Ca2+-Induced Gelation of Pre-heated Whey Protein Isolate

Addition of CaCl₂ to pre-heated whey protein isolate (WPI) suspensions caused an increase in turbidity when pre-heating temperatures were ≥ 64°C. Pre-heating to ≥ 70°C was required for gelation. WPI suspensions which contained CaCl₂ became turbid at 45°C and formed thermally induced gels at 66°C. Thermally and Ca²⁺-induced gels showed significant time/temperature effects but the penetration force values in the Ca²⁺-induced gels were always lower. However, Ca²⁺-induced gels were higher in shear stress at fracture. The Ca²⁺-induced gels had a fine-stranded protein matrix that was more transparent than the thermally induced gels, which showed a particulate microstructure.     

Effects of thiol reagents and ethanol on strength of whey protein gels

Chemical reagents which interfere with secondary interactions affected gel network formation and the properties of gels made from 20% whey protein isolates. Thus, p-hydroxymercuribenzoate and N-ethylmaleimide 0–16 mmol/dm³ and dithiothreitol 0–32 mmol/dm³ reduced the hardness and cohesiveness of whey protein gels apparently by interfering with formation of disulfide bonds. The addition of ethanol (0–15%) increased the hardness of gels presumably by enhancing electrostatic interactions and hydrogen bonding.     

Shear-induced structuring of particulate whey protein gels

The effects of steady shear on particulate whey protein isolate (WPI) gels, at pH 5.4, have been investigated by light microscopy (LM) and dynamic oscillatory measurements. The steady shear was performed on suspensions at constant rates between 0.5 and 126/s. The gel point under static conditions (T_g) was around 78 °C and the shearing was performed during heating from 20 to 76 or to 82 °C. The gel point was postponed by the shear up to 82 °C. Steady shear up to 76 °C, at rates less than 6/s, resulted in a weaker storage modulus (G′), less frequency dependence and a higher stress at fracture compared to the unsheared gel. Steady shear up to 82 °C, at rates below 6/s, resulted in the formation of two different types of network structure. One structure was similar in appearance to the unsheared network, showing pores in the range of 50 μm. The other structure was dense, composed of smaller particles than the unsheared network and with pores in the range of 10 μm. The gels composed of two structures showed a lower G′ and stress at fracture compared to the unsheared gel. A shear rate above 24/s up to 76 °C resulted in irregular networks, which were composed of two different types of structures. One was loose and open, similar in appearance to the unsheared network structure. The other structure was dense and compact, and was present as individual aggregates. These gels also showed a weaker G′ than the unsheared gel. A shearing up to 82 °C at rates above 24/s resulted in a coarse, inhomogeneous network structure. The gels showed a weak G′, indicating aggregate break-up during the steady shear.     

Effects of drying on the gel strength and cation mobility of furcellaran

Thermal stability, by means of air drying a furcellaran powder, and its impact on gel strength and cation mobility were studied. Halogen heating in the temperature range 90–115°C for 15 min resulted in loss on drying (L_D, %). These results can be described by polynom L_D=−9.583+2.989τ−0.249τ²+0.00729τ³+0.1034t (R²=0.9976), indicating a gradual decomposition of carbohydrates. Air-drying induced a decrease in gel strength and the partial removal of potassium, calcium and sodium ions from the matrix. Air drying above 115°C yielded a remarkable destruction of polysaccharides with a total collapse in gelling power.     

Aerated whey protein gels as a controlled release system of creatine investigated in an artificial stomach

A controlled creatine-release system has been developed from whey protein-based gels. Their functionalization was carried out by aeration and sodium ions induced “cold gelation” processes. The effect of protein concentration in the aerated whey protein gels at pH 7.0 and 8.0 was analyzed. Physicochemical properties of the aerated gels were evaluated. It was possible to obtain the ions induced whey protein aerated gel with well distributed creatine and different microstructure as well as rheological properties. Different protein concentrations and pH enabled obtaining gels with different rheological properties, texture, air fraction, diameter of air bubbles, microstructure and surface roughness. An increase in the protein concentration enhanced the hardness of the samples, regardless of their pH. The mechanical strength of gels prepared at pH 8 were higher than those obtained at pH 7, as was manifested by the smaller storage modulus of the latter. The former gel exhibited a microstructure between particulate and fine-stranded. A stronger gel matrix produced smaller air bubbles. Aerated gels produced at pH 7.0 had higher roughness than those obtained at pH 8.0. Optimal conditions for inclusion of air bubbles into the gel matrix were: 9% protein concentration at pH 8.0 and this aerated gel was selected for digestion in the artificial stomach. There is a small conversion of creatine to creatinine in the artificial stomach digestion process (9.6% after 6 h). The diffusion of creatine crystals from the aerated gel matrix was the mechanism responsible for the release process. Aerated whey protein gels can be used as matrices for time extended releasing of creatine in the stomach.     

Structural properties of single and mixed milk/soya protein systems

Single-component gels were prepared by cold-setting aqueous preparations of thermally processed milk and soya proteins. Small deformation mechanical measurements on soya protein samples showed a strong elastic response (G′) even at the hydration temperature (50°C). Both proteins produced an initial monotonie increase in G′ on cooling, followed by a relatively constant modulus during a subsequent time sweep at the setting temperature (5°C). Networks were fully reversible on heating; the milk protein gels melting out completely at temperatures >60°C, whereas the soya protein gels maintained significant structure even at the highest accessible temperature (95°C). The lack of thermal hysteresis or of sharp, cooperative melting was also confirmed by differential scanning calorimetry. Further investigation of the macromolecular properties of the gels, comprising G′ dependence as a function of frequency of oscillation and creep experiments, suggests that gels remain stable within the time scale of the measurements (90 min). Finally, under increasing amplitude of oscillation, networks withstood structural breakdown up to strain levels of ~70%; behaviour anticipated for biopolymer gels. Mixed gels were studied using a fixed amount of milk protein (10% w/w) with soya protein concentrations from 6 (minimum gelling requirement) to 16% w/w (solubility limit). Comparison of melting profiles (G′ vs. T) for the phase separated systems with those obtained for the individual components indicated phase-inversion from a milk protein continuous network to a soya continuous system at a soya protein concentration of ~11%. Analysis of solvent partition between the constituent phases utilized classical theory of network deswelling for polymer combinations below the phase inversion point and phase equilibria treatment for the soya continuous network with milk protein inclusions. In the case of equilibrium separation of the two components, results were expressed in terms of a single adjustable parameter, p (the ratio of solvent to polymer in one phase divided by the corresponding ratio in the other phase), indicating a soya hydrophilicity of ~1.25 times that of milk protein.     

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.     

Formation of Elastic Whey Protein Gels at Low pH by Acid Equilibration

Abstract: Whey protein gels have a weak/brittle texture when formed at pH ≤ 4.5, yet this pH is required to produce a high-protein, shelf-stable product. We investigated if gels could be made under conditions that produced strong/elastic textural properties then adjusted to pH ≤ 4.5 and maintain textural properties. Gels were initially formed at 15% w/w protein (pH 7.5). Equilibration in acid solutions caused gel swelling and lowered pH because of the diffusion of water and H⁺ into the gels. The type and concentration of acid, and presence of other ions, in the equilibrating solutions influenced pH, swelling ratio, and fracture properties of the gels. Swelling of gels decreased fracture stress (because of decreased protein network density) but caused little change to fracture strain, thus maintaining a desirable strong/elastic fracture pattern. We have shown that whey protein isolate gels can be made at pH ≤ 4.5 with a strong/elastic fracture pattern and the magnitude of this pattern can be altered by varying the acid type, acid concentration, pH of equilibrating solution, and equilibrating time. Practical Application: Low-pH shelf-stable whey protein gels having the strong/elastic texture can be made by forming gels at high pH and equilibrating in acid solutions. Acid equilibration causes the gel to swell and lower the gel pH. Moreover, gel properties can be altered by varying the acid type, acid concentration, pH of equilibrating solution, and equilibrating time.     

Gel formation by β-conglycinin and glycinin and their mixtures

Gel formation and gel properties of β-conglycinin, glycinin and their mixtures were studied as a function of pH using small and large deformation rheology and differential scanning calorimetry. We conclude that heat denaturation is a prerequisite for gel formation. Gelation temperatures of β-conglycinin were lower than those of glycinin and more dependent on protein concentration. At pH 7.6, protein solutions gelled at a higher temperature than at pH 3.8.Glycinin gels were stiffer than β-conglycinin gels at the same pH and protein concentration, and fractured at a higher strain and stress. At pH 7.6, G′ is lower than at pH 3.8 for both proteins and the gels could be deformed to a larger extent. Based on the appearance of the gels (turbid at pH 7.6, white at pH 3.8) and the fracture properties, we conclude that different network structures are formed as a function of pH. The reason why glycinin gives a better gel than β-conglycinin is believed to be due to a difference in network structure as well as in strength of interaction between the protein molecules.Mixing of both soy proteins resulted in improved gelling properties at pH 3.8. The elastic modulus of the mixture was larger than the weighed sum of the separate contributions. Furthermore, mixing reduced the protein dispersability at pH 7.6. This strongly indicates the presence of an interaction between the proteins. Gels of the 1:1 mixture (pH 3.8) had a fracture stress and strain in between those of the gels of the separate proteins.     

In situ tensile deformation of zein films with plasticizers and filler materials

Material deformation is a dynamic process. Visualisation of this deformation can help to understand the local deformation and fracture behaviour. Zein (the prolamin protein from maize) films with different amount of plasticizers (0–25%) and different filler materials (maize oil, Dimodan^®, Vestosint^®, at 25% (w/w) to protein) were deformed under tension and observed at micron scale in real time by a confocal laser scanning microscope (CLSM). The addition of plasticizers increased strain and decreased stress of zein films. At low level of plasticizers (6.25% and 12%), zein films deformed and fracture through micro-crack formation and propagation normal the tensile axis. At high Plasticization, only micro-pores were observed during tensile deformation. The filler material oil and Dimodan^® increased, but Vestosint^® decreased tensile strain in comparison to the control. This shows that the fracture dynamic is affected by the filler materials and is indeed observed by the CLSM. Analysis of local strain by Fluospheres^® as particle tracking showed a good linear correlation with the tensile strain of the plasticized zein films. The local strains of filler materials and zein matrix in the films were different from the overall tensile strain. The combination of CLSM with a fluospheres^® as particle tracking is a good method to study local deformation in biomaterials to understand the deformation and fracture behaviour of biomaterials.     

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.     

Deformation and fracture of emulsion-filled gels: Effect of oil content and deformation speed

The large deformation properties of gelatine, κ-carrageenan and whey protein isolate (WPI) gels filled with bound and unbound oil droplets were studied as a function of compression speed. The rheological properties of the gel matrices controlled the compression speed-dependency of the gels containing oil droplets. Polymer gels (gelatine and κ-carrageenan gels) showed a predominantly elastic behaviour. Their Young's modulus was not affected by the compression speed. The increase of fracture stress and strain observed with increasing compression speed was related to friction between the structural elements of the gels and, for gelatine, to the unzipping of physical bonds. Particle gels (WPI gels) showed a more viscoelastic behavior. Their Young's modulus and fracture stress increased with compression speed. This was attributed to the viscous flow of the matrix and friction phenomena between structural elements of the gel. The effect of an increase in the oil volume fraction (φ) on the Young's modulus was for all gels according to the Van der Poel theory. In addition, oil droplets embedded in the gel matrix acted as stress concentration nuclei and increased friction. The relative impact of these two effects was related to the viscoelastic properties of the gels and to droplet–matrix interaction. For polymer gels and gels with bound droplets, stress concentration phenomena played a relatively larger role. For particle gels and gels with unbound droplets, friction phenomena were relatively more important, increasing the viscoelastic character of the gels. As a result, an increase in φ resulted in a decrease of both fracture stress and fracture strain for polymer gels and in an increase of the fracture stress and a decrease of fracture strain for particle gels.     

Monitoring the effects of divalent ions (Mn and Ca) in heat-set whey protein gels

Exploring the effects of cations in whey protein-based gels (WPG) is of importance when these gels are used for controlled release applications in food systems. The objective of this study was to evaluate both water uptake and cation release from heat-set WPGs. Magnetic Resonance Imaging and NMR relaxometry were employed to study the uptake and release. A non-paramagnetic (Ca⁺²) and a paramagnetic cation (Mn⁺²) were incorporated into the WPG as model divalent cations. Cylindrical pieces of WPGs with manganese were immersed in water (pH 2.40, 7.00, 10.40) or in EDTA solution whereas WPGs with calcium were immersed in water at pH 2.40. Water uptake by the gels was influenced by both ionic environment and pH. The release of Mn⁺² from WPG was enhanced by the presence of EDTA. Relaxation spectra of Mn⁺²-loaded gels were significantly influenced by pH of the suspending fluid and by the presence of EDTA. Results of relaxometry experiments, obtained with Ca⁺²-loaded gels immersed in water at pH 2.40, indicated a strong correlation (R² > 0.99) between relative areas of the proton pools and the amount of calcium released to the medium. Results support the use of MRI and NMR to monitor cation release and water uptake in WPG, non-destructively.     

Structure–fracture relationships in gas-filled gelatin gels

Food aeration has become one of the fastest growing unit operations practiced in the food industry. Dispersed air (or other gases) provides an additional phase within the gel that may accommodate new textural and functional demands. This paper addresses the relationships between structural characteristics and fracture properties of gas-filled gelatin gels (GGG), and compare these properties with those of control gelatin gels (CGG). Three gases were used in the fabrication of GGG: air, nitrogen and helium. Experimental methods to determine density, gas hold-up, bubble sizes and bubble size distributions as well as fracture properties of GGG are presented. Increasing protein concentration produced higher density, lower gas hold-up and decreased polydispersity of bubbles due to its effect on increased solution viscosity. Type of gas affected density and gas hold-up due to the different diffusivities of gases and structures (bubble size, size distribution and number of bubbles per area) formed in GGG. Fracture values increased for both GGG and CGG with increasing protein concentration for the three gases used. GGG were weaker and less ductile than CGG, the decrease in stress and strain at fracture being between 70 and 80%, and 40 and 65%, respectively. A power law relationship (σ_f = 2.73 × 10⁻¹²ρ_G^4.76) was found between the fracture stress and gel density for the three gases studied. This study shows that the presence of bubbles in gel-based food products results in unique textural properties conferred by the additional gaseous phase.     

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.     

ELECTROSTATIC EFFECTS ON PHYSICAL PROPERTIES OF PARTICULATE WHEY PROTEIN ISOLATE GELS

Physical properties of particulate whey protein isolate gels formed under varying electrostatic conditions were investigated using large strain rheological and microstructural techniques. The two treatment ranges evaluated were adjusting pH (5.2‐5.8) with no added NaCl and adjusting the NaCl (0.2‐0.6 M) at pH 7. Gels (10% protein w/v) were formed by heating at 80C for 30 min. The large strain properties of fracture strain (γ_f), fracture stress (σ_f), and a measure of strain hardening (R_0.3) were determined using a torsion method. Gel microstructure was evaluated using scanning electron microscopy (SEM) and gel permeability (B_gel). Overlaying σ_f and γ_f curves for pH and NaCl treatments demonstrated an overlap where gels of equal σ_f and γ_f could be formed by adjusting pH or NaCl concentration. The high fracture stress (σ_f～ 23 kPa and γ_f～ 1.86) pair conditions were pH 5.47 and 0.25 M NaCl, pH 7.0. The low fracture stress (σ_f～ 13 kPa and γ_f～ 1.90) pair conditions were pH 5.68 and 0.6 M NaCl, pH 7.0. The 0.25 M NaCl, pH 7 treatment demonstrated higher R_0.3 values than the pH 5.47 treatment. When the sulfhydryl blocker n‐ethylmaleimide was added at 2 mM to the 0.25 M NaCl, pH 7 gel treatment, its rheological behavior was NSD (p>0.05) to the pH 5.47 gel treatment, indicating disulfide bond formation regulated strain hardening. Altering surface charge or counterions, and disulfide bonding, was required to produce gels with similar large strain rheological properties. An increase in gel permeability coincided with an increase in pore size as observed by SEM, independent of rheological properties. This demonstrated that at the length scales investigated, microstructure was not linked to changes in large strain rheological properties.     

Inhibition of Clostridium sporogenes growth in mascarpone cheese by co-inoculation with Streptococcus thermophilus under conditions of temperature abuse

The ability of a biological control system to inhibit the outgrowth of Clostridium sporogenes spores during storage of mascarpone cheese under temperature-abuse conditions was investigated. Challenge studies were carried out on mascarpone cheese artificially contaminated with spores of C. sporogenes (10 cfu g⁻¹), and with or without the coinoculum of a Streptococcus thermophilus strain (10⁵cfu g⁻¹). During storage at 4, 12, and 25°C, the outgrowth of clostridia spores, the growth of S. thermophilus, and the pH changes were evaluated at 10, 20, 30, and 40 days. In mascarpone cheese stored at 4° and 12°C, S. thermophilus and C. sporogenes did not show any growth. The initial pH (6·14) of the product also remained unchanged. During storage at 25°C S. thermophilus grew up to about 10⁷cfu g⁻¹after 10 days, resulting in a pH decrease of mascarpone cheese to values close to 4·5. The cell number decreased progressively during storage reaching values near to 10¹cfu g⁻¹after 40 days, whereas product acidity remained constant. C. sporogenes, when inoculated alone, also grew at 25°C. The cell number increased to levels of about 10⁷cfu g⁻¹after 20–40 days of storage according to the different mascarpone cheese lots used. No growth was found when C. sporogenes was co-inoculated in mascarpone cheese with S. thermophilus and stored at 25°C. The study on the behaviour of C. sporogenes, known as a non-toxigenic variant of Clostridium botulinum, allowed us to obtain useful information for setting up an effective biological control system to inhibit growth of the toxigenic species as well. The use of an additional barrier, besides refrigerated storage, may help to maintain the safety of mascarpone cheese in the event it was exposed to elevated temperatures.     

Isothermal gelation of proteins. 1. Urea-induced gelation of whey proteins and their gelling mechanism

The changes in dynamic elastic moduli of whey proteins [whey protein isolates, β-lactoglobulin (B-Lg), α-lactalbumin (A-La) and bovine serum albumin (BSA)] at various concentrations in the presence of 8 molldm³ urea with time were measured at 25°C, because whey protein-urea systems set to gels automatically at room temperature without heating. From the time dependence behavior of elastic moduli for the proteins, the individual proteins were characterized as BSA having good, B-Lg intermediate and A-La poor urea-induced gelation. The disulfide bonds and hydrogen bonds played important roles in the formation the urea-induced gels.     

Ultrasonic generation of aerated gelatin gels stabilized by whey protein β-lactoglobulin

Dispersed air provides an additional phase within gel-type foods may accommodate new textural and functional demands. This paper addresses the effect of using whey protein β-lactoglobulin (β-lg), with different degrees of denaturation, as stabilizing agent in the formation of aerated gelatin gels using ultrasound as a novel method to incorporate bubbles in model foods. The heat denaturation, aggregate formation and surface properties of β-lg dispersions were studied at three pHs (6.0, 6.4 and 6.8) and at a heating temperature of 80 °C. β-Lg dispersions with four degrees of denaturation (0%, 20%, 40% and 60%) were used to stabilize bubbles generated by high intensity ultrasound in aerated gelatin gels. Experimental methods to determine gas hold-up, bubble size distributions and fracture properties of aerated gelatin gels stabilized by β-lg (AG), as well as control gels (CG), aerated gelatin gels without β-lg, are presented. Gas hold-up of AG peaked at a degree of denaturation of 40% when AG were fabricated using β-lg heated at pH 6.4 and 6.8, whereas using β-lg heated at pH 6.0 gas hold-up decreased constantly with increasing degree of denaturation. The use of β-lg as surfactant at pH 6.8 and 6.4 reduced the bubble sizes of AG compared with CG, but no effect was observed at pH 6.0. AG showed values of stress and strain at fracture lower than CG (5.86 kPa and 0.62), probably because of the lower gas hold-up of CG. However, both type of aerated gels were weaker and less ductile than non-aerated gels, with a decrease in stress and strain at fracture for AG between 56–71% and 33–43%, respectively. This study shows that the presence of bubbles in gel-based food products results in unique rheological properties conferred by the additional gaseous phase.     

A comparative study of heat and high pressure induced gels of whey and egg albumen proteins and their binary mixtures

Large deformation rheological studies of either egg albumen or whey protein isolate (15% protein w/w) gels induced by heating at 90 °C for 30 min were compared to those induced by a range high pressures (400–800 MPa for 20 min). Gels made by heating indicated higher gel strength and Young's modulus values for whey protein from pressures of 400–600 MPa for 20 min but similar values at 650–800 MPa. In contrast, egg albumen showed no gelation below 500 MPa for 20 min, but there was an increase in both gel strength and Young's modulus with increasing pressure, although values remained lower than those of the heat-induced gels. A mixture of 10:5 whey/egg albumen showed the highest gel strength and Young's modulus for both heated and high pressure-treated (400–600 MPa) gels, although, the heated mixture had the highest values. Electron micrographs indicated that high pressure-treated gels had a porous aggregated network for egg albumen while whey proteins showed a continuous fine stranded network. The heated mixtures of whey:egg albumen (7.5:7.5) showed large dense aggregates whereas high pressure-treated mixtures produced smaller aggregates. Raman spectroscopy of both heated and high pressure-treated whey and egg albumen (15% w/w in D₂O pD7) and their binary mixtures (7.5:7.5, protein w/w) indicated changes in β-sheet structures in the Amide 111′ region (980–990 cm⁻¹); however, peak intensity was reduced for high pressure-treated samples. β-Sheet structure (1238–1240 cm⁻¹) present in heated whey was absent in high pressure-treated whey and in egg albumen. Involvement of hydrophobic regions was reflected by changes in the CH (1350 cm⁻¹) and CH₂ (1450 cm⁻¹) bending vibrations. In addition to the Trp residues at 760 cm⁻¹, there were broad peaks at 874–880 cm⁻¹ and tyrosine 1207 cm⁻¹ in the high pressure-treated samples. Disulphide bands (500–540 cm⁻¹) in heated whey and egg albumen proteins showed higher peak intensities compared to high pressure-treated samples. Differences in the experimental and theoretical spectra indicated changes in the hydrophobic regions, tyrosine (1207 cm⁻¹) and tryptophan (880 cm⁻¹) and CH₂ bending in high pressure-treated samples, whereas heated samples indicated marked changes in β-sheet structures (987 and 1238 cm⁻¹) as well as hydrophobic regions CH (1350 cm⁻¹) and CH₂ (1450 cm⁻¹) bending vibrations.