Heat-induced Whey Protein Aggregates Research Articles

Rehydration Properties of Whey Protein Isolate Powders Containing Nanoparticulated Proteins

The rehydration properties of original whey protein isolate (WPIC) powder and spray-dried WPI prepared from either unheated (WPIUH) or nanoparticulated WPI solutions were investigated. Nanoparticulation of whey proteins was achieved by subjecting reconstituted WPIC solutions (10% protein, w/w, pH 7.0) to heat treatment at 90 °C for 30 s with no added calcium (WPIH) or with 2.5 mM added calcium (WPIHCa). Powder surface nanostructure and elemental composition were investigated using atomic force microscopy and X-ray photoelectron spectroscopy, followed by dynamic visualisation of wetting and dissolution characteristics using environmental scanning electron microscopy. The surface of powder particles for both WPIUH and WPIC samples generally appeared smooth, while WPIH and WPIHCa displayed micro-wrinkles with more significant deposition of nitrogen and calcium elements. WPIH and WPIHCa exhibited lower wettability and solubility performance than WPIUH and WPIC during microscopic observation. This study demonstrated that heat-induced aggregation of whey proteins, in the presence or absence of added calcium, before drying increases aggregate size, alters the powder surface properties, consequently impairing their wetting characteristics. This study also developed a fundamental understanding of WPI powder obtained from nanoparticulated whey proteins, which could be applied for the development of functional whey-based ingredients in food formulations, such as nanospacers to modulate protein–protein interactions in dairy concentrates.

Bulk and interfacial properties of milk fat emulsions stabilized by whey protein isolate and whey protein aggregates

Whey protein is widely used in the food industry as an emulsion stabilizer because of its outstanding emulsifying ability. Recent studies have shown that heat-induced whey protein aggregates may also have potential to stabilize emulsions. The interfacial behavior of whey protein and whey protein aggregates adsorbed at the milk fat-water interface has not been well investigated, especially not in the nonlinear regime, which is highly relevant for the preparation of products such as recombined dairy cream.In this study, the interfacial properties of milk fat-water interfaces stabilized by whey protein isolate (WPI) and whey protein aggregates (WPA) at different bulk concentrations (0.1 wt% - 4.0 wt%) were studied by Large Amplitude Oscillatory Dilatation (LAOD). Lissajous plots were used to analyse the nonlinear response of the interfaces as a function of strain amplitude and frequency. The elastic modulus was quantified based on the tangent modulus at zero instantaneous strain in expansion and in compression. Bulk stability of creams stabilized with the mentioned proteins was studied by determining creaming rate, droplet size distribution, ζ-potential and viscosity of the continuous phase.At low concentrations (<2.0 wt%), WPI-stabilized cream had smaller oil droplets than WPA-stabilized cream, indicating that at these concentrations WPI had better emulsifying ability. For concentrations higher than 2.0 wt%, WPA was a better emulsifier in terms of creaming stability because of the higher viscosity of the continuous phase of the emulsions. Both WPI and WPA could prevent coalescence equally well if the concentration was higher than 0.5 wt%. LAOD measurements showed that at a protein concentration of 0.1 wt%, there was little difference between WPI- and WPA-stabilized interfaces. At 4.0 wt%, WPI showed abrupt intra-cycle yielding followed by a predominantly viscous behavior at large expansion. The WPA interfacial layer had a larger maximum linear strain, and showed a more gradual softening in expansion and mild strain hardening in compression. We hypothesize that WPI formed denser and more brittle (quasi-) 2d structures at the interface, while the interfaces formed by WPA might have a thicker and more stretchable 3d structure. The WPA-stabilized emulsion was less resistant to coalescence upon drastic stirring, which can be explained with its different large deformation behavior, and is relevant for applications where the cream is subjected to large deformations (whipping or stirring).

Physical Properties of Heat-induced Whey Protein Aggregates Formed at pH 5.5 and 7.0

Physical Properties of Heat-induced Whey Protein Aggregates Formed at pH 5.5 and 7.0

CLABSI Reduction using Novel Technology and Nursing Best Practice

Ultrafiltration (UF) is used for the concentration of whey proteins from whey. Membrane fouling during processing results in a decrease in flux, while the predominant molecular fouling reaction and fouling species are still largely unknown. Heat-induced whey protein aggregates are inevitably generated in industrial cheese-making processes, and were found to accelerate membrane fouling during whey concentration. By means of upstream microfiltration prior to UF, these aggregates can be selectively removed. By this means, UF flux could be increased up to twofold for the UF of sweet whey and threefold for acid whey. For filtration of sweet whey, significant calcium retention was observed. Therefore, membrane fouling for both whey types seem follow different molecular mechanisms. Besides the removal of whey protein aggregates, the retention of microorganisms and a log-reduction of up to log 3.5 was achieved by pre-filtration. Shelf-life of whey could be increased to two weeks without pasteurisation.

Spatial arrangement of casein micelles and whey protein aggregate in acid gels: Insight on mechanisms

Skim milk used for the yoghurt manufacture contains 2 main colloidal particles, the native micellar casein (NMC) and the heat-induced whey protein aggregates (WPA). The aim of this study was to understand how these 2 colloids organize in space to form binary acid gels. While acid milk gels were considered homogeneous for scales typically above ∼10 μm, shorter length scales were examined to investigate the early stages of particle aggregation. The NMC and WPA were dispersed in milk permeate at different protein concentrations, separately or in an 80/20 w/w mixture (MIX). Acidification was performed at 35 °C with glucono-δ-lactone to achieve a final pH at ∼4.5 in 6 h. Acid gelation was studied by rheology, while the microstructure of the gels at pH 4.5 was studied by confocal scanning laser microscopy and transmission electron microscopy (TEM). Using Shih et al.'s (1990) model on the rheological data, it seemed that aggregation in the NMC and MIX mixtures was driven by the casein micelles and therefore differed from that of the WPA suspension. The different organizations were confirmed using image analysis of confocal or TEM images. The differences in gel formation were discussed in terms of the different interactive properties of the surface of these 2 colloids, together with their different internal structure.

Enhancement of ultrafiltration-performance and improvement of hygienic quality during the production of whey concentrates

Ultrafiltration (UF) is used for the concentration of whey proteins from whey. Membrane fouling during processing results in a decrease in flux, while the predominant molecular fouling reaction and fouling species are still largely unknown. Heat-induced whey protein aggregates are inevitably generated in industrial cheese-making processes, and were found to accelerate membrane fouling during whey concentration. By means of upstream microfiltration prior to UF, these aggregates can be selectively removed. By this means, UF flux could be increased up to twofold for the UF of sweet whey and threefold for acid whey. For filtration of sweet whey, significant calcium retention was observed. Therefore, membrane fouling for both whey types seem follow different molecular mechanisms. Besides the removal of whey protein aggregates, the retention of microorganisms and a log-reduction of up to log 3.5 was achieved by pre-filtration. Shelf-life of whey could be increased to two weeks without pasteurisation.

Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk

The objective of this study was to determine the contribution of phospholipids from buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The aggregation mechanisms were characterised using analysis with Ellman's reagent, one-dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that phospholipids from buttermilk were involved in the formation of protein aggregates through the MFGM fragments at a low temperature, whereas phospholipids could interact directly with the proteins at a higher temperature (80 °C).

On the use of buttermilk components as aggregation nuclei during the heat-induced denaturation of whey proteins

The aim of this study was to gain a better understanding of the effect of buttermilk constituents on the heat-induced aggregation of whey proteins. Spray-dried buttermilk protein concentrates were prepared and powders were hydrated in liquid whey protein concentrate. Samples were heated up to 90°C (pH 4.6) in the presence or absence of a thiol-blocking agent. SDS–PAGE gels under reducing conditions showed that considerable amounts of protein had already lost the native form prior to heating. Confocal images confirmed the presence of protein aggregates prior to heating. The results suggest overall that reactive particles from buttermilk were formed during the processing steps, leading to interactions between protein and components from milk fat globule membrane through both thiol/disulfide exchanges and hydrophobic interactions. It appeared that these “pre-formed” particles could act as aggregation nuclei during heat-induced denaturation of whey proteins.

Heat-Induced Aggregation of Whey Proteins in Aqueous Solutions below Their Isoelectric Point

Processing beverages containing high concentrations of globular proteins represents a technological challenge due to their instability during heating caused by protein aggregation and gelation. Aggregation of whey protein mixtures was investigated in aqueous model systems at pH 3.5, 4.0, and 4.5 at heating conditions resembling conventional industrial treatment (90 °C for 30 s). The extent of aggregation progressively decreased moving away from the pI. Protein aggregates became smaller and had a more open structure compared to higher pH values. Significant loss of protein dispersibility occurred at pH 4.0 and 4.5 above the denaturation T of whey protein (∼70 °C), at which aggregation was caused by intermolecular hydrophobic interactions. Accessible thiol groups were detected in the heat-treated systems, with a higher intensity at higher pH and increasing extent of aggregation. Intermolecular -S-S- bonding played only a minor role in the aggregation at all conditions studied.

Model mixtures evidence the respective roles of whey protein particles and casein micelles during acid gelation

In acidified milk, heat-induced whey protein aggregates and casein micelle particles assemble to form a soft gel. The present study was set to evaluate the respective roles of whey protein aggregates (WPIA) and native casein micelles (NMC) during acid gelation by means of changing their ratio in model systems. NMC and WPIA were dispersed in milk permeate at different weight ratios ranging from 0% to 100% NMC for a total protein concentration of ∼45 g kg−1. Acidification was performed at 35 °C by addition of glucono-δ-lactone to achieve the same final pH of 4.5 in 6 h. Acid-induced gelation of these systems was followed using small deformation rheology followed by large deformation test and whey retention measurement at pH 4.5, while their microstructure was investigated microscopically. The results showed that higher content in WPIA promoted faster gelation and led to more elastic gels with smaller pore size and increased whey retention. The effects were particularly dramatic up to ∼10% w/w WPIA, where the aggregates were about equimolar to the casein micelles and covered ∼8% of the micellar surface. The results were discussed in terms of the physical interactions between two populations of colloids of different abilities for acid gelation. It seemed likely that a preferred interaction exists between the casein micelles and the aggregates, and directs the structural and mechanical properties of the acid gel.

Formation of β-lactoglobulin aggregates during thermomechanical treatments under controlled shear and temperature conditions

Tailoring of heat-induced whey protein aggregates is a constant concern for the dairy industry. In this study, an original laboratory-scale device was used to study the effect of process and composition variables on β-lactoglobulin aggregation. Experiments were carried under processing-like conditions: high protein concentration (2–10% w/w), high heating (1°Cs−1) and cooling (2°Cs−1) rates, concentrated ionic environment (2.2–11mM of CaCl2) and shear (0–400s−1). Different holding times (0–240s) and temperatures (67–95°C) have been tested. The use of two distinct granulometric methods (laser granulometry and Focused Beam Reflectance Measurement) enabled monitoring of the kinetics of aggregate formation with information on size and concentration. The process conditions have an important influence on the functional properties of the suspension: the median volume diameter goes from less than 2μm for short time and static heat treatment up to 80μm for long time. Applying shear during heat treatment decreases the concentration of small aggregates (1–10μm) and increases the concentration of large aggregates (20–200μm). Using the quantitative characterization of temperature and time impact, it is possible to select the optimal temperature to maximize denaturation and minimize size. With the β-lg solution used in this study, the optimal temperature is around 88°C.

Improved heat stability by whey protein–surfactant interaction

One of the main changes that occur during heat treatment of milk is whey protein denaturation, which in its turn may lead to protein aggregation and gelation. In this contribution, the effect of lysophospholipids, the main components of lysolecithins, as well as alternative surfactants, on heat-induced whey protein aggregation has been studied. Hereby, attention was paid to the relation between polar lipid molecular structure (e.g. effect of alkyl chain length, effect of polar head group) and heat-stabilising properties. Residual protein determination in the supernatant obtained after centrifugation of heated whey protein solutions learned that whey protein aggregation was at least partly prevented in the presence of surfactants. As the short alkyl chain lysophospholipids were particularly effective heat stabilisers, hydrophilic surfactants seemed to be most effective, which may be ascribed to their higher critical aggregation concentration. Upon more severe heat treatment, protein aggregation was probed either in-situ by oscillatory rheology, or ex-situ by yield rheometry. As some surfactants significantly reduced the gel strength, or even prevented heat-induced gel formation, these experiments corroborated the heat-stabilising effect of hydrophilic surfactants. Nuclear Magnetic Resonance (NMR) enabled a more direct evaluation of the protein–surfactant interaction. A strong hydrophobic interaction between small molecular weight surfactants and whey proteins became obvious from the chemical shift of the surfactant hydrophobic groups in the NMR spectrum and could be quantified by pulsed field gradient NMR (pfg-NMR) diffusiometry. The results indicated that protein–surfactant interaction did not occur upon thermal denaturation, but already took place at room temperature. However, the effect of this interaction became mainly obvious during thermal treatment. Overall, this work indicated that bound surfactants largely minimise heat-induced protein intermolecular interactions and hence prevent heat-induced protein aggregation. As the surfactant molecular structure plays a decisive role, it follows that the heat stability of whey protein containing products may be optimised by appropriate selection of ingredients such as (lyso)phospholipids.

Influence of Cross-linked Waxy Maize Starch on the Aggregation Behavior of Casein Micelles During Acid-induced Gelation

The influence of cross-linked waxy maize starch on the aggregation behavior of casein micelles was investigated using a combination of physico-chemical techniques. Milk was homogenized at two different temperatures (55 and 65 °C) and then heated at 95 °C for 5 min in a pilot scale system. The possible interactions between modified starch and milk proteins during lactic acid fermentation were evaluated. While 1% starch did not show differences in the whey protein complexes formed during heating compared to milk with no starch (as measured by size exclusion chromatography), a higher (2.5%) concentration of starch clearly showed an increased amount of heat-induced whey protein aggregates. The gelation pH also increased significantly with 2.5% starch compared to that of the control samples. The storage modulus (G′) increased with increasing levels of starch, and confocal microscopy confirmed that the microstructure of the casein gels was altered by the presence of modified starch. Milk-starch mixtures preheated and homogenized at 55 or 65 °C exhibited similar physico-chemical behavior during acidification. The results suggested a lack of interaction between starch granules and casein micelles during acidification, and scanning electron microscopy images collected with a self-assembled monolayer technique also confirmed that starch granules were not attached to milk caseins but only embedded in the protein gel matrix.

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.

Control of Heat-Induced Aggregation of Whey Proteins Using Casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P<or=0.05) increased from an initial diameter of 165.5 nm in the unheated mixture to 272 nm following heating at 85 degrees C for 10 min at pH 6. Whey protein denaturation was significantly (P<or=0.05) promoted when heated in the presence of micellar casein, but whey protein aggregation was controlled down to pH 5.4. The protective behavior of alphas1/beta-casein and micellar casein differed in that the former inhibited denatured whey protein aggregation, whereas the latter system promoted denaturation but controlled aggregation.

Characterization of soluble aggregates from whey protein isolate

The aggregation behavior of a commercially available whey protein isolate upon heat treatment at neutral pH and low ionic strength was determined using a combination of size exclusion chromatography, multi-angle laser light scattering, dynamic light scattering and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The objective of the work was to examine the effect of heating on the denaturation and subsequent aggregation of 10% (w/v) WPI solutions at neutral pH and low ionic strength. Previous work on heat-induced whey protein aggregation has focused in particular on β-lactoglobulin (β-lg), which is the main protein responsible for heat-induced aggregation; however, because of the presence of α-lactalbumin (α-lac) in WPI, differences in the aggregation behavior occur. Time and temperature affected the amount of residual native α-lac and β-lg, showing a decrease in native protein as aggregates formed. The aggregates differed in molecular weight and hydrodynamic diameter depending on temperature of heating. Electrophoresis of soluble aggregates, collected by preparative size exclusion chromatography, showed a ratio of about 2.0 β-lg/α-lac, regardless of temperature or time of heating. On the other hand, the composition of the residual native protein suggested higher α-lac reactivity at 65 °C than at 75 and 85 °C compared to β-lg, with generally faster rates of denaturation for both proteins with increasing temperature of heating.

Recent advances in the characterisation of heat-induced aggregates and intermediates of whey proteins

This article reviews the latest developments in understanding the mechanisms of heat-induced aggregation of whey proteins, in particular β-lactoglobulin. The influences of the reaction conditions (pH, concentration, temperature, ionic strength) on protein aggregation are briefly described. The aggregation process can be interpreted using reaction schemes consisting of several, complex intermediate states. The aggregates and intermediates are held together via disulphide bridges and non-covalent interactions, mainly hydrophobically driven associations.

Heat-Induced Aggregation of Whey Proteins Is Enhanced by Addition of Thiolated β-Casein

Bovine β-casein has been chemically thiolated by reaction with thiolactone. The purified product was found to contain a variable number of free thiol groups in addition to a number of disulfide bridges. Gel electrophoresis under nonreducing conditions showed the presence of monomeric, dimeric, and tetrameric forms of the protein. Heating whey protein concentrate in the presence of this thiolated casein resulted in a significant increase in the rate of aggregation of all of the whey proteins with the exception of α-lactalbumin, where in general the rate of aggregation decreased. This enhancement of the heat-induced aggregation was found to vary with the concentration of thiolated casein. The results are discussed with relevance to decreasing the costs of manufacture of whey protein concentrate. Keywords: Whey protein; β-casein; thiolation; heating; aggregation

Fractal aggregation of whey proteins

Heat-induced aggregation of whey proteins is investigated in the intermediate pH regime (pH 4–6). Dynamic light scattering was used to monitor the apparent hydrodynamic radii of the aggregates during aggregation, and static light scattering was applied to study the structure of the aggregates. The structure is characterized by a fractal dimension, D, which depends on pH, electrolyte concentration and temperature of denaturation. In the pH range 4–6 whey proteins agglomerate in fractal floes, the properties of which are in agreement with predictions of computer simulations for diffusion and reaction-limited aggregation. In addition, rheological data for whey protein gels show that elastic moduli and yield stresses vary with protein concentration according to a power-law. This power-law dependence is also interpreted in the framework of fractal theories, resulting in fractal dimensions which compare very well with those derived from the light scattering data.

Effect of Calcium, N-Ethylmaleimide and Casein upon Heat-Induced Whey Protein Aggregation

Low- and high-speed centrifugation, Sephadex gel-filtration and polyacrylamide gel electrophoresis were employed to further investigate the nature and extent of protein aggregation produced by heating whey and skimmilk systems.Results indicated that protein aggregation in heated whey systems is a multi-reaction process involving a) denaturation of whey proteins through thiol-disulfide group, hydrogen and hydrophobic bond reactions; b) aggregation to form intermediate-sized protein particles which were excluded from Sephadex G-100 and G-200 gels and sedimentable at 144,000g but not sedimentable at 1,000g, primarily through thiol-disulfide group reactions; and c) gross aggregation of the above protein particles in the presence of calcium ions to sizes sufficient to be sedimentable at 1,000g.n-Ethylmaleimide prevented heat-induced, irreversible whey protein denaturation and interaction of whey proteins with casein by thiol-disulfide group reactions, but did not interfere with the ability of whole casein to stabilize the whey proteins against gross aggregation. Therefore, it is proposed that in addition to the more specific thiol-disulfide group reaction between β-lactoglobulin and κ-casein, denatured whey proteins are stabilized against heat-induced gross aggregation in skimmilk by complexing with casein micelles through calcium-dependent linkages.