RESUMEN

We analyze stress distributions in a two-dimensional bidisperse cemented granular packing for a broad range of the values of particle-size ratio, the volumes of large and small particles, and the amount of cementing matrix. In such textured porous materials, the stress concentration, which controls the fracture and fragmentation of the material under tensile loading or in grinding processes, reflects not only the porosity but also the contact network of the particle phase and the resulting stress chains. By means of peridynamic simulations under tensile loading, we show how both the texture and stress distribution depend on size ratio, volume ratio, and the amount of the cementing matrix. In particular, the volume fraction of the class of small particles plays a key role in homogenizing stresses across the system by reducing porosity. Interestingly, the texture controls not only the porosity but also the distribution of pores inside the system with its statistical variability, found to be strongly correlated with the homogeneity of stresses inside the large particles. The most homogeneous stress distribution occurs for the largest size ratio and largest volume fraction of small particles, corresponding to the lowest pore size dispersion and the cushioning effect of small particles and its similar role to the binding matrix for stress redistribution across the packing. At higher porosity, the tensile stresses above the mean stress fall off exponentially in all phases with an exponent that strongly depends on the texture. The exponential part broadens with decreasing matrix volume fraction and particle-size ratio. These correlations reveal the strong interplay between size polydispersity and the cohesive action of the binding matrix for stress distribution, which is significant for the behavior of textured materials in grinding operations.

RESUMEN

Contact-resonance atomic force microscopy (CR-AFM) is of great interest and very valuable for a deeper understanding of the mechanics of biological materials with moduli of at least a few GPa. However, sample surfaces can present a high topography range with significant slopes, where the local angle can be as large as  ±â€¯50°. The non-trivial correlation between surface slope and CR-frequency hinders a straight-forward interpretation of CR-AFM indentation modulus measurements on such samples. We aim to demonstrate the significant influence of the surface slope on the CR-frequency that is caused by the local angle between sample surface and the AFM cantilever and present a practical method to correct the measurements. Based on existing analytical models of the effect of the AFM set-up's intrinsic cantilever tilt on CR-frequencies, we compute the non-linear variation of the first two (eigen)modes CR-frequency for a large range of surface angles. The computations are confirmed by CR-AFM experiments performed on a curved surface. Finally, the model is applied to directly correct contact modulus measurements on a durum wheat starch granule as an exemplary sample.

Asunto(s)

RESUMEN

KEY MESSAGE: Genetic (Pinb-D1 alleles) and environment (through vitreousness) have important effects on bread wheat milling behavior. SKCS optimal values corresponding to soft vitreous or hard mealy grains were defined to obtain the highest total flour yield. Near-isogenic lines of bread wheat that differ in hardness, due to distinct puroindoline-b alleles (the wild type, Pinb-D1a, or the mutated forms, Pinb-D1b or Pinb-D1d), were grown in different environments and under two nitrogen fertilization levels, to study genetic and environmental effects on milling behavior. Milling tests used a prototype mill, equipped with two break steps, one sizing step, and two reduction steps, and this enabled 21 individual or aggregated milling fractions to be collected. Four current grain characters, thousand grain weight, test weight, grain diameter, and protein content, were measured, and three characters known to influence grain mechanical resistance, NIRS hardness, SKCS hardness index, and grain vitreousness (a character affecting the grain mechanical behavior but generally not studied). As expected, the wild type or mutated forms of Pinb-D1 alleles led to contrasted milling behavior: soft genotypes produced high quantities of break flour and low quantities of reduction flour, whereas reverse quantities were observed for hard genotypes. This different milling behavior had only a moderate influence on total flour production. NIRS hardness and vitreousness were, respectively, the most important and the second most important grain characters to explain milling behavior. However, contrary to NIRS hardness, vitreousness was only involved in endosperm reduction and not in the separation between the starchy endosperm and the outer layers. The highest flour yields were obtained for SKCS values comprised between 30 and 50, which corresponded either to soft vitreous or hard mealy grains. Prediction equations were defined and showed a good accuracy estimating break and reduction flours portions, but should be used more cautiously for total flour.

Asunto(s)

RESUMEN

KEY MESSAGE: Genetic (different forms of puroindoline-b) and environment (through variations in vitreousness), have important effects on wheat grain mechanical properties. The two methods of hardness measurements (NIRS, SKCS) do not give the same information. Bread wheat near-isogenic lines differing in hardness, due to distinct puroindoline-b alleles (the wild type, Pinb-D1a, or the mutated forms, Pinb-D1b or Pinb-D1d), were grown for three years in seven sites and under two nitrogen fertilization levels, to study genetic and environmental effects on grain mechanical properties. Two methods, Near-Infrared Reflectance Spectroscopy (NIRS) and Single Kernel Characterization System (SKCS), currently used for grain hardness characterization, were carried out. Grain vitreousness, which is known to affect the grain mechanical behavior but is generally not studied, was also measured, as well as three other characters (Thousand Grain Weight, Test Weight and protein content). The relationships between the different characters were studied. Results revealed a clear effect of the different Pinb-D1 alleles on NIRS hardness, and a marked impact of the environmental conditions on vitreousness. SKCS hardness was influenced by both Pinb-D1 alleles and environmental conditions. The relationship between SKCS and NIRS hardness was strong when considering together soft and hard genotypes, but moderate within a class of genetical hardness. Vitreousness had only a weak effect on NIRS hardness, whereas vitreousness and SKCS values were strongly correlated, with two distinct regressions for soft and hard genotypes. Vitreousness was positively related to protein content, especially in the case of hard genotypes, which were able to reach high vitreousness values never observed for soft genotypes.

Asunto(s)

RESUMEN

The milling behaviour of two naturally infected samples of durum wheat grain with contrasting levels of mycotoxins was studied. Although the two samples showed a similar milling behaviour, an increase of approximately 20% in deoxynivalenol (DON) levels was found in semolina from the sample containing the higher level of mycotoxin. However, even if the highest concentration of DON was found in fractions originating from the grain outer layers, the mycotoxin contamination in semolina and flours were not related to the amount of two compounds (ash or phytic acid) used to monitor these external tissues. The presence of the trichothecene-producing fungi in the inner-most semolina fraction was also shown using specific DNA primers and PCR amplification. Comparison of DON concentrations in the feed stock and corresponding output at each milling step or grinding of semolina fractions followed by sizing showed that concentration of mycotoxin occurs in the finest particles at the first processing steps. Therefore, DON contamination of milling fractions is not simply due to the presence of peripheral grain tissues.

Asunto(s)

RESUMEN

Wheat grain hardness is a major factor affecting the milling behaviour and end-product quality although its exact structural and biochemical basis is still not understood. This study describes the development of new near-isogenic lines selected on hardness. Hard and soft sister lines were characterised by near infrared reflectance (NIR) and particle size index (PSI) hardness index, grain protein content, thousand kernel weight and vitreousness. The milling behaviour of these wheat lines was evaluated on an instrumented micromill which also measures the grinding energy and flour particle size distribution was investigated by laser diffraction. Endosperm mechanical properties were measured using compression tests. Results pointed out the respective effect of hardness and vitreousness on those characteristics. Hardness was shown to influence both the mode of fracture and the mechanical properties of the whole grain and endosperm. Thus, this parameter also acts on milling behaviour. On the other hand, vitreousness was found to mainly play a role on the energy required to break the grain. This study allows us to distinguish between consequences of hardness and vitreousness. Hardness is suggested to influence the adhesion forces between starch granules and protein matrix whereas vitreousness would rather be related to the endosperm microstructure.

Asunto(s)

RESUMEN

The structure of the wheat gamma 46 gliadin was investigated, in aqueous solutions, under high pressure or temperature by the use of ultraviolet and fluorescence spectroscopic techniques. We found that high pressure (above 400 MPa) induces a change in the protein conformation that results in a decrease of the polarity of the environment of aromatic amino acids. This new conformation was able to bind the hydrophobic probe, 8-anilino-1-naphtalene-sulfonic acid (ANS), indicating an increase in the gliadin surface hydrophobicity. Thermodynamic parameters of this conformational change were measured and infrared spectroscopy studies were used to probe the potential secondary structure modifications. The high stability of gamma 46 gliadin could be related to its elastic character, as the observed changes were always found to be reversible.

Asunto(s)

RESUMEN

We have recently purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, which protects thylakoids isolated from the leaves of nonacclimated spinach (Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin showed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised against two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparations. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structurally and functionally different from WAX9 isolated from the surface wax of cabbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprotective, but no lipid transfer activity. The cryoprotective activity of cryoprotectin was strictly dependent on Ca(2+) and Mn(2+) and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher in the presence of ethylenediaminetetraacetate than in the presence of Ca(2+) and Mn(2+).

Asunto(s)

RESUMEN

Modifications of mechanical properties of wheat dough during thermal treatments depend mainly on the capacity of wheat gluten proteins to establish intra- and intermolecular interactions when subjected to high-temperature processing. The present study investigates the effect of thermal treatments on the mechanical properties and protein solubility of wheat gluten-based network. The increase in treatment temperatures (from 80 to 135 C) induces an increase in mechanical resistance of the gluten network (tensile strength increases from 0.26 to 2.04 MPa) and a decrease in deformability (elongation decreases from 468 to 236%). The increase in temperature (from 80 to 135 C) also induces a very strong reduction of protein solubility in 2% SDS (from 68 to 0%) that could be correlated to the mechanical changes observed. It was concluded that the modifications of the wheat gluten network properties seem to depend mainly on the temperature level, as temperatures >108-116 C allow activation of thermosetting reactions.

Asunto(s)

RESUMEN

The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.

Asunto(s)

RESUMEN

Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h, respectively, were isolated from mid-maturation seed cDNA libraries. The T aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5% similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three-step procedure including heat treatment, anion-exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13,391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N-terminal end sequence AASAAT. Using the 5,5'-dithiobis(2-nitrobenzoic acid)-reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP:thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed alpha-amylase inhibitors (chloroform/methanol-soluble proteins). The presence of a putative transmembrane domain at the N-terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.

Asunto(s)

RESUMEN

The potential role of a molecular chaperone on the rate of production of extensively altered Bacillus thuringiensis Cry1C proteins was investigated. Analysis of the proteins produced by the recombinant B. thuringiensis strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from B. thuringiensis ssp. israelensis in tandem with the truncated-cry1C genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.

Asunto(s)

RESUMEN

The cDNA encoding a wheat (Triticum durum) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage. Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion. However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.

Asunto(s)

RESUMEN

The CM (chloroform-methanol-soluble) proteins are low-molecular-weight cysteine-rich proteins that are found in wheat and barley endosperms. A cDNA clone encoding a Triticum durum (T. durum) CM3 protein has been isolated from a mid-maturation seed cDNA library. The T. durum CM3 protein is synthesized as a precursor including a signal peptide (SP) of 25 residues. Northern blot analysis shows that in developing seed the highest level of CM3 protein mRNA is detected at mid-maturation. The hybridization patterns obtained by Southern blot analysis indicated that T. durum CM proteins are encoded by a small multigene family. The similarity between the wheat and barley CM proteins encoded by homologous chromosomes is much higher than that between each of the three members of the T. durum family. All CM proteins contain ten cysteine residues organized in a conserved cysteine motif.

Asunto(s)

RESUMEN

The wheat kernel CM16 protein, a subunit of the heterotetrameric insect alpha-amylase inhibitor that has been involved in the technological quality of wheat-products, was produced in Escherichia coli. Cloning of the cDNA part encoding the mature protein in a pET expression plasmid, under the control of a promoter for the bacteriophage T7 RNA polymerase, allows the synthesis of large amounts of the CM16 protein in the bacteria. Upon induction with isopropyl thiogalactopyranoside the recombinant protein accumulates in insoluble inclusion bodies. Solubilization with 6 M urea containing 0.5 mM dithiothreitol, followed by slow elimination of the denaturing agents by step dialysis, results in a significant recovery of the recombinant protein in a soluble, monomeric form. Characterization of the protein was done by automated Edman degradation and total amino acid determination. The recombinant protein in comparison with the one isolated from wheat exhibits a Met extension at the N-terminus that was introduced in the construction for translation initiation. The CM16 protein produced in this manner has the advantage over wheat purified protein of not being contaminated with other proteins from the same family and constitutes adequate material for further analysis of the technological properties of the protein in wheat-derived products.

Asunto(s)