crust.

In this work, water sorption kinetics of bread crust are described using an oscillatory sorption test in combination with a Langmuir type equation. Both kinetic and thermodynamic information could be obtained at the same time. An advantage of applying a Langmuir type equation for a quantitative description of the water uptake kinetics is that no prior knowledge is necessary with respect to shape and surface area of the sample. It was shown that adsorption and desorption of water to the bread crust particle surface is much faster than the experimental time used (15 min at minimum). From this, we may conclude that diffusion of water into the solid matrix is the rate-limiting step in the water sorption process. The method also allows one to calculate a Gibbs free energy. The method is suitable for use up to relative humidities of 60%.

Aggregation of beta-lactoglobulin regulated by glucosylation.

A large number of proteins are glycosylated, either in vivo or as a result of industrial processing. Even though the effect of glycosylation on the aggregation of proteins has been studied extensively in the past, some reports show that the aggregation process is accelerated, whereas others found that the process is inhibited by glycosylation. This paper investigates the reasons behind these controversial results as well as the potential mechanism of the effect of glucosylation on aggregation using bovine beta-lactoglobulin as a model. Glucosylation was found to inhibit denaturant-induced aggregation, whereas heat-induced aggregation was accelerated. It was also found that the kinetic partitioning from an unfolded state was driven toward refolding for glucosylated protein, whereas aggregation was the preferred route for the nonglucosylated protein.

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

BACKGROUND: Bread 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 alpha-gliadins contain several peptides that are associated to the disease. RESULTS: We obtained 230 distinct alpha-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 alpha-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, alpha-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-alpha9 and glia-alpha20, but never the intact epitopes glia-alpha and glia-alpha2. 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. CONCLUSION: Our analysis shows that alpha-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.

Do sulfhydryl groups affect aggregation and gelation properties of ovalbumin?

The aim of this work is to evaluate the impact of sulfhydryl groups on ovalbumin aggregation and gelation. Ovalbumin was chemically modified to add sulfhydryl groups in various degrees. The rate of aggregation was not affected by the introduction of sulfhydryl groups, and disulfide bond formation was preceded by physical interactions. Hence, disulfide interactions may not be the driving force for the aggregation of ovalbumin. Investigation of the aggregates and gels by electron microscopy and rheology suggested that a critical number of sulfhydryl groups can be introduced beyond which the microstructure of the aggregates transforms from fibrillar into amorphous. Rheological studies further suggested that covalent networks, once formed, do not have the possibility to rearrange, reducing the possibility to attain a stronger network. These results show that, even though aggregation of ovalbumin may be primarily driven by physical interactions, formed disulfide bonds are important to determine the resulting aggregate morphology and rheological properties.

Coeliac Disease: background and biochemical aspects.

Coeliac Disease has to be considered a main food related affliction, with life long consequences for the people having the disease. Coeliac Disease patients suffer from adverse effects that can be related to specific gluten peptide sequences that trigger a sequence of immune related reactions leading to damage of the intestine and related malabsorption symptoms. Recently, detailed information has come available on peptide sequences that are toxic for Coeliac Disease patients. This information is discussed in relation to prevention of the disease and the development of safe cereals for Coeliac Disease patients.

Cold-set globular protein gels: interactions, structure and rheology as a function of protein concentration.

We identified the contribution of covalent and noncovalent interactions to the scaling behavior of the structural and rheological properties in a cold gelling protein system. The system we studied consisted of two types of whey protein aggregates, equal in size but different in the amount of accessible thiol groups at the surface of the aggregates. Analysis of the structural characteristics of acid-induced gels of both thiol-blocked and unmodified whey protein aggregates yielded a fractal dimension (2.3 +/- 0.1), which is in line with other comparable protein networks. However, application of known fractal scaling equations to our rheological data yielded ambiguous results. It is suggested that acid-induced cold-gelation probably starts off as a fractal process, but is rapidly taken over by another mechanism at larger length scales (>100 nm). In addition, indications were found for disulfide cross-link-dependent structural rearrangements at smaller length scales (<100 nm).

Acid-induced cold gelation of globular proteins: effects of protein aggregate characteristics and disulfide bonding on rheological properties.

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.

Gluten protein composition in several fractions obtained by shear induced separation of wheat flour.

Recently, it was found that applying curvilinear shear flow in a cone-cone shearing device to wheat flour dough induces separation, resulting in a gluten-enriched fraction in the apex of the cone and gluten-depleted fraction at the outer part. This article describes whether fractionation of the various proteineous components occurs during and after separation of Soissons wheat flour. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high performance liquid chromatography (SE-HPLC) were found to be suitable techniques for this. It is concluded that all protein fractions migrate to the center of the cone as a result of which the composition of the gluten-enriched fraction remains rather similar to that in the original flour. However, the larger glutenin polymer fraction migrated faster, as a result of which the concentration of large polymers was increased with a factor 2.4 compared to that of Soissons flour. The concentration of monomers in the gluten-enriched fraction was decreased to 70% of the original concentration in the original wheat flour.

Colloidal aspects of texture perception.

Recently, considerable attention has been given to the understanding of texture attributes that cannot directly be related to physical properties of food, such as creamy, crumbly and watery. The perception of these attributes is strongly related to the way the food is processed during food intake, mastication, swallowing of it and during the cleaning of the mouth after swallowing. Moreover, their perception is modulated by the interaction with other basic attributes, such as taste and aroma attributes (e.g. sourness and vanilla). To be able to link the composition and structure of food products to more complicated texture attributes, their initial physical/colloid chemical properties and the oral processing of these products must be well understood. Understanding of the processes in the mouth at colloidal length scales turned out to be essential to grasp the interplay between perception, oral physiology and food properties. In view of the huge differences in physical chemical properties between food products, it is practical to make a distinction between solid, semi-solid, and liquid food products. The latter ones are often liquid dispersions of emulsion droplets or particles in general. For liquid food products for instance flow behaviour and colloidal stability of dispersed particles play a main role in determining their textural properties. For most solid products stiffness and fracture behaviour in relation to water content are essential while for semi-solids a much larger range of mechanical properties will play a role. Examples of colloidal aspects of texture perception will be discussed for these three categories of products based on selected sensory attributes and/or relevant colloidal processes. For solid products some main factors determining crispness will be discussed. For crispiness of dry cellular solid products these are water content and the architecture of the product at mesoscopic length scales (20-1000 microm). In addition the distribution of water at mesoscopic length scales was found to be important. For semi-solid foods, sensory characteristics as spreadability, watery and crumbliness are primarily determined by food properties at mesoscopic length scales. Crumbliness is directly related to the formation of free running cracks that occur during eating of the product. Exudation of the continuous liquid phase of gels during compression gives rise to watery/juicy sensory attributes. For liquid food products, colloidal interactions of emulsion droplets, particles, proteins, and polysaccharides with saliva and oral surfaces were found to affect texture characteristics as creaminess, fattiness, roughness and astringency.

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.

Water uptake mechanism in crispy bread crust.

Crispness is an important quality characteristic of dry solid food products such as crispy rolls. Its retention is directly related to the kinetics of water uptake by the crust. In this study, a method for the evaluation of the water sorption kinetics in bread crust is proposed. Two different sorption experiments were used: an oscillatory sorption test and a sorption test in which the air relative humidity (RH) was increased stepwise. These two experiments had different time scales, which made it possible to get a better understanding of the mechanisms involved. Results show that the adsorption and desorption dynamics of the oscillatory sorption test could be described by a single exponential in time. The water uptake rate ( k) was one of the fitting parameters. A maximum in the water uptake rate was found for a RH value between 50 and 70%. The rate parameters of the experiment where RH was increased stepwise were around a factor 10 lower than those derived from oscillatory sorption experiments. This is an important factor when designing experiments for the determination of water uptake rates. In addition, also a parameter describing the time dependence of the rate parameters of the oscillatory sorption experiment was calculated (C), again by fitting a single exponential to the rate parameters. C was in the same range as the rate parameter of the isotherm experiment. This indicates that different (relaxation) processes are acting at the same time in the bread crust during water uptake.

Effect of protein charge on the generation of aggregation-prone conformers.

This study describes how charge modification affects aggregation of ovalbumin, thereby distinguishing the role of conformational and electrostatic stability in the process. Ovalbumin variants were engineered using chemical methylation or succinylation to obtain a range of protein net charge from -1 to -26. Charge modification significantly affected the denaturation temperature. From urea-induced equilibrium denaturation studies, it followed that unfolding proceeded via an intermediate state. However, the heat-induced denaturation process could still be described as a two-state irreversible unfolding transition, suggesting that the occurrence of an intermediate has no influence on the kinetics of unfolding. By monitoring the aggregation kinetics, the net charge was found not to be discriminative in the process. It is concluded that the dominant factor determining ovalbumin aggregation propensity is the rate of denaturation and not electrostatic repulsive forces.

Glycoforms of beta-lactoglobulin with improved thermostability and preserved structural packing.

In this article we show how various degrees of glycosylation can be used to control the thermal stability of proteins. The primary amines of beta-lactoglobulin were glycosylated with glucose or fructose within a range of non-denaturing reaction parameters. The modified fractions were characterized and analyzed for structural stability and hydrophobic exposure. The modification procedure gave rise to the production of glycoproteins with a well-defined Gaussian distribution, where glucose appeared more reactive than fructose. The integrity of the secondary, tertiary, and quaternary structures remained unaffected by the modification procedure. However, upon heating the stability of the modified fractions increased up to 6 K. Here we demonstrate the effects on the thermodynamic properties of proteins by glycosylation; this work serves as a first step in understanding and controlling the process underlying aggregation of glycosylated proteins.