ROLE OF pH IN GEL FORMATION OF WASHED CHICKEN MUSCLE AT LOW IONIC STRENGTH

This work was designed to test the hypothesis that it is not solubilization of the myofibrillar proteins per se that is required to form good gels at low salt concentrations, but the protein‐containing structures must be disorganized. Gels were made from washed minced chicken breast muscle at 0.15, 0.88, and 2.5% sodium chloride. The gels made with varying salt concentrations were evaluated either at pH 6.0–6.5 or pH 7.0–7.4. Strain values, an indicator of protein quality, were high only at neutral pH in the gels containing 0.15 or 0.88% salt. At 2.5% salt, strain values of gels made at acid pH were superior to those at the low salt concentrations at acid pH, but inferior to gels with 2.5% salt at neutral pH. Poor gels were obtained at 0.15% salt and low pH whether or not there was an intermittent adjustment to neutral pH. A neutral salt wash markedly increased the water content of the mince, suggesting that solubility‐inhibiting proteins were removed. Good quality gels were obtained in the absence of any detectable solubilization of myosin and only minimal solubilization of actin.     

Heat Stability of Oil-in-Water Emulsions Containing Milk Proteins: Effect of Ionic Strength and pH

Emulsions (20 wt% soybean oil; 2 wt% protein) made with caseinate at pH 7 and with whey protein isolate (WPI) at pH 7 and 3 were stable to heating at 90 and 121°C. WPI emulsions destabilized at pH values between 3.5 and 4.0. In the presence of KCI (12.5–200 mM), large particles were formed in WPI emulsions at pH 3 and the emulsions were viscous. At pH 7, moderate concentrations of KCI decreased the heat stability and gels were formed. KCI had less effect on WPI emulsions made at pH 3. Combining the emulsions with caseinate allowed some control of the heat-induced gelation.     

Hydroxyl Radical-Stressed Whey Protein Isolate: Functional and Rheological Properties

The objective of the study was to examine the sensitivity of whey protein functionality to oxidizing radicals. Whey protein isolate (WPI) was oxidatively stressed by incubation at 20 °C for 3, 5, and 10 h in hydroxyl radical-generating media containing 0.1 mM ascorbic acid, 0.1 mM FeCl₃, and 1–10 mM H₂O₂. Protein solubility decreased (P?<?0.05) with increasing H₂O₂ concentrations and oxidation time. Surface properties of WPI, including both emulsifying and foaming activities, exhibited significant improvements (P?<?0.05) at H₂O₂ concentrations up to 5 mM and oxidation time up to 5 h. The longer oxidation time or higher H₂O₂ concentrations tended to diminish the surface functionality. However, the oxidative stress, though decreasing the onset gelling temperature, had a general detrimental effect on WPI gelation (hardness, springiness, and storage modulus). The results indicated opposing effects of oxidation on WPI: detrimental to hydrodynamic properties (solubility, gelation) but beneficial to surface properties (emulsification, foaming).     

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.     

Cold‐set whey protein gels induced by calcium or sodium salt addition

Cold‐set whey protein isolate (WPI) gels formed by sodium or calcium chloride diffusion through dialysis membranes were evaluated by mechanical properties, water‐holding capacity and microscopy. The increase of WPI concentration led to a decrease of porosity of the gels and to an increase of hardness, elasticity and water‐holding capacity for both systems (CaCl₂ and NaCl). WPI gels formed by calcium chloride addition were harder, more elastic and opaque, but less deformable and with decreased ability to hold water in relation to sodium gels. The non linear part of stress–strain data was evaluated by the Blatz, Sharda, and Tschoegl equation and cold‐set gels induced by calcium and sodium chloride addition showed strain‐weakening and strain‐hardening behaviour, respectively. The fractal structure of the gels indicated a weak‐link behaviour. For WPI gels results suggest intrafloc links, formed at heating step, which were more rigid than the interfloc links, promoted by salt addition.     

Oxidation promotes cross-linking but impairs film-forming properties of whey proteins

The objective of the study was to investigate the impact of oxidation on the film-forming properties of whey protein isolate (WPI). Sequential heating (70–90 °C) then oxidation (0.1 mM FeCl₃/1 mM ascorbate/0–20 mM H₂O₂) (H → O) or vice versa (O → H) were conducted to oxidize/unfold WPI at pH 6.8 and 8.0 before casting. The resulting films were characterized through mechanical, microstructural, and protein electrophoretic analyses. Oxidation promoted protein cross-linking mainly through disulfide bonds. Tensile strength (TS) and elongation at break (EAB) of films decreased for WPI oxidized by higher concentrations of H₂O₂. Film solubility (protein leachability) at pH 3–7, ranging from 20 to 40%, was unaffected by H₂O₂ up to 5 mM but reached almost 100% at above 5 mM H₂O₂ except at pH 4–5. β-Lactoglobulin dimers and its complex with α-lactalbumin were abundant in O → H WPI films and polymers of WPI dominated in H → O films. Microstructural images confirmed that oxidation promoted crumbly structures thereby explaining the reduced film-forming capability.     

Compression Strength of Dairy Gels and Microstructural Interpretation

Contour plots were developed for the compression stress (at 20% deformation) of single-component, mixed and filled protein gels. Samples were made by heating and acidification from skim milk powder, SMP (0–20% TS), whey protein isolate, WPI (0–10% TS), and recombined cream, within pH 3.6–3.9, 4.6–4.8 and 5.1–5.3. At higher pH, WPI gels were stronger than SMP gels. WPI had a reinforcing effect on SMP gels, while small additions of SMP to WPI gels resulted in weaker mixed gels. Filled gels containing cream had higher compression strengths than mixed gels. Micrographs showed linking of casein chains by WPI strands in mixed gels and compatibility of fat globules with casein micelles in the protein network of filled gels.     

EFFECT OF MgCl2/SODIUM PYROPHOSPHATE ON CHICKEN BREAST MUSCLE MYOSIN SOLUBILIZATION AND GELATION

Sodium chloride (0.29 M) at pH 7 solubilized about 24% of the myosin of washed, minced chicken breast muscle. At a similar pH, 0.2 M sodium chloride in the presence of 10 mM sodiumpyrophosphate and 10 mM magnesium chloride solubilized almost 60% of the myosin. In spite of the greater solubility of myosin under the latter conditions, when gels were prepared with these concentrations of salt at pH 7, the gels without the sodium pyrophosphate and magnesium chloride were slightly superior in both stress (39.3 kPa vs 28.3 kPa) and true strain (2.3 vs 2.0) values. Gels made at a lower pH (6.1–6.5) made much poorer gels. This was true whether the low pH was obtained naturally in the preparation of the sample or re‐adjusted after bringing the mince to a neutral pH. It appears that conditions of pH and salt content that cause solubilization of myosin at more dilute conditions does not contribute to gel quality, but the neutral pH is an important factor for obtaining good gels at ionic strengths <0.3.     

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.     

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.     

Rheology of galactomannan–whey protein mixed systems

The rheological behaviour of whey protein/galactomannan mixtures in aqueous solutions was studied under gelling conditions of the protein component, at neutral pH and at a pH close to the protein isoelectric point. The presence of the neutral polysaccharide had significant effects on the formation and viscoelastic behaviour of the cured gels. This effect was dependent on the structural organisation of the protein network. At pH 7 the galactomannan had a general positive effect on WPI gel formation. It is suggested that under these conditions, the protein network forms a continuous phase that accommodate the polysaccharide chains, acting as a filler of the protein network. The minimum protein concentration for gelation to occur, the gelation temperature and time all decrease in the presence of the galactomannan. Under pH conditions near the whey protein isoelectric point, different effects were observed as a result of the galactomannan addition. At low WPI concentration, the galactomannan had a detrimental influence on the protein network formation, but a negligible effect or even a positive influence on the gelation process at higher concentrations.     

Factors Influencing Whey Protein Gel Rheology: Dialysis and Calcium Chelation

Influence of dialyzable compounds on the Theological properties (shear stress and shear strain at failure) of heat-induced whey protein concentrate (WPC) and whey protein isolate (WPI) gels was examined. Dialyzing WPC and WPI suspensions prior to gelation increased the stress of two of three WPC gels and a WPI gel. Dialysis also significantly increased the strain of the same two WPC gels, normalizing all strain values. Replacement of calcium lost through dialysis did not significantly change gel rheology. However, chelating calcium caused a significant decrease in the stress of all gels: a minimum amount of calcium and/or a calcium complex appears to have a major role in whey protein gelation.     

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.     

Effect of temperature,calcium and protein concentration on aggregation of whey protein isolate: Formation of gel-like micro-particles

Protein aggregation occurs in biological systems and industrial processes, affecting protein solubility and functional properties. In this study, whey protein isolate (WPI) obtained from bovine milk was used as a model to study the dependence of aggregation on pre-heating temperature and on protein and calcium concentrations. WPI solutions (0.1–5.0%, w/v) were heated at 25–85 °C for 30 min prior to cooling and calcium addition. Tryptophan shifted to a more hydrophilic environment as WPI concentrations and pre-heating temperatures increased. Pre-heated WPI solutions yielded soluble particles, which aggregated to form porous gel-like particles by addition of calcium chloride. WPI microgel particles could be prepared by using a cold gelation method and preheated the protein above 65 °C. The particle size was monodisperse with sizes of about 190 nm and 255 nm, respectively in solutions pre-heated to 75 or 85 °C and containing 5 mm calcium.     

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     

A COMPARISON OF THE VISCOELASTIC PROPERTIES OF CONVENTIONAL AND MAILLARD PROTEIN GELS

The properties of gels prepared by heating solutions of bovine serum albumin (BSA) for 30 min at 121C in the presence and absence of glucono-delta-lactone (GDL gels) and xylose (Maillard gels) were compared. During formation the pH of the Maillard and GDL gels decreased to 4.9 whereas the pH of the gels formed in the absence of GDL or xylose remained near neutral. Maillard gels show much less syneresis compared with the GDL gels and contained nondisulphide covalent crosslinks as evidenced by very low protein solubilities in mixtures of sodium dodecyl sulphate and β-mercaptoethanol. Both the GDL and Maillard gels could be formed at much lower protein concentrations than the neutral conventional gels. The stress relaxation of the gels in compression was measured and the response analyzed using Peleg's equation. The parameters in this equation were not strongly dependent on protein concentration or degree of deformation. The neutral pH gels were far more elastic than the low pH gels, but despite the difference in crosslinking mechanisms the viscoelastic behaviour of the Maillard and GDL gels was similar. However, the break strength and asymptotic residual modulus of the Maillard gels were higher. It is suggested that the stress relaxation occurs in weaker, noncovalently linked regions of the gel, whereas the nondisulphide covalent crosslinks in the Maillard gels reinforce strong regions already containing disulphide linkages.     

Oil-in-Water Emulsions Stabilized by Sodium Caseinate or Whey Protein Isolate as influenced by Glycerol Monostearate

Competitive adsorption between glycerol monostearate (GMS) and whey protein isolate (WPI) or sodium caseinate was studied in oil-in-water emulsions (20 wt % soya oil, deionized water, pH 7). Addition of GMS resulted in partial displacement of WPI or sodium caseinate from the emulsion interface. SDS-PAGE showed that GMS altered the adsorbed layer composition in sodium caseinate stabilized emulsions containing < 1.0 wt % protein. Predominance of β-casein at the interface in the absence of surfactant was reduced in the presence of GMS. The distribution of α-lactalbumin and β-lactoglobulin between the aqueous bulk phase and the fat surface in emulsions stabilized with WPI was independent of the concentration of added protein or surfactant.     

Effects of heating rate and pH on fracture and water-holding properties of globular protein gels as explained by micro-phase separation

The effect of heating rate and pH on fracture properties and held water (HW) of globular protein gels was investigated. The study was divided into 2 experiments. In the 1st experiment, whey protein isolate (WPI) and egg white protein (EWP) gels were formed at pH 4.5 and 7.0 using heating rates ranging from 0.1 to 35 °C/min and holding times at 80 °C up to 240 min. The 2nd experiment used one heating condition (80 °C for 60 min) and probed in detail the pH range of 4.5 to 7.0 for EWP gels. Fracture properties of gels were measured by torsional deformation and HW was measured as the amount of fluid retained after a mild centrifugation. Single or micro-phase separated conditions were determined by confocal laser scanning microscopy. The effect of heating rate on fracture properties and HW of globular protein gels can be explained by phase stability of the protein dispersion and total thermal input. Minimal difference in fracture properties and HW of EWP gels at pH 4.5 compared with pH 7.0 were observed while WPI gels were stronger and had higher HW at pH 7.0 as compared to 4.5. This was due to a mild degree of micro-phase separation of EWP gels across the pH range whereas WPI gels only showed an extreme micro-phase separation in a narrow pH range. In summary, gel formation and physical properties of globular protein gels can be explained by micro-phase separation. PRACTICAL APPLICATION: The effect of heating conditions on hardness and water-holding properties of protein gels is explained by the relative percentage of micro-phase separated proteins. Heating rates that are too rapid require additional holding time at the end-point temperature to allow for full network development. Increase in degree of micro-phase separation decreases the ability for protein gels to hold water.     

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.     

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.