The rôle of non-covalent forces in micelle formation by vicilin from Vicia faba. The effect of pH variations on protein interactions

The influence of environmental pH on the capacity of vicilin from fababeans to self-associate into a micelle arrangement was investigated. Various types of micelle-based protein interactions were identified; these responses were related to certain vicilin conformational parameters, specifically, thermal stability and surface hydrophobicity (So). The most extensive micelle responses were observed from pH 6·0 to 6·8; a gradual deterioration in the degree of protein self-association occurred with pH increases from 6·8 to 8·0. These results corresponded with significant decreases in vicilin So, i.e. from 296 at pH 6·5 to 158 at pH 8·0. Thermal parameters over this range reflected minimal conformational disturbances. The observed importance of molecular surface hydrophobicity was supportive of the original premise that micelle structures are products of hydrophobic associative forces.     

The rôle of non-covalent forces in micelle formation by Vicilin from Vicia faba. II. The effect of stabilizing and destabilizing anions on protein interactions

The effect of a variety of stabilizing and destabilizing anions on the capacity of vicilin from fababeans to self-associate into a micelle arrangement was examined; these observations were related to protein conformational parameters, specifically, thermal stability and surface hydrophobicity (So). Extensive vicilin interactions occurred with moderately stabilizing environments (denaturation temperature (Td) values of 87·7 to 92·5°C) in which the protein So values were not extreme (240 ± 40). In contrast, highly stabilized molecules (Td > 95°C) with low So values (e.g. 137) or destabilized molecules (Td < 80°C) with high So values (e.g. 572) did not self-associate into a micelle arrangement. From these relationships, the micelle response appeared to be dependent on a critical balance of non-covalent forces operative at several levels. A specific intramolecular hydrophilic-hydrophobic balance was essential for protein association; similarly, intermolecular attractive hydrophobic forces had to be dominant over electrostatic repulsive forces for micelle formation to occur.     

Legumin-T from faba bean legumin: Isolation,partial characterization and surface functional properties

Legumin-T was prepared by limited tryptic hydrolysis of legumin from faba beans (Vicia faba L.) and was chromatographically purified. It is composed of two principle subunits consisting each of an intact β-polypeptide chain and a nicked α-chain of legumin besides minor polypeptides. The molecular mass was (250 ± 11) kDa estimated by gel chromatography. Amino acid analysis showed that the transformation of legumin to legumin-T resulted in a loss of polar amino acids and an increase of non-polar hydrophobic amino acids. The time-dependent surface tension and the surface compressions isotherms showed a rather similar behavior of legumin and legumin-T. The calculated ratio of the surface areas A/A₀ revealed, however, difference between both proteins (legumin: A/A₀ = 1, legumin-T: A/A₀ < 1) pointing to an irreversible desorption of legumin-T from the surface due to compression. The emulsifying activity of legumin-T was higher than that of the parent legumin.     

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.     

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.     

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.     

Heat Induced Gelation of Pea (Pisum sativum) Mixed Globulins, Vicilin and Legumin

Mixed globulins (MG) were extracted from ground dry peas (Pisum sativum, B-160) with 0.5M NaCl, 50 mM potassium phosphate, pH 7.2, and isolated by precipitation at pH 4.5. Crude vicilin and legumin were fractionated from the MG by dialysis against 0.2M NaCl, pH 4.8, and centrifugation, then further purified using DEAE-cellulose chromatography. Conditions for maximum gel hardness of heat induced MG gel, as determined with an Instron Universal Testing Machine, were heating for 20 min at pH 7.1 at 87°C. Purified vicilin, but not legumin, formed heat induced gels. The relationship was linear between protein (globulin) concentration and log gel hardness. At all protein concentrations studied, as proportion of legumin decreased, gel hardness increased.     

Thermal aggregation of globulin from an indigenous Chinese legume,Phaseolus angularis (red bean)

The thermal aggregation behavior of red bean (Phaseolus angularis) globulin (RBG) was studied at ≈1% (w/v) protein concentration in 0.01 M phosphate buffer, pH 7.4. The percentage of protein precipitated was affected by heating temperature, heating time and salt concentration. The influences of several salts of the chaotropic series and protein structure-modifying agents on thermal coagulation of RBG were also investigated. The effects of chaotropic salts did not follow the lyotropic series of anions. Sodium dodecyl sulfate caused a more pronounced reduction in heat-induced aggregation of RBG than did dithiothreitol, while N-ethylmaleimide did not affect aggregation until after a long heating period. Differential scanning calorimetric (DSC) data showed that heat aggregation of RBG was preceded by thermal denaturation. SDS-PAGE showed that heating led to the disappearance of some protein bands, and the basic polypeptide of 11S globulin (legumin) was not found in the buffer-soluble aggregates. Heating caused increases of surface hydrophobicity, again suggesting protein unfolding prior to aggregate formation. The buffer-insoluble aggregates did not show any DSC response, indicating extensive denaturation, and had a lower surface hydrophobicity and higher disulfide content than the buffer-soluble aggregates. The data suggest that electrostatic and hydrophobic interactions may play an important role in thermal aggregation of RBG, with disulfide bonds playing a limited role.     

Differential scanning calorimetric study of meals and constituents of some food grain Legumes

Two thermal transitions were observed in soya bean meal by differential scanning calorimetry (DSC) and on the basis of previous data were ascribed to the denaturation of the 11S globulin, glycinin, and the 7S globulin, β-conglycinin. Three DSC transitions were apparent in Vicia faba meal; one was associated with starch gelatinisation, and was absent from thermograms of soya bean because the latter stores oil rather than starch. The remaining two transitions were identified with the 11S globulin, legumin, and a 7S globulin, vicilin. The denaturation parameters of legumin and glycinin were very similar, in contrast to those of vicilin and β-conglycinin. From this it was concluded that legumin and glycinin probably represent homologous proteins, whereas the two 7S proteins possess intrinsically different structures to one another. Cowpeas (Vigna unguiculata) and dry beans (Phaseolus vulgaris) afforded two major transitions which were assigned to 7S globulins and starch. From its DSC profile, the 7S globulins of cowpea appeared heterogeneous, but nevertheless possessed denaturation characteristics similar to those of vicilin of V. faba. In contrast, the thermal behaviour of the 7S globulin of dry beans (glyco-protein II) was distinct from that of any of the other 7S globulins investigated. Three protein transitions were observed in the three lupin species examined. As in the case of soya bean, no starch transition was present. On the basis of the correspondence in transition temperature, one of the protein transitions was assigned to the 11S globulin component. Overall, this study indicates the potential of DSC as a means for obtaining data on seed protein homology from whole meals rather than the extracted proteins.     

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.     

Molecular Forces in Thermal Association-Dissociation and Gelation of Legumin from Broad Beans

A 0.5% legumin solution when heated showed neither dissociation into constituent subunits of the legumin nor gel formation, but association of the legumin molecules to form soluble aggregates and network-like structures occurred. Sulfhydryl content of the protein were shown to possibly be responsible for the depression of dissociation. In the 12.5% legumin solution, gelling process and gel properties were affected by reagents such as NaSCN, N-ethylmaleimide and 2-mercaptoethanol. Results indicated that hydrophobic interactions and disulfide bonding helped to enhance the gelling process, where contributions of those forces to the rate of soluble aggregate formation and their junctures were notable.     

Amino acid composition of storage proteins of a promising chickpea (Cicer arietinum L) cultivar

The amino acid compositions of flours made from the cotyledons and from the whole seeds of a disease-resistant, stable and high-yielding cultivar of chickpea (Cicer arietinum L) cv H75–35, known locally as Gaurav, have been analysed. These, together with similar analyses of the albumin, globulin, legumin and vicilin protein fractions, have been compared with those of other legumes. Seed protein content was 19·8 % with globulin constituting 62·4 % of the total seed protein. The ratios of albumin to globulin and legumin to vicilin were 1:4 and 6:1, respectively. The proportion of basic amino acids in the albumin was low whereas the reverse was true in the globulin. The legumin fraction seems to be superior in terms of total essential amino acids to those from other sources. Sulphur amino acids were the most limiting, followed by tryptophan or threonine depending on the fraction. However, the ratio of methionine to cystine was high (2·76:1). The extent of the sulphur amino acid deficiency was assessed, and possible approaches for its improvement are outlined.     

Production of ace inhibitory peptides by digestion of chickpea legumin with alcalase

Short peptides from different sources have proved to be very efficient inhibitors of the angiotensin I-converting enzyme, an enzyme with a major role in the regulation of blood pressure. These peptides are of therapeutic value, so that the possibility of obtaining such peptides by treatment of chickpea legumin with the protease alcalase has been explored. Legumin is the main storage protein in chickpea seeds. Treatment of legumin with alcalase yielded a hydrolysate that inhibited the angiotensin I-converting enzyme with an IC₅₀ of 0.18 mg/ml. Fractionation of this hydrolysate by reverse phase chromatography afforded six inhibitory peptides with IC₅₀ values ranging from 0.011 to 0.021 mg/ml. All these peptides contain the amino acid methionine and are also rich in other hydrophobic amino acids. These results demonstrate that hydrolysates of chickpea legumin obtained by treatment with alcalase are a good source of peptides with angiotensin I-converting enzyme inhibitory activity.     

Protein–protein interactions of a whey–pea protein co-precipitate

The aim of this study was to investigate the mechanisms behind protein–protein interactions in a co-precipitate of whey protein isolate (WPI) and pea protein isolate (PPI). A co-precipitate and blend, consisting of 80% WPI and 20% PPI, were compared. Covalent disulphide interactions were studied by blocking of free thiols with N-Ethylmaleimide (NEM), while electrostatic interactions were studied in systems with 0.5 m NaCl and hydrophobic interactions with 0.2% SDS. Protein solubility, stability and secondary, tertiary and quaternary protein structures were analysed. Co-precipitation did not introduce different protein–protein interactions than the direct blending of proteins. SDS affected solubility (P < 0.05), secondary and tertiary structure. However, the effects of NEM and NaCl were significant greater (P < 0.05) on the same parameters and thermal stability, especially when combined (P < 0.01). Thus, the protein–protein interactions in a whey–pea co-precipitate and protein blend consisted of disulphide bonds and electrostatic interactions.     

Kappa-casein interactions in the suspension of the two major calcium-sensitive human beta-caseins

The possible effects of both the beta-casein (beta-CN) phosphorylation level and the kappa-CN glycosylation level on micelle formation were studied using the doubly-phosphorylated form (beta-CN-2P) and the quadruply-phosphorylated form (beta-CN-4P) of human beta-CN, along with bovine kappa-CN to compare with previous studies using the more highly glycosylated human kappa-CN. Addition of bovine kappa-CN to human beta-CN-2P, beta-CN-4P, or a 1/1 (wt/wt) mixture of the two was at kappa/beta molar ratios from 0.0 to approximately 0.6 and micelles were reconstituted by addition of Ca+2 either directly at 37 degrees C for determination of the fraction suspended or at an initial temperature of 4 degrees that was gradually increased to 37 degrees C with the change in particle size monitored by turbidity measurements. Analysis of the data indicates that the 4P form requires more kappa-CN for stabilization than the 2P form but that the mixture of the two is more like the 4P form in that lateral kappa-kappa interactions may enhance beta-kappa interactions and micelle formation. Above a kappa/beta molar ratio of about 0.2, the caseins were fully suspended into reconstituted micelles. However, micelle size decreased at a higher ratio, indicating that the kappa-CN probably occupies a surface position and may regulate micelle size by its relative abundance. A comparison with published results suggests that the higher glycosylation level of human kappa-CN may protect a larger surface area and result in smaller micelles. Changes in reconstituted micelle size with pH indicate that positively charged groups in the kappa-CN may interact with the negatively charged phosphate esters in the beta-CN moieties in addition to kappa-beta hydrophobic interactions.     

Restricted proteolysis of legumin of broad beans: Effect on thermodynamic properties of aqueous solutions and interaction with ficoll

Thermodynamic properties of aqueous solutions of both native and modified legumin of broad beans (Vicia faba L.) have been examined. A restricted trypsin-induced proteolysis was used to modify protein structure. Evaluation of protein affinity to ficoll in aqueous solutions showed that modified protein possessed higher hydrophilicity. Thermodynamic properties of diluted solutions were used to predict the phase behaviour in concentrated systems containing protein and ficoll. At specific concentrations of native legumin, the system can separate in two phases, whereas in the case of modified protein the single-phase behaviour of the system was predicted for any concentrations of both components. The experimental data obtained in concentrated systems confirmed predictions of thermodynamic analysis of diluted solutions.     

Use of casein micelles to improve the solubility of hydrophobic pea proteins in aqueous solutions via low-temperature homogenization

The dairy industry struggles to maintain consumer attention in the midst of declining fluid milk sales. Current trends create an opportunity to incorporate plant-based proteins with milk to produce a high-protein, multisourced, functional food product. Plant-based proteins, such as those in peas, can be challenging to use in food systems because of their low solubility and undesirable off-flavors. Casein micelles have unique structural properties that allow for interactions with small ions and larger macromolecules that aid in their noteworthy ability as a nanovehicle for hydrophobic compounds. The objective of this study was to use the inherent structure of the casein micelle along with common dairy processing equipment to create a stable colloidal dispersion of casein micelles with pea protein to improve its solubility in aqueous solutions. We created 3 blends with varying ratios of casein-to-pea protein (90:10, 80:20, 50:50). We subjected the mixtures to 3 cycles of homogenization using a bench-top GEA 2-stage homogenizer at 27,580 kPa maintained at 4°C, followed by pasteurization at 63°C for 30 min. The resulting blends were homogeneous liquids with increased stability due to the lack of protein precipitation. Further protein analysis by HPLC and AA sequencing revealed that vicilin, an insoluble storage protein, was the main pea protein incorporated within the casein micelle structure. These results supported our hypothesis that low-temperature homogenization can successfully be used to create a colloidal dispersion with increased stability, in which insoluble plant-based proteins may be incorporated with casein micelles in an aqueous solution. Additionally, 3-dimensional microscope images of the blends indicated a noticeable difference between the surface roughness upon addition of pea protein to the casein micelle matrix. This research highlights a promising application for other plant-based proteins to be used within the dairy industry to help drive future product innovation while also meeting current processing conditions and consumer demands.     

Kinetics of Protein Extraction in Reverse Micelle

Sodium bis (2-ethylhexyl) sulfosuccinate (AOT)-sodium dodecyl sulfate (SDS)/isooctane-octanol reverse micelle extraction was tested an efficient and effective approach to separate peanut protein from full-fat peanut powder. Here, important kinetic factors including pH, ion strength, and temperature were studied during reverse micelle backward extraction. The extraction conditions were obtained by response surface experiments as follows: pH 7.5, ion concentration 1.1 mol/L at temperature 35°C. Under these optimum extraction conditions, the extraction rate of protein reached 79.03%. A model on the kinetic partitioning of peanut protein was also developed. The backward extraction in this reverse micelle system was controlled by interfacial resistance instead of diffusion resistance in reverse micelle and aqueous phase with the total mass transfer rate of 0.8×10^?5 m³·s^?1. A two-film theory may be the mechanism for flat interface. Results of mass transfer process are helpful for creating an reverse micelle extraction process, and used for purification of peanut proteins, promoting the development of food industry.     

Inhibition of Bacterial Pathogens in Medium and on Spinach Leaf Surfaces using Plant‐Derived Antimicrobials Loaded in Surfactant Micelles

Encapsulation of hydrophobic plant essential oil components (EOC) into surfactant micelles can assist the decontamination of fresh produce surfaces from bacterial pathogens during postharvest washing. Loading of eugenol and carvacrol into surfactant micelles of polysorbate 20 (Tween 20), Surfynol® 485W, sodium dodecyl sulfate (SDS), and CytoGuard® LA 20 (CG20) was determined by identification of the EOC/surfactant‐specific maximum additive concentration (MAC). Rheological behavior of dilute EOC‐containing micelles was then tested to determine micelle tolerance to shearing. Antimicrobial efficacy of EOC micelles against Escherichia coli O157:H7 and Salmonella enterica serotype Saintpaul was first evaluated by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Pathogen‐inoculated spinach was treated with eugenol‐containing micelles applied via spraying or immersion methods. SDS micelles produced the highest MACs for EOCs, while Tween 20 loaded the lowest amount of EOCs. Micelles demonstrated Newtonian behavior in response to shearing. SDS and CG20‐derived micelles containing EOCs produced the lowest MICs and MBCs for pathogens. E. coli O157:H7 and S. Saintpaul were reduced on spinach surfaces by application of eugenol micelles, though no differences in numbers of surviving pathogens were observed when methods of antimicrobial micelle application (spraying, immersion) was compared (P ≥ 0.05). Data suggest eugenol in SDS and CG20 micelles may be useful for produce surface decontamination from bacterial pathogens during postharvest washing.