Use of Whey Protein Soluble Aggregates for Thermal Stability—A Hypothesis Paper

Forming whey proteins into soluble aggregates is a modification shown to improve or expand the applications in foaming, emulsification, gelation, film‐formation, and encapsulation. Whey protein soluble aggregates are defined as aggregates that are intermediates between monomer proteins and an insoluble gel network or precipitate. The conditions under which whey proteins denature and aggregate have been extensively studied and can be used as guiding principles of producing soluble aggregates. These conditions are reviewed for pH, ion type and concentration, cosolutes, and protein concentration, along with heating temperature and duration. Combinations of these conditions can be used to design soluble aggregates with desired physicochemical properties including surface charge, surface hydrophobicity, size, and shape. These properties in turn can be used to obtain target macroscopic properties, such as viscosity, clarity, and stability, of the final product. A proposed approach to designing soluble aggregates with improved thermal stability for beverage applications is presented.     

Functional properties of whey protein concentrate texturized at acidic pH: Effect of extrusion temperature

Reactive supercritical fluid extrusion (RSCFX) process at acidic condition (pH 3.0) was used to generate texturized whey protein concentrate (TWPC) and the impacts of process temperature on product's physicochemical properties were evaluated. TWPC extruded at 50 and 70 °C formed soft-textured aggregates with high solubility than that extruded at 90 °C that formed protein aggregates with low solubility. Total free sulfhydryl contents and solubility studies in selected buffers indicated that TWPC is primarily stabilized by non-covalent interactions. Proteins texturized at 90 °C showed an increased affinity for 1-anilino-naphthalene-8-sulfonate (ANS) and a decreased affinity for cis-parinaric acid (CPA), indicating changes in protein structure. Water dispersion of TWPC at room temperature showed thickening function with pseudoplastic behavior. Secondary gelation occurred in TWPC obtained at 50 and 70 °C by heating the cold-set gels to 95 °C. TWPC texturized at 90 °C produced cold-set gels with good thermal stability. Compared to control, TWPC formed stable oil-in-water emulsions. Factors such as degree of protein denaturation and the balance of surface hydrophobicity and solubility influenced the heat- and cold-gelation and emulsifying properties of the protein ingredients. TWPC generated by low and high temperature extrusions can thus be utilized for different products requiring targeted physicochemical functionalities.     

Characterisation of soluble aggregates from commercial whey protein concentrate suspensions: Effect of protein concentration,pH, and heat treatment conditions

Soluble aggregates obtained from heat‐treated suspensions of commercial whey protein concentrate with 74.4% w/w protein were characterised. The effect of protein concentration (7 and 8% w/w), pH (7.0, 7.5 and 8.0), and heating time (0, 5, 10, 15, 20 and 30 min) at 80 °C were evaluated. Whey protein concentrate suspensions with the highest protein concentration (8% w/w) and the lowest pH (pH 7.0) had the highest steady shear viscosity and absorbance values, indicating the effect of the soluble aggregate content (high concentration) and the aggregate size (at lower pH values). According to principal component analysis, samples with 8% w/w and pH 7.0 were grouped in a plot region that confirmed the behaviour observed by confocal microscopy. Those whey protein concentrate suspensions could have soluble aggregates with a strong probability of interacting with cations (in cold gelation applications such as microencapsulation) and with each other (in film‐formation during coating).     

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

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

Combined effect of dynamic heat treatment and ionic strength on the properties of whey protein foams – Part II

The aim was to investigate the effect of dynamic thermal treatment in a tubular heat exchanger on the denaturation and foaming properties of whey proteins, such as overrun, foam stability and texture. A 2% w/v WPI solution (pH 7.0), with and without NaCl addition (100 mM), was submitted to heat treatment at 100 °C. The results demonstrated that heat treatment slightly reduced overrun, whereas NaCl and heat treatment improved foam stability, enhanced texture and provided smaller bubble diameters with more homogeneous bubble size distributions in foams. The foaming properties of proteins, especially stability, were shown to depend not only on the amount of protein aggregates, but also on their size. While insoluble aggregates (larger than 1 μm diameter) accelerated drainage, soluble aggregates (about 200 nm diameter) played a key role on the stabilization of gas–liquid interfaces.     

Effect of dynamic heat treatment on the physical properties of whey protein foams

The influence of dynamically heat-induced aggregates on whey protein foams was investigated as a function of the thermal treatment applied with the aim of determining the optimal temperature for the production of heat-induced aggregates dedicated to foaming. The native protein solutions (2% w/v WPI; 50 mM NaCl) at neutral pH were heat-treated using a tubular heat exchanger between 70 °C and 100 °C. Protein denaturation and aggregation were followed by micro-differential scanning calorimetry, size exclusion chromatography, laser diffraction and dynamic light scattering. The protein solutions were whipped using a kitchen mixer to produce foams. Foam overrun, stability against drainage, texture and bubble size distribution were measured.     

Preparation of spherical aggregates of taro starch granules

Taro starch spherical aggregates were prepared by including a spray drying step in a conventional pilot plant-scale starch isolation procedure. The aim of this study was to characterize these spherical aggregates. The procedure was repeated three times to verify the spherical aggregates formation, which was shown by scanning electron microscopy. Amylose was not be detected in the aggregates, and a protein content of 4.5 g/100 g was recorded. Aggregation of taro starch granules did not affect the X-ray diffraction pattern. The maximum peak viscosity of the aggregate preparation was obtained at a relatively high temperature (i.e. start of the temperature holding step). The phase transition of taro starch aggregates in excess water showed high temperatures, with low molecular reorganization during storage. During water retention capacity (WRC) and solubility tests, taro starch aggregates were stable until 70 °C. The spray drying conditions used produced spherical aggregates of taro starch that presented physicochemical and functional characteristics with potential for encapsulation of substances.     

Microemulsions as nanoreactors to produce whey protein nanoparticles with enhanced heat stability by thermal pretreatment

Native whey proteins (NWPs) may form gels or aggregates after thermal processing. The goal of this work was to improve heat stability of NWPs by incorporating protein solutions in nanoscalar micelles of water/oil microemulsions to form whey protein nanoparticles (WPNs) by thermal pretreatment at 90 °C for 20 min. The produced WPNs smaller than 100 nm corresponded to a transparent dispersion. The WPNs produced at NWP solution pH of 6.8 had a better heat stability than those produced at pH 3.5. The salt concentration (0–400 mM NaCl) in NWP solutions did not significantly change the size of corresponding WPNs. Compared to NWPs, the 5% (w/v) dispersion of WPNs at pH 6.8, 100 mM NaCl did not form a gel after heating at 80 °C for 20 min. The improved heat stability and reduced turbidity of WPNs may enable novel applications of whey proteins in beverages.     

The effect of protein profile and preheating on denaturation of whey proteins and development of viscosity in milk protein beverages during heat treatment

The effect of preheat temperature (63 or 77 °C for 30 s; final heat 120 °C for 30 s) and casein to whey protein ratio on the physical characteristics of 3.3%, w/w, dairy protein beverages was investigated. Dispersions preheated at 77 °C had lower viscosity than dispersions preheated at 63 °C. Casein‐containing dispersions had significantly lower levels of α‐lactalbumin denaturation than whey protein‐only dispersions. A higher proportion of casein improved the thermal stability of protein dispersions. Overall, alteration of preheat temperature and casein to whey protein ratio can influence dairy beverage quality, with increasing levels of casein reducing physical changes due to heat treatment.     

Whey protein aggregate formation during heating in the presence of κ-carrageenan

The effect of a negatively charged polymer, κ-carrageenan, on the aggregation behaviour of whey proteins during heating was studied. Aqueous solutions of whey protein isolate (WPI) at 0.5% were heated in the presence of κ-carrageenan (0.1%) at pH 7.0. This concentration was chosen as optimal in the detection of the intermediate aggregates during chromatographic analysis. The residual unaggregated protein, the intermediate aggregates and the soluble aggregates were all examined as a function of heating time and temperature, using size-exclusion chromatography coupled with light scattering detection. The presence of κ-carrageenan did not affect the aggregation of whey proteins heated at 75 °C; however, a change in the mechanism of aggregation seemed to occur at higher temperatures, and intermediates with higher molecular mass formed at 85 °C. At 90 °C, in the presence of κ-carrageenan, the extent of WPI aggregation was much larger, as soluble aggregates were no longer present and less residual protein was recovered in the unaggregated peak.     

Influence of calcium-binding salts on heat stability and fouling of whey protein isolate dispersions

The effect of the calcium-binding salts (CBS), trisodium citrate (TSC), tripotassium citrate (TPC) and disodium hydrogen phosphate (DSHP) at concentrations of 1–45 mm on the heat stability and fouling of whey protein isolate (WPI) dispersions (3%, w/v, protein) was investigated. The WPI dispersions were assessed for heat stability in an oil bath at 95 °C for 30 min, viscosity changes during simulated high-temperature short-time (HTST) and fouling behaviour using a lab-scale fouling rig. Adding CBS at levels of 5–30 mm for TSC and TPC and 25–35 mm for DSHP improved thermal stability of WPI dispersions by decreasing the ionic calcium (Ca²⁺) concentration; however, lower or higher concentrations destabilised the systems on heating. Adding CBS improved heat transfer during thermal processing, and resulted in lower viscosity and fouling. This study demonstrates that adding CBS is an effective means of increasing WPI protein stability during HTST thermal processing.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Heat-induced aggregation of whey proteins in the presence of κ-casein or sodium caseinate

Aggregates were formed by heating mixtures of whey protein isolate (WPI) and pure κ-casein or sodium caseinate at pH 7 and 0.1 M NaCl. The aggregates were characterized by static and dynamic light scattering and size exclusion chromatography. After extensive heat-treatment at 80 °C for 24 h, almost all whey proteins and κ-casein formed mixed aggregates, but a large proportion of the sodium caseinate did not aggregate. At a given WPI concentration the size of the aggregates decreased with increasing κ-casein or sodium caseinate concentration, but the overall self-similar structure of the aggregates was the same. The presence of κ-casein or caseinate therefore inhibited growth of the heat-induced whey protein aggregates. The results were discussed relative to the reported chaperone-like activity of casein molecules towards heat aggregation of globular proteins.     

Impact of protein self-assemblages on foam properties

The influence of dynamically heat-induced aggregates on whey protein foams was investigated as a function of the thermal treatment applied to WPI using a bubbling technique. The aim was to determine the interplay between the size/shape/proportion of the heat-induced aggregates and the properties of protein foams (formation and stability). Results showed that insoluble protein aggregates were highly branched and cohesive, whereas soluble aggregates were constituted by subunits, associated by hydrophobic bonds and formed by α-La and β-Lg monomers linked by disulfide bridges. Using the bubbling procedure, protein aggregates were shown to slow down significantly foam formation. However, the rate of foam formation remained nearly unchanged for wet foams when the amount of insoluble aggregates was inferior to 5% and when their size remained lower than 100 μm. Similarly, protein aggregates did not seem to affect the destabilisation kinetics of wet foams, regardless of amount, size, shape and proportion.     

Heat stability of whey protein ingredients based on state diagrams

A state diagram approach was used to compare heat stability among whey protein ingredients. Solutions were thermal processed and solubility, turbidity, and colloidal structure (sol, precipitate, or gel) were plotted against the variables of pH (3–7) and protein concentration (1–10%, w/w) to form state diagrams. Each of the whey protein ingredients produced state diagrams with unique patterns in the protein concentrations associated with colloidal structures and in heat stability regions. Relative heat stability among ingredients depended on the pH-thermal processing combination, in that patterns observed at pH ≤ 4.5 were not equivalent to patterns produced at pH > 4.5. Heating at temperatures of <100 °C for extended time did not produce the same results as heating at > 100 °C for short times. State diagrams were able to differentiate among the whey protein ingredients, but absolute values were determined by the heating treatment.     

Functional properties of whey proteins as affected by dynamic high-pressure treatment

An ultra high-pressure homogenizer was used to treat whey protein isolate solutions (3%, w/w). The treated solutions (up to 300 MPa) were characterised for both physico-chemical properties (particle size distribution, surface hydrophobicity and structural conformation) and functional properties (solubility, foaming stability and interfacial rheology). Dynamic high-pressure treatment did not affect the conformation of the proteins (determined by micro-calorimetry, size-exclusion chromatography and electrophoretic technique). This treatment dissociated large protein aggregates leading to unmasking of the buried hydrophobic groups without affecting protein solubility. Interactions may then occur between these groups that enhance the viscoelasticity of air-water interfaces (assessed by drop tensiometry) and improve foam stability (evaluated by sparging method). Dynamic high-pressure-treated whey proteins showed better foaming and stabilising properties.     

The physicochemical parameters during dry heating strongly influence the gelling properties of whey proteins

In this study we determined the composition (proportion of native proteins, soluble and insoluble aggregates) and quantified the gelling properties (gel strength and water holding capacity) of pre-texturized whey proteins by dry heating under controlled physicochemical conditions. For this purpose, a commercial whey protein isolate was dry heated at 80 °C (up to 6 days), 100 °C (up to 24 h) and 120 °C (up to 3 h) under controlled pH (2.5, 4.5 or 6.5) and water activity (0.23, 0.32, or 0.52). Gelling properties were quantified on heat-set gels prepared from reconstituted pre-texturized proteins at 10% and pH 7.0. The formation of dry-heat soluble aggregates enhanced the gelling properties of whey proteins. The maximal gelling properties was achieved earlier by increasing pH and water activity of powders subjected to dry heating. An optimized combination of the dry heating parameters will help to achieve better gelling properties for dry heated whey proteins.     

Newtonian viscosity behavior of dilute solutions of polymerized whey proteins. Would viscosity measurements reveal more detailed molecular properties?

Dilute solution Newtonian viscosity of whey protein polymers at different temperatures has not been assessed in literature. In this work, a thermally-treated whey protein solution at 8% w/w and pH 6.8 was prepared. Dilute solution viscometry was investigated at different temperatures from 30 °C to 65 °C. Intrinsic viscosity and voluminosity data indicate slight shrinkage in molecular size upon temperature increase. The temperature dependence of viscosities was expressed by the Arrhenius–Frenkel–Eyring equation and the thermodynamic parameters of viscous flow of polymer solutions were calculated. Results show a positive entropy change of viscous flow, indicating ordered structures. Chromatographic separation results prove that although disulphide bonds form the polymer backbone chain, both hydrophobic associations and hydrogen bonding still play a role in molecular structuring, even at very low protein concentrations. The calculated shape factor indicates spherical polymer molecules over the entire investigated temperature range.     

Influence of green tea polyphenols on the colloidal stability and gelation of WPC

Green tea extracts are being widely used in food products due to their health-promoting properties. Polyphenols can interact with food proteins leading to the formation of soluble or insoluble complexes; therefore they could alter functional properties of proteins. The objective of the present work was to study the colloidal stability and gelation characteristics of a whey protein concentrate (WPC) in the presence of green tea polyphenols. Mixtures of WPC35 (8 and 30% w/v) and green tea polyphenols (0.25–1% w/v) were prepared at pH 4.5 and 6.0. The size of particles formed was analyzed by light scattering, while gelation was characterized by means of dynamic rheometry and texture analysis of gels. At pH 6.0, the particles were smaller and had a higher net charge than at pH 4.5, which accounted for by a less precipitation of the system at pH 6.0. The G′ parameters of gels upon cooling at 35 °C increased with increasing polyphenols concentration at both pH values. However, the relative viscoelasticity decreased. The texture analysis indicated that the addition of polyphenols improved the firmness and adhesiveness of the gels at pH 6.0, while no significant differences were seen at pH 4.5. The results obtained in this work indicate that pH-dependent interaction between green tea polyphenols and WPC induces the formation of aggregates that modifies the viscoelastic and texture properties of the gels.     

Effect of succinylation on physicochemical and functional properties of milk protein concentrate

Milk protein concentrate (MPC) is a complete dairy protein ingredient (containing both caseins and whey proteins) available with protein concentrations ranging from 40 to 90%. However, MPC powders are poorly soluble which thus, restricts their potential use for food application. Succinylation involves chemical derivatisation of ϵ-amino group of lysine in proteins and enhances solubility of less soluble proteins. In the present investigation, highest degree of succinylation was achieved at the level of 4 mol of succinic anhydride/mole of lysine in MPC. Findings from intrinsic tryptophan intensities stated that succinylation of milk proteins showed structural modification and increase in hydrophobicity. Further, the effects of succinylation on the functional properties (solubility, water- and oil-binding capacities, viscosity, foaming capacity and stability, emulsion activity and stability) of MPC were evaluated. Succinylation of proteins significantly (P < 0.05) increased solubility as a result of altered charge on protein and reduced particle size of native MPC, which in turn improved other functional properties of native MPC. The microstructure of succinylated MPC at different degrees of succinylation by scanning electron microscopy revealed that the size and number of white patch like structures present on protein increased with degree of succinylation.