Preliminary study of the influence of dextran on the precipitation of legumin from aqueous salt solutions

Ultrasound has been used to study the behaviour of dextran, legumin (11s globulin of the broad bean Vicia faba ) and their mixtures in aqueous salt solutions under different physico-chemical and thermodynamic conditions. The attenuation coefficient of the solutions was measured as a function of pH (3–10) and ionic strength (0, 0.025, 0.05 M NaCl). The attenuation in the dextran solutions was independent of pH and ionic strength, whereas that in the solutions containing legumin had a maximum value near to the isoelectric point of the legumin (∼φ 5). The magnitude of this maximum depended on the ionic strength of the solution. The cause of the observed attenuation was scattering of ultrasound by precipitated protein. The addition of dextran to solutions where the biopolymers are thermodynamically compatible decreases the attenuation, whereas its addition to solutions where the biopolymers are incompatible increases it. This is because protein precipitation is greater in incompatible systems.     

Interfacial behaviour of succinylated faba bean legumin at low ionic strength

Changes in the tensio-active properties of the main storage protein from faba beans (legumin) after succinylation were studied at a low salt concentration. Surface tension, surface dilatational properties of monolayers and emulsifying activity were measured at a ionic strength of I = 0.02. The results were compared with those at a high ionic strength of I = 0.3. Parameters of the Gibbs' adsorption isotherm indicate that the most surface-active derivatives are legumins with a moderate degree of succinylation (34% and 65%). The equilibrium surface pressure, π_e, inreased from 18.47 (native legumin) to 20.72 mN/m (65% succinylation). The critical association concentration, CAC, i. e., the subphase concentration at which the plateau of π_e was reached, decreased from 15.9·10^–6 to 7.12·10^–6 g/ml after 34% succinylation. The film forming properties differed from the adsorption behaviour. Only monolayers of the 65% succinylated legumin exhibited viscoelastic behaviour. By contrast, the emulsifying activity, EAI, reached the highest values for the 65% and 95% succinylated legumins. Low salt concentrations favour the adsorption of the native legumin and reduce the surface activity of succinylated legumin. Monolayer formation and especially the ability to form elastic networks seems to be diminished by the repulsive interaction of like-charged molecules. The emulsifying properties of the higher succinylated legumins are not influenced by the ionic strength whereas those of the native and low succinylated legumin are distinctly lower at I = 0.02. This result points to different adsorption and stabilizing processes during emulsion formation.     

Functional properties of the α-chain of a major legumin subunit of Pisum sativum legumin

The constituting polypeptides of a heavy pea legumin subunit were separated. The interfacial and emulsifying properties of the α-chains were studied and compared with those of the respective subunit and the native legumin molecules. The α-polypeptides show modified interface behaviour as compared to the legumin. Faster diffusion and higher adsorption barriers during the phase of rearrangement, higher interface coverage in the equilibrium state and improved emulsifying properties were measured.     

Restricted enzymatic hydrolysis of legumin of broad beans (Vicia faba L.) by trypsin in concentrated solutions. Control of hydrolysis process at the expense of change of enzyme-substrate ratio

Kinetics and mechanism of proteolysis of broad bean (Vicia faba L.) legumin by trypsin in concentrated solutions at different enzyme-substrate ratios (E/S = 1/40, 1/100, 1/200, 1/1000) were studied. By the method of HPLC it was established that during proteolysis process (E/S = 1/40) take place both protein content decrease in hydrolysate and change of its molecular mass (from 360 to 280 kD), which evidences for the mixed type of proteolysis, including cooperative and non-cooperative mechanisms. Decrease of E/S from 1/40 up to 1/100–1/1000 leads not only to unproportional drop in proteolysis rate, but also to decrease of the relative contribution of cooperative proteolysis which is, possibly, connected with presence in protein molecule of at least two groups of attack sites, differentiating by their affinity with enzyme. Change of enzyme-substrate ratio is accompanied also by change of subunit composition of product of non-cooperative proteolysis (‘modified protein’) and also of temperature and enthalpy of its denaturation.     

Electrostatic effects on β-lactoglobulin transitions during heat denaturation as studied by differential scanning calorimetry

This work comprises the study of the thermal treatment of β-lg and its denaturation as a function of pH and ionic strength followed by differential scanning calorimetry. The concentration of protein was 14 (w/v)% in order to study the behaviour of highly concentrated β-lg solutions during heating. The denaturation temperature of β-lg was dependent on both pH and ionic strength, meaning that electrostatic interactions between protein monomers in the native state were important for the denaturation of β-lg. The thermograms from the calorimetric measurements also revealed that the quarternary structure of β-lg at pH-values close to the isoelectric point was influenced by the presence of salt and the nature of the salt (NaCl, KI and LiI). Small exotherms emerged in the thermograms at the low temperature side of the denaturation temperature for β-lg. The presences of these exotherms are probably caused by restructuring of the quarternary structure of native β-lg prior to denaturation, due to dissociation into smaller entities and possible also formation of a liquid crystalline-like structure in the highly concentrated protein solution. The present study provides a contribution to the understanding of the importance of the electrostatic interactions between native β-lg molecules and how different salts and ionic strengths affect the denaturation properties of the protein in concentrated systems.     

Protein aggregation induced by phase separation in a pea proteins–sodium alginate–water ternary system

The physicochemical properties of a native, globular plant protein–linear anionic polysaccharide aqueous system at 20 °C were investigated in conditions where biopolymers carry a net negative charge (pH 7.2, 0.1 M NaCl). The pea proteins–sodium alginate mixtures showed a phase separation mainly by thermodynamic incompatibility, characterized at both the macroscopic and microscopic scale. Phase diagram was established and confocal laser scanning microscopy (CLSM) provided accurate data on the microstructure morphology of the system, regarding its phase behavior. In admixture, sodium alginate induced a protein aggregation, certainly by a local depletion of the polysaccharide. Protein aggregates were present in both single-phase and biphasic mixtures, while increasing the sodium alginate concentration provided larger and denser protein microdomains, leading to a non-equilibrium state. By phase separation, the pea protein aggregates entrapped a part of the sodium alginate phase, thus modifying the protein volume fraction. Along a tie-line, a phase inversion phenomenon was detected, from a sodium alginate to a pea proteins-continuous phase. Rheological properties of the mixed systems depended on the biopolymer composition and were modified with respect to individual components. The protein-enriched phase volume fraction modified the flow behavior to non-Newtonian for starting concentrated mixtures; a shear-thinning at high shear rates was evidenced, while mixtures with a particularly high sodium alginate concentration (>0.8 wt%) had an apparent yield stress for low shear rates. Mechanical spectra showed that both protein enrichment within microdomains in the presence of alginate and stronger protein–protein interaction with time impacted the viscoelastic properties (G′ and G″ moduli) of the whole mixture.     

The effect of shear rate on the viscosity of solutions of sodium carboxymethylcellulose and k-carrageenan

The apparent viscosities of aqueous solutions of sodium carboxymethylcellulose (NaCMC) and k-carrageenan have been measured over a range of shear rates from 14 to 1142 s^?1. Comparable measurements were also made on gum solutions to which glucose, sucrose, or glucose syrup had been added. For all the solutions the variation of relative viscosity with shear rate fits a power law equation. Addition of sugars to the most concentrated solutions of NaCMC has no effect on the non-Newtonian behaviour but addition of glucose syrup to the most dilute solutions of NaCMC decreased the non-Newtonian behaviour. However, the general pattern of behaviour showed close similarity with that of guar gum and locust bean gum. In the more concentrated glucose and sucrose solutions the non-Newtonian behaviour of k-carrageenan was increased appreciably. There was no comparable effect on k-carrageenan in glucose syrup solutions. This different behaviour of k-carrageenan is attributed to the development of solute-solute interactions.     

Thermodynamic approach to the comparative analysis of integral hydrophobicity of legumin and legumin-T in native and denaturated forms. 1. Broad bean legumin

The specific integral hydrophobicity of denatured forms of legumin and legumin-T from broad beans was evaluated through their hydration heat capacities. These were calculated as differences between partial heat capacities obtained from differential scanning microcalorimetry data and heat capacities in the gas phase, which were calculated by the use of the method of additive group contributions, taking into account the expansion of protein molecule in the gas phase. It is shown that there is no significant difference between the data for denatured forms of both proteins. The comparative evaluation of the hydration heat capacities of native forms of legumin and legumin-T was carried out. It is shown that the native legumin-T has a greater value of the hydration heat capacity and respectively is more hydrophobic than the native legumin. On the basis of the values of differences between the heat capacities in denatured and native states it is shown that the accessible hydrophobic surface of the native legumin-T related to a mean amino acid residue is 8-13 Ų greater than that of the native legumin.     

Selected physico-chemical properties of succinylated legumin from pea (Pisum sativum L.)

Selected physico-chemical properties of pea legumin before and after succinylation have been investigated using isoelectric focusing, PAGE, SDS-PAGE, hydrophobicity measurements, SE-HPLC and RP-HPLC. Exhaustive succinylation shifted the I.P. of legumin from 4.75 to 3.5. The stepwise dissociation of legumin by increasing succinylation has been confirmed both by means of PAGE in a nondenaturing system, and by SE-HPLC. The results of SDS-PAGE provided evidence for the exposure of α-polypeptide chains in the native legumin. High succinylation resulted in a decrease of the surface hydrophobicity (S₀) measured by both fluorescence probes (cis-parinaric acid and anilino-naphtalene sulfonic acid). RP-HPLC gave a response both to conformational changes and the introduced succinyl residues.     

Gelling properties of kefiran, a food-grade polysaccharide obtained from kefir grain

The physicochemical and gelling properties of kefiran, a water-soluble glucogalactan with probed health-promoting properties, were investigated. Gel permeation chromatograms revealed a single distribution of molecular weight corresponding to 10⁷ Da. Intrinsic viscosity of kefiran determined using Huggins extrapolations was 6.0 dl/g and using Kramer approximations was 5.95 dl/g.Kefiran has a Newtonian behaviour in diluted solutions, which becomes pseudoplastic at higher concentrations. Rheological behaviour of the solution before and after freeze drying was evaluated by small deformation oscillatory rheological measurements. The mechanical spectrum of solution corresponded to an entangled network behaviour. After freeze–thaw treatment of the solution, a rheological behaviour transition from a liquid-like system to a gel was observed. The storage modulus (G′) in cryogels was 35 times higher than the value obtained for the solution. Rheological characteristics of the cryogel were influenced by kefiran concentration. As the polymer concentration increased, higher number of interactions was evident for the increment in both moduli (G′ and G″). The behaviour of kefiran cryogels about 37 °C determines its ability to melt at mouth temperature. These results suggest that kefiran cryogels could be an interesting alternative for its application in food formulations.     

Role of Noncovalent Forces in Micellization Using Legumin from Vicia faba as a Study System

The seed storage protein, legumin, from Vicia faba interacted to form micelles and elaborate protein networks under varying conditions. Several molecular parameters correlated with observed micelle interaction parameters; these included thermal properties as indicators of protein stability and surface hydrophobicity as an assessment for potential hydrophobic interactions. The optimal pH for micelle formation ranged from 5.5 to 6.5, values at which electrostatic repulsions were minimal and surface hydrophobicites were adequate to allow hydrophobic interactions. The micelle response was affected by anions both in terms of concentration and type of anion. Finally, gradual denaturation of legumin with increasing urea concentrations had a negative impact on the micelle response.     

Some viscosity characteristics of faba bean protein isolates within a pH range relevant for foods

The rheological behaviour of concentrated aqueous dispersions of unmodified (FBPI I) and acetylated (FBPI I) faba bean (Vicia faba L. minor) protein isolates are investigated within a pH range of 4.0 up to 7.5. Besides the fact that equal concentrated dispersions of FBPI I show a much lower apparent viscosity η than those prepared with FBPI II, η and the flow behaviour index n depend also on the kind of preparation. Especially the direction of pH shifting and the mode of pH adjustment lead to marked differences of the rheological properties. In the case of FBPI II dispersions a maximum of η and n is shown at pH 5.5 to 6.5, seldom at pH 7.0, probably connected with a maximum of the hydrodynamic volume. Holding the aqueous dispersions for 24h causes mostly an increase of η and n.     

Expression of legumin and vicilin genes in pea mutants and the production of legumin in transgenic plants

Pea seeds contain two major storage proteins, legumin and vicilin, in proportions that are genetically and environmentally determined. They are synthesized from at least 40 genes and at least 10 different genetic loci. Mutant alleles at loci involved in starch synthesis, which result in perturbations in starch accumulation, also affect the expression of legumin genes, thereby influencing the legumin : vicilin ratio within the total seed protein. Examples of such alleles includer(starch‐branching enzyme) and rb(ADP‐glucose pyrophosphorylase( both of which result in a reduction in legumin synthesis; double mutants (rrb) show a particularly severe reduction in the amount of legumin. The effects of such mutations are specific to legumins. The amounts of vicilin are unaffected by mutations at rorrb. One of the consequences of the production of legumin from many genes is structural heterogeneity that is believed to preclude the purification of homogeneous legumin for crystallization and 3D‐structure determination. Expression of cloned legumin cDNA in E. coli can result in sequence homogeneity, butE. coli is unable to carry out the normal proteolytic processing of legumin precursors and consequently such material is different from that produced in pea seeds. This paper describes the high‐level synthesis, processing and assembly of pea legumin in transgenic wheat seeds, leading to the spontaneous in vitroformation of paracrystalline arrays of legumin, which may be attributed to the fact that the legumin consists of a single type of subunit. Such material might be used as a source of single‐sequence, processed and assembled pea legumin for structural investigation.     

Interfacial adsorption and shear flow properties of gum arabic-sodium caseinate mixtures

This work discusses rheological and physicochemical properties of sodium caseinate and gum arabic, in aqueous media (25 °C). All the systems showed low viscosity values and Newtonian behaviour in the shear rate range studied. From particle size distribution, a limited thermodynamic compatibility was observed since hydrocolloids mixtures showed particles size between the values from the systems alone. Interfacial tension measurements at equilibrium at the air-water interface, revealed that the binding affinity of arabic to the air water interface is very low compared to that of caseinate. The adsorption critical concentration could be deduced as a function of aqueous solutions composition. In competitive adsorption experiments involving simultaneous adsorption of the hydrocolloids from a bulk mixture, sodium caseinate displaced gum arabic from the interface at both polysaccharide concentrations, changing the adsorption critical concentration.     

Modulation of rheological properties by heat-induced aggregation of whey protein solution

Heat-induced protein aggregation at low protein concentrations generally leads to higher viscosities. We here report that aggregated protein can yield weaker gels than those from native protein at the same concentration. Aggregated protein was produced by heating a solution of whey protein isolate (WPI) at 3% and 9% w/w. The higher protein concentration resulted in a larger aggregate size and a higher intrinsic viscosity. The protein fraction in native WPI had the smallest size and the lowest intrinsic viscosity. The same trend was observed for the shear viscosity after concentrating the suspensions containing aggregates to around 15% w/w. Suspensions containing aggregates that were produced from a higher concentration possessed a higher viscosity. After reheating the concentrated suspensions, the suspension from the 9% w/w aggregate system produced the weakest gel, followed by the one from 3% w/w, while the native WPI yielded the strongest gel. Reactivity of the aggregates was also an important factor that influenced the resulting gel properties. We conclude that aggregation of whey protein solution is a feasible route to manipulate the gel strength of concentrated protein systems, without having to alter the concentration of the protein.     

Functional Properties of Flax Seed Mucilage

Flax seed (Linwn usitatissimum L.) mucilage was prepared by extraction of seeds with water followed by evaporation, precipitation with ethanol and freeze drying of extract. Proximate composition, solubility, foamability and moisture sorption characteristics were determined. The mucilage contained less carbohydrates, more minerals and more protein than commercial locust bean and guar gums. Its solubility, however, was higher than locust bean and guar gums, and lower than gum arabic. Flax seed mucilage exhibited good foam stability properties in aqueous solutions at 1.0% (w/v). Very diluted solutions exhibited Newtonian-like behavior while shear thinning was shown at concentrations above 0.2% (w/v). The viscosity was maximum at a pH range 6.0–8.0 and it was reduced in solutions containing NaCl.     

小麦面筋蛋白分子聚集态对脱酰胺程度的影响

本研究以从稀到浓的小麦面筋蛋白溶液为对象,对其进行湿热脱酰胺处理,分析了湿热脱酰胺改性过程中当蛋白浓度从极稀到亚浓变化时,小麦面筋蛋白分子结构和聚集态变化对脱酰胺程度的影响。实验结果表明:小麦面筋蛋白脱酰胺程度、水解度和Zeta电位随着蛋白原始聚集态的增大而显著降低,且脱酰胺程度和水解度随小麦面筋蛋白原始聚集态变化呈现线性相关性。小麦面筋蛋白分子内作用力随着蛋白原始聚集态的增强而显著增大,红移程度则减小。小麦面筋蛋白分子内部非共价键包括疏水键和氢键在蛋白分子中起主导作用,二硫键作用微小。以上结果说明小麦面筋蛋白聚集形态与湿热有机弱酸脱酰胺速率具有较强的相互关系,蛋白原始的聚集形态是决定分子结构伸展程度和脱酰胺改变趋势的最重要因素。     

Quantitative studies on the cotyledonary proteins in the genus Pisum

Forty-five lines of peas including primitive or wild forms, field peas, and round and wrinkled garden peas, were grown under uniform conditions and the seeds examined for variation in protein characteristics likely to influence nutritional value. The characters measured were crude protein, extractable protein, globulins and albumins, the percentages of legumin, total sulphur and protein sulphur, carbon: nitrogen and nitrogen: sulphur ratios. The extractable protein was separated quantitatively into an albumin fraction (20–35%) and a globulin fraction (legumin and vicilin). Without exception lines high in albumin content were low in legumin content (correlation coefficient r= ?0.757). As both the albumin fraction and legumin are rich in sulphur amino acids, this negative correlation has important implications for attempts through plant breeding to improve the nutritional quality of legume seed proteins, by increasing the sulphur amino acid content. Total sulphur was not correlated with any other protein character.     

Functional properties of the system gelatine/Vicia faba protein in dependence on the acetylation degree of the Vicia faba protein

By acetylation of Vicia faba protein it becomes in aqueous solution compatible with gelatine up to a substitution degree of about 50%. The swellability and brittleness of foils and the viscosity of aqueous solution of Vicia faba protein are increased by its acetylation and these changes can be observed in mixtures of gelatine with acetylated Vicia faba protein, too. A heat denaturation of aqueous solutions of acetylated Vicia faba protein or mixtures with gelatine leads to an increase of the viscosity; all these solutions show pseudo-plastic flow behaviour.     

Linear viscoelasticity of carob protein isolate/locust bean gum blends

Linear viscoelastic properties of locust bean gum (LBG), carob protein isolate (CPI) and LBG–CPI aqueous dispersions were studied at different concentration and pH values. LBG and CPI dispersions show marked different linear viscoelasticity behaviour. The former system exhibits a fluid-like behaviour for all the concentrations considered (2–4 wt.%), whereas the later shows gel-like mechanical spectra. An increase in linear viscoelastic functions, G′ and G″, takes place as protein concentration becomes higher, as a consequence of a strengthening of the gel network. CPI dispersions were affected by the pH value, in agreement with the CPI solubility profile. The behaviour observed for CPI–LBG dispersions shows an apparent evolution with time, suggesting occurrence of thermodynamic incompatibility between both biopolymers that finally leads to a segregative separation of LBG-rich and CPI-rich phases. Both microstructure and viscoelastic properties of the final LBG–CPI systems are highly dependent on pH value.