Article pubs.acs.org/JAFC
Heat-Induced Aggregation of Whey Proteins in Aqueous Solutions below Their Isoelectric Point Leonardo Cornacchia,*,† Cécile Forquenot de la Fortelle,†,§ and Paul Venema§ †
Nutricia Research, Uppsalalaan 12, 3584 CT Utrecht, The Netherlands Laboratory of Physics and Physical Chemistry of Foods, Department of Agrotechnology and Food Sciences, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands
§
S Supporting Information *
ABSTRACT: 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. KEYWORDS: whey protein, aggregation, thiol reactivity, surface hydrophobicity
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INTRODUCTION The use of whey proteins in beverages represents an important opportunity due to their high nutritive and biological values,1 which can fulfill the dietary requirements of various categories of individuals (e.g., the elderly and the frail).2 However, the production of stable whey protein-based nutritional beverages is a great technological challenge. Processing (i.e., thermal treatment to ensure microbial safety) and product formulation affect protein solubility, which can lead to turbidity and/or sedimentation3,4 or compromise manufacturing due to fouling of the heating equipment during pasteurization/sterilization processes.5 To reduce the effect of the heat load the whey protein undergoes, whey-based beverages can be formulated at acidic pH (below pH 4.6) as, for their microbial safety, a milder thermal treatment compared to pH-neutral recipes is possible.6 Whey protein of bovine milk is a mix of globular proteins (βlactoglobulin, β-LG, 50%; α-lactalbumin, α-LA, 20%; bovine serum albumin, BSA, 5%; immunoglobulins, IGs, 10%; lactoferrin, LF, 2.5%; lactoperoxidase, LP, 0.5%) with a high level of secondary, tertiary, and in some cases quaternary structure.7 Far from their isoelectric point (pI), proteins have a net charge that leads to electrostatic repulsion, resulting in stable protein solutions.8,9 At the pI (β-LG, pI = 5.1; α-LA, pI = 4.3; BSA, pI = 4.1) where the net charge of the proteins equals zero, the electrostatic repulsive forces are minimal, and intermolecular interactions, such as hydrophobic interaction and hydrogen bonds, may lead to aggregation.9−11 The solubility of whey proteins is highly dependent on the modification of the protein structure caused by heat treatment. Native globular proteins unfold upon heating and, depending on the balance of attractive and repulsive interactions, can form aggregates or gels with various structures (i.e., opaque particulates or transparent filaments).9,12,13 When whey © 2013 American Chemical Society
proteins are heated in an aqueous solution, the globular structure progressively unfolds, exposing hydrophobic groups and free thiol groups (when present, as in the case of β-LG and BSA) which are normally buried in the interior of the native protein molecule away from the protein−water interface. The exposed hydrophobic domains are driven toward their association, thereby leading to noncovalent protein aggregation (physical aggregation). At the same time, reactive −SH groups can lead to intermolecular covalent cross-linking (chemical aggregation) via oxidation or, as thiolate groups −S−, via sulfhydryl/disulfide interchange reactions. The −S−S− bridges can also rearrange within the same molecule via an intraprotein thiol−disulfide exchange reaction (i.e., disulfide bond shuffling).14,15 As Ako et al.16 pointed out, aggregation and gelation of denatured whey proteins can even occur at conditions of strong electrostatic repulsion. The mechanism of heat-induced aggregation and gelation of whey proteins has been extensively studied for model systems at neutral pH4,17−20 or at high acidity (pH ∼2), where finestranded fibrillar structures are formed.12,21,22 At intermediate pH values, near the pI, the structural characteristics of particulate β-LG gels have been studied. It was found that between pH 4.05 and 5.55, β-LG heated at 60 °C for 24 h formed particulate aggregates without evidence for fibrillar structures. However, near the pI, spherical crystalline structures, called spherulites, can be formed by fibrillar structures growing radially from a central amorphous nucleus.13 Recently, β-LG close to the pI was thoroughly investigated as a way to obtain Received: Revised: Accepted: Published: 733
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Protein Dispersibility after Heat Treatment. Whey protein dispersibility before and after heat treatment was measured in 1, 5, and 10% protein solutions at pH 3.5, 4.0, and 4.5. Solutions with a volume of 15 mL were transferred into sealed screw-capped glass test tubes and heated in a water bath at 92 °C. The temperature increase of the sample was monitored with a thermocouple inserted in a reference tube containing 15 mL of deionized water. The initial temperature of the sample was 20 °C, and heat treatment was immediately stopped when the sample temperature had reached 50, 60, 70, 80, or 90 °C. All of these temperatures were reached within 5 min. One of the samples was held at 90 °C for 30 s (this latter heat treatment is indicated as 90* °C). After heat treatment, the test tubes were cooled to room temperature using running tap water. This heating protocol was chosen to mimic temperature treatments of acidic formulations on liquid processing lines. At the end of the heat treatment protein aggregates were removed by centrifuging the samples at 3500g for 30 min using a refrigerated centrifuge (Biofuge Fresco, Heraeus Sepatech GmbH, Osterode, Germany). The supernatant was filtered through a 0.2 μm pore size syringe filter (Minisart; Sartorius Stedim Biotech GmbH, Goettingen, Germany), and the protein concentration was analyzed by Dumas. The dispersibility was expressed as the percentage of the protein concentration in the supernatant, relative to the initial total protein concentration of the solution. The heat treatment was repeated in three replicate experiments, and values were reported as an average and standard deviation. Accessibility of Thiol Groups. The accessibility of the free thiol groups in protein was determined by their reactivity with 5,5′dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, Inc., St. Louis, MO, USA) according to the method described by Ellman,28 adapted for the composition of the system.19 A 0.1 M sodium citrate at pH 3.5, 4.0, and 4.5 buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, Inc.) was prepared and diluted 1:1 with demineralized water. Ellman’s reagent was prepared by dissolving 8 mg of DTNB (Sigma-Aldrich, Inc.) in 2 mL of buffer. The reaction mix for the analysis of accessible reactive thiol groups was prepared by adding 2.5 mL of reaction buffer to 50 μL of Ellman’s reagent solution and 250 mL of protein solution (or protein gels) and was left to react overnight. After complete reaction, solutions were spun at 16000g for 100 min at 20 °C (Biofuge Fresco, Heraeus Sepatech GmbH, Osterode, Germany) to remove the aggregates. The supernatant was collected, and the reactive thiol concentration was measured spectrophotometrically at 412 nm and determined using an extinction coefficient of 13600 M−1 cm−1.4,28−30 This method is based on the quantification of p-nitrothiophenol derived from the equimolar reaction between DTNB and −SH. Blank samples were prepared by replacing 250 μL of protein solutions with 250 μL of reaction buffer. The heat treatment was repeated in three replicated experiments, and values were reported as an average and standard deviation. Surface Hydrophobicity. Surface hydrophobicity was determined using the fluorescent probe 1-(anilino)-naphthalene-8-sulfonate (ANS) (Sigma-Aldrich, Inc.), according to the method described by Monahan, German, and Kinsella.4 Protein solutions of 1, 5, and 10% at pH 3.5, 4.0, and 4.5 were heat treated as described above and centrifuged at 3500g for 30 min at 20 °C, and the protein concentration in the supernatant was measured by Dumas. ANS [10% (w/v)] was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Inc.) and diluted in 30 mM citrate buffer (pH 4.0) to obtain a stock solution containing 0.5% (w/v) ANS; 0.2 mL of the 0.5% (w/v) ANS solution was added to 0.3 mL of supernatant and diluted with 30 mM citrate buffer (pH 4.0) to a final volume of 3 mL. Samples were analyzed by a fluorescence spectrometer (LS50B; Perkin-Elmer, Norwalk, CT, USA), with an emission wavelength from 400 to 580 nm, an excitation wavelength at 350 nm, and both slits widths (emission and excitation) at 4 nm, as described in the literature.31 Surface hydrophobicity was expressed as the value at the maximum emission wavelength (475 nm) and normalized relative to protein concentration. The background signal as caused by the presence of dispersed aggregates in systems with no added ANS was
stable dispersions of spherical protein particles, called microgels.23,24 Furthermore, micrometer-size highly-dense spherical whey protein particles were prepared25,26 by heat-induced gelation of whey protein solutions dispersed as droplets in water-in-oil emulsions. Although the literature has provided an in-depth characterization of the structures formed when β-LG is heated in aqueous solutions around the pI, the mechanism of aggregation of whey protein mixtures at moderately acidic conditions (3.5 < pH < 4.5) has received less attention. Therefore, in this study we chose to focus on whey protein aggregation and dispersibility at heating conditions resembling the manufacturing of acidic beverages. The relevance of intermolecular disulfide bonds and hydrophobic interactions on the heat-induced aggregation of whey protein isolate in aqueous solutions at pH 3.5, 4.0, and 4.5 was investigated. The microstructure of the systems was also characterized. The results of this study contribute to a better understanding of the mechanism leading to whey protein instability at acidic pH and can be used to develop novel strategies for improving the stability and processability of concentrated whey protein aqueous formulations.
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MATERIALS AND METHODS
Materials. A commercial whey protein isolate (BiPRO, Davisco Foods International Inc., Le Sueur, MN, USA) was used. The protein content was 92.84% (w/w) (all percentages used are w/w, unless stated otherwise) protein on a wet basis (N × 6.25), as measured by Dumas nitrogen analysis (Thermo Fisher Scientific Inc., Flash 4000 N/Protein Analyzer, Waltham, MA, USA). All salts and reagents used for the thiol reactivity and the hydrophobicity study were of reagent grade. HPLC grade chemicals were used for ζ-potential measurements, transmission electron microscopy (TEM), and electrophoresis experiments. Preparation of Protein Solutions. Protein solutions were freshly prepared for each set of experiments, similar to methods reported in the literature.16,27 Protein powder was dispersed in demineralized water to obtain a homogeneous stock solution of 25%. The concentrated protein solution was stirred at room temperature for 3 h and stored overnight to allow for complete hydration of the protein. The next day, the 25% solution (approximately 400 mL) was transferred into a 3.5 kDa cutoff dialysis tubing (35 mm dry SnakeSkin, Thermo Scientific, Rockford, IL, USA) and dialyzed against 10 L of demineralized water for 2 h. After washing with demineralized water, the dialysis tubing was transferred into 10 L containing 3% of 12 kg mol−1 polyethylene glycol (PEG) (Sigma-Aldrich, Steinheim, Germany) solution, and the dialysis was continued for 24 h. The overall preparation was performed at 4 °C, and after dialysis no sign of bacterial growth was observed. Addition of PEG was necessary to compensate for osmotic pressure, thereby avoiding bursting of the dialysis tube. After dialysis, the concentration of the protein in the dialyzed solution was determined by the UV absorbance at 280 nm (kinetics spectrophotometer, LKB Biochron, Ultrospec K 4053, Cambridge, UK) using calibration curves prepared by dissolving known amounts of whey protein. At the dialysis conditions used in this study, the final protein concentration was approximately 14%. This stock solution was divided into various aliquots, and the pH was adjusted to pH 3.5, 4.0, and 4.5 using 2 N HCl. Finally, the solutions were diluted to the different protein concentrations (1, 5, and 10%). Mineral and Lactose Contents. The concentrations of Ca, Cl, P, K, Mg, Mn, Na, and lactose were measured in the nondialyzed and dialyzed solutions with the same protein concentration. Minerals were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-OES) (iCAP 6300, Thermo Fisher Scientific Inc.), whereas lactose was determined by high-performance anion-exchange chromatography (HPAEC) and pulsed electrochemical detection (PED) (Dionex ICS-5000, Thermo Scientific, Sunnyvale, CA, USA). 734
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found to be negligible relative to the signal when ANS was added (85% purity, Sigma-Aldrich), β-LG (>90% purity, Sigma-Aldrich), and BSA (>98% purity, Sigma-Aldrich) standards. Protein concentrations were obtained using
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RESULTS AND DISCUSSION Effect of Dialysis on Minerals and Lactose. The data for nondialyzed and dialyzed protein dispersions obtained by ICPOES (for minerals) and HPAEC-PED (for lactose) are compared in Table 1. Nondialyzed protein solutions contained Table 1. Compositional Data for 10% (w/w) Whey Protein Solutions before and after Dialysis through a 3.5 kDa Cutoff Membrane element
control
3.5 kDa dialyzed system
Ca Cl P K lactose Mg Mn Na
9 1 5 3 5.43 1 0.005 54
9 0 5 2 0.55 1 0.003 50
unit mg/100 mg/100 mg/100 mg/100 μg/mL mg/100 mg/100 mg/100
g g g g g g g
only residual amounts of lactose and minerals. At the conditions used in the present study, dialysis did not lead to a significant loss of minerals, but reduced the lactose content 10-fold. This shows that most of the ions are present as counterions or associated with the protein structure (e.g., Ca2+ in α-LA). ζ-Potential and Visual Appearance. The ζ-potential of dialyzed whey protein solutions was measured upon titration with 1 M HCl from pH 7 to 2 using Zetasizer Nano cells (DTS1060, Malvern Instruments Inc., Worcestershire, UK), and the pI of the whey protein mix was determined. pI was reached around pH 4.8 (Supporting Information, Figure S1). The whey protein solutions showed an increased turbidity as the pH was close to the pI. The visual appearance of the 5 and 10% freshly prepared whey protein samples at pH 4.5, 4.0, or 3.5 are shown in Figure 1. After 2 h at room temperature
C U = (CS/IS)IU where, CU and CS are the concentrations, whereas IS and IU are the measured intensities of the unknown sample (U) and reference (S), respectively. The heat treatment of each system was performed in duplicate, and the heat-treated samples were run on two different lanes in the same gel. The average values of band intensity calculated from all of the previous were then quantified with a calibration curve obtained on a different gel (five concentrations in three replicates) and corrected according to the band intensity of an internal standard. The concentrations of α-LA, β-LG, and BSA in the WPI were obtained by analyzing unheated aqueous solutions containing whey protein by SDS-PAGE. The relative concentrations of α-LA, β-LG, and BSA derived from a calibration curve were 24.0 ± 0.3, 68.0 ± 1.0, and 3.0 ± 0.2%, respectively.
Figure 1. Whey protein solutions, 10 and 5% (w/w), at pH 4.5, 4.0, and 3.5, before and after 2 h of storage at room temperature.
(Figure 1), the 5% whey protein samples at pH 4.5 phaseseparated into an opaque protein-rich white sediment and a (protein-depleted) clear top layer. Less extensive separation was observed in the 10% whey protein samples at pH 4.5. No macroscopic phase separation was observed at pH 4.0 or 3.5, showing a cloudy or translucent appearance, respectively. At pH 4.5, close to the isoelectric point, the whey protein molecules form (reversible upon pH change) aggregates that 735
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The lower pH values investigated gave significantly higher dispersibility. The 1 and 5% protein systems at pH 3.5 did not lead to any significant dispersibility loss when heated from 20 °C to 90* °C. At higher protein concentration (10% protein), however, a limited loss in dispersibility (∼10%) was observed. A similar trend was observed for systems at pH 4.0. In this latter case 10% protein systems showed a lower dispersibility between 50 and 80 °C (dispersibility of ∼80%) and formed a gel at 90 °C. At pH values between pH 3.5 and 4.5 a significant effect of the pH of the system on protein dispersibility was found. At all protein concentrations and heat treatments studied, the protein dispersibility increased progressively as the pH was decreasing below the pI. This is due to the higher net positive charge on the protein surface at low pH (Supporting Information, Figure S1), which leads to a stronger intermolecular electrostatic repulsion. Accessibility of Thiol Groups. During the heating of whey protein, the formation of intermolecular disulfide bridges has been found to occur as a result of thiol−disulfide exchange reactions or oxidation. As explained by Visschers and de Jongh15 and De Wit,38 this process is driven by free sulfhydryl groups located on the cysteine of β-LG and BSA and is regulated by its pKa (≈8.3), with the thiolate ion (−S−) being the reactive species. Moreover, reactive thiols should be free from steric hindrance and be exposed and accessible to complete the reaction.39 In this study, the concentration of accessible thiol groups was monitored in 10% whey protein systems at pH 3.5, 4.0, and 4.5 during heating from 20 to 90 °C. To evaluate the accessibility of thiol groups, the reaction mixes in which the DTNB was allowed to react with proteins were buffered at pH 4.0 for all three systems (Figure 3A) and at pH 3.5, 4.0, 4.5 (Figure 3B). As shown in Figure 3A, the concentration of accessible thiol groups in the three different systems followed the same trend. No thiol groups were accessible to react with DTNB below 80 °C. As the proteins formed aggregates above the denaturation temperature, molecular rearrangement might have led to the exposure of the thiol groups to the solvent or to an open structure that allowed diffusion of DTNB (as already postulated by McGuffey et al.19). This implies the presence of accessible thiol groups. The experiment conducted in the three different buffer mixes (Figure 3B) revealed a pH-dependent accessibility of thiols on the protein aggregates. Again, reactive free thiol goups were detected with DTNB, and systems at pH 4.5 showed higher residual thiol group reactivity than systems at pH 4.0 and 3.5 (0.425 ± 0.003, 0.953 ± 0.014, and 4.134 ± 0.019 nmol/mg protein, in systems heated at 90* °C at pH 3.5, 4.0, and 4.5, respectively). As this might be due both to the increased reactivity of thiols to DTNB with increasing pH and to the different structural reorganization of the aggregates resuspended in different buffers, an additional experiment was performed. Solutions at the same concentration of free cysteine (0.4 mM) in citrate buffers at pH 3.5, 4.0, and 4.5 were tested with DTNB. Results showed values of absorbance that increased with increasing pH 3.5 < pH 4.0 < pH 4.5 (Supporting Information, Figure S2). However, the increased absorbance due to the sole effect of pH was significantly lower than the combined effect of pH and structural rearrangement observed for heat-treated (aggregated) whey proteins. Therefore, it can be concluded that free thiol groups were accessible to DTNB only above 80 °C, when protein unfolding and aggregation occurred. Furthermore, the thiol accessibility
are large enough to be prone to sedimentation. This is often ascribed to the interactions between non-native protein fractions present in the whey protein isolate powders.33,34 Whey Protein Dispersibility. The results obtained for the protein dispersibility on systems containing 1, 5, and 10% protein are shown in Figure 2. In line with previous work,8 a
Figure 2. Dispersibility of 1, 5, and 10% (w/w) whey protein solutions at pH 4.5, 4.0, and 3.5 as a function of heat treatment.
limited dispersibility of unheated whey protein at pH 4.5 was found. Dispersibility in the non-heat-treated systems was 93.88 ± 0.71, 93.22 ± 3.79, and 90.15 ± 1.26% at 1, 5, and 10% protein, respectively. This loss of dispersibility is in line with the visual observations described in the previous section. At increasing temperature, the protein dispersibility progressively decreased until a sharp decrease occurred above 70 °C. This temperature is close to the peak of whey protein denaturation curve as measured by differential scanning calorimetry by Potes et al.35 According to the pathway suggested by Lumry,36 upon heating, native proteins undergo structural changes, which can be followed by (irreversible) non-native protein association to form aggregates. Aggregation affects protein dispersibility so that protein clusters precipitate as they exceed a critical size37 or are able to form a stable microgel. 736
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Figure 3. Temperature-dependent thiol group concentration of 10% whey protein solutions at pH 4.5, 4.0, and 3.5: (A) reaction with DTNB performed in citrate buffer, pH 4.0; (B) reaction with DTNB performed in citrate buffer, pH 3.5, 4.0, and 4.5.
increased rapidly with increasing pH from pH 3.5 to 4.5, whereas their reactivity was only slightly enhanced by increasing the pH from pH 3.5 to 4.5. This in agreement with earlier results30,40 where the presence of exposed reactive of thiol groups was observed at acidic pH, in acid-induced coldset gels of whey proteins. However, Alting et al.41 observed a decrease of detectable thiol groups when aggregation was caused by a long heating period (>6 h). A long heat treatment might induce a compacting of the aggregate structure, which was not detected during the short heating time used in this study. Surface Hydrophobicity. Heating globular proteins (such as whey proteins) causes conformational changes (denaturation), which can lead to the exposure of hydrophobic sites that are buried inside the native structure of the protein and are inaccessible to the surrounding.42 Hydrophobicity is one of the major factors controlling intermolecular association of proteins, which results in the formation of (soluble or insoluble) aggregates.43 Hence, studying the evolution of exposed hydrophobicity as a function of heating can provide useful information on the mechanism regulating whey protein aggregation and the impact on dispersibility. Here we assume that even if large aggregates are present, a discussion of the hydrophobicity on a molecular level is still meaningful. The effect of pH and heating on whey protein hydrophobicity in 1, 5, and 10% protein solutions, as determined using the aromatic probe ANS, is reported in Figure 4. Independent of protein concentration, systems at pH 4.5 showed the lowest fluorescence intensity (70 °C. The 10% WPI solutions at pH 4.0 showed gelation at 90 °C, making the measurement of the hydrophobicity impossible. The increasing hydrophobicity with increasing heat treatment was also observed by Moro et al.42 in 737
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(∼10−50 nm). This particle size distribution can explain the dispersibility value for the heat-treated pH 3.5 system (89.29 ± 3.66%). Whereas small aggregates (∼10−50 nm) remained dispersed, large aggregates (∼200−400 nm) precipitated, causing a ∼10% dispersibility loss. Heat-treated systems at pH 4.0 could be analyzed by TEM only before reaching the gelation temperature. Figure 5 shows the TEM results for pH 4.0 systems heated at 70 °C. The TEM picture gives an impression of the structure immediately before the formation of a gel network, with the presence of spherical particles with a radius of ∼50 nm. Heat-induced (90* °C) aggregation of systems at pH 4.5 resulted in the formation of large aggregates (size ∼2000 × 4000 nm), which seem to result from the association of spherical smaller aggregates. No indication for the presence of single smaller aggregates was found. Similar structures were described by Jung et al.46 for β-LG systems at pH 5.8 and by Sullivan et al.47 using TEM and SEM, respectively. PAGE. To investigate the mechanism leading to heatinduced aggregation and the relative contribution of the α-LA, β-LG, and BSA to aggregation, the water dispersible fractions of thermally treated 10% whey protein solutions were analyzed by native PAGE, native PAGE under reducing conditions (DTT), SDS-PAGE, and SDS-PAGE under reducing conditions (SDS + DTT) (Figure 6). The three major protein bands (α-LA, β-LG, and BSA) were observed after electrophoresis. The gels were scanned and the protein concentrations quantified on the basis of the intensity of the bands. Results of the intensity analysis are summarized in Figure 7. Analysis of gels showed that the aggregation of α-LA, β-LG, and BSA was negligible in all of the systems heated to 80 °C. Samples heat-treated at 90 and 90* °C showed significant differences depending on pH and type of electrophoresis used. As observed in Figure 7 the concentration (%) of α-LA, β-LG, and BSA on native PAGE was reduced compared to the control at all pH values (pH 3.5: α-LA = 28.35 ± 8.50, 19.05 ± 8.52; βLG = 38.13 ± 14.58, 27.86 ± 12.01; BSA = 46.79 ± 27.36, 22.07 ± 5.01. pH 4.0: α-LA = 23.44 ± 0.31, 21.22 ± 6.01; β-LG = 38.26 ± 0.15, 34.56 ± 12.13; BSA = 53.87 ± 12.04, 50.20 ± 1.57. pH 4.5: α-LA = 16.27 ± 1.50, 9.65 ± 2.13; β-LG = 22.02 ± 1.15, 13.40 ± 0.73; BSA = 101.32 ± 20.52, 57.91 ± 18.13, at 90 and 90* °C, respectively). This set of data shows that the amount of aggregated protein increased with increasing heating time and at pH 4.5, thus implying a more extensive aggregation close to the pI (in line with the results for dispersibility). The contributions of the various whey protein fractions in the formation of aggregates were proportional to their concentrations in the initial solution. By analyzing the data from native PAGE (Figure 7), in the aggregation at the three different pH values, α-LA, β-LG, and BSA participated in the following proportions at 90 °C: 28.50 ± 1.15, 69.89 ± 2.37, 1.60 ± 1.45. α-LA, β-LG, and BSA participated in the following proportions at 90*: 27.69 ± 1.33, 69.93 ± 1.79, 2.38 ± 0.88. At these temperatures, systems at pH 3.5 also aggregated into small clusters (SA band on the top of the gel in Figure 6). The SA band progressively reduced with increasing pH due to the larger size of the aggregates, which hindered their transport through the gel. Unfortunately, the relevance of intermolecular disulfide bonds on aggregation and the relative contributions of α-LA, β-LG, and BSA could not be quantitatively determined on DTT-PAGE for systems at pH 4.0 and 4.5. This was due to the presence of a smeared band between 70 and 30 kDa, which
a similar study on interactions between WPC and ANS in aqueous systems treated at 85 °C for periods from 0 to 60 min. The change of surface hydrophobicity in the various systems closely followed the behavior observed for dispersibility. As pointed out earlier,43 a low hydrophobicity leads to a higher dispersibility in an aqueous environment. Accordingly, the present study showed that the loss of dispersibility can be explained with the increasing surface hydrophobicity, which resulted in the formation of insoluble whey protein aggregates. Microstructure. TEM images showed significant differences in the microstructure of 10% whey protein solutions at pH 3.5, 4.0, and 4.5 both before and after heat treatment (Figure 5). Aggregates were present in the samples before heat
Figure 5. TEM micrographs showing untreated 10% (w/w) whey protein solutions at pH 3.5, 4.0, and 4.5 (left) and after heat treatment (right).
treatment. This was expected for systems at pH 4.0 and 4.5 due to their turbid appearance and reduced dispersibility. At pH 3.5, further away from the pI, unheated samples were translucent and the dispersibility was high (∼100%); however, still some large aggregates were present. These can be attributed to the structural changes (i.e., denaturation) occurring during the production of the whey protein powder. In Figure 5, the three different morphologies of the systems are shown. At pH 4.0 the size and the distribution of the aggregates were more homogeneous than at pH 3.5 and 4.5. The pH 3.5 system showed that larger aggregates coexist with smaller aggregates, whereas at pH 4.5, larger aggregates (∼1000 nm) could be observed. The larger aggregates seem to result from the aggregation of smaller spherical particles and are surrounded by numerous smaller (
