Interfacial shear rheology of aged and heat-treated beta-lactoglobulin films: displacement by nonionic surfactant

Interfacial shear rheology of adsorbed beta-lactoglobulin films (bulk protein concentration 10(-)(3) wt %) has been studied over the temperature range 20-90 degrees C using a two-dimensional Couette-type viscometer. Effects of the type of interface (air-water, triolein-water, and n-dodecane-water), the pH (2.0, 5.6, 6.0, 7.0, and 9.0), and the extent of the heat treatment have been assessed via measurements of changes in the apparent interfacial shear viscosity and elasticity before and after the addition of increasing amounts of nonionic surfactant Tween 20 (polyoxyethylene sorbitan monolaurate). The highest interfacial viscosities were obtained at the n-dodecane-water interface and the lowest at the air-water interface. Competitive displacement of protein from the interface by Tween 20 was easier at the air-water and n-dodecane-water interfaces as compared to the triolein-water interface. The surface shear viscosity was higher and the displacement by Tween 20 more difficult as the isoelectric point of the protein was approached, which is in agreement with the presence of a more strongly cross-linked protein network at the interface. The effect of heat treatment was dependent on the pH of the aqueous solution. No simple relationship between the surface rheological characteristics and the ease of displacement by Tween 20 could be inferred.     

Adsorption of whey protein isolate at the oil-water interface as a function of processing conditions: a rheokinetic study

In this paper we present surface dynamic properties (interfacial tension and surface dilational properties) of a whey protein isolate with a high content of beta-lactoglobulin (WPI) adsorbed on the oil-water interface as a function of adsorption time. The experiments were performed at constant temperature (20 degrees C), pH (5), and ionic strength (0.05 M). The surface rheological parameters and the interfacial tension were measured as a function of WPI concentration (ranging from 1 x 10(-)(1) to 1 x 10(-)(5)% w/w) and different processing factors (effect of convection and heat treatment). We found that the interfacial pressure, pi, and surface dilational modulus, E, increase and the phase angle, phi, decreases with time, theta, which should be associated with WPI adsorption. These phenomena have been related to diffusion of the protein toward the interface (at short adsorption time) and to the protein unfolding and/or protein-protein interactions (at long-term adsorption) as a function of protein concentration in solution and processing conditions.     

Charge modifications to improve the emulsifying properties of whey protein isolate

Whey protein isolate was modified by ethylene diamine in order to shift its isoelectric point to an alkaline pH. The extent of the modification was studied using SDS-PAGE and MALDI-TOF mass spectrometry. The modified whey proteins were used as an emulsifier to stabilize oil-in-water emulsions at acidic and neutral pH ranges, and their emulsifying properties were compared with that of the unmodified whey proteins and with the previously studied ethylene diamine modified sodium caseinate. The emulsifying activity of the modified whey proteins was similar to that of the unmodified ones, but the stability of an emulsion at pH 5 was significantly improved after the modification. Charge and coverage of droplet surface and the displacement of the interfacial proteins by surfactant Tween 20 were further studied as a function of pH. As compared with the unmodified whey proteins, the modified ones were proven to cover the interface more efficiently with extensive surface charge at pH 5, although the interfacial layer was less resistant to the surfactant displacement.     

Effect of conductivity control on the separation of whey proteins by bipolar membrane electroacidification

Since the limiting factor of the bipolar membrane electroacidification (BMEA) process at 20% WPI (whey protein isolate) was hypothesized to be the lack of mobile ion inherent to the protein solution at pH 5.0, the aim of the present work is to study the effect of the conductivity control on the precipitation behavior of whey protein. BMEA performances were evaluated by measuring electrodialytic parameters, protein kinetic precipitation, molecular profiles, and isolate chemical composition and purity. The highest protein precipitation with 10% WPI solution was obtained at pH 4.6 and at a conductivity level of 200 microS/cm maintained with many 0.4-mL additions of 1.0 M KCl (200 microS[+]), with a 46% precipitation of the total protein, beta-lg composing the main part of the precipitated protein. With a 20% WPI solution, it was possible to reach pH 4.65 with conductivity control at 350 microS/cm. However, the 27% protein precipitation was still low. The changes in viscosity as pH decreases observed at 20% WPI would decreased the final precipitation rate of beta-lg, since the viscosity of the 20% WPI dispersion was very different.     

Light-scattering study of the structure of aggregates and gels formed by heat-denatured whey protein isolate and beta-lactoglobulin at neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.     

Heat-induced changes in the ultrasonic properties of whey proteins

The physical aggregation of commercial whey protein isolate (WPI) and purified beta-lactoglobulin was studied by ultrasound spectroscopy. Protein samples were dialyzed to achieve constant ionic strength backgrounds of 0.01 and 0.1 NaCl, and gelation was induced in situ at constant temperatures (from 50 to 75 degrees C) or with a temperature ramp from 20 to 85 degrees C. Changes in the ultrasonic properties were shown in the early stages of heating, at temperatures below those reported for protein denaturation. During heating, the relative ultrasound velocity (defined as the difference between sample velocity and reference velocity) decreased continuously with temperature, indicating a rearrangement of the hydration layer of the protein and an increase in compressibility of the protein shell. At temperatures <50 degrees C the ultrasonic attenuation decreased, and <65 degrees C both velocity and attenuation differentials showed increasing values. A sharp decrease in the relative velocity and an increase in the attenuation at 70 degrees C were indications of "classical" protein denaturation and the formation of a gel network. Values of attenuation were significantly different between samples prepared with 0.01 and 0.1 M NaCl, although no difference was shown in the overall ultrasonic behavior. WPI and beta-lactoglobulin showed similar ultrasonic properties during heating, but some differences were noted in the values of attenuation of WPI solutions, which may relate to a less homogeneous distribution of aggregates caused by the presence of alpha-lactalbumin and other minor proteins in WPI.     

Heat-induced soy-whey proteins interactions: formation of soluble and insoluble protein complexes

The aggregation behavior during heating of a solution containing soy protein and whey protein isolate (WPI) was studied using rheology, confocal microscopy, gel filtration, and electrophoresis. Soy/WPI mixtures formed gels at 6% total protein concentration with a high elastic modulus (G') and no apparent phase separation. The ratio of soy to WPI was fundamental in determining the type of network formed. Systems containing a high soy to WPI ratio (>70% soy protein) showed a different evolution of the elastic modulus during heat treatment, with two apparent stages of network development. Whey proteins formed disulfide bridges with soy proteins during heating, and at low ratios of soy/WPI, the aggregates seemed to be predominantly formed by 7S, the basic subunits of 11S and beta-lactoglobulin. Size exclusion chromatography indicated the presence of high molecular weight soluble complexes in mixtures containing high soy/WPI ratios. Results presented are the first evidence of interactions between soy proteins and whey proteins and show the potential for the creation of a new group of functional ingredients.     

Protein-peptide interactions in mixtures of whey peptides and whey proteins

The effects of several conditions on the amounts and compositions of aggregates formed in mixtures of whey protein hydrolysate, made with Bacillus licheniformis protease, and whey protein isolate were investigated using response surface methodology. Next, the peptides present in the aggregates were separated from the intact protein and identified with liquid chromatography-mass spectrometry. Increasing both temperature and ionic strength increased the amounts of both intact protein and peptides in the aggregates. There was an optimal amount of added intact WPI that could aggregate with peptides, yielding a maximal amount of aggregated material in which the peptide/protein molar ratio was around 6. Under all conditions applied, the same peptides were observed in the protein-peptide aggregates formed. The dominant peptides were beta-lg AB [f1-45], beta-lg AB [f90-108], and alpha-la [f50-113]. It was hypothesized that peptides could form a kind of glue network that can include beta-lactoglobulin via hydrophobic interactions at the hydrophobic binding sites at the surface of the protein.     

Influence of competitive adsorption on flocculation and rheology of high-pressure-treated milk protein-stabilized emulsions

The effect of high-pressure treatment (HPT) on the droplet-size distribution and small-deformation rheology of oil-in-water emulsions containing beta-lactoglobulin and a nonionic surfactant or sodium caseinate has been investigated at neutral pH. Addition of Tween 20 (polyoxyethylenesorbitan monolaurate) to a beta-lactoglobulin-stabilized emulsion results in competitive displacement of the adsorbed globular protein film and, following HPT, the formation of a less flocculated emulsion. The age of the beta-lactoglobulin-stabilized emulsion prior to addition of sodium caseinate influences the competitive adsorption behavior. The strengthening of the beta-lactoglobulin layer with time makes it more resistant to disruption by sodium caseinate. The level of pressure-induced flocculation of beta-lactoglobulin-coated oil droplets depends on the intensity of processing conditions and on the degree of interfacial displacement. In contrast, beta-lactoglobulin added after emulsification appears to show little evidence of competitive adsorption behavior at the caseinate oil-water interface. Changes in the rheological properties of these latter systems following HPT can be attributed to pressure-induced denaturation and gelation of beta-lactoglobulin in the continuous phase of the emulsion.     

Formulation engineering can improve the interfacial and foaming properties of soy globulins

In this contribution, we have analyzed the effect of different strategies, such as change of pH (5 or 7) or ionic strength (at 0.05 and 0.5 M), and addition of sucrose (at 1 M) and Tween 20 (at 1 x 10(-4) M) on interfacial characteristics (adsorption, structure, dynamics of adsorption, and surface dilatational properties) and foam properties (foam capacity and stability) of soy globulins (7S and 11S at 0.1 wt %). We have observed that (1) the adsorption (presence of a lag period, diffusion, and penetration at the air-water interface) of soy globulins depends on the modification in the 11S/7S ratio and on the level of association/dissociation of these proteins by varying the pH and ionic strength (I), the effect of sucrose on the unfolding of the protein, and the competitive adsorption between protein and Tween 20 in the aqueous phase. The rate of adsorption increases at pH 7, at high ionic strength, and in the presence of sucrose. (2) The surface dilatational properties reflect the fact that soy globulin adsorbed films exhibit viscoelastic behavior but do not have the capacity to form a gel-like elastic film. The surface dilatational modulus increases at pH 7 and at high ionic strength but decreases with the addition of sucrose or Tween 20 into the aqueous phase. (3) The rate of adsorption and surface dilatational properties (surface dilatational modulus and phase angle) during adsorption at the air-water interface plays an important role in the formation of foams generated from aqueous solutions of soy globulins. However, the dynamic surface pressure and dilatational modulus are not enough to explain the stability of the foam.     

Impact of whey protein emulsifiers on the oxidative stability of salmon oil-in-water emulsions

To obtain a better understanding of how the interfacial region of emulsion droplets influences lipid oxidation, the oxidative stability of salmon oil-in-water emulsions stabilized by whey protein isolate (WPI), sweet whey (SW), beta-lactoglobulin (beta-Lg), or alpha-lactalbumin (alpha-La) was evaluated. Studies on the influence of pH on lipid oxidation in WPI-stabilized emulsions showed that formation of lipid hydroperoxides and headspace propanal was much lower at pH values below the protein's isoelectric point (pI), at which the emulsion droplets were positively charged, compared to that at pH values above the pI, at which the emulsion droplets were negatively charged. This effect was likely due to the ability of positively charged emulsion droplets to repel cationic iron. In a comparison of lipid oxidation rates of WPI-, SW-, beta-Lg-, and alpha-La-stabilized emulsions at pH 3, the oxidative stability was in the order of beta-Lg > or = SW > alpha-La > or = WPI. The result indicated that it was possible to engineer emulsions with greater oxidative stability by using proteins as emulsifier, thereby reducing or eliminating the need for exogenous food antioxidants.     

Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high-pressure homogenization

The conformational changes of whey proteins upon adsorption at the soy oil/water interface were investigated using Fourier transform infrared (FT-IR) spectroscopy. Significant changes were observed in the bands assigned to beta-sheets and alpha-helix structures following the adsorption of proteins at the oil/water interface. The remaining interfacial proteins after Tween 20 desorption revealed small changes in beta-sheet and alpha-helical structures, whereas in the desorbed whey proteins the unordered structures largely increased, and beta-sheet structures almost disappeared. These FT-IR results provide important knowledge about the conformational modifications in whey proteins occurring upon adsorption at the oil/water interface. Finally, specific conformational changes are necessary to stabilize emulsions: adsorption-induced unfolding, increase in alpha-helical structures to establish interactions with the oil phase, and aggregation between adsorbed whey proteins to form protein membranes. Moreover, the structural changes in whey protein adsorbed at the oil/water interface under high-pressure homogenization are irreversible.     

Lateral phase separation in adsorbed binary protein films at the air-water interface.

Lateral phase separation in two-dimensional mixed films of soy 11S/beta-casein, acidic subunits of soy 11 (AS11S)/beta-casein, and alpha-lactalbumin/beta-casein adsorbed at the air-water interface has been studied using an epifluorescence microscopy method. No distinct lateral phase separation was observed in the mixed protein films when they were examined after 24 h of adsorption from the bulk phase. However, when the soy 11S/beta-casein and AS11S/beta-casein films were aged at the air-water interface for 96 h, phase-separated regions of the constituent proteins were evident, indicating that the phase separation process was kinetically limited by a viscosity barrier against lateral diffusion. In these films, beta-casein always formed the continuous phase and the other globular protein the dispersed phase. The morphology of the dispersed patches was affected by the protein composition in the film. In contrast with soy 11S/beta-casein and AS11S/beta-casein films, no lateral phase separation was observed in the alpha-lactalbumin/beta-casein film at both low and high concentration ratios in the film. The results of these studies proved that proteins in adsorbed binary films exhibit limited miscibility, and the deviation of competitive adsorption behavior of proteins at the air-water interface from that predicted by the ideal Langmuir model (Razumovsky, L.; Damodaran, S. J. Agric. Food Chem. 2001, 49, 3080-3086) is in fact due to thermodynamic incompatibility of mixing of the proteins in the binary film. It is hypothesized that phase separation in adsorbed mixed protein films at the air-water and possibly oil-water interfaces of foams and emulsions might be a source of instability in these dispersed systems.     

Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.     

Dynamic interfacial rheology as a tool for the characterization of whey protein isolates gelation at the oil-water interface.

Heat-induced interfacial aggregation of a whey protein isolate (WPI), previously adsorbed at the oil-water interface, was studied by interfacial dynamic characteristics coupled with microscopic observation and image analysis of the drop after heat treatment. The experiments were carried out at temperatures ranging from 20 to 80 degrees C with different thermal regimes. During the heating period, competition exists between the effect of temperature on the film fluidity and the increase in mechanical properties associated with the interfacial gelation process. During the isothermal treatment, the surface dilational modulus, E, increases, and the phase angle, delta, decreases with time to a plateau value. The frequency dependence of E and delta is characteristic of viscoelastic films with increasing delta and decreasing E at lower frequencies. The effects of heat treatment depend on the conditions at which the gelation process takes place. Microscopic observation of gelled films gives complementary information on the effect of heat treatment on WPI adsorbed films.     

Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate

The effects of heat-induced denaturation and subsequent aggregation of whey protein isolate (WPI) solutions on the rate of enzymatic hydrolysis was investigated. Both heated (60 °C, 15 min; 65 °C, 5 and 15 min; 70 °C, 5 and 15 min, 75 °C, 5 and 15 min; 80 °C, 10 min) and unheated WPI solutions (100 g L(-1) protein) were incubated with a commercial proteolytic enzyme preparation, Corolase PP, until they reached a target degree of hydrolysis (DH) of 5%. WPI solutions on heating were characterized by large aggregate formation, higher viscosity, and surface hydrophobicity and hydrolyzed more rapidly (P < 0.001) than the unheated. The whey proteins exhibited differences in their susceptibility to hydrolysis. Both viscosity and surface hydrophobicity along with insolubility declined as hydrolysis progressed. However, microstructural changes observed by light and confocal laser scanning microscopy (CLSM) provided insights to suggest that aggregate size and porosity may be complementary to denaturation in promoting faster enzymatic hydrolysis. This could be clearly observed in the course of aggregate disintegration, gel network breakdown, and improved solution clarification.     

Principal component similarity analysis of Raman spectra to study the effects of pH,heating, and kappa-carrageenan on whey protein structure

Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.     

Binding of 2-nonanone and milk proteins in aqueous model systems

Interactions of the model flavor compound 2-nonanone with individual milk proteins, whey protein isolate (WPI), and sodium caseinate in aqueous solutions were investigated. A method to quantify the free 2-nonanone was developed using headspace solid-phase microextraction followed by gas chromatography with flame ionization detection. Binding constants (K) and numbers of binding sites (n) for 2-nonanone on the individual proteins were calculated. The 2-nonanone binding capacities decreased in the order bovine serum albumin > beta-lactoglobulin > alpha-lactalbumin > alpha s1-casein > beta-casein, and the binding to WPI was stronger than the binding to sodium caseinate. All proteins appeared to have one binding site for 2-nonanone per molecule of protein at the flavor concentrations investigated, except for bovine serum albumin, which possessed two classes of binding sites. The binding mechanism is believed to involve predominantly hydrophobic interactions.     

Preparation and characterization of beta-carotene nanodispersions prepared by solvent displacement technique

This work demonstrated the preparation of protein-stabilized beta-carotene nanodispersions using the solvent displacement technique. The emulsifying performance of sodium caseinate (SC), whey protein concentrate (WPC), whey protein isolate (WPI), and a whey protein hydrolysate (WPH, 18% degree of hydrolysis) was compared in terms of particle size and zeta-potential of the nanodispersions. SC-stabilized nanodispersions exhibited a bimodal particle size distribution: large particles (stabilized by casein micelles) with a mean particle size of 171 nm and small particles (stabilized by casein submicelles) of 13 nm. This was confirmed with transmission electron microscopy analysis. Most of the beta-carotene precipitated (87.6%) was stabilized in the small particles. On the other hand, the nanodispersions stabilized by the whey proteins were polydispersed with larger mean particle sizes. The mean particle size of WPC and WPI was 1730 and 201 nm, respectively. The SC-stabilized nanodispersion was expected to be more stable as indicated by its higher absolute zeta-potential value (-31 mV) compared to that of WPC (-15 mV) and WPI (-16 mV). Partially hydrolyzed whey protein possessed improved emulsifying properties as shown by WPH-stabilized samples. It was interesting to note that increasing the SC concentration from 0.05 to 0.5 wt % increased the particle size of beta-carotene stabilized by casein micelles, while the reverse was true for those stabilized by SC submicelles. Microfluidization at 100 MPa of SC solution dissociated the casein micelles, resulting in a decrease in mean particle size of the casein micelle-stabilized particles when the SC solution was used to prepare nanodispersions. The results from this work showed that protein-stabilized beta-carotene nanodispersions could be prepared using the solvent displacement technique.     

Rheology of mixed beta-casein/beta-lactoglobulin films at the air-water interface

The adsorption of dilute mixtures of beta-casein/beta-lactoglobulin to the air-water interface was investigated using surface dilatation and surface shear rheology. The data were fitted to simple rheological models to try to gain further information regarding the composition and nature of the interface. The dilatational measurements suggested that the composition of the interface could be determined using these models and that the surface concentration was dominated by the beta-casein in the early stages of adsorption but that high levels of beta-lactoglobulin were present in the final stages. Surface shear rheological measurements showed a similar trend. However, the shear measurements appeared to be more sensitive to the strength of the network than to the composition of the interface. Fluorescence microscopy supported the findings and demonstrated that any "phase separation" capable of affecting the surface rheological measurements occurred at the sub-micrometer scale. The results also demonstrated that the heterogeneity of the interface, once formed, is kinetically trapped, and no further phase separation occurs over the time span of the experiments.