Ultrasonic spectroscopy study of relaxation and scattering in whey protein solutions

Ultrasonic attenuation spectroscopy was used to investigate the influence of pH on fast chemical reactions and aggregation of whey proteins in aqueous solution. Ultrasonic attenuation spectra (1–100 MHz) of 2.5 wt% aqueous solutions containing either ‘native’ or ‘alkali-denatured’ proteins were measured as a function of pH (2–12). Peaks in the attenuation occurred at pH 2.8 and 11.6 due to proton transfer equilibria, ie  CO₂H ↔  CO₂⁻ + H⁺ and  NH₂ + H⁺ ↔  NH₃⁺ respectively. Attenuation at other pH values was attributed to a hydration relaxation mechanism. Relaxation times for the equilibria were of the order of 10⁻⁸ s. There was an additional attenuation peak at the isoelectric point of the proteins (pH 5) for solutions containing ‘alkali-denatured’ protein, which was due to scattering of ultrasound by aggregated proteins. The particle size distribution of the aggregates could be determined using ultrasonic scattering theory to analyse the attenuation spectra. Ultrasonic spectroscopy is an extremely valuable tool for probing the molecular characteristics of proteins in solution. © 1999 Society of Chemical Industry     

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

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

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.     

Optimisation of mild-acidic protein extraction from defatted sunflower (Helianthus annuus L.) meal

For protein isolation from defatted sunflower meal, mild-acidic extraction was investigated to minimise concomitant oxidation and polymerisation of phenolic compounds and their irreversible binding to proteins. Because of the impaired solubility of sunflower proteins at low pH, the potential of sodium chloride (NaCl) to improve protein extractability was firstly screened for pH 2–11. Increasing NaCl concentrations of the aqueous solvent (c_NaCl) up to 2.8 mol/L enhanced the relative protein yield to almost 80% at ambient temperature and pH 5.6–7.4. As to improved protein recovery at minimal interactions with phenolic acids, the concerted effects of pH (3.2–7.4), c_NaCl (1–3 mol/L), temperature (T, 15–45 °C), and meal-to-solvent ratio (MSR, 0.03 and 0.05 g/mL) on the protein concentration of the extract (c_PE) and the relative protein yield (RPY) were examined, using response surface methodology (RSM). Aside from the prevailing influence of pH value and salt concentration, elevated temperature slightly enhanced protein extraction, whereas MSR mainly influenced c_PE, but hardly RPY. Calculated models proved suitable for the evaluation of extraction processes and the prediction of optimum conditions in terms of high protein yields at the lowest pH possible. Extraction at pH 6.0 was shown to be an appropriate compromise yielding 76–83% of the meal protein, depending on the constraints given. With elevated NaCl concentrations compensating for unfavourable pH conditions, mild-acidic extraction was found to be suitable for the recovery of high-quality sunflower protein in terms of light-coloured protein isolates.     

Effect of whey protein enzymatic hydrolysis on the rheological properties of acid-induced gels

A protein dispersion blend of β-lactoglobulin and α-lactalbumin was heat-denatured at pH 7.5, hydrolyzed by α-chymotrypsin and then acidified with glucono-δ-lactone to form gels at room temperature. Heat treatment induced the formation of whey protein polymers with high concentration of reactive thiol groups (37 μmol/g). The reactive thiol group concentration was reduced by half after 40 min enzymatic hydrolysis. It was further reduced after enzyme thermal deactivation. During acidification, the first sign of aggregation for hydrolyzed polymers occurred earlier than for non hydrolyzed polymers. Increasing the hydrolysis duration up to 30 min resulted in more turbid gels characterized by an open microstructure. Elastic and viscous moduli were both reduced, while the relaxation coefficient and the stress decay rate constants were increased by increasing the hydrolysis duration. After one week storage at 5 °C, the hardness of gels made from hydrolyzed polymers increased by more than 50%. The effect of polymer hydrolysis on acid-induced gelation is discussed in relation to the availability and reactivity of thiol groups during gel formation and storage.     

Kinetics of phase separation during pressure-induced gelation of a whey protein isolate

The kinetic process of pressure-induced gelation of whey protein isolate (WPI) solutions (20–28%, w/v) was studied using in situ light scattering. The gelation of WPI solutions could be induced by pressurization at 250 MPa, a pressure lower than that reported in other studies. The gelation time decreased with increasing WPI concentration and followed an exponential rule. The relationship of the logarithm of scattered light intensity (I) versus time (t) was linear after the induced time and could be described by the Cahn–Hilliard linear theory. With increasing time, the scattered intensity deviated from the exponential relationship, and the time evolution of the scattered light intensity maximum I_m and the corresponding wavenumber q_m could be described in terms of the power-law relationship as I_m  t^β and q_m  t^−α, respectively. These results indicated that phase separation occurred during the gelation of WPI solutions under high pressure.     

Effect of ultrasonication on the properties of skim milk used in the formation of acid gels

Skim milk was ultrasonicated for times up to 30 min either with or without temperature control. Ultrasonication (US) without temperature control resulted in the generation of considerable heat, with the milk reaching  95 °C within 15 min of treatment. The whey proteins were denatured. Changes to the casein micelle size were observed, with decreases during the early stages of US and increases (because of aggregation) on prolonged treatment. Significant κ-casein dissociated from the micelles. Acid gels prepared from these ultrasonicated samples increased in firmness (final G′) up to a maximum final G′ after  15 min of US, followed by a decrease from this maximum on prolonged treatment. US with temperature control demonstrated that the denaturation of the whey proteins was entirely due to the heat generated during US, although the casein micelle size was still reduced. Acid gels prepared from ultrasonicated skim milk in which the temperature remained below the denaturation temperature of the whey proteins had low final G′, although a small increase was observed with increasing US time. Acid gels prepared from the samples that were ultrasonicated at temperatures above the denaturation temperature of the whey proteins had higher final G′, which could reach values similar to those obtained by the conventional heating of milk. The results of this study indicate that, in skim milk, most of the effect of US can be related to the heat generated from the treatment, with US itself having only a small effect on the milk when the temperatures are controlled.

Industrial relevance

The control and the manipulation of the firmness of acid skim milk gels are important in many dairy food applications such as yoghurts and some types of cheese. US is an emerging technology that could be used to process skim milk for use in acid gelled products. This study has demonstrated that acid gel firmness can be substantially manipulated when skim milk is ultrasonically treated before acidification; however, most of the effect is due to the heat generated during US treatment. As the effects of US are similar to those obtained through conventional heating processes, and as US can control spoilage microorganisms, using US under controlled temperature conditions could be an alternative to conventional heating to give desired functional properties and storage stability to milk products. However, the temperature/denaturation/aggregation would need to be carefully controlled to minimize the detrimental effects of excessive heating.     

Thermal and mechanical behavior of laminated protein films

The whey protein and zein films plasticized by glycerol and olive oil were prepared by casting method and then were laminated. The thermal behavior of the whey protein, zein and whey protein-zein laminated films was investigated by the dynamic mechanical thermal analysis (DMTA) and differential scanning calorimetry (DSC). Both techniques showed that the films containing olive oil had higher glass transition temperature (T_g) than the films containing glycerol (e.g.115 and 88 °C in the zein-olive oil and zein-glycerol films, respectively). As well as, the laminated films had higher T_g than the whey protein films (e.g.82 and 31 °C in the whey-zein-glycerol and whey-glycerol films, respectively). The T_g values obtained from two different methods were close. The results showed that the T_gs of the zein-glycerol films predicted by Couchman and Karasz equation were very close to the values obtained by DSC experiments. The tensile tests showed that the laminated films had higher tensile strength than the single whey protein films and the single zein films plasticized by olive oil (260% and 200% in the whey-zein-glycerol and whey zein-olive oil films in comparison to single whey-glycerol films, respectively).     

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.     

Characterisation of sodium caseinate as a function of ionic strength, pH and temperature using static and dynamic light scattering

Extensive static and dynamic light scattering (DLS) measurements were done on sodium caseinate solutions as a function of the ionic strength (3–500 mM NaCl), pH (5–8) and temperature (10–70 °C). DLS results were analysed in terms of two populations: the caseinate and a small weight fraction of large particles with a hydrodynamic radius (R_h) of about 65 nm that was independent of the ionic strength, pH and temperature. Caseinate was present as individual molecules at low ionic strength (3 mM), but formed small aggregates (R_h=11 nm) at high ionic strength (>100 mM). The aggregation number (N_agg) increased weakly with decreasing pH between pH 8 and 6, but extensive acid-induced aggregation occurred below pH 5.4 at 250 mM and below pH 6.0 at 3 mM. N_agg increased reversibly with increasing temperature.     

Physicochemical changes in whey protein concentrate texturized by reactive supercritical fluid extrusion

The mechanisms of interactions in whey protein concentrate (WPC) texturized by reactive supercritical fluid extrusion and pH modifications were evaluated in terms of protein solubility in different extraction buffers, electrophoresis, free sulfhydryl (SH) groups, and apparent viscosity. The soluble protein content and free SH groups of the texturized WPC (tWPC) produced at pH 2.89 decreased by 20% and 16% relative to the unextruded control. It was completely soluble in the presence of urea and SDS, indicating the importance of non-covalent interactions in maintaining the structure of this product. Its dispersion (20% w/w) yielded a creamy texture with a particle size in the micron-range (mean diameter 5 μm) and contributed 258 times higher viscosity compared to the unextruded control. The tWPC produced at pH 8.16 was soluble only in the presence of a reducing agent. It yielded a grainy texture with a high proportion of large particles due to an extensive aggregation via intermolecular disulfide formations.     

Stability and mechanism of whey protein soluble aggregates thermally treated with salts

The formation of whey protein aggregates, often termed soluble aggregates, with specific physicochemical properties has been shown to result in improved functionality in gels, foams, emulsions, encapsulation, films and coatings. This work evaluated the potential of whey protein soluble aggregates to improve thermal stability in the presence of salts and determine the mechanism of improved thermal stability. Solutions of whey protein isolate (WPI) or β-lactoglobulin (β-lg) (7% w/w, pH 6.8) were heated for 10 min at 90 °C to form soluble aggregates. Native proteins and soluble aggregates were diluted to 3% w/w in solutions containing 0–108 mM NaCl and thermally treated (90 °C, 5 min). Turbidity, solubility, and viscosity were evaluated, in addition to ζ-potential and S_o (surface hydrophobicity). Size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and dynamic light scattering were used to determine aggregate size and transmission electron microscopy (TEM) was used to evaluate aggregate shape. Use of soluble aggregates improved thermal stability due to their altered aggregate shape and higher charge, and resulted in final aggregates that were smaller and less dense, leading to reduced viscosity and turbidity, and increased solubility compared to native proteins. It is concluded that soluble aggregates formed under the appropriate conditions to produce the desirable physicochemical properties can be used to improve whey protein thermal stability with a possible application in beverages.     

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.     

Rheology and microstructure of cross-linked waxy maize starch/whey protein suspensions

The objective of the present work was to investigate the effect of the heating process on the structural and rheological properties of whey protein isolate/cross-linked waxy maize starch (WPI/CWMS) blends depending upon the concentration and the starch/whey protein ratio. Starch concentration ranged from 3 to 4% (w/w) and the protein content was of 0.5, 1 and 1.5% (w/w). The blend (pH 7, 100 mM ionic strength) was heated using a jacketed vessel at two pasting temperatures: 90 and 110 °C. The particle size distribution of the WPI suspension (1.5%) displayed three distinct classes of aggregates (0.3, 65 and 220 μm), whereas the size of swollen starch granules varied from 48 to 56 μm according to the pasting temperature. When the two components were mixed together, the peak attributed to swollen starch granules was attenuated and broadened towards higher values (up to 88 μm) due to protein aggregates (260–410 μm). This effect was more pronounced as the protein concentration increased. When compared to starch alone, the rheology of the mixed system was dramatically modified for the flow behaviour as well as for the viscoelastic properties which changed from a solid-like (3–4% starch) to a liquid-like behaviour (3–4% starch/1.5% protein). Microscopic observations showed aggregated proteins located in the continuous phase and swollen starch granules as the dispersed phase. Protein aggregates were of different sizes, part of them appeared adsorbed onto swollen starch granules while another part was unevenly distributed in the continuous phase, yielding discontinuous network which could explain the peculiar viscoelastic behaviour of such suspensions.     

High pressure microfluidization treatment of heat denatured whey proteins for improved functionality

Solutions (5% protein) of a whey protein concentrate (WPC) in fresh acid whey or in water, as well as the fresh whey alone, were adjusted to pH 5.8, 4.8 or 3.8, heat treated at 90 °C for 10 min and further exposed to high pressure (150 MPa) microfluidization treatment. The volumes of sediment after centrifugation were recorded as a measure of the degree of insolubility of the proteins. Microfluidization disrupted the heat-induced aggregates into non-sedimenting whey protein polymers so that in some cases, especially at pH 3.8, the products studied were almost completely resistant to sedimentation after the microfluidization treatments. Heat denatured/microfluidized whey proteins reaggregated upon subsequent heating, with the pH having a major impact on the amount of sediment produced. Microfluidization of aqueous WPC solutions heat-treated before spray- or freeze-drying substantially increased the solubility of the powders upon reconstitution. Heat-induced viscoelastic gels were produced from freeze-dried microfluidized samples processed at pH 3.8 and reconstituted to solutions containing 12% (w/w) protein.     

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.     

Effect of surface-active stabilizers on the surface properties of coconut milk emulsions

Previously we have demonstrated improved stability of coconut milk emulsions homogenized with various surface-active stabilizers, i.e., 1 wt% sodium caseinate, whey protein isolate (WPI), sodium dodecyl sulfate (SDS), or polyoxyethylene sorbitan monolaurate (Tween 20) [Tangsuphoom, N., & Coupland, J. N. (2008). Effect of surface-active stabilizers on the microstructure and stability of coconut milk emulsions. Food Hydrocolloids, 22(7), 1233–1242]. This study examines the changes in bulk and microstructural properties of those emulsions following thermal treatments normally used to preserve coconut milk products (i.e., −20 °C, −10 °C, 5 °C, 70 °C, 90 °C, and 120 °C). Calorimetric methods were used to determine the destabilization of emulsions and the denaturation of coconut and surface-active proteins. Homogenized coconut milk prepared without additives was destabilized by freeze–thaw, (−20 °C and −10 °C) but not by chilling (5 °C). Samples homogenized with proteins were not affected by low temperature treatments while those prepared with surfactants were stable to chilling but partially or fully coalesced following freeze–thaw. Homogenized coconut milk prepared without additives coalesced and flocculated after being heated at 90 °C or 120 °C for 1 h in due to the denaturation and subsequent aggregation of coconut proteins. Samples emulsified with caseinate were not affected by heat treatments while those prepared with WPI showed extensive coalescence and phase separation after being treated at 90 °C or 120 °C. Samples prepared with SDS were stable to heating but those prepared with Tween 20 completely destabilized by heating at 120 °C.     

Effect of inulin on the rheological properties of silken tofu coagulated with glucono-δ-lactone

This study investigated the rheological properties of inulin-containing silken tofu coagulated with glucono-δ-lactone (GDL) upon heating. Inulin (Raftiline^® HP-gel) was added to a soy protein isolate-enriched cooked soymilk at 0%, 1%, 2%, 3% and 4% (w/v) levels along with 0.4% (w/v) GDL to prepare acid-induced silken tofu. Gelation was induced by heating the soymilk mixture from 20 to 90 °C at a constant rate (1 °C/min) or isothermally at 90 °C for 30 min. The gelling properties were measured with dynamic small-deformation mechanical analysis and static large-deformation compression tests. The rheological changes in soymilk during gelation were dependent upon both the pH decline (hydrolysis of GDL) and the specific temperature of heating. Control samples heated to 50 °C, with the pH lowered to 5.95, started to gel, showing a rapid increase in storage (G′) and loss (G″) moduli afterwards. The addition of 2% inulin lowered the on-set gelling temperature by 2.8 °C and improved (P < 0.05) both rheological parameters of the tofu gel as well as hardness and rupture force (textural profile analysis) of the formed silken tofu. The results indicated that inulin enhances the viscoelastic properties of GDL-coagulated silken tofu, and the textural effect of inulin is an added benefit to its current application mainly as a prebiotical ingredient in food.     

The antigenic response of<Emphasis Type="Italic"> β</Emphasis>-lactoglobulin is modulated by thermally induced aggregation

The antigenic response of thermally denatured and aggregated -lactoglobulin was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from the egg yolk of chicken immunized with heat-denatured -lactoglobulin as a measure for the potential antigenicity/allergenicity. The heat denaturation and the aggregation of heated whey protein isolate solutions were followed by reversed-phase high-performance liquid chromatography and photon correlation spectroscopy. Thermally modified whey proteins showed a remarkable increase of antigenicity when heated to 90 °C, possibly as a consequence of the exposure or formerly hidden epitopes. Above 90 °C, the antigenic response decreased owing to the loss of conformational epitopes and masking of sequential epitopes in the course of aggregation to particles. When large and compact particles were formed, the antigenicity was reduced remarkably. Depending the heating conditions applied, the structure and the size of whey protein particles and thus the potential allergenicity may be modulated in a wide range.     

Heat denaturation of Brazil nut allergen Ber e 1 in relation to food processing

Ber e 1, a major allergen from Brazil nuts, is very stable to in vitro peptic digestion. As heat-induced denaturation may affect protein digestibility, the denaturation behaviour of Ber e 1 was investigated. The denaturation temperature of Ber e 1 varies from approximately 80–110 °C, depending on the pH. Upon heating above its denaturation temperature at pH 7.0, the protein partly forms insoluble aggregates and partly dissociates into its polypeptides, whereas heating at pH 5.0 does neither induce aggregation, nor dissociation of the protein. The denaturation temperature of approximately 110 °C at pH values corresponding to the general pH values of foods (pH 5–7) is very high and is expected to be even higher in Brazil nuts themselves. As a result, it is unlikely that heat processing causes the denaturation of all Ber e 1 present in food products. Consequently, the allergen is assumed to be consumed (mainly) in its native form, having a high stability towards pepsin digestion.