Whey Protein Isolate Aggregates Research Articles

Fabrication of Whey Protein Isolate-Pectin Nanoparticles by Thermal Treatment: Effect of Dynamic High-Pressure Treatment.

This study investigated the impact of dynamic high-pressure (DHP) treatment on the ability of whey protein isolate (WPI) to form associative complexes with pectin and to form aggregate particles after their subsequent heat treatment. Light scattering showed that DHP treatments disrupted preexisting WPI aggregates and assembled pectin chains. Complexes formed from WPI/pectin mixtures at pH 4.5 were an order of magnitude smaller when formed after DHP treatment, regardless of the degree of esterification. WPI/pectin complexes formed after DHP treatment were more stable against subsequent pH neutralization than complexes formed without DHP treatment, and WPI/high-methoxyl pectin (HMP) complexes had greater stability than WPI/low-methoxyl pectin (LMP) complexes. WPI/pectin particles prepared by thermal treatment of complexes at pH 4.5 were also smaller when prepared after DHP treatment. WPI/HMP particles were stable to subsequent pH neutralization, while WPI/LMP particles became larger after neutralization.

Effect of chitosan and chitosan oligosaccharide on the interaction and in vitro gastric digestive behavior of whey protein isolate

Carbohydrate-protein interactions can improve the properties and texture of proteins. The objective of this study was to investigate the effects of pH and preheating to 90 °C on the interaction between chitosan (CS)/chitosan oligosaccharide (COS) and whey protein isolate (WPI). Additionally, by simulating in vitro gastric digestion to evaluate the digestive behavior of the CS/COS-WPI complexes when applied to protein-supplemented beverages. The results demonstrated that only CS exhibited greater efficacy in preventing the denaturation and aggregation of WPI caused by heat treatment at pH 4.0. As pH continued to expand to 6.0, changes in the strength of electrostatic interactions and the tertiary structure of proteins influenced the formation of the complex system structure. The in vitro digestion experiments revealed that CS and COS inhibition of WPI digestion in the gastric resulted in an opposite trend with an increasing ratio. We hope that this work will offer promising strategies for developing protein-enrich beverages.

Effects of Various Types of Vacuum Cold Plasma Treatment on the Chemical and Functional Properties of Whey Protein Isolate with a Focus on Interfacial Properties

Vacuum cold plasma (VCP), a novel non-thermal processing technology used to modify the physicochemical properties and functionalities of food materials, was applied to whey protein isolate (WPI). The treatment affects the protein chemistry and, as a result, leads to differences in the behavior in solution and at interfaces. To minimize the undesirable effects of high oxidation and to increase the effectiveness of reactive species, the VCP treatment was applied at low pressure using different types of gases (air, combination of argon and air, and sulfur hexafluoride (SF6)). The treatment led to a decrease in the sulfur content and an increase in the carbonyl content, evidenced by oxidation reactions and enhanced disulfide bond formation, as well as cross-linking of protein molecules. Fluorescence-based indicators suggest that the hydrophobicity of the proteins as well as their aggregation increase after VCP treatment with an argon–air gas mixture; however, it decreases after VCP treatments with air and SF6. The chemical modifications further lead to changes in the pH of aqueous WPI solutions, as well as the average size and ζ-potential of WPI aggregates. Moreover, the dynamic surface tension, surface dilational elasticity, and the thickness of the WPI adsorption layers at the air/water interface depend on the VCP type. SF6 plasma treatment leads to a significant decrease in pH and an increase in the ζ-potential, and consequently to a significant increase in the aggregate size. The dynamic surface tension as well as the adsorption rates increase after SF6VCP treatment, but decrease after air–VCP and argon–air–VCP treatments. The adsorbed WPI aggregates form strong viscoelastic interfacial layers, the thickness of which depends on the type of VCP treatment.

Impacts of the Dynamic High-Pressure Pre-Treatment and Post-Treatment of Whey Protein Aggregates on Their Physicochemical Properties and Emulsifying Activities.

The impacts of dynamic high-pressure (DHP) pretreatment and post-treatment (100 MPa) on the physicochemical and functional properties of whey protein isolate (WPI) aggregates formed by thermal treatment were investigated in this study. When WPI aggregates were formed by thermal treatment, the size of the aggregates formed with the DHP pretreated WPI was smaller than that of the aggregates formed with the original WPI. The size of the WPI aggregates formed by thermal treatment decreased with DHP post-treatment. The conformational parameters (ζ-potential, surface hydrophobicity, and intrinsic fluorescence intensity) of the WPI subjected to DHP pretreatment were not significantly influenced by thermal treatment. However, DHP post-treatment affected these parameters for the WPI aggregates formed during thermal treatment because of dissociation caused by intense shear and cavitation forces during DHP treatment. The emulsifying activity index (EAI) of the WPI aggregates slightly improved with DHP treatment, but its order had little effect on the magnitude of the EAI increase. DHP pretreatment or post-treatment can modulate the conformational structures and the physicochemical properties of protein aggregates.

The Effects of Ethanol and Rutin on the Structure and Gel Properties of Whey Protein Isolate and Related Mechanisms

The effects of different levels of rutin (0, 0.05%, 0.1%, 0.2% and 0.3% w/v) and ethanol on the structure and gel properties of whey protein isolate (WPI) were examined. The results showed that the addition of ethanol promoted the gel formation of WPI. The addition of rutin increased the gel strength of WPI and maintained the water-holding capacity of the gel. Ethanol caused an increase in thiol content and surface hydrophobicity, but rutin decreased the thiol content and surface hydrophobicity of WPI. The particle size, viscosity and viscoelasticity of WPI increased at rutin levels of 0.2% and 0.3%, indicating that rutin caused cross-linking and aggregation of WPI, but rutin had no significant effect on the zeta-potential, indicating that electrostatic interactions were not the main force causing the changes in protein conformation and gel properties. Ethanol and rutin improved the gel properties of WPI possibly by inducing cross-linking of WPIs via hydrophobic and covalent interactions.

Physicochemical properties and formation mechanism of whey protein isolate-sodium alginate complexes: Experimental and computational study

The study explored the interaction mechanism between whey protein isolate and polysaccharide at the molecular level. Firstly, the effect of pH on the formation of whey protein isolate-sodium alginate complex was investigated. The particle size and turbidity were reduced in the presence of sodium alginate over a wide pH range. They were remarkably decreased (by 65% and 68.5%, respectively) at pH 5.0 (near the isoelectric point), which was confirmed by fluorescence microscopy. Raman spectroscopy results showed that the presence of sodium alginate resulted in more compact structure (α-helix content increased by 7.53% at pH 5.0) of whey protein isolate at pH 5.0. Then, the interaction and formation mechanism of the complex at the molecular level were explored by computational approaches. Molecular docking study showed that the amino acid residues of β-lactoglobulin formed a complex with –COOH and –OH of sodium alginate. Molecular dynamics simulation analysis illustrated that the binding energy (−338.17 kJ/mol) of the complex was relatively low at pH 5.0, indicating that β-lactoglobulin and sodium alginate had relatively strong binding capacity and stability. Electrostatic interactions which belong to non-bonding contribution played a major role in the formation of β-lactoglobulin-sodium alginate complex. This study provided theoretical basis for further research on the interactions between protein and polysaccharide, improving the stability of protein in acidic food. • Alginate inhibited the aggregation of whey protein isolate near the isoelectric point. • Spectroscopic and molecular dynamics simulation approaches were used. • Relatively strong binding of sodium alginate to whey protein at pH 5.0 • Electrostatic forces dominated the binding of sodium alginate to whey protein isolate.

Effect of low-methoxy pectin on interfacial and emulsion stabilizing properties of heated whey protein isolate (WPI) aggregates

• QCM-D study of heated WPI and heated biopolymer complexes on hydrophobic surface. • A higher ratio of pectin to WPI increased the adsorbed mass on the surface. • A higher ratio of pectin to WPI decreased the protein load from 15.11 to 9.19 mg/m 2 . • Interfacial properties of the heated WPI-LMP affected the emulsion stability. In this study, we have investigated the interfacial and emulsion stabilizing properties of heat-induced whey protein isolate (WPI) aggregates prepared in the presence or absence of low-methoxy pectin (LMP). Sub-micron complex particles were formed by heating of oppositely charged WPI and LMP at different WPI:LMP mass ratios. The Quartz Crystal Microbalance with Dissipation (QCM-D) technique was used to better understand the adsorption properties of these biopolymeric particles at a model hydrophobic surface. Clear correlations were observed between the interfacial properties of WPI and WPI:LMP aggregates and the resulting emulsion characteristics. Compared to WPI aggregates (which led to emulsions containing larger flocculated droplets), the heated complexes (formed at a higher ratio of LMP to WPI) were highly efficient as Pickering stabilizers leading to emulsions with smaller droplets (5.5–6.8 μm). Cryo-SEM analysis clearly revealed the manipulation of the emulsions’ interfacial microstructure due to the adsorption of a WPI:LMP inter-polymeric film onto the surface of the droplets. The effectiveness of the colloidal particles in stabilizing emulsions depended largely on the formation of a sufficiently ‘dense layer’ of particles at the oil-water interface where these properties were controlled by the composition of WPI and LMP at the oil droplet surface. Moreover, the emulsion physical stability was greatly influenced by the formation of an inter-connected film at the oil droplet interface, whereby these viscoelastic films increased the steric hindrance against coalescence of emulsion droplets. This work provides the mechanism of emulsion stabilization by WPI particles formed by heating in the absence or presence of pectin.

Morphological and physicochemical properties of rice starch dry heated with whey protein isolate

The effect of dry heating treatment with whey protein isolate (WPI) on the morphological and physicochemical properties of rice starch (RS) was investigated. Scanning electron microscopy results demonstrated that WPI-mediated aggregation of RS granules was more pronounced as the dry heating time increased, which was further confirmed by analysis of the particle size distribution. Differential scanning calorimetry results showed that dry heating treatment in the presence of WPI significantly decreased the gelatinization temperature of RS but without affecting the gelatinization enthalpy change. X-ray diffraction and Fourier transform infrared spectroscopy analysis demonstrated that the long and short range orders of RS were partially damaged following dry heating treatment and the incorporation of WPI. In addition, a significant increase in the oil-binding ability was observed in the dry heated starch samples, which was more pronounced under the combination of dry heating with WPI. These phenomena could be attributed to the interaction of RS with WPI, the aggregation and denaturation of WPI, and the disruption of hydrogen bonds within starch during the treatment process.

Influence of high-pressure homogenization on structural properties and enzymatic hydrolysis of milk proteins

This study investigated the effects of HPH processing parameters on the conformational structure of bovine serum albumin (BSA) and whey protein isolate (WPI).BSA and WPI dispersions (1% w/v in phosphate buffer, pH = 7.5) were treated in a bench-scale HPH unit at different pressures (100, 150, 200 MPa) and number of passes (1, 2, 3, 5). The modifications of proteins' primary structure (carbonyl groups), secondary structure (α-helix, β-sheet, turn), tertiary and quaternary structure (free -SH groups) were analyzed together with their particle size distribution (PSD). HPH-assisted hydrolysis with trypsin and α-chymotrypsin was performed (200 MPa, 1 pass). Hydrolysis degree and molecular weight distribution of peptides were determined and compared with those of untreated protein dispersions.HPH treatments did not affect proteins' primary structure while slight modifications of secondary structure were detected. Interestingly, free - SH groups in BSA increased with increasing the pressure due to partial unfolding, while decreased in WPI, possibly due to disaggregation and compaction of native WPI aggregates. HPH pre-treatment allowed enhancing BSA hydrolysis reaction rate, while the compaction of WPI aggregates caused a reduced hydrolysis extent. In conclusion, HPH caused an appreciable modification of protein conformation and affected the degree of enzymatic hydrolysis.

Nonlinear interfacial rheology and atomic force microscopy of air-water interfaces stabilized by whey protein beads and their constituents

In recent years, food-grade Pickering particles have gained considerable interest, because of their ability to form stable emulsions and foams. Such Pickering stabilizers are often produced by aggregation of proteins, which typically results in a mixture of cross-linked particles and unbound proteins (smaller constituents). This study focuses on the possible contribution to the interfacial behaviour of these smaller constituents in whey protein isolate (WPI) bead suspensions, which are produced by cold-gelation of WPI aggregates. To understand the interfacial properties of the total mixture, we have studied the involved structures and interactions hierarchically, from native WPI, to aggregates, and finally gel beads.Air-water interfaces were subjected to large amplitude oscillatory dilatation (LAOD) and shear (LAOS) using a drop tensiometer and a double wall ring geometry. The non-linear responses were analysed using Lissajous plots. The plots of native WPI- and aggregates-stabilized interfaces showed a rheological behaviour of a viscoelastic solid, while bead-stabilized interfaces tended to have a weaker and more fluid-like behaviour.The interfacial microstructure was analysed by imaging Langmuir-Blodgett films of the protein systems using atomic force microscopy (AFM). The native WPI and aggregate films had a highly heterogeneous structure in which the proteins form a dense clustered network. The beads are randomly distributed throughout the film, separated by large areas, where smaller proteinaceous material is present. This smaller and surface-active material present in the bead suspensions plays an important role in interface stabilization, and could also largely influence the macroscopic properties of interface-dominated systems.

Effect of cysteine addition and heat treatment on the properties and microstructure of a calcium-induced whey protein cold-set gel

A model gel of whey protein isolate (WPI) was prepared by cold gelation with calcium. This system was modified by the addition of free cysteine residues (Cys) at different steps of the process. The WPI cold-set gels obtained were then subjected to heat treatment at 90°C. First, the effect of Cys addition on the heat-induced aggregation of WPI was studied through Atomic Force Microscopy (AFM) and infrared spectroscopy (ATR-FTIR), while Cys' effect on cold gelation was observed by AFM, Confocal Laser Scanning Microscopy (CLSM) and oscillatory rheology (amplitude sweeps). The impact of heating on the microstructure and the viscoelastic properties of the WPI cold-set gels were finally investigated through several techniques, including DSC, ATR-FTIR, CLSM, cryo-SEM, and rheological measurements (temperature sweeps). When added during the first step of cold gelation, Cys modified heat-induced aggregation of WPI, resulting in the formation of a denser gel network with a fractal dimension (Df) of 2.8. However, the addition of Cys during the second step of cold gelation led to the formation of highly branched clusters of WPI and a looser gel network was observed (Df = 2.4). In this regard, the use and limitations of oscillatory rheology and the “Kraus model” to determine the Df of WPI cold-set gels was discussed. The viscoelastic properties and the microstructure of the WPI cold-set gels were irreversibly modified by heating. Gels were stiffer, more brittle, and coarser after heat treatment. New disulfide bonds and calcium bridges formed, as well as H-bonded β-sheets, all contributing to the formation of the final gel network structure.

Heat-induced gelation of mixtures of casein micelles with whey protein aggregates

Gelation of aqueous solutions of casein micelles (MC) in mixtures with fractal aggregates of whey protein isolate (WPI) has been studied first by addition of different amounts of aggregates at a fixed concentration of micelles and then by substitution of micelles with aggregates at a fixed total protein concentration. Addition of small amounts of aggregates (1–5 g/L) to suspensions of micelles (20–60 g/L) at a fixed pH led to a strong decrease of the gelation temperature (Tg) and to an increase of the elastic modulus (G′) of the mixed gels. A minimum in Tg and a maximum in G′ as a function of the WPI fraction were obtained in mixtures with fixed total protein concentration and fixed pH and explained by the opposing influence of increasing WPI aggregate concentration and decreasing concentration of micelles. Increasing the total protein concentration at the same WPI fraction caused a decrease of Tg and increase of G′. Decreasing the pH at the same protein composition led to a decrease of Tg but caused an increase of G′. Tg decreased sharply if a small amount of CaCl2 was added, but if more than about 30 mM of calcium was added, Tg increased again. Gelation of mixtures did not depend significantly on the size of the WPI aggregates, but if fractal aggregates were replaced by microgels, weaker gels were produced and the temperature of gelation was higher. Potentiometric titration curves showed that protonation of the micelles and of the WPI aggregates in the mixtures was independent. It is concluded that MC and WPI aggregates co-aggregate during gelation and there is a transition between gelation at a critical temperature for pure MC suspensions and gelation controlled by an activation energy for pure WPI aggregate suspensions.

Gelation of oil-in-water emulsions stabilized by heat-denatured and nanofibrillated whey proteins through ion bridging or citric acid-mediated cross-linking

This work addresses the fabrication and characterization of gels from emulsions stabilized by heat-denatured and nanofibrillated whey protein isolate (WPI) using citric acid and CaCl2 as cold-gelling agents. Nano-thick fibrils and wormlike aggregates were produced by heating of WPI at pH 2.0 and 8.0, respectively. Fibrillated WPI, heat-denatured WPI, and their mixture at a concentration of 65 mg mL−1 and pH 8.0 were used to stabilize oil-in-water emulsions. Emulsions stabilized by fibrillated WPI showed a higher creaming stability than samples stabilized by heat-denatured and mixed fibrillated/heat-denatured WPI. However, they were more polydisperse and had larger droplets. Gelation of emulsions by citric acid through the generation of covalent bonds was confirmed by OPA colorimetric method and FT-IR spectroscopy. Emulsion gels prepared by citric acid-mediated cross-linking were firmer than those formed by CaCl2 and also showed a higher ability to hold water and retain oil. Scanning electron microscopy also demonstrated a more open and porous structure for ion-induced emulsion gels compared to citric acid-induced counterparts. Generally, the results indicated that the citric acid as a new gelling agent and different forms of WPI aggregates can be used in the formulation of edible emulsion gels with modifiable functional properties.

Influence of pulsed light treatment on the aggregation of whey protein isolate

The effect of pulsed light (PL) on the aggregation of whey protein isolate (WPI) solutions was investigated. PL fluence values from 4 to 16J/cm2 were used to treat WPI (1% w/v) solutions in sodium phosphate buffer (pH=7.5). Whey protein structural modification and aggregation were assessed through the determination of free SH-groups and UV-absorption spectra. Additionally, covalent and non-covalently linked protein-protein interactions were identified through the measurement of turbidity, aggregation index, particle size distribution, and SDS-PAGE.WPI upon PL treatment showed structural changes as demonstrated by the immediate increase of free SH-group content (unfolding) and the subsequent formation of a small fraction of aggregation of unfolded proteins, due to both hydrophobic interactions and the formation of disulphide bonds. Turbidity, mean particle size, and aggregation index increased in samples treated at PL fluence from 4 to 16J/cm2. Furthermore, particle size distribution analysis of samples treated at higher fluence indicated that WPI dimer dissociation and formation of larger particles were likely to occur. The association of intermediate and larger protein molecules as well as the formation of soluble aggregates between β-lactoglobulin and α-lactalbumin were also observed in gel electrophoresis analysis. In conclusion, the results of this investigation demonstrated the potential of PL treatments to induce protein denaturation, with a minimal formation of soluble protein aggregates.

Interactions in protein mixtures. Part I: Second virial coefficients from osmometry

The interaction of proteins (β-lactoglobulin, Bovine Serum Albumin (BSA), gelatins and whey protein isolate (WPI)) in solution was quantified by measuring their second virial coefficient using membrane osmometry. At neutral pH below 20–40 mM ionic strength, electrostatic repulsion dominated the interaction. At higher ionic strength, BSA, WPI and whey protein aggregates (WPA) were well approximated as hard spheres. On the other hand β-lactoglobulin behaves as an adhesive hard sphere for which the stickiness parameter τ (known from the Baxter model for sticky hard spheres) and depth and width of the adhesive part of the interaction potential were calculated.

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.

Binding of retinyl acetate to whey proteins or phosphocasein micelles: Impact of pressure-processing on protein structural changes and ligand embedding

A whey protein isolate (WPI) and native phosphocaseins (PC) at pH6.5–6.6 were processed with retinyl acetate (RAC) using pressure-assisted technological tools to improve RAC embedding through processing-induced protein structural changes. To this end, protein-RAC dispersions were submitted to ultra-high pressure homogenisation (UHPH) at 300MPa and an initial fluid temperature (Tin) of 14°C or 24°C, or isostatic high-pressure at 300MPa and 14°C or 34°C for 15min. A short-time thermal treatment (STTT, 73°C for 4s) able to generate WPI aggregates was assessed for comparison. Processing effects were investigated in terms of protein particle sizes and molecular weights (Mw). Mw calculated using protein size determination obtained from light scattering measurements were in agreement with the known values. The amounts of RAC retained in WPI particles (unfolded and/or aggregated proteins) or in PC assemblies were quantitated after protein precipitation by ammonium sulphate. A 2.3–3.7nmol RAC was carried per mg of pressure-denatured whey proteins, significantly less than after STTT (6.3nmol RAC per mg of heat-denatured whey proteins) indicating that RAC embedding varied according to the technological tool, pressure or temperature. A 3.8–5.4nmol RAC was carried per mg of PC assemblies through pressure-induced dissociation/reassociation of PC micelles. Combined pressure and mild temperature increased RAC embedding in PC assemblies.

Design and characterization of soluble biopolymer complexes produced by electrostatic self-assembly of a whey protein isolate and sodium alginate

The aim of this study was to design and characterize soluble biopolymer complexes formed by self-assembly of an anionic polysaccharide (sodium alginate, SA) and a whey protein isolate (WPI). First, conditions for producing stable protein aggregates were studied by analysing the effect of concentration (2, 4, 6% w/w) and heating temperature (55, 70, 85 °C) on WPI structural characteristics, using spectroscopic techniques UV–Vis and fluorescence. As heat treatment of WPI at the highest temperature induced the formation of soluble protein aggregates with a greater exposure of hydrophobic patches on their surface, this condition was selected to form protein particles. Secondly, from aqueous solutions of 6% w/w heat-treated WPI at 85 °C, WPI/SA systems were obtained at different ratios (2:1, 4:1 and 6:1) and transmittance measurements as a function of pH (6.0–3.0) were made. Dynamic behaviour of biopolymers in WPI/SA systems was discussed in terms of transition pHs, pHc and pHϕ, corresponding to the formation of WPI-SA soluble complexes and the beginning of associative phase separation (coacervation), respectively. Finally, with these pH values, phase diagrams were constructed for each system. From these diagrams, pH 4.0 was selected to perform deposition of SA onto WPI aggregates surface, as at this pH value formation of WPI-SA soluble complexes was observed for all WPI:SA ratios. Resultant self-assembled biopolymer complexes were then characterized by the same set of spectroscopic techniques. Higher WPI:SA ratios produced soluble self-assembled biopolymer particles where hydrophobic patches of protein aggregates would be more occluded ensuring the protection of potential lipophilic bioactive agents attached inside.

Raman Spectroscopic Characterization of Structural Changes in Heated Whey Protein Isolate upon Soluble Complex Formation with Pectin at Near Neutral pH

The mechanism leading to an alteration of heat aggregation of whey protein isolate (WPI) in the presence of pectin was investigated by assessing structural changes of proteins using Raman spectroscopy. WPI solutions were heated without or with pectin at 0.015-0.2 pectin to WPI weight ratios and pH 6.0-6.4. In the absence of pectin, thermal denaturation resulted in a loss of α-helical structure and an increase in β-structure and random coils of protein. At pH 6.0 and 6.2, heat aggregation of WPI was suppressed when pectin (0.05-0.15 pectin to WPI ratios) was present as shown by a decrease in turbidity and particle size. Concomitantly, changes in the secondary structures were reduced, indicating the enhanced stability of protein structure by pectin. Raman results also revealed that α-helix and β-sheet are dominant structures in heated WPI--pectin soluble complexes, and hydrogen bonding between biopolymers increased. The effect of pectin was pH dependent, indicating the involvement of electrostatic interaction.

Enzymatic Hydrolysis of Heat-Induced Aggregates of Whey Protein Isolate

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