Characterisation and foaming properties of hydrolysates derived from rapeseed isolate.

Two hydrolysis methods used to obtain rapeseed isolate derivates were compared: chemical hydrolysis performed under alkaline conditions and pepsic proteolysis performed under acidic conditions. The mean molecular weights obtained for the hydrolysates varied from 26 to 2.5 kDa, depending on the level of hydrolysis. Further characterisation showed that, at the same level of hydrolysis, the chemical hydrolysates differed by their charges and hydrophobicity from those derived from enzymatic digestion. Analysis of the foaming properties showed, for both cases, that a limited degree of hydrolysis, around 3%, was sufficient to optimise the foaming properties of the isolate despite the different physicochemical properties of the peptides generated. The study of foaming properties at basic, neutral and acidic pHs showed that the hydrolysate solutions yielded dense foams which drained slowly and which maintained a very stable volume under the three pH conditions tested.

Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.

Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.

Conformation of wheat gluten proteins. Comparison between functional and solution states as determined by infrared spectroscopy.

The conformation of wheat gluten proteins in their functional hydrated solid state (doughy state) has been studied for the first time using attenuated total reflection infrared spectroscopy. The amide I band of functional gluten proteins reveals that, in addition to beta-turns and alpha-helices, these proteins contain a significant amount of intra- and intermolecular extended beta-sheet structures. It appears that the solubilization of gluten proteins results in a major decrease of the amount of beta-sheet structures accompanied by an increase of the content of the beta-turn and alpha-helical conformations. In addition, the alpha-helices appears to be more distorted in solution than in the functional state. Furthermore, spectra of omega- and gamma-gliadins, which are two types of prolamins of differing amino acid sequence and conformation, confirm the results obtained on the functional protein system. These results suggest that viscoelastic gluten proteins may interact through aligned beta-sheets corresponding to their repetitive domains.

Molecular flexibility in wheat gluten proteins submitted to heating.

Prolamin proteins are responsible for the network that gives wheat dough its viscoelastic properties. Non-prolamin depleted gluten was prepared under conditions that preserve its functionality. Electron Spin Resonance (ESR) was used to provide information about the dynamics of the protein at temperatures between 5 and 90 degrees C by specific spin labelling of its cysteine residues. The spectra were of a composite type, resulting from at least two populations of spin labels largely differing in molecular mobility. The correlation time of the less mobile nitroxide radicals was determined by saturation transfer ESR. Upon heating there was a transfer from the slow to the fast moving population of radicals, and an increase of mobility of this last catagory that followed the Arrhenius law. The effect of temperature on molecular flexibility was reversible. This was not the case for purified, polymerised glutenin subunits extracted from gluten. Urea created similar modifications on gluten as heat.

Separation of pure toxic peptides from a beta-gliadin subfraction using high-performance liquid chromatography.

The beta v subfraction was isolated from peptic-tryptic digests of beta-gliadin by chromatography on Biogel P-10 and applied to a Lichrosorb RP-18 or a mu-Bondapak C-18 column. Fractionation was achieved using reverse-phase high-performance liquid chromatography with a linear gradient of acetonitrile in ammonium acetate. A better resolution was obtained with the mu-Bondapak column. The first-eluted peptides a, b and c1 appeared to be well purified and apparently uncontaminated. Analysis of peptides a and b showed that they contained 40 to 42% glutamine/glutamic acid, 20 to 23% proline, 14 to 16% valine and 8 to 10% leucine. They had valine as the N-terminal amino acid and their molecular mass was estimated as 5500 using sodium dodecylsulfate electrophoresis after dansylation. Peptide c1 differed from peptides a and b in containing less valine and leucine and additional amino acids such as threonine, phenylalanine and tyrosine. In addition, it had a lower molecular mass (approximately 5000) and serine as the N-terminal amino acid. Peptide b exhibited an obvious cytotoxicity for cultured coeliac jejunal mucosa at a very low concentration (0.01 g/l) and was the most toxic peptide.

Evolution of wheat gliadins conformation during film formation: a fourier transform infrared study.

The secondary structures of wheat gliadins (a major storage protein fraction from gluten) in film-forming solutions and their evolution during film formation were investigated by Fourier transform infrared spectroscopy. In the film-forming solution, wheat gliadins presented a mixture of different secondary structures, with an important contribution of beta-turns induced by proline residues. The presence of plasticizer did not have any influence on protein secondary structure in the film-forming solution. The evolution of protein conformation was followed during drying; the major feature of this evolution was a clear growing of the infrared band at 1622 cm(-1), characteristic of intermolecular hydrogen-bonded beta-sheets. This revealed the formation of protein aggregates during film drying. The influence of the drying temperature on film properties and gliadin secondary structures was also investigated. Higher drying temperatures induced an increase of both the tensile strength of the films and the amount of beta-sheets aggregates. Although the appearance of heat-induced disulfide bridge cross-links has already been described, there is clear evidence that hydrogen-bonded beta-sheets aggregates are also induced by thermal treatment. It was not possible, however, to determine whether there is a direct relationship between the occurrence of these aggregates and the increase of the tensile strength of the films.

Molecular determinants of the influence of hydrophilic plasticizers on the mechanical properties of cast wheat gluten films.

The influence of a set of hydrophilic plasticizers varying in their chain length (ethyleneglycol and longer molecules) on the tensile strength and elongation at break of cast gluten films was studied. When considered on a molar basis (moles of plasticizer per mole of amino acid), the effect of the different plasticizers depended on their respective molecular weights for plasticizer/amino acid ratios in the range from 0.10 to 0.40. However, above a ratio of 0.40-0.50 mol/mol of amino acid, these differences were abolished and both stress and strain reached a plateau value, with all plasticizers studied. In fact, when a homologous series of molecules was considered, the ability for plasticizer to decrease stress and increase strain was closely related to the number of hydrogen bonds the molecule was able to share with the protein network. Ethyleneglycol's efficiency was, however, lower than expected from its hydrogen-bonding potential; a comparison with other diols demonstrated that this was due to the small size of this molecule. The particular effect of glycerol concentration on the films' mechanical properties suggested that other molecular features of the plasticizer, such as the number and position of hydroxide groups in the molecule, were involved in the plasticization mechanism.

Prolamin aggregation, gluten viscoelasticity, and mixing properties of transgenic wheat lines expressing 1Ax and 1Dx high molecular weight glutenin subunit transgenes.

The composition of high molecular weight (HMW) subunits of glutenin determines the gluten strength and influences the baking quality of bread wheat. Here, the effect of transgenes coding for subunits 1Ax1 and 1Dx5 was studied in two near-isogenic wheat lines differing in their HMW subunit compositions and mixing properties. The subunits encoded by the transgenes were overexpressed in the transformed lines and accounted for 50-70% of HMW subunits. Overexpression of 1Ax1 and 1Dx5 subunits modified glutenin aggregation, but glutenin properties were much more affected by expression of the 1Dx5 transgene. This resulted in increased cross-linking of glutenin polymers. In dynamic assay, the storage and loss moduli of hydrated glutens containing 1Dx5 transgene subunits were considerably enhanced, whereas expression of the 1Ax1 transgene had a limited effect. The very high strength of 1Dx5 transformed glutens resulted in abnormal mixing properties of dough. These results are discussed with regard to glutenin subunit and glutenin polymer structures.

Biopolymeric colloidal carriers for encapsulation or controlled release applications.

Biopolymers represent an interesting alternative to synthetic polymers in order to be used as structured carriers for controlled release and encapsulation applications. In particular, the ability of these carriers to entrap both hydrophilic and hydrophobic drugs may be very promising for many applications. In addition, the absence of chemical compounds and organic solvents used to produce biopolymeric matrices could be very interesting for some industrial applications. Simple or complex coacervation methods involving proteins or protein and polysaccharide mixtures were used to create new matrices dedicated to controlled release applications. Controlled release experiments with model compounds were conducted in order to evaluate the performance of such matrices. An alternative and promising research field deals with particles obtained from hydrogel systems. Totally transparent solid matrices resulting from the dehydration of new protein gels were formed and swelling capacities of these matrices were studied.

Preparation of Ulex europaeus lectin-gliadin nanoparticle conjugates and their interaction with gastrointestinal mucus.

One approach to improve the bioavailability and efficiency of drugs consists of the association of a ligand (i.e. lectins), showing affinity for biological structures located on the mucosa surfaces, to nanoparticulate drug delivery systems. In this context, Ulex europaeus lectin-gliadin nanoparticle conjugates (UE-GNP) were prepared with the aim of evaluating their in vitro bioadhesive properties. The lectin was fixed by a covalent procedure to gliadin nanoparticles by a two-stage carbodiimide method. Typically, the amount of bound lectin was calculated to be approximately 15 microg lectin/mg nanoparticle, which represented a coupling efficiency of approximately 16% of the initial lectin concentration. In addition, the activity of these conjugates was tested with bovine submaxillary gland mucin (BSM) and the level of binding to this mucin was always much greater with UE-GNP than with controls (gliadin nanoparticles). However, the presence of 50 micromol fucose, which is the reported specific sugar for U. europaeus lectin, specifically inhibited the activity of these conjugates and, therefore, the UE-GNP binding to BSM was attenuated by 70%. These results clearly showed that the activity and specificity of U. europaeus lectin was preserved after covalent coupling to these biodegradable carriers.

Soy glycinin microcapsules by simple coacervation method.

Encapsulation of a dispersed oil phase (hexadecane) was realized by simple coacervation method using soy glycinin as the wall forming material. Suitable emulsification and coacervation conditions, that favor the formation of microcapsules wall, were identified and investigated. Mild acid (pH 2.0) and heat (55 degrees C) treatments of the reaction medium during the emulsification step enhanced significantly the deposition of coacervated glycinin around oil droplets. A pronounced correlation between glycinin concentration in the continuous phase, specific surface of the dispersed phase and the microencapsulation efficiency was also observed. Coacervation step study concerned the morphology and the stability of microcapsules. Controlled initiation of the coacervation, by slow readjustment of the pH, allowed a homogeneous precipitation of glycinin around oil droplets as well as the absence of aggregation phenomena. Since the morphology of microcapsules was considerably affected by a prolonged stirring of the reaction medium, the coacervation and reticulation time were optimized in order to preserve the homogeneity of the microcapsules size distribution and the microencapsulation efficiency.

Relations between aggregative, viscoelastic and molecular properties in gluten from genetic variants of bread wheat.

Glutens differing in their low- and high-molecular-weight glutenin subunits (LMW-GS and HMW-GS) were extracted from genetic variants of bread wheat. Their composition was analysed by electrophoresis, the glutenin size distribution was determined by size-exclusion chromatography. Rheological measurements in the dynamic regime and electron spin resonance spin-labelling experiments gave data on the viscoelasticity and the protein flexibility of the glutens, respectively. In glutens differing in their HMW-GS, a relation is observed between the composition, aggregative properties, segmental flexibility and viscoelastic behaviour. This is not found in glutens with different LMW-GS. However, the proportion of rigid polypeptide segment is related to the height of the elastic plateau in all cases. The organisation of the protein network in gluten is discussed in reference to this data.

Rheological interfacial properties of plant protein-arabic gum coacervates at the oil-water interface.

This study concerns the interfacial properties of the plant proteins-arabic gum coacervates, which are involved in encapsulation processes based on complex coacervation. The results make it possible to deduce the prerequisite characteristics of the protein, which are involved in the coacervate interfacial properties. The influence of pH and concentration on protein interfacial properties was also studied so as to enable us to predict the best conditions to achieve encapsulation. It has been established that, to obtain a good encapsulation yield, the coacervate must show high surface-active properties and its adsorption on the oil droplets must be favored compared to the free protein adsorption. On the other hand, mechanical properties of the interfacial film made of the coacervate, appear to be a key parameter, as reflected by the dilational viscoelasticity measurements. When compared to the properties of the proteins films, an increase of the rigidity of the interfacial film was shown with the coacervates. It was also observed that viscoelastic properties of the coacervate film were strongly reduced, as well as the associated relaxation times. In acidic conditions, the coacervates containing alpha-gliadin are characterized by an interfacial viscoelastic behavior. This behavior reflects the softness of the interfacial film. This viscoelasticity allows also the formation of a continuous layer around the oil droplets to be encapsulated. Drop tensiometry is shown to be a method that could allow the most adapted protein to be selected and the conditions of the coacervation process to be optimized with regard to concentration and pH.

Identification of IgE-binding epitopes on gliadins for patients with food allergy to wheat.

BACKGROUND: Food allergy to wheat induces different symptoms as atopic eczema/dermatitis syndrome (AEDS), urticaria and more severe reactions as wheat-dependent exercise-induced anaphylaxis (WDEIA). Different gliadin classes are involved in this allergy but IgE-binding epitopes are known only on omega5-gliadins and for WDEIA cases. OBJECTIVES: The aim of the study was to identify IgE-binding epitopes on several gliadin classes and for several patients with different symptoms and ages. METHODS: Eleven sera were analysed by pepscan with overlapping synthetic peptides. RESULTS: Sera from five patients with anaphylaxis, urticaria or WDEIA, displayed strong IgE-binding to sequential epitopes of the repetitive domains of alphabeta, gamma, omega2 or omega5-gliadins with two immunodominant epitopes on omega5-gliadin and a consensus motif of the type QQX1PX2QQ (X1 being L, F, S or I and X2 Q, E or G). One patient allergic to deamidated wheat proteins also had IgE to a repetitive peptide of gamma and omega2-gliadins of the type QPQQPFP. Sera from four patients with AEDS detected no linear epitopes on gliadins, despite the fact that they contained specific IgE to alpha, beta, gamma or omega-gliadins. One child with AEDS recognized cysteine-containing sequences in the nonrepetitive domains of alphabeta and gamma-gliadins. CONCLUSION: B epitopes in wheat allergy were different from B epitopes of coeliac disease. Differences exist in IgE-binding epitopes between patients with food allergy to wheat. IgE from those suffering from WDEIA, anaphylaxis and urticaria detected sequential epitopes in the repetitive domain of gliadins whereas IgE from AEDS patients probably recognized conformational epitopes.

[Application of hydrophobic chromatography to the fractionation of wheat prolamins]. / Application de la chromatographie d'interaction hydrophobe au fractionnement des prolamines du blé tendre.

Hydrophobic chromatography is applied to the fractionation of wheat prolamins. Proteins are separated on "Phenyl Sepharose CL 4B" column. They are eluted by variations of pH and polarity of solvent. Components with the same electrophoretic mobility appear in several chromatographic fractions and gliadin groups, as indicated by Woychick classification, are heterogeneous. This method is excepted to give new information about interaction properties of gluten proteins.

Hydrophobic-cluster analysis of plant protein sequences. A domain homology between storage and lipid-transfer proteins.

Hydrophobic-cluster analysis was used to characterize a conserved domain located near the C-terminal amino acid sequence of wheat (Triticum aestivum) storage proteins. This domain was transformed into a linear template for a global search for similarities in over 5200 protein sequences. In addition to proteins that had already been found to exhibit homology to wheat storage proteins, a previously unreported homology was found with non-specific lipid-transfer proteins from castor bean (Ricinus communis) and from spinach (Spinacia oleracea) leaf. Hydrophobic-cluster analysis of various members of the present protein group clearly shows a typical domain structure where (i) variable and conserved domains are located along the sequence at precise positions, (ii) the conserved domains probably reflect a common ancestor, and (iii) the unique properties of a given protein (chain cut into subunits, repetitive domains, trypsin-inhibitor active site) are associated with the variable domains.

Adsorption kinetics and rheological interfacial properties of plant proteins at the oil-water interface.

Adsorption and rheological properties of plant proteins were determined by means of the dynamic pendant drop technique. The plant protein properties were compared with the interfacial properties of gelatin, which is well-known for its surface-active properties and is commonly used in food and health products. The results showed that alpha gliadins (wheat proteins) and pea globulins have the highest surface active properties at the oil-water interface, even higher than gelatin at the same concentration (weight/volume). After a short time of adsorption, alpha gliadin interfacial behavior is characterized by a pronounced viscoelasticity, which was confirmed with time whereas pea protein interfacial behavior became elastic after a long initial adsorption period. Finally, the behavior of gelatin is very close to the alpha gliadin behavior for the short initial adsorption period, whereas it looks like the behavior of legume seed proteins for longer times of the adsorption kinetics. This study emphasizes the importance of the choice of the proteins and the emulsification time in the encapsulation process, according to the interfacial behavior.

Microcapsules based on glycinin-sodium dodecyl sulfate complex coacervation.

Microcapsules processed by complex coacervation were prepared using hexadecane for the oil phase and glycinin (a soybean storage protein)-sodium dodecyl sulfate (SDS) as the main wall-forming material. The study underlines the essential role of SDS, which, by the way of [glycinin(+)-SDS(-)] insoluble complex formation, allowed the precipitation of proteins around oil droplets. Moreover, particular attention was attributed to the study of suitable conditions of glycinin cross-linking with glutaraldehyde. The reticulation step was performed at pH 4.0 and it was observed that the precipitated state of proteins increased considerably the efficiency of the cross-linking reaction. Analysis of the reactional medium after each main step of the process (emulsification, complex coacervation, cross-linking) allowed the follow-up and characterization of microcapsule formation. Optimization of different process parameters such as glycinin concentration, glycinin/SDS/glutaraldehyde ratios, pH and the kinetics of cross-linking allowed the encapsulation of the totality of oil and the use of more than 98% of initially introduced proteins for the microcapsule wall formation.

Development of an in vitro system simulating bucco-gastric digestion to assess the physical and chemical changes of food.

The release of nutrients from solid food depends on the physical and chemical characteristics of substrates, and on dynamic physiological events including pH, gastric emptying and enzymatic secretion. Our laboratory has developed an in vitro digestive system mimicking mouth and stomach processes to determine physical and chemical changes of bread during digestion. To simulate oral-phase digestion, bread was minced and subjected to in vitro amylase digestion, releasing 219 +/- 11 g oligosaccharides/kg total carbohydrate. During the gastric phase, bread proteins, which are converted into insoluble aggregated proteins during breadmaking, were emptied in various states of peptic digestion: undigested aggregated proteins and degraded proteins of intermediate and low molecular weight. The mean particle size of ground bread decreased progressively to the end of the gastric digestion (from 292 to 109 microm). The in vitro digestive system proved to be a useful tool for understanding the dynamic digestion of various food components held within the structure of a food matrix.

Properties and microstructure of thermo-pressed wheat gluten films: a comparison with cast films.

Wheat gluten films were prepared by thermo-pressing, and their mechanical properties were compared to those of cast films. The stress-strain relationship was established for films with various amounts of glycerol. Both relationships were quite different, revealing a different network organization. Thermo-pressed films presented higher stress values than cast films, but the effect of the glycerol amount was similar in both cases, an increase of the glycerol amount leading to a decrease of both films stress. The glycerol influence on the strain at break of thermo-pressed films was very limited, with strain values reaching a maximum around 200%. The role of disulfide bridges on themomoulded films mechanical properties was investigated, and it was shown that some rearrangements and a significative protein insolubilization occurred during the process. The effective flow porosity of the protein network for thermo-pressed films was estimated by water capillary rise measurements to about 7%. Scanning electron microscopy was used to obtain some information about the microstructure of both cast and thermo-pressed films.