Role of polysaccharides in food, digestion, and health.

Polysaccharides derived from plant foods are major components of the human diet, with limited contributions of related components from fungal and algal sources. In particular, starch and other storage carbohydrates are the major sources of energy in all diets, while cell wall polysaccharides are the major components of dietary fiber. We review the role of these components in the human diet, including their structure and distribution, their modification during food processing and effects on functional properties, their behavior in the gastrointestinal tract, and their contribution to healthy diets.

Energy utilization and growth performance of chickens fed novel wheat inbred lines selected for different pentosan levels with and without xylanase supplementation.

Different F5 recombinant inbred lines from the cross Yumai 34×Ukrainka were grown in replicated trials on a single site in one harvest year at Rothamsted Research. A total of 10 samples from those lines were harvested and used in a broiler experiment. Twenty nutritionally complete meal-form diets that had 630 g/kg of wheat with different amounts of pentosan, with and without exogenous xylanase supplementation, were used to compare broiler growth performance and determine apparent metabolizable energy corrected for N retention (AMEn). We examined the relationship between the nutritive value of the wheat samples and their chemical compositions and results of quality tests. The amounts of total and water soluble pentosans in wheat samples ranged from 36.7 to 48.0 g/kg DM, and 6.7 to 11.6 g/kg DM, respectively. The mean crude oil and protein contents of the wheat samples were 10.5 and 143.9 g/kg DM, respectively. The average determined value for the kinematic viscosity was 0.0018 mPa.s, and 2.1 mPa.s for the dynamic viscosity. The AMEn of the wheat-based diets had a maximum range of 0.47 MJ/kg DM within the ten wheat samples that were tested. Xylanase supplementation improved (P<0.05) dietary AMEn, dry matter, and fat digestibility coefficients. There was a positive (P<0.05) relationship between in vitro kinematic viscosity of the wheat samples and the total pentosan content. There was a negative relationship between the total pentosan content in the wheat and broiler growth performance. An increase by 10 g of pentosan per kg of wheat reduced (P<0.001) daily feed intake and weight gain by 2.9 g and 3.5 g, respectively. The study shows that the feeding quality of wheat samples can be predicted by their total pentosan content. Supplementary xylanase improved energy and nutrient availability of all wheat samples that was independent of differences in pentosan content.

In vitro screening for putative psoriasis-specific antigens among wheat proteins and peptides.

BACKGROUND: Patients with psoriasis who had raised IgG and/or IgA antigliadin antibodies showed clinical improvement in a trial with a gluten-free diet. The selection of patients for the diet treatment was based on the presence of specific antibodies, i.e. the result of humoral immunity. OBJECTIVES: As psoriasis is now considered to be a T cell-mediated disease we decided to challenge peripheral blood mononuclear cells (PBMCs) in vitro from randomly selected patients with well-defined wheat proteins/peptides to explore the possibility of identifying a specific antigen with T cell activating properties in a subgroup of patients. METHODS: PBMCs from 37 patients (20 female and 17 male; mean age 49years) and 37 healthy controls (12 female and 25 male; mean age 57years) were included. Not all patients participated in all experiments. The PBMCs were exposed in vitro with the following wheat proteins/peptides in various concentrations: total albumins, 0·28 α-amylase inhibitor and the synthetic peptides, p31-43, p57-68 and p62-75, based on coeliac-active sequences of α-gliadin. The proliferative response was measured as counts per minute after the cells had been pulsed with methyl-(3) H-thymidine. RESULTS: Albumin, α-amylase inhibitor, p31-43 and p57-68 elicited a significant response in both patients and controls but showed no differences between the groups. The response induced by the α-amylase inhibitor was higher than that induced by the albumin fraction and the p31-43 and p57-68 peptides. At a concentration of 25µgmL(-1) , five of 36 patients with psoriasis responded positively to the p62-75 peptide and none of the 33 controls, using a stimulation index of 2·4 as the cut-off level (P<0·05). These five patients did not show clinical features that differed from the remaining patients. Among the responding patients the relative number of CD4+ cells increased in some but not all after in vitro challenge with the albumins, 0·28 α-amylase inhibitor, and p62-75. These antigens could also induce in vitro the expression of the homing antigen cutaneous lymphocyte antigen (CLA) in a few patients and controls. CONCLUSIONS: The wheat protein antigens, especially the p62-75 peptide, might be of interest in a subgroup of patients with psoriasis.

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward.

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with beta-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.

Do modern types of wheat have lower quality for human health?

Wheat is the major staple food in Western Europe and an important source of energy, protein, dietary fibre, minerals, B vitamins and phytochemicals. Plant breeders have been immensely successful in increasing yields to feed the growing global population. However, concerns have been expressed that the focus on increasing yield and processing quality has resulted in reduced contents of components that contribute to human health and increases in adverse reactions. We review the evidence for this, based largely on studies in our own laboratories of sets of wheats bred and grown between the 18th century and modern times. With the exception of decreased contents of mineral micronutrients, there is no clear evidence that intensive breeding has resulted in decreases in beneficial components or increases in proteins which trigger adverse responses. In fact, a recent study of historic and modern wheats from the UK showed increases in the contents of dietary fibre components and a decreased content of asparagine in white flour, indicating increased benefits for health.

Wheat.

Wheat is the dominant crop in temperate countries being used for human food and livestock feed. Its success depends partly on its adaptability and high yield potential but also on the gluten protein fraction which confers the viscoelastic properties that allow dough to be processed into bread, pasta, noodles, and other food products. Wheat also contributes essential amino acids, minerals, and vitamins, and beneficial phytochemicals and dietary fibre components to the human diet, and these are particularly enriched in whole-grain products. However, wheat products are also known or suggested to be responsible for a number of adverse reactions in humans, including intolerances (notably coeliac disease) and allergies (respiratory and food). Current and future concerns include sustaining wheat production and quality with reduced inputs of agrochemicals and developing lines with enhanced quality for specific end-uses, notably for biofuels and human nutrition.

Elastomeric proteins: biological roles, structures and mechanisms.

Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain beta-turns as a structural motif within the elastomeric domains.

Improving wheat as a source of iron and zinc for global nutrition.

Wheat is the staple food crop in temperate countries and increasingly consumed in developing countries, displacing traditional foods. However, wheat products are typically low in bioavailable iron and zinc, contributing to deficiencies in these micronutrients in countries where wheat is consumed as a staple food. Two factors contribute to the low contents of bioavailable iron and zinc in wheat: the low concentrations of these minerals in white flour, which is most widely consumed, and the presence of phytates in mineral-rich bran fractions. Although high zinc types of wheat have been developed by conventional plant breeding (biofortification), this approach has failed for iron. However, studies in wheat and other cereals have shown that transgenic (also known as genetically modified; GM) strategies can be used to increase the contents of iron and zinc in white flour, by converting the starchy endosperm tissue into a 'sink' for minerals. Although such strategies currently have low acceptability, greater understanding of the mechanisms which control the transport and deposition of iron and zinc in the developing grain should allow similar effects to be achieved by exploiting naturally induced genetic variation. When combined with conventional biofortification and innovative processing, this approach should provide increased mineral bioavailability in a range of wheat products, from white flour to wholemeal.

Estimation of the iron bioavailability in green vegetables using an in vitro digestion/Caco-2 cell model.

It is estimated that over 30% of the global population is anaemic, half of which is due to iron deficiency. The bioavailability of iron from vegetables is low and variable, and influenced by food composition and matrix. We have therefore determined the relative bioavailability of iron in five types of green vegetable, spinach, broccoli, savoy cabbage, curly kale and green pepper, by measuring the ferritin response in a simulated digestion/Caco-2 cell model. Savoy cabbage gave the highest ferritin response and analysis of the digest showed that the iron was present in low molecular weight fractions which contained glucose, fructose, organic acids and amino acids. The addition of fructose 1,6-biphosphate to the Caco-2 cells increased iron uptake 2-fold. These results demonstrate that cabbage was the best source of bioavailable iron out of the vegetables studied and suggest that the formation of complexes with fructose derivatives contribute to increase the iron bioavailability.

Allergens to wheat and related cereals.

Wheat is one of the major crops grown, processed and consumed by humankind and is associated with both intolerances (notably coeliac disease) and allergies. Two types of allergy are particularly well characterized. The first is bakers' asthma, which results from the inhalation of flour and dust during grain processing. Although a number of wheat proteins have been shown to bind IgE from patients with bakers' asthma, there is no doubt a well-characterized group of inhibitors of alpha-amylase (also called chloroform methanol soluble, or CM, proteins) are the major components responsible for this syndrome. The second well-characterized form of allergy to wheat proteins is wheat-dependent exercise-induced anaphylaxis (WDEIA), with the omega(5)-gliadins (part of the gluten protein fraction) being the major group of proteins which are responsible. Other forms of food allergy have also been reported, with the proteins responsible including gluten proteins, CM proteins and non-specific lipid transfer proteins. Processing of wheat and of related cereals (barley and rye, which may contain related allergens) may lead to decreased allergenicity while genetic engineering technology offers opportunities to eliminate allergens by suppressing gene expression.

Detailed analysis of the expression of an alpha-gliadin promoter and the deposition of alpha-gliadin protein during wheat grain development.

BACKGROUND AND AIMS: Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development. METHODS: A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences. KEY RESULTS: GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream. CONCLUSIONS: The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

Transformation of wheat with high molecular weight subunit genes results in improved functional properties.

The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods. There are both quantitative and qualitative effects of HMW subunits on the quality of the grain, the former being related to differences in the number of expressed HMW subunit genes. We have transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour. A range of transgene expression levels was obtained with some of the novel subunits present at considerably higher levels than the endogenous subunits. Analysis of T2 seeds expressing transgenes for one or two additional HMW subunits showed stepwise increases in dough elasticity, demonstrating the improvement of the functional properties of wheat by genetic engineering.

In vitro fermentation by human fecal microflora of wheat arabinoxylans.

The fermentation of three arabinoxylan (AX) fractions from wheat by the human fecal microflora was investigated in vitro. Three AX fractions, with average molecular masses of 354, 278, and 66 kDa, were incorporated into miniature-scale batch cultures (with inulin as a positive prebiotic control) with feces from three healthy donors, aged 23-29. Microflora changes were monitored by the culture-independent technique, fluorescent in situ hybridization, and short chain fatty acid (SCFA) and lactic acid production were measured by high-performance liquid chromatography. Total cell numbers increased significantly in all treated cultures, and the fermentation of AX was associated with a proliferation of the bifidobacteria, lactobacilli, and eubacteria groups. Smaller but statistically significant increases in bacteroides and clostridia groups were also observed. All AX fractions had comparable bifidogenic impacts on the microflora at 5 and 12 h, but the 66 kDa AX was particularly selective for lactobacilli. Eubacteria increased significantly on all AX fractions, particularly on 66 kDa AX. As previously reported, inulin gave a selective increase in bifidobacteria. All supplemented cultures showed significant rises in total SCFA production, with a particularly high proportion of butyric acid being produced from AX fermentation. The prebiotic effect, that is, the selectivity of AX for bifidobacteria and lactobacilli groups, increased as the molecular mass of the AX decreased. This suggests that molecular mass may influence the fermentation of AX in the colon.

Do we need to worry about eating wheat?

Wheat is a staple food throughout the temperate world and an important source of nutrients for many millions of people. However, the last few years have seen increasing concerns about adverse effects of wheat on health, particularly in North America and Europe, with the increasing adoption of wheat-free or gluten-free diets. This relates to two concerns: that wheat products are disproportionally responsible for increases in obesity and type 2 diabetes and that wheat gluten proteins cause a range of adverse reactions, including allergies, coeliac disease and 'non-coeliac gluten sensitivity'. The first concern has been refuted in previous publications, and we therefore focus on the second here. Current evidence indicates that allergy to ingested wheat and coeliac disease (and related intolerances) each occur in up to 1% of the population. The extent to which their prevalence has increased is difficult to quantify due to improved diagnosis and increased awareness. However, neither appears to be increasing disproportionally when compared with other immunologically mediated adverse reactions to food. Other adverse reactions to wheat are more difficult to define as their mechanisms are not understood and they are therefore difficult to diagnose. In particular, 'non-coeliac wheat sensitivity' has been reported to occur in 6% or more of the population in the US. However, the application of more rigorous diagnostic criteria is likely to give substantially lower estimates of prevalence. It is therefore unlikely that the health of more than a small proportion of the population will be improved by eliminating wheat or gluten from the diet. In fact, the opposite may occur as wheat is an important source of protein, B vitamins, minerals and bioactive components.

Purification and characterization of an insect alpha-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix lachryma-jobi).

A protein inhibitor of locust gut alpha-amylase was purified from seeds of Job's Tears (Coix lachryma-jobi) using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two major isomers, each a dimer of two closely similar or identical subunits of Mr about 26,400, and associated by inter-chain disulphide bonds. These isomers also had closely similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, Bacillus subtilis, Aspergillus oryzae and barley malt. The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform. This showed a high degree of homology with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean), and endochitinase activity was demonstrated by following the release of radioactivity from [3H]chitin. This novel combination of functions may be relevant to protection of the grain from insect feeding and fungal infection.

Wheat seed proteins exhibit a complex mechanism of protein elasticity.

Elastomeric proteins are found in a number of animal tissues (elastin, abductin and resilin), where they have evolved to fulfil a range of biological functions. All exhibit rubber-like elasticity, undergoing deformation without rupture, storing the energy involved in deformation, and then recovering to their initial state when the stress is removed. The second part of the process is passive, entropy decreasing when the proteins are deformed, with the higher entropy of the relaxed state providing the driving force for recoil. In plants there is only one well-documented elastomeric protein system, the alcohol-soluble seed storage proteins (gluten) of wheat. The elastic properties of these proteins have no known biological role, the proteins acting as a store for the germinating seed. Here we show that the modulus of elasticity of a group of wheat gluten subunits, when cross-linked by gamma-radiation, is similar to that of the cross-linked polypentapeptide of elastin. However, thermoelasticity studies indicate that the mechanism of elastic recoil is different from elastin and other characterized protein elastomers. Elastomeric force, f, has two components, an internal energy component, f(e), and an entropic component, f(s). The ratio f(e)/f can be determined experimentally; if this ratio is less than 0.5 the elastomeric force is predominantly entropic in origin. The ratio was determined as 5.6 for the cross-linked high M(r) subunits of wheat glutenin and near zero for the cross-linked polypentapeptide of elastin. Tensile stress must be entropic or energetic in origin, the results would suggest that elastic recoil in the wheat gluten subunits, in part, may be associated with extensive hydrogen bonding within and between subunits and that entropic and energetic mechanisms contribute to the observed elasticity.

Identification of a novel beta-turn-rich repeat motif in the D hordeins of barley.

The amino acid sequence of the C-terminal part of a barley D hordein seed protein was deduced from the nucleotide sequence of a partial cDNA. It showed high homology with the HMW glutenin subunits of wheat, both proteins consisting predominately of repeated sequences. Whereas the wheat repeats are based on tri-, hexa- and nonapeptides that are rich in glycine, proline and glutamine, the D hordein also contains eleven copies of a novel unrelated motif: Thr-Thr-Val-Ser. The repeated sequences in the wheat glutenin subunits have been demonstrated to form an unusual spiral supersecondary structure based on beta-turns. Conformational analysis of the Thr-Thr-Val-Ser motif by secondary structure prediction and by circular dichroism spectroscopy of an 18 residue synthetic peptide demonstrates that it also forms beta-turns. Thus, D hordein may also have a spiral structure like that of HMW glutenin, despite the presence of a different repeat motif. This conservation of protein conformation in D hordein and the wheat glutenin subunits may indicate a structural role, perhaps in packing of the proteins within the protein bodies of the developing grain.

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein.

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.

Molecular cloning of the barley seed protein CMd: a variant member of the alpha-amylase/trypsin inhibitor family of cereals.

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of alpha-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with alpha-amylases of bacterial origin.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein.

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.