How micro-phase separation alters the heating rate effects on globular protein gelation

This study was conducted to determine how the combination of heating rate and pH can be used to alter viscoelastic properties and microstructure of egg white protein and whey protein isolate gels. Protein solutions (1% to 7% w/v protein, pH 3.0 to 8.5) were heated using a range of heating rates (0.2 to 60 °C/min) to achieve a final temperature of 80 °C. The gelation process and viscoelastic properties of formed gels were evaluated using small strain rheology. Single phase or micro-phase separated solution conditions were determined by confocal laser scanning microscopy. In the single phase region, gels prepared by the faster heating rates had the lowest rigidity at 80 °C; however, a common G' was achieved after holding for 4 h at 80 °C . On the other hand, under micro-phase separation conditions, faster heating rates allowed phase separated particles to be frozen in the network prior to precipitation. Thus, gels produced by slower heating rates had lower rigidities than gels produced by faster heating rates. The effect of heating rate appears to depend on if the solution is under single phase or micro-phase separated conditions. PRACTICAL APPLICATION: The effect of heating rate and/or time on protein gel firmness can be explained based on protein charge. When proteins have a high net negative charge and form soluble aggregates, there is no heating rate effect and gels with equal firmness will be formed if given enough time. In contrast, when electrostatic repulsion is low, there is a competition between protein precipitation and gel formation; thus, a faster heating rate produces a firmer gel.     

The effect of pH on gel structures produced using protein–polysaccharide phase separation and network inversion

Forming heat-induced gels through combined effects of micro-phase separation of whey protein isolate (WPI; 5%, w/v, 100 mm NaCl) by pH change (5.5, 6.0, and 6.5), and addition of κ-carrageenan (0–0.3%, w/w), were evaluated. The microstructure of WPI gels was homogeneous at pH 6.0 and 6.5 and micro-phase separated at pH 5.5. Addition of 0.075% κ-carrageenan to WPI solutions caused the microstructure of the gel to switch from homogeneous (pH 6.0 and 6.5) to micro-phase separated; and higher concentrations led to inversion of the continuous network from protein to κ-carrageenan. Protein solutions containing 0.075% (w/w) κ-carrageenan produced gels with increased storage modulus (G′) at pH 6.5 and decreased G′ at pH 5.5. All gels containing 0.3% (w/w) κ-carrageenan had κ-carrageenan-continuous networks. It was shown that microstructural and rheological changes were different in WPI and κ-carrageenan mixed gels when micro-phase separation was caused by pH rather than ionic strength.     

Effect of CMC Molecular Weight on Acid‐Induced Gelation of Heated WPI‐CMC Soluble Complex

Acid‐induced gelation properties of heated whey protein isolate (WPI) and carboxymethylcellulose (CMC) soluble complex were investigated as a function of CMC molecular weight (270, 680, and 750 kDa) and concentrations (0% to 0.125%). Heated WPI‐CMC soluble complex with 6% protein was made by heating biopolymers together at pH 7.0 and 85 °C for 30 min and diluted to 5% protein before acid‐induced gelation. Acid‐induced gel formed from heated WPI‐CMC complexes exhibited increased hardness and decreased water holding capacity with increasing CMC concentrations but gel strength decreased at higher CMC content. The highest gel strength was observed with CMC 750 k at 0.05%. Gels with low CMC concentration showed homogenous microstructure which was independent of CMC molecular weight, while increasing CMC concentration led to microphase separation with higher CMC molecular weight showing more extensive phase separation. When heated WPI‐CMC complexes were prepared at 9% protein the acid gels showed improved gel hardness and water holding capacity, which was supported by the more interconnected protein network with less porosity when compared to complexes heated at 6% protein. It is concluded that protein concentration and biopolymer ratio during complex formation are the major factors affecting gel properties while the effect of CMC molecular weight was less significant.     

Modification of rheological properties of whey protein isolates by limited proteolysis

Whey protein isolate (WPI) was subjected to limited tryptic hydrolysis and the effect of the limited hydrolysis on the rheological properties of WPI was examined and compared with those of untreated WPI. At 10% concentration (w/v in 50 mM TES buffer, pH 7.0, containing 50 mM NaCl), both WPI and the enzyme-treated WPI (EWPI) formed heat-induced viscoelastic gels. However, EWPI formed weaker gels (lower storage modulus) than WPI gels. Moreover, a lower gelation point (77 °C) was obtained for EWPI as compared with that of WPI which gelled at 80 °C only after holding 1.4 min. Thermal analysis and aggregation studies indicated that limited proteolysis resulted in changes in the denaturation and aggregation properties. As a consequenece, EWPI formed particulated gels, while WPI formed fine-stranded gels. In keeping with the formation of a particulate gel, Texture Profile Analysis (TPA) of the heat-induced gels (at 80 °C for 30 min) revealed that EWPI gels possessed significantly higher (p < 0.05) cohesiveness, hardness, gumminess, and chewiness but did not fracture at 75% deformation. The results suggest that the domain peptides, especially β-lactoglobulin domains released by the limited proteolysis, were responsible for the altered gelation properties.     

Structure and rheological properties of acid-induced egg white protein gels

This study compares the rheological properties of acid-induced gels prepared of industrial spray-dried egg white proteins (EWP) with the acid-induced gels prepared of ovalbumin (OA) and whey protein isolate (WPI). Also we aimed to form transparent gels of EWP by means of the cold-gelation process. We showed that it was not possible to prepare cold-set gels because ovotransferrin (OT), present in EWP, was found to interfere with fibril formation. Therefore, we developed a new purification method in which first OT was selectively denatured by a heating step, subsequently precipitated by acidification and removed by centrifugation. Finally, the supernatant was desalted by ultra filtration. This resulted in a preheated EWP preparation, which mainly contains OA (>80%). By removing OT using this new preheat procedure transparent gels were obtained after acid-induced gelation. Fracture properties of various EWP preparations were determined and compared with those of acid-induced gels of OA and WPI. Gels formed from different EWP preparations were weak (fracture stress 1–15 kPa, fracture strain 0.3–0.7), and the networks consisted of thin strands with hardly any additional disulphide bonds formed during the gelation step. In conclusion, the microstructure of the aggregates formed in the first step of the cold-gelation process and the amount of additional disulphide bonds formed during the second step appeared to be the determining factors contributing to the hardness and deformability of acid-induced gels of egg white proteins.     

Effect of heating temperature,pH, concentration and starch/whey protein ratio on the viscoelastic properties of corn starch/whey protein mixed gels

The viscoelastic properties of corn starch (CS) gels were more dependent on heating temperature, while the properties of whey protein isolate (WPI) gels were more dependent on pH. Thus heating temperature (75, 85, 95 °C) and pH (5, 7, 9) were varied to obtain a series of mixed gels with interesting viscoelastic properties. WPI gels showed extensive stress relaxation (SR) indicative of a highly transient network structure, while CS gels relaxed very little in 2000 s. Based on SR results, it appeared that CS/WPI mixed gels with 25 and 50% CS formed compatible network structures at 15% total solids only at pH 9. This supposition was supported by SEM microstructures obtained for dehydrated gels and a synergistic increase in the large‐strain fracture stress for these gels. Some synergy was also found for mixed gels at 30% total solids at pH 9, while at pH 7 the mixed gels seemed to contain separate additive WPI and CS networks unlike the case for pH 7 at 15% total solids. In both cases (15 and 30% total solids) the degree of elasticity of the mixed gels decreased as the WPI content increased. Mixed gels (CS:WPI = 0.5) at pH 9 showed increased fracture stress and fracture strain relative to the same gels at pH 7. This suggests that a unique chemical compatibility exists at pH 9 and results in gels that combine the elasticity of CS and the internal stress dissipation of WPI. © 2001 Society of Chemical Industry     

Foams Prepared from Whey Protein Isolate and Egg White Protein: 2. Changes Associated with Angel Food Cake Functionality

ABSTRACT:  The effects of sucrose on the physical properties and thermal stability of foams prepared from 10% (w/v) protein solutions of whey protein isolate (WPI), egg white protein (EWP), and their combinations (WPI/EWP) were investigated in wet foams and angel food cakes. Incorporation of 12.8 (w/v) sucrose increased EWP foam stability (drainage 1/2 life) but had little effect on the stability of WPI and WPI/EWP foams. Increased stability was not due to viscosity alone. Sucrose increased interfacial elasticity ( E  ') of EWP and decreased E' of WPI and WPI/EWP combinations, suggesting that altered interfacial properties increased stability in EWP foams. Although 25% WPI/75% EWP cakes had similar volumes as EWP cakes, cakes containing WPI had larger air cells. Changes during heating showed that EWP foams had network formation starting at 45 °C, which was not observed in WPI and WPI/EWP foams. Moreover, in batters, which are foams with additional sugar and flour, a stable foam network was observed from 25 to 85 °C for batters made from EWP foams. Batters containing WPI or WPI/EWP mixtures showed signs of destabilization starting at 25 °C. These results show that sucrose greatly improved the stability of wet EWP foams and that EWP foams form network structures that remain stable during heating. In contrast, sucrose had minimal effects on stability of WPI and WPI/EWP wet foams, and batters containing these foams showed destabilization prior to heating. Therefore, destabilization processes occurring in the wet foams and during baking account for differences in angel food cake quality.     

Combining protein micro-phase separation and protein–polysaccharide segregative phase separation to produce gel structures

The ability of protein micro-phase separation and protein–polysaccharide segregative phase separation to generate a range of gel structures and textures was evaluated. Whey protein isolate/κ-carrageenan mixed gels were prepared with 13% (w/v) whey protein isolate, 0–0.6% (w/w) κ-carrageenan and 50, 100 or 250 mM NaCl. The microstructure of gels, determined by confocal laser scanning microscopy, varied from homogenous to protein continuous, bicontinuous, coarse stranded or κ-carrageenan continuous, depending on the κ-carrageenan concentration. Microstructure also varied from stranded to particulate (micro-phase separated) depending on the salt concentration. The rheological behavior of mixed gels corresponded to the shift in the continuous phase from protein to κ-carrageenan. At small concentrations of κ-carrageenan, where carrageenan-rich droplets were dispersed in a continuous protein-rich matrix, gel strength (fracture stress) and firmness (G′) increased due to increased local concentration of proteins caused by phase separation. At higher κ-carrageenan concentrations, gels were substantially less firm, weaker and less deformable (fracture strain). The change in the continuous phase from protein continuous to carrageenan continuous explained the major change in mechanical properties and water-holding properties. The shift in microstructure occurred at lower concentrations of κ-carrageenan when whey proteins were under micro-phase separation conditions. The results demonstrated how the combined mechanisms of ion-induced micro-phase separation of proteins and protein–polysaccharide phase separation and inversion can be used to alter gel structure and texture.     

Gelation of whey protein and xanthan mixture: Effect of heating rate on rheological properties

The effects of heating rate and xanthan addition on the gelation of a 15% w/w whey protein solution at pH 7 and in 0.1 M phosphate buffer were studied using small-amplitude oscillatory shear (SAOS) rheological measurements and uniaxial compression tests. WPI solutions were heated from 25 to 90 °C at five heating rates (0.1, 1, 5, 10 and 20 °C/min). Gelation temperature of WPI decreased with decreasing of heating rates and with xanthan addition. Under uniaxial compression, the WPI gels prepared with no more than 0.2% w/w xanthan exhibited distinct fracture point and were tougher (i.e. higher fracture stress and fracture strain) than the gels prepared with no less than 0.5% w/w xanthan. In general, the fracture strain of WPI gels increased with heating rate, though not significantly, at all xanthan contents investigated. However, the fracture stress of WPI gels, generally, decreased with heating rate when xanthan content was 0–0.2% and increased with heating rate when xanthan content was 0.5 and 1%.     

Gelation of single heated vs. double heated whey protein isolate

Gelation of single and double heated whey protein dispersions was investigated using Ca²⁺ as inducing agents. Whey protein isolate (WPI) dispersions (10% w/w) were single heated (30 min, 80 °C at pH 7.0) or double heated (30 min, 80 °C at pH 8.0 and 30 min, 80 °C at pH 7.0) and diluted to obtain the desired protein and/or calcium ions concentration (4–9% and 5–30 mm, respectively). Calcium ions were added directly or by using a dialysis method. Double-heated dispersions gelled faster at lower protein and calcium ion concentrations than single-heated dispersions. Gels obtained from double-heated dispersions had lower values of shear strain and shear stress at fracture than gels obtained from single-heated dispersions. Double heating caused a significant complex modulus (G*) increase at 4% WPI and 15 mm calcium ions in comparison with gels obtained from single-heated dispersion. Less significant differences between gels made from double and single-heated dispersions were observed at 6% WPI, however a higher value of complex modulus was obtained for 8% protein gels from the single-heated solution. Native and non-reduced SDS–PAGE did not show clearly the effect of different procedures of heating on the quantities of polymerised proteins. Proteins in double-heated dispersions had higher hydrophobicity. Increased calcium concentration caused decreased protein hydrophobicity for both single and double-heated solutions.     

Comparative effect of thermal treatment on the physicochemical properties of whey and egg white protein foams

The optimization of the functionalities of commercial protein ingredients still constitutes a key objective of the food industry. Our aim was therefore to compare the effect of thermal treatments applied in typical industrial conditions on the foaming properties of whey protein isolate (WPI) and egg white proteins (EWP): EWP was pasteurized in dry state from 1 to 5 days and from 60 °C to 80 °C, while WPI was heat-treated between 80 °C and 100 °C under dynamic conditions using a tubular heat exchanger. Typical protein concentrations of the food industry were also used, 2% (w/v) WPI and 10% (w/v) EWP at pH 7, which provided solutions of similar viscosity. Consequently, WPI exhibited a higher foamability than EWP. For WPI, heat treatment induced a slight decrease of overrun when temperature was above 90 °C, i.e. when aggregation reduced too considerably the amount of monomers that played the key role on foam formation; conversely, it increased foamability for EWP due to the lower aggregation degree resulting from dry heating compared to heat-treated WPI solutions. As expected, thermal treatments improved significantly the stability of WPI and EWP foams, but stability always passed through a maximum as a function of the intensity of heat treatment. In both cases, optimum conditions for foam stability that did not impair foamability corresponded to about 20% soluble protein aggregates. A key discrepancy was finally that the dry heat treatment of EWP provided softer foams, despite more rigid than the WPI-based foams, whereas dynamically heat-treated WPI gave firmer foams than native proteins.     

Isostrength Comparison of Large-Strain (Fracture) Rheological Properties of Egg White and Whey Protein Gels

The effects of protein concentration and heating conditions on the physical properties of whey protein isolate (WPI) (in 50 mM NaCl, pH 7) and egg white (pH 9) gels were examined. Egg white and WPI gels had similar values for shear stress at fracture (i.e., isostrength), while trends for shear strain at fracture were protein-type specific. The rigidity ratio (R_0.3), ratio of the rigidity at fracture (G_f) to the rigidity at 30% of fracture strain, measured departure from the stress-strain relationship of an ideal Hookean solid. All gels fit master curves of G_f vs R_0.3, which were described by a power law model of R_0.3=A(G_f)-°19, where "A" showed protein type-specific characteristics.     

Enhancement of the functional properties of whey protein by conjugation with maltodextrin under dry‐heating conditions

Conjugation of whey protein isolate (WPI) and maltodextrin (MD, dextrose equivalent of 6) was achieved by dry‐heating at an initial pH of 7.0, at 60 °C and 79% relative humidity, with WPI: MD6 ratio of 1:1, for up to 24 h. Conjugation was achieved with limited development of colour and advanced Maillard products on 24 h of heating. Conjugation increased the protein solubility at pH 4.5, by 7.1–8.5%, compared to the unheated and heated WPI controls. Conjugation of WPI with MD6 enhanced the stability and retention of clarity in protein solutions heated at 85 °C for 10 min with 50 mM added NaCl.     

Rheology of whey protein isolate-xanthan mixed solutions and gels. Effect of pH and xanthan concentration

Flow properties at pH 5.5-7.5 of whey protein isolate (WPI)-xanthan solutions containing 0-0.5 w/w% xanthan were studied by viscosimetry, although rigidity and fracture properties of the corresponding heat-set gels (90°C, 30 min) were determined by uniaxial compression. All the studied solutions displayed generalized shearthinning flow behaviour. Synergistic WPI-xanthan interactions has been revealed by observing that rheological parameters [σ_msf, K, n, η (γ)] characterizing blends were larger than those calculated from the two separated solutions. Such a behaviour was attributed to segregative phase separation of whey proteins and xanthan. Effects of xanthan on WPI-xanthan gel properties both depended on pH and xanthan concentration. Simultaneous increased xanthan concentration and decreased pH inhibited gelation of WPI-xanthan blends. Regarding gel strength, synergistic WPI-xanthan interactions were observed at pH >7.0 and low xanthan concentration (0.05 or 0.1 w/w%). Antagonism between the two macromolecules occurred at low xanthan concentration and pH ≤6.5, and high xanthan concentration (0.2 or 0.5 w/w%) at all pH tested. Low xanthan concentration rendered mixed gels more brittle than protein gels, and high xanthan concentration decreased pH effects on gel stress-strain relationships. The balance between strong thermal aggregation of concentrated whey proteins - in presence of incompatible xanthan -, high viscosity of blends and repulsive surface forces of protein molecules was thought to be at the origin of WPI-xanthan gel mechanical properties.     

Thermodynamic incompatibility between denatured whey protein and konjac glucomannan

The current study investigated the effect of a neutral polysaccharide, konjac glucomannan, on the heat-induced gelation of whey protein isolate (WPI) at pH 7. Oscillatory rheology (1 rad/s; 0.5% strain), differential scanning calorimetry and confocal laser scanning microscopy were used to investigate the effect of addition of konjac in the range 0-0.5% w/w, on the thermal gelation properties of WPI. The minimum gelling concentration for WPI samples was 11% w/w; the concentration was therefore held constant at this value. Gelation of WPI was induced by heating the samples from 20 to 80 °C, holding at 80 °C for 30 min, cooling to 20 °C, and holding at 20 °C for a further 30 min. On incorporation of increasing concentrations of konjac the gelation time decreased, while the storage modulus (G′) of the mixed gel systems increased to ∼1450 Pa for 11% w/w WPI containing 0.5% w/w konjac gels, compared to 15 Pa for 11% w/w WPI gels (no konjac). This increase in gel strength was attributed to segregative interactions between denatured whey proteins and konjac glucomannan.     

Rapid Heating of Alaska Pollock and Chicken Breast Myofibrillar Proteins as Affecting Gel Rheological Properties

Surimi seafoods (fish/poikilotherm protein) in the U.S.A. are typically cooked rapidly to 90+°C, while comminuted products made from land animals (meat/homeotherm protein) are purposely cooked much more slowly, and to lower endpoint temperatures (near 70 °C). We studied heating rate (0.5, 25, or 90 °C/min) and endpoint temperature (45 to 90 °C) effects on rheological properties (fracture, small strain) of washed myofibril gels derived from fish (Alaska pollock) compared with chicken breast at a common pH (6.75). This was contrasted with published data on gelation kinetics of chicken myosin over the same temperature range. Heating rate had no effect on fracture properties of fish gels but slow heating did yield somewhat stronger, but not more deformable, chicken gels. Maximum gel strength by rapid heating could be achieved within 5 min holding after less than 1 min heating time. Dynamic testing by small strain revealed poor correspondence of the present data to that published for gelling response of chicken breast myosin in the same temperature range. The common practice of reporting small‐strain rheological parameters measured at the endpoint temperature was also shown to be misleading, since upon cooling, there was much less difference in rigidity between rapidly and slowly heated gels for either species.     

EFFECT OF EMULSION DROPLETS ON THE RHEOLOGY OF WHEY PROTEIN ISOLATE GELS

The effects of droplet size and emulsifier type on the rheology of whey protein isolate (WPI) gels containing emulsion droplets was studied. Gels were prepared by dispersing droplets of corn oil (20 wt%, d₃₂= 0.7 – 4 μm) in a 10 wt% WPI solution (pH 7.0, 50 mM NaCl), and heating at 90C for 15 min. Gel strength was determined by measuring the stress of gels at 20% compression using an Instron Universal Testing Machine. Droplets stabilized by WPI increased the gel strength, those stabilized by non-ionic surfactants (Tween 20 and Triton X-100) decreased it slightly, and those stabilized by SDS decreased it drastically. Gel strength increased as the droplet size decreased for droplets stabilized by WPI, but was relatively insensitive to the size of droplets stabilized by the small molecule surfactants. These observations may be explained in terms of the interactions between the emulsifiers and the protein network. Droplets coated with emulsifiers which can be incorporated into the protein network reinforce the structure and so increase gel strength, whereas droplets coated with emulsifiers which cannot be incorporated into the protein network disrupt the three dimensional structure of the gel and decrease its strength.     

Foams Prepared from Whey Protein Isolate and Egg White Protein: 1. Physical, Microstructural, and Interfacial Properties

ABSTRACT:  Foams were prepared from whey protein isolate (WPI), egg white protein (EWP), and combinations of the 2 (WPI/EWP), with physical properties of foams (overrun, drainage 1/2 life, and yield stress), air/water interfaces (interfacial tension and interfacial dilatational elasticity), and foam microstructure (bubble size and dynamic change of bubble count per area) investigated. Foams made from EWP had higher yield stress and stability (drainage 1/2 life) than those made from WPI. Foams made from mixtures of EWP and WPI had intermediate values. Foam stability could be explained based on solution viscosity, interfacial characteristics, and initial bubble size. Likewise, foam yield stress was associated with interfacial dilatational elastic moduli, mean bubble diameter, and air phase fraction. Foams made from WPI or WPI/EWP combinations formed master curves for stability and yield stress when normalized according to the above-mentioned properties. However, EWP foams were excluded from the common trends observed for WPI and WPI/EWP combination foams. Changes in interfacial tension showed that even the lowest level of WPI substitution (25% WPI) was enough to cause the temporal pattern of interfacial tension to mimic the pattern of WPI instead of EWP, suggesting that whey proteins dominated the interface. The higher foam yield stress and drainage stability of EWP foams appears to be due to forming smaller, more stable bubbles, that are packed together into structures that are more resistant to deformation than those of WPI foams.     

pH and Heat Treatment Effects on Foaming of Whey Protein isolate

The overrun obtained by whipping whey protein isolate (WPI) was significantly (p<0.05) affected by changing pH. Heating WPI at pH 4.0 reduced rate and amount of overrun. The highest overrun values for unheated WPI were observed at pH 5.0 and 7.0 after heating at 55°C for 10 min. The maximum foam stability for unheated WPI was obtained at pH 5.0. Heat treatment had little effect on stability at pH 4.0 or 7.0 but at pH 5.0, 80°C for 10 min improved stability by 65%. Based on surface pressure data, the rate of adsorption of β-lactoglobulin interfacial films and the work of compression correlated with overrun, maximum overrun, overrun development and foam stability.     

Quantitative analysis of confocal laser scanning microscopy images of heat-set globular protein gels

Gels of the globular protein β-lactoglobulin were made by heating solutions at pH 7 and different NaCl concentrations (C_s). The influence of the ionic strength on the gel structure was studied by confocal laser scanning microscopy (CLSM). For C_s < 0.2 M the images were homogeneous, but at higher NaCl concentrations micro-phase separation was observed. The protein concentration in the two phases was determined from the images. It is shown how CLSM images can be quantitatively analysed in terms of the pair correlation function of the protein concentration fluctuations, yielding results that can be directly compared to those obtained from light scattering. The transition between so-called finely stranded and particulate gels is explained by a switch from net repulsive to net attractive interaction between growing protein aggregates.