Physicochemical basis for cosolvent modulation of β-lactoglobulin functionality: Interfacial tension study

The impact of neutral cosolvents on the thermal stability of globular proteins in aqueous solutions depends on the nature of the cosolvent, e.g., sorbitol causes a pronounced increase in the thermal denaturation temperature (T_m) of β-lactoglobulin (β-lg), whereas glycerol does not. When a protein unfolds there is a change in the exposed surface area and in the type of molecular interactions that occur at the protein–solvent–cosolvent interface. These changes contribute to the free energy change associated with protein denaturation and depend on cosolvent type. In this study we measured the equilibrium interfacial tensions of aqueous glycerol (0–70% w/w) and sorbitol (0–55% w/w) solutions as a function of temperature to provide insights into the role of the interfacial energy on the thermal stability of β-lg. There was a slight increase in interfacial tension with increasing sorbitol concentration, indicating its preferential exclusion from the oil–water interface. On the other hand, there was an appreciable decrease in interfacial tension with increasing glycerol concentration, indicating its preferential accumulation at the oil–water interface. These changes were largely independent of the measurement temperature (30–80 °C). Our results suggest that sorbitol increases the T_m of β-lg mainly through a steric exclusion effect, whereas glycerol has little effect on T_m because the steric exclusion effect is counter-balanced by a differential interaction effect.     

Modulation of thermal stability and heat-induced gelation of β-lactoglobulin by high glycerol and sorbitol levels

The influence of glycerol and sorbitol on the thermal stability and heat-induced gelation of β-lactoglobulin (β-lg) in aqueous solutions was investigated. The thermal stability of β-lg was characterized by measuring the thermal denaturation temperature (T_m) using differential scanning calorimetry, while its gelation properties were characterized by measuring the gelation temperature (T_gel) and final gel rigidity (G^∗) using dynamic shear rheology. All experiments were carried out using aqueous solutions containing 10% (w/w) β-lg, glycerol (0–70% w/w) or sorbitol (0–55% w/w), and 5 mM phosphate buffer (pH 7.0). No salt was added to these solutions so that there was a relatively strong electrostatic repulsion between the protein molecules, which usually prevents gelation. When the cosolvent concentration was increased from 0% to 50%, T_m increased from 74 to 86 °C for sorbitol, but only from 74 to 76 °C for glycerol, which indicated that sorbitol was much more effective at stabilizing the native state of the globular protein than glycerol. Protein solutions containing sorbitol (0–55%) did not form a gel after heating, but those containing glycerol formed gels when the cosolvent concentration exceeded about 10%, with G^∗ increasing with increasing glycerol concentration. We attribute these effects to differences in the preferential interactions of polyols and water with the surfaces of native and heat-denatured proteins, and their influence on the protein–protein collision frequency.     

Inhibition of droplet flocculation in globular-protein stabilized oil-in-water emulsions by polyols

The influence of neutral cosolvents (polyols) on the stability of hydrocarbon oil-in-water emulsions stabilized by a globular protein was investigated. Glycerol (0–40 wt%) and sorbitol (0–35 wt%) were added to n-hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-lg, pH 7.0, 150 mM NaCl), either before or after incubation at 30 °C for 24 h. The stability of the emulsions to flocculation and creaming improved when neutral cosolvents were added, with the effectiveness of the cosolvents depending on their type, concentration and time of addition. Emulsion stability was better for sorbitol than glycerol, improved with increasing cosolvent concentration, and was better when the cosolvents were added immediately after homogenization than when they were added 24 h later. The influence of the cosolvents on emulsion stability is interpreted in terms of their effect on the conformation and interactions of the adsorbed proteins, as well as on the droplet–droplet collision frequency. This study has implications for the development of protein stabilized oil-in-water emulsions for utilization in industrial products.     

Impact of cosolvents on formation and properties of biopolymer nanoparticles formed by heat treatment of β-lactoglobulin–Pectin complexes

The purpose of this study was to determine the influence of neutral cosolvents on the formation and properties of biopolymer nanoparticles formed by thermal treatment of protein–polysaccharide electrostatic complexes. Biopolymer particles were formed by heating (85 °C, 20 min) an aqueous solution containing a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin) above the thermal denaturation temperature (T_m) of the protein under pH conditions where the biopolymers formed electrostatic complexes (pH 5). The impact of two neutral cosolvents (glycerol and sorbitol) on the self-association of β-lactoglobulin and on the formation of β-lactoglobulin–pectin complexes was examined as a function of solution pH (3–7) and temperature (30–95 °C). Glycerol had little impact on the pH-induced self-association or aggregation of the biopolymers, but it did increase the thermal aggregation temperature (T_a) of the protein–polysaccharide complexes, which was attributed to its ability to increase aqueous phase viscosity. Sorbitol decreased the pH where insoluble protein–polysaccharide complexes were formed, and greatly increased their T_a, which was attributed to its ability to increase T_m, alter biopolymer–biopolymer interactions, and increase aqueous phase viscosity. This study shows that neutral cosolvents can be used to modulate the properties of biopolymer nanoparticles prepared by thermal treatment of protein–polysaccharide electrostatic complexes.     

The effect of binary cosolvent systems (glycerol–sucrose mixtures) on the heat-induced gelation mechanism of bovine serum albumin

The influence of cosolvent composition on the thermal denaturation and gelation of bovine serum albumin (BSA) has been studied. Cosolvent composition was varied by changing the ratio of glycerol to sucrose in 40 wt% cosolvent solutions containing BSA. Differential scanning calorimetry measurements on 0.5 wt% BSA solutions showed that the thermal denaturation temperature of the protein increased with increasing sucrose content. Temperature scanning dynamic shear rheology and turbidity measurements on 4 wt% BSA solutions showed that the gelation temperature and final gel strength increased with increasing sucrose concentration. These observations were attributed to the fact that sucrose was more effective than glycerol at increasing the thermal stability and attraction between globular BSA molecules through a steric exclusion effect. The molecular origin of these effects is the tendency for the system to minimize the contact area of the protein molecules with the surrounding cosolvent solution.     

Protein–polysaccharide interactions: The determination of the osmotic second virial coefficients in aqueous solutions of β-lactoglobulin and dextran

Solutions containing dextran and solutions containing mixtures of dextran +β-lactoglobulin are studied by membrane osmometry. The low concentration range of these solutions is considered. From the measured osmotic pressures the virial coefficients are obtained. These are analyzed using the osmotic virial coefficient of β-lactoglobulin solutions published earlier by us [Schaink, H.M., & Smit, J.A.M. (2000). Determination of the osmotic second virial coefficient and the dimerization of beta-lactoglobulin in aqueous solutions with added salt at the isoelectric point. PCCP, 2, 1537–1541]. The second cross-virial coefficient A₁₂ is found to be positive indicating a repulsive and probably mainly steric interaction between neutral in nature dextran and and practically uncharged β-lactoglobulin (pH=5.18). The measurements show that the β-lactoglobulin has only a small tendency to form multimers in the presence of dextran. The phase diagram of solutions of dextran+Whey Protein Isolate (appr. 60% β-lactoglobulin) is also presented. The McMillan–Mayer equation of state that considers only the second virial coefficients is found to be unreliable for the extrapolation up to the concentrations at which phase separation is expected.     

Influence of glycerol and sorbitol on thermally induced droplet aggregation in oil-in-water emulsions stabilized by β-lactoglobulin

The influence of polyol cosolvents (glycerol and sorbitol) on the flocculation stability of hydrocarbon oil-in-water emulsions stabilized by a globular protein was examined. Salt (150 mM NaCl) and polyols (0–40 wt%) were added to n-hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-Lg, pH 7.0) either before or after isothermal heat treatments (30–90 °C for 20 min). When salt was added to emulsions before heat treatment, appreciable droplet flocculation was observed below the thermal-denaturation temperature of the adsorbed β-Lg (T_m∼70 °C), and more extensive flocculation was observed above T_m. On the other hand, when salt was added after heat treatment, appreciable droplet flocculation still occurred below T_m, but little flocculation was observed above T_m. Addition of cosolvents to the emulsions increased the temperature where extensive droplet flocculation was first observed when they were heated in the presence of salt, which was attributed to their ability to increase T_m and to reduce the droplet collision frequency, with sorbitol being more effective than glycerol. Our results are interpreted in terms of the influence of the cosolvents on protein conformational stability, protein-protein interactions and the physiochemical properties of aqueous solutions. This study has important implications for the formulation and production of protein stabilized oil-in-water emulsions for industrial applications, such as foods, pharmaceuticals and cosmetics.     

On solid-like rheological behaviors of globular protein solutions

Dynamic viscoelastic and steady flow properties of β-lactoglobulin, bovine serum albumin, ovalbumin, and α-lactalbumin aqueous solutions were investigated at 20°C. When a sinusoidal strain in the linear viscoelastic region was applied, the solutions of the globular proteins except for α-lactalbumin showed typical solid-like rheological behavior: the storage modulus G′ was always larger than the loss modulus G″ in the entire frequency range examined (0.1–100 rad/s). Under a steady shear flow, strong shear thinning behavior was observed with increasing shear rate from 0.001 to 800 s⁻¹, for the globular proteins except for α-lactalbumin. The values of the steady shear viscosity η were lower than those of the dynamic shear viscosity η* at a comparable time scale of observation, violating the Cox–Merz rule, and thus suggesting that a solid-like structure in a globular protein solution was susceptible to a steady shear strain. During isothermal gelation of the protein colloids at 70°C, no crossover between G′ and G″ was observed so that the gelation point was judged by an abrupt increase in the modulus or a sudden decrease in tanδ.     

Polyelectrolyte screening effects on the dissolution of whey protein gels at high pH conditions

The literature reports an optimum NaOH concentration for the alkaline cleaning of whey deposits or gels; at NaOH concentrations higher than this optimum, cleaning proceeds much more slowly. Although this phenomenon is of great importance in the cleaning of dairy equipment, no conclusive physical explanation has yet been presented. In this study, we present strong evidence that the dissolution rate is affected by the equilibrium-swelling ratio in β-lactoglobulin (βLg) gels. The swelling ratio is greatly reduced in the presence of salts due to the polyelectrolyte screening effect of the cations. This has been observed in free-swelling βLg gels using gravimetrical analysis and in the uniaxial swelling of WPC gel deposits using fluid dynamic gauging. At high dissolution pH (>13.3), the high Na⁺ concentration reduces swelling in spite of the high surface charge of the protein. It is proposed that the reduction of the free volume inside the gel impedes the transport of the protein aggregates out of the NaOH penetration zone. We have also observed that the final dissolution rate of gels pre-soaked in 1 M NaOH or NaCl is similar, despite the difference in pH, and much lower than for untreated gels: the high Na⁺ concentration in the soaked gels hinders swelling, inhibiting the disentanglement of the protein clusters regardless of the high pH.     

Quality characteristics of spent layer<Emphasis Type="Italic"> surimi</Emphasis> during frozen storage

The effects of different washing solutions and cryoprotectants on quality characteristics of spent layer surimi were studied during frozen storage at –18 °C. The highest (P<0.05) process yield was obtained with 0.5% sodium bicarbonate and 0.04 M sodium phosphate buffer solutions. While washing resulted in decreases (P<0.05) in protein, fat, cholesterol and ash contents, collagen and myofibrillar protein showed increases compared to the unwashed mince. A cryoprotectant mixture of 2% sucrose+2% sorbitol+0.3% sodium pyrophosphate resulted in higher water-holding capacity, except for surimi washed with 0.04 M sodium phosphate buffer solution, and lower cooking loss (P<0.05) than a cryoprotectant mixture of 4% sucrose+2.8% sorbitol. Increased lightness and decreased redness was observed as a result of washing with all solutions (P<0.05).     

Microstructure and rheology of β-lactoglobulin–galactomannan aqueous mixtures

The effect of the addition of a galactomannan (locust bean gum, LBG, or tara gum, TG) on the microstructure and rheological properties of a globular protein (β-lactoglobulin, β-Lg) solution was studied at pH 7.0, when the protein bears a net negative charge. Confocal laser scanning microscopy was used to explore the microstructure. Steady shear and dynamic oscillatory measurements were performed with a controlled stress rheometer AR2000 (TA Instruments) fitted with a cone-and-plate geometry. Mixtures were prepared with 6.5 wt% β-lactoglobulin concentration and 0.31–0.82 wt% LBG or 0.23–0.71 wt% TG concentration. All mixed systems were two-phase. The microstructure was clearly dependent on the concentration of the galactomannan in the mixture: the systems evolved from a continuous matrix of β-lactoglobulin enriched phase containing some small inclusions of the galactomannan, to a matrix of galactomannan-enriched continuous phase containing aggregates of β-lactoglobulin. Modifications of the flow and viscoelastic properties with respect to the individual components were clearly evidenced for the mixed systems. Phase inversion detected by microscopy could also be detected by rheology as a modification in the flow/viscoelastic behaviour.     

Impact of cosolvent (glucose) on the stabilization of ovalbumin

Proteins are stabilized by glucose against denaturation due to extremes of pH. This was studied by means of density, ultrasonic velocity, viscosity and surface tension measurements in the ovalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12). Few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, the partial apparent specific volume and the partial apparent specific adiabatic compressibility were calculated for the said systems. Obtained results suggest that the stabilization of ovalbumin occurs in the presence of glucose through strengthening of hydrophobic interactions supported by other non-covalent interactions and the steric exclusion effect of the cosolvent molecules.     

Cold-set thickening mechanism of β-lactoglobulin at low pH: Concentration effects

There is an interest in developing protein based thickening agents for nutritional considerations. A procedure to convert whey protein concentrates or isolates into a pH modified cold-thickening ingredient was developed. Concentration effects on thickening mechanism of this whey protein ingredient were studied with a β-lactoglobulin model system at the pH of the modification procedure, 3.35. In this study, concentration effects on thermal aggregation of β-lactoglobulin were studied at low pH using capillary and rotational viscometry, transmission electron microscopy (TEM), and high performance liquid chromatography coupled with multi-angle laser light scattering (HPLC-MALS). From the results of capillary viscometry, a critical concentration (C_c  6.9% w/w) was identified below which no significant thickening functionality could be achieved. Microscopy revealed formation of flexible fibrillar network at pH 3.35 during heating at all concentrations. These flexible fibrils had a diameter of about 5 nm and persistence length of about 35 nm as compared to more linear and stiff fibrils formed at pH 2 and low ionic strength conditions. Under similar heating conditions at concentration above C_c, larger aggregates similar to microgels were observed compared to the concentration below C_c, where isolated fibrils with an average contour length of about 130 nm were observed. These microgels and apparently stronger interactions between aggregates at concentrations above C_c were seemingly responsible for thickening functionality of heated β-lactoglobulin solutions and subsequently modified powders. Further investigation of β-lactoglobulin aggregation at this pH may provide capability to mechanistically tailor the functional attributes of modified ingredients.     

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Factors influencing the production of o/w emulsions stabilized by β-lactoglobulin–pectin membranes

A primary emulsion was prepared by homogenizing 10 wt% corn oil with 90 wt% aqueous β-lactoglobulin solution (0.5 wt% β-lg, pH 3 or 7) using a two-stage high-pressure valve homogenizer. This emulsion was mixed with aqueous pectin (citrus, 59% DE) stock solution (2 wt%, pH 3 or 7) and NaCl solution to yield secondary emulsions with 5 wt% corn oil, 0.225 wt% β-lactoglobulin, 0.2 wt% pectin and 0 or 100 mM NaCl. The final pH of the emulsions was then adjusted (3–8). Primary and secondary emulsions were ultrasonically treated (30 s, 20 kHz, 40% amplitude) to disrupt any flocculated droplets. Secondary emulsions were more stable than primary emulsions at intermediate pHs. Secondary emulsions prepared at pH 7 had smaller particle diameters (0.35 to 6 μm) than those prepared at pH 3 (0.42 to 18 μm) across the whole pH range studied, and also had smaller diameters than the primary emulsions (0.35 to 14 μm). Ultrasound treatment reduced the particle diameter of both primary and secondary emulsions and lowered the rate of creaming. The presence of NaCl screened the charges and thus the electrostatic interaction between biopolymer molecules and primary emulsion droplets. Secondary emulsions were more stable to the presence of 100 mM NaCl at low pHs (3–4) than primary emulsions. This study shows that stable emulsions can be prepared by engineering their interfacial membranes using the electrostatic interaction of natural biopolymers, especially at intermediate pHs where proteins normally fail to function.     

Response surface methodology for optimization of extraction yield, viscosity, hue and emulsion stability of mucilage extracted from Lepidium perfoliatum seeds

Response surface methodology was used to determine the optimum processing conditions that give maximum extraction yield, viscosity, hue and emulsion stability, as well as, minimum protein content for the gum extracted from Lepidium perfoliatum seed. Temperature (45–75 °C), processing time (1.5–3.5h), pH (5–8) and water to seed ratio (30:1–60:1) were the factors investigated. Experiments were designed according to Central Composite Rotatable Design with these four factors, including central and axial points. For each response, a second-order polynomial model was developed using multiple linear regression analysis. Applying desirability function method, optimum operating conditions were found to be extraction temperature of 48.1 °C, pH of 8, water to seed ratio of 30:1 and process time of 1.5 h. At this optimum point, extraction yield, viscosity, protein content, hue and emulsion stability were found to be 17.36%, 463.07 mPa s, 2.84%, 60.47 and 88.96 %, respectively.     

Sweet Basil (Ocimum basilicum) Extracts Obtained by Supercritical Fluid Extraction (SFE): Global Yields, Chemical Composition, Antioxidant Activity, and Estimation of the Cost of Manufacturing

In this work, supercritical technology was used to obtain extracts from Ocimum basilicum (sweet basil) with CO₂ and the cosolvent H₂O at 1, 10, and 20% (w/w). The raw material was obtained from hydroponic cultivation. The extract’s global yield isotherms, chemical compositions, antioxidant activity, and cost of manufacturing were determined. The extraction assays were done for pressures of 10 to 30 MPa at 303 to 323 K. The identification of the compounds present in the extracts was made by GC-MS and ESI-MS. The antioxidant activity of extracts was determined using the coupled reaction of beta-carotene and linolenic acid. At 1% of cosolvent, the largest global yield was obtained at 10 MPa and 303 K (2%, dry basis—d.b.); at 10% of cosolvent the largest global yield was obtained at 10 and 15 MPa (11%, d.b.), and at 20% of cosolvent the largest global yield was detected at 30 MPa and 303 K (24%, d.b.). The main components identified in the extracts were eugenol, germacrene-d, epi–alpha–cadinol, malic acid, tartaric acid, ramnose, caffeic acid, quinic acid, kaempferol, caffeoylquinic acid, and kaempferol 3-O-glucoside. Sweet basil extracts exhibited high antioxidant activity compared to beta-carotene. Three types of SFE extracts from sweet basil were produced, for which the estimated cost of manufacturing (class 5 type) varied from US$ 47.96 to US$ 1,049.58 per kilogram of dry extract.     

Formation of biopolymer particles by thermal treatment of β-lactoglobulin–pectin complexes

The purpose of this study was to prepare and characterize biopolymer particles based on thermal treatment of protein–polysaccharide electrostatic complexes formed from a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin). Initially, the optimum pH and pectin concentration for forming protein–polysaccharide complexes were established by mixing 0.5 wt% β-lactoglobulin solutions with beet pectin (0–0.5 wt%) at different pH values (3–7). Biopolymer complexes in the sub-micron size range (d = 100–300 nm) were formed at pH 5.0 and 0.1 wt% pectin. These particles were then subjected to a thermal treatment (30–90 °C at 0.8 °C min⁻¹). The presence of pectin increased the thermal aggregation temperature of the protein, although aggregate formation was still observed when the protein–polysaccharide systems were heated above about 70 °C. The impact of pH (3–7) on the properties of heat-treated biopolymer particles (83 °C, 15 min, pH 5) was then established. The biopolymer particles were stable to aggregation over a range of pH values, which increased as the amount of pectin was increased. The biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components, or as substitutes for lipid droplets.     

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO₂, ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO₂ in comparison to the other strains of Dekkera. These strains were inoculated (10³–10⁴ cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 °C and 21 °C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10⁵ cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 °C while slower growth was observed at 15 °C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 °C compared to 21 °C.     

Factors that determine the fracture properties and microstructure of globular protein gels

Protein gel matrices are responsible for the texture of many foods. Therefore an understanding of the chemical reactions and physical processes associated with fracture properties of gels provides a fundamental understanding of select mechanical properties associated with texture. Globular proteins form thermally induced gels that are classified as fine-stranded, mixed or particulate, based on the protein network appearance. The fundamental properties of true shear stress and true shear strain at fracture, used to describe the physical properties of gels, depend on the gel network. Type and amount of mineral salt in whey protein and β-lactoglobulin protein dispersions determines the type of thermally induced gel matrix that forms, and thus its fracture properties. A fine-stranded matrix is formed when protein suspensions contain monovalent cation (Li⁺, K⁺, Rb⁺, Cs⁺) chlorides, sodium sulfate or sodium phosphate at ionic strengths ≤0.1 mol/dm³. This matrix has a well-defined network structure, and varies in stress and strain at fracture at different salt concentrations. At ionic strengths >0.1 mol/dm³ the matrix becomes mixed. This network appears as a combination of fine strands and spherical aggregates, and has high stress values and minimum strain values at fracture. Higher concentrations of monovalent cation salts cause the formation of particulate gels, which are high in stress and strain at fracture. The salt concentration required to change microstructure depends on the salt's position in the Hofmeister series. The formation of a particulate matrix also occurs when protein suspensions contain low concentrations (10–20 mol/dm³) of divalent cation (Ca²⁺, Mg²⁺, Ba²⁺) chloride salts or di-cationic 1,6-hexanediamine at pH 7.0. The divalent cation effect on β-lactoglobulin gelation is associated with minor changes in tertiary structure involving amide—amide interproton connectivities (determined by ¹H NMR) at 40–45°C, increasing hydrophobicity and intermolecular aggregation. The type of matrix formed appears to be related to the dispersed or aggregated state of proteins prior to denaturation. Mixed and particulate matrices result from conditions which favor aggregation at temperatures (25–45°C) which are much lower than the denaturation temperature (~65°C). Therefore, general (e.g. Hofmeister series) and protein-specific factors can affect the dispersibility of proteins and thereby determine the microstructure and fracture properties of globular protein gels.