Effect of Calcium on the Morphology and Functionality of Whey Protein

Article pubs.acs.org/Biomac

Effect of Calcium on the Morphology and Functionality of Whey Protein Nanofibrils Simon M. Loveday,*,† Jiahong Su,† M. Anandha Rao,‡ Skelte G. Anema,§ and Harjinder Singh† †

Riddet Institute, Massey University, Private Bag 11 222, New Zealand Department of Food Science, Cornell University, Geneva, New York 14456, United States § Fonterra Research Centre, Palmerston North, New Zealand ‡

S Supporting Information *

ABSTRACT: Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl2, but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl2 on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl2 on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Smallamplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl2 is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.

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long, semiflexible morphology3 seen under “default” conditions. Fibrils made in the presence of salts are shorter and more flexible5,9 than at low ionic strength, while those assembled at 100−120 °C are stiffer11 than at lower temperatures. A given fibril morphology can often be obtained using several alternative sets of conditions.10 Self-assembly of β-lactoglobulin is thought to require hydrolysis of peptide bonds,12 and a mechanistic model was recently proposed13 under the assumption that fibrils are composed of peptides, but some evidence to the contrary also exists.11,14 The thioflavin T assay, which measures fibril length concentration, typically shows a sigmoid time course, with a lag phase, growth phase, and plateau phase characteristic of nucleation-limited reactions. The lag phase can be eliminated by shearing and/or seeding.15 Certain β-sheet-rich regions of the β-lactoglobulin molecule are found in abundance in heatinduced fibrils,12 and peptides homologous with these regions nucleate de novo fibril formation very effectively.8 Association of building blocks into fibril nuclei is thought to occur via hydrophobic interactions, 16 which may also drive the association of individual fibrils into multifibril bundles.16,17 There is relatively little information in the literature on the rheological properties of β-lactoglobulin self-assembling fibril dispersions,18 with the notable exception of work from King’s College, London.1,19,20 That work developed a method for tracking the mechanical properties of self-assembling protein

INTRODUCTION A number of globular proteins self-assemble in vitro under denaturing conditions into fibrils several nanometers thick and several micrometers long. These fibrils share certain structural characteristics with the pathogenic amyloid fibrils formed in vivo in association with certain diseases, so the fibrils formed in vitro are often called “amyloid-like”. The high aspect ratio of these fibrils means that they can form entangled networks or “fine-stranded” gels at much lower protein concentrations than particle aggregate gels at higher pH.1 This makes protein fibrils efficient thickeners and gelling agents with potential uses in food and pharmaceutical applications. Proteins from egg, soybean, and milk sources will assemble into fibrils in vitro under conditions that facilitate denaturation but inhibit random aggregation.2 In practice, this usually means heating at low ionic strength and at a pH far from the protein’s isoelectric point or incubation at 20−37 °C in concentrated chemical denaturants. Bovine β-lactoglobulin is often used as a model protein for amyloid-like self-assembly because it is readily available, relevant to food applications, and structurally well-characterized.3 Heat-induced self-assembly of β-lactoglobulin has been studied for about a decade. Many studies have used solutions of 1−2% protein at pH 2 and low ionic strength, and the heating temperature has almost invariably been 80 °C. Under these conditions, the yield of self-assembled protein reaches a maximum at around 10 h.4,5 However, self-assembly can be accelerated considerably by stirring,6,7 seeding,6,8 or heating at lower pH.5 Adding NaCl9 and/or CaCl25,10 or heating at >80 °C11 accelerates self-assembly, while also modifying the © 2011 American Chemical Society

Received: July 21, 2011 Revised: September 1, 2011 Published: September 6, 2011 3780

dx.doi.org/10.1021/bm201013b | Biomacromolecules 2011, 12, 3780−3788

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Prior to measurement, each sample was presheared at 200 s−1 for 2 min to erase its shear history. After that the shear rate was varied from 0.01 to 200 s−1 in steps that gave five points per decade, evenly spaced on a logarithmic scale. Each shear step lasted 10 s, and data were collected only during the last 5 s of the shear step. This avoided making measurements during the first few seconds after the shear rate was changed, when instabilities sometimes occurred. For the in situ rheometer heating experiments, a 2.5 mm thick protective cover made from 316 stainless steel was fitted over the base plate, and measurements were made with a 40 mm cone (angle 4°, truncation length 97 μm), also made from 316 stainless steel. Both cover plate and cone were supplied by TA Instruments. The temperature at the upper surface of the protective plate was slightly lower than the temperature given by the rheometer software, and software settings were adjusted to compensate for this. After the sample was transferred and the cone lowered into place, silicone oil (200 Fluid, 1000 CST, Dow Corning, Midland, MI) was applied to the exposed edges of the sample and a close-fitting steel moisture trap (TA Instruments) was put in place. A collar on top of the cone was filled with silicone oil, and the edges of the moisture trap dipped into the oil, forming an airtight seal. During heating, oscillatory measurements were made at a frequency of 1 Hz and strain of 0.1%. Linear regression of oscillatory data was done with Minitab 15 (Minitab Inc., State College, PA). Dialysis. Dialysis used Spectra/Por 1 membrane with 6000−8000 Da molecular weight cutoff (Spectrum Laboratories, Rancho Dominguez, CA). Sections of membrane were rinsed in water and soaked in buffer for an hour prior to use. The buffers were HCl in water for pH 2, 10 mM acetic acid/sodium acetate for pH 4, and 20 mM Na2HPO4/NaH2PO4 for pH 6.7, all containing 0.05% sodium azide. Samples were dialyzed at 4 °C with gentle stirring in the buffer outside the membrane. Buffer was changed after the first 4 h and then once daily for the next 4 days.

systems at high temperature and low pH. Protein solutions were heated on the base plate of a rheometer while smallamplitude oscillatory measurements were made in situ.1 The primary focus of that work was elucidating the mechanism of network assembly by experimental validation of theoretical models. Protein concentrations in the range of 7.5−16.9% in studies of heat-induced self-assembly,1,19 and concentrations as low as 5% were tested during solvent-induced assembly.20 The in situ rheometer heating method is an excellent tool for noninvasively probing network assembly in real time, but no other group has used it at such extreme temperatures and pH, perhaps because it demands special materials and painstaking experimental precautions. Salts are ubiquitous in food and biological systems, and in this work we have sought to better understand their effect on fibril network assembly during heating. To date, almost all investigations into the effect of salts on β-lactoglobulin selfassembly have used only NaCl. We chose CaCl2 because it accelerates self-assembly much more effectively than NaCl,5 and significant amounts of Ca2+ are present in many dairy-based food products. We focused on rheological properties because they are sensitive to network assembly and highly relevant to potential applications of fibrils, but we also sought to relate them to fibril morphology. We used whey protein isolate (WPI), which is readily available and industrially relevant, while still comprising ∼90% protein21 and up to 80% β-lactoglobulin (Singh, H., personal communication). The in situ rheometer heating method gave a very sensitive measure of fibril network mechanical properties in real time.

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MATERIALS AND METHODS

RESULTS Behavior of 2% w/w Whey Protein Isolate Solu tions. WPI solutions were heated at pH 2 and 80 °C in all cases. Under these conditions, viscosity increased during the first 3 h of heating, and was approximately constant thereafter. For that reason, viscosities after 3 h heating are shown in Figure 1 as

Chemicals. CaCl2 was AnalaR grade from BDH (Poole, England), and Milli-Q water was used throughout the study. WPI was supplied by Fonterra Cooperative Ltd., Auckland, New Zealand. Sample Preparation. WPI was added to Milli-Q water with stirring, pH was adjusted to 2 with 6 M HCl, and the sample was stirred overnight at 4 °C. The pH was checked the next day and adjusted if necessary. WPI solutions were heated at 80 ± 0.2 °C in a Lab Companion BS-11 water bath (Jeio Tech, Seoul, Korea). Negative Stain Transmission Electron Microscopy. An ultrafiltration method4 was used to purify fibrils and reduce the background in TEM images. 100 μL of heated protein solution was added to 2 mL of pH 2 water in a centrifuge filter (100 kDa cutoff, Amicon Ultra-4, Millipore), which had been previously washed with 2 mL of pH 2 water. The filter was centrifuged at 3000g for 15 min, the filtrate was discarded, and 2 mL of pH 2 water was added to the retentate. The sample was centrifuged again, retentate topped up with 2 mL of pH 2 water, centrifuged a third time, then 1 mL of pH 2 water was added to the retentate, mixed by inversion, and transferred to a 1.5 mL of plastic tube (Eppendorf, Hamburg, Germany). The final dilution of heated protein was ∼10-fold. A copper TEM grid coated with Formvar was placed on a droplet of sample for 5 min. The grid was removed, touched against filter paper to soak away excess sample, and then placed on a drop of 2% uranyl acetate in water for 5 min. Excess stain was soaked away with filter paper. The negatively stained grid was examined with a Philips CM10 electron microscope (Eindhoven, The Netherlands). Image contrast was improved by contrast-stretching with Adobe Photoshop Elements 2.0 (Adobe Systems Inc., San Jose, CA). Rheometry. Continuous rotational flow data were collected at 20 °C using an AR-G2 rheometer (TA Instruments, New Castle, DE) fitted with a 60 mm diameter stainless steel cone with angle 4° and truncation length 112 μm. After heating for the required time, sample tubes were withdrawn from the water bath and placed in ice water for 5−10 min. A sample was transferred from the tube onto the rheometer base plate with a disposable plastic transfer pipet.

Figure 1. Effect of CaCl2 on the viscosity of 2% WPI dispersions at pH 2 heated for 3 h at 80 °C. Different symbols show apparent viscosity at different shear rates, and vertical bars are standard errors of 5−8 replicates.

a function of shear rate and added CaCl2. Adding up to 40 mM CaCl2 decreased viscosity slightly, and increasing CaCl2 to 3781


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80 mM increased viscosity by a decade and a half. A further increase to 120 mM CaCl2 gave a modest increase in viscosity. Interestingly, WPI samples heated with 80 mM CaCl2 showed time-dependent changes in viscosity during the preshear step of 2 min at 200 s−1, whereas samples at other CaCl2 levels showed little or no change in viscosity during preshearing. WPI samples with 80 mM CaCl2 heated for 3 h were slightly antithixotropic (rheopectic) during 120 s of shearing, in that apparent viscosity increased from 0.148 ± 0.005 to 0.201 ± 0.014 Pa s after 70 s shearing and remained constant thereafter. WPI fibrils formed without CaCl2 were long and semiflexible and associated in large entangled networks more than 10 μm across on the TEM grid (Figure 2A). For TEM images of this

and were bent and twisted (Figure 2B). The same morphology was seen with 60 mM CaCl2 (Figure 2C), but fibrils were more numerous and intertwined more in local networks. These morphologies were very similar to β-lactoglobulin fibrils seen in earlier work,5 except that the transition from straight and semiflexible to short and bent occurred at ≤40 mM CaCl2 with WPI, whereas with β-lactoglobulin it occurred between 40 and 80 mM CaCl2. For β-lactoglobulin there was a mixture of the two fibril types between 40 and 80 mM CaCl2, and the ratio depended on the CaCl2 concentration,5 whereas no long, semiflexible fibrils were seen in WPI heated with 40 mM CaCl2 in the present work. Behavior of 10% w/w Whey Protein Isolate Solutions. Whey protein self-assembly was tested at higher concentration by tracking gelation in real time with an in situ heating method developed by Kavanagh et al.1 and used extensively by Gosal et al.19 Samples of WPI solution were transferred onto the rheometer stage at 60 °C, heated rapidly to 80 °C, and held at that temperature while collecting oscillatory shear data, i.e., a “gel-cure” experiment. The rheometer software recorded the displacement and torque for each pseudo-straincontrolled oscillation and attempted to fit a sine curve to the torque waveform in order to calculate oscillatory parameters. Torque waveforms were quite jagged at G′ < 20 Pa, so calculation of oscillatory parameters may not have been very reliable prior to the rapid increase in G′. Time course data were analyzed according to the method of Scott Blair and Burnett,22 using eq 1, in which G′ is the storage modulus, G′inf is the limit of G′ as time tends toward infinity, B is the time taken for G′ to reach G′inf/e, and t is time. Others who have used eq 1 derived values of G′inf and B by linearizing it to give eq 2,19,22 whose parameters can be calculated by linear regression analysis of data plotted as ln(G′) vs (1/t). (1) (2)

However, we used nonlinear regression of G′ vs t in order to avoid data transformations which would have distorted the nature of errors. Nonlinear regression used only G′ and t data for which ln(G′) vs 1/t was linear. Reciprocal weighting of G′ was necessary to avoid large G′ values having undue influence on the fit line. The linear and nonlinear regression analyses gave G′ values up to 5% different and tgel values up to 3% different. Structure development rate during gelation, defined as dG′/dt, can be determined under different gelling conditions. Pronounced concentration dependence of dG′/dt has been reported for a wide range of polymers. In high-methoxyl pectin/sucrose systems, dG′/dt was strongly dependent upon the temperature, showing a complex behavior due to the several kinds of intermolecular interactions involved.23 Here dG′/dt was calculated by differentiating eq 1 with respect to t, yielding eq 3. Its maximum value, (dG′/dt)max, was calculated from eq 4, which is derived analytically from eq 3, as described in the Supporting Information. Figure 2. Morphology of whey protein fibrils with no CaCl2 (A), with 40 mM added CaCl2 (B), or with 60 mM added CaCl2 (C). Fibrils were created by heating 2% w/w WPI at pH 2 for 3 h at 80 °C.

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type of fibril at high magnification and a detailed analysis of fibril morphology, the reader is referred to our earlier work. 5 Fibrils formed in the presence of 40 mM CaCl2 were shorter

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Time course data and model fits are plotted in Figure 3 on log−log axes, and the inset of Figure 3 shows the linear region

Table 1. Effect of CaCl2 on Gel Times, tgel (in s), for 10% WPI Heated at 80 °C on the Rheometera CaCl2

0 mM

40 mM

80 mM

120 mM

tgel when G′ > 10 Pa tgel from manual selection tgel from eq 1

637 491 471

334 271 208

209 156 126

115 73 83

a

Three different methods were used for estimating tgel, as explained in the text.

slower rate. Plotting data as log(tgel) vs CaCl2 concentration gave highly linear correlations, with linear regression yielding R 2 values of 0.997, 0.995, and 0.974 for tgel(G′ > 10 Pa), tgel(manual), and tgel(eq 1), respectively. G′inf increased to a maximum with 80 mM CaCl2 and then decreased with more CaCl2 (Figure 4), so that G′inf with 40 mM

Figure 3. Gel cure data for 10% w/w WPI with 0−120 mM CaCl2 heated at 80 °C on the rheometer. Lines show the fit of eq 1 to data, and the inset shows the linear fitting (eq 2).

on “log(G′) vs 1/t” axes. All data sets had an initial lag phase, during which G′ was below 10 Pa, followed by a very rapid increase in G′, and then further increase in G′ but at a slowing rate. CaCl2 had a marked impact on the duration of the lag phase, shortening it in a concentration-dependent manner. For 80 and 120 mM data sets there was dip in G′ after long heating times, similar to that seen by Gosal et al.19 They suggested that the dip could be caused by slippage due to gel shrinkage and syneresis after extended heating or the infiltration of silicone oil between the cone and the sample. The model in eq 1 was an excellent fit for 0 and 120 mM data, which had clear linear regions on “log(G′) vs 1/t” axes (Figure 3 inset). The linear regions were less well-defined with 40 and 80 mM data, and in choosing which points to include in the regression, preference was given to the later points, for which G′ was higher and could therefore be measured more accurately. Scott Blair and Burnett22 advocated experimental measurement of the gel time, tgel, and Gosal et al.19 defined tgel as the time where a discontinuity in slope occurred on a plot of log(G′) vs t. We could not pinpoint the discontinuity in our data with much precision because prior to the discontinuity, variation in G′ was quite noisy and there were too few data points to find a trend. We also considered using the crossover of G′ and G″ as an indication of gelling,24 but data were too noisy for the sample without CaCl2, and G′ and G″ were almost equal prior to gelation of the sample with 80 mM CaCl2. We tried three other approaches to determining tgel : (i) extrapolating eq 1 to the time at which it predicted G′ = 1 Pa (i.e., the x-intercept of eq 2); (ii) manually selecting the point immediately prior to the rapid increase in G′; (iii) choosing the first time at which G′ exceeded 10 Pa. Values of the three variants of tgel as a function of CaCl2 concentration are shown in Table 1. They generally followed the order tgel(G′ > 10 Pa) > tgel(manual) > tgel(eq 1). All measures of tgel decreased by ∼50% between 0 and 40 mM CaCl2 and then decreased further with more CaCl2 but at a

Figure 4. Effect of CaCl2 on G′inf and (dG′/dt)max for 10% w/w WPI solutions at pH 2, heated at 80 °C.

and G′inf with 120 mM CaCl2 were approximately equal. A repeat experiment gave slightly different values of G′inf, but the same trend as in Figure 4. Values of (dG′/dt)max also increased to a maximum with 80 mM CaCl2 and decreased with more CaCl2, although in a repeat experiment (dG′/dt)max increased by another 35% when CaCl2 was increased to 120 mM. The time at which the maximum in (dG′/dt) occurred was between 4tgel and 5tgel in all cases. Plotting the time course data on reduced coordinates of G′/G′inf vs t/tgel (where tgel was calculated with eq 1) resulted in a good superposition (Figure 5), particularly at 2 < t/tgel < 10. Effect of Dialysis and pH Adjustment on Whey Protein Isolate Fibrils. The propensity of WPI fibrils for electrostatic aggregation was tested by increasing the pH at 20 °C. Fibril dispersions were adjusted to a range of pHs with NaOH, left overnight at 4 °C, and then centrifuged at low speed. Fibrils formed without CaCl2 (Figure 6A) precipitated between pH 4.4 and pH 5.8, and a small amount of translucent precipitate was visible at pH 4.0, 4.2, 6.0, and 6.2. Fibrils formed with 80 mM CaCl2 precipitated over a wider pH range (Figure 6B), with a small translucent precipitate at pH 2.5−3.0, a larger and less compact precipitate at pH 3.5, and a compact, opaque precipitate at pH 4.0−7.0. The aggregation induced by CaCl2 is probably due to two effects: bridging of negatively charged regions by Ca2+ cations and shielding of positively charged regions by Cl− anions. 3783


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Figure 7. Effect of dialysis on the solubility of WPI fibrils made with CaCl2. Tubes of 2% w/w WPI with 80 mM CaCl2 were heated at 80 °C for 1 h, treated as described in the figure, stored overnight at 4 °C, and then centrifuged for 30 min at 1000g. Figure 5. Superposition of time course data by normalizing against G′inf and tgel. Equation 1 was used to calculate G′inf and tgel.

and there were clumps of stained material that may have been either tightly entwined fibrils or aggregated nonfibrillar material. The effects of dialysing and neutralizing on the rheological properties of fibril dispersions were examined using samples heated with 0, 40, or 120 mM CaCl2 (Figure 9). The WPI concentration with 40 mM CaCl2 was increased to 4% in order to obtain viscosities well above the lower measurement limit with the cone and plate geometry. Dialysing samples with 40 and 120 mM CaCl2 resulted in a relatively small decrease in apparent viscosity, which was more prominent at shear rates of 1 s−1 and below. Dialysis may have slightly diluted fibril dispersions, contributing to the decrease in viscosity. Neutralizing had relatively little effect on the viscosity of the control sample made without CaCl2, but viscosity decreased by over a decade for fibrils made with 40 mM CaCl2. Adjusting pH also affected the morphology of these fibrils (Figure 10). Fibrils were long, semiflexible, and with a smooth appearance at pH 2, whereas at pH 6.7 they appeared to be shorter, thinner, and with a rough or irregular surface. There were generally fewer fibrils at pH 6.7, and a lot of small nonfibrillar protein aggregates were also present, suggesting that some fibrils had disintegrated into small aggregates.

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Figure 6. Effect of pH on the physical stability of whey protein fibrils made without added CaCl2 (A) or with 80 mM CaCl2 (B). All tubes were initially pH 2, and labels indicate the final pH to which they were adjusted. Solutions were stored overnight at 4 °C after pH adjustment and then centrifuged for 30 min at 1000g.

DISCUSSION

We previously reported that calcium accelerated β-lactoglobulin self-assembly (measured via thioflavin T fluorescence) by both shortening the lag phase and increasing the fibril growth rate. 5 The effect on rheological properties of WPI fibril dispersions was somewhat different, in that all samples had reached a plateau in viscosity within 3 h heating, and only the height of the plateau was affected by CaCl2. In earlier work11 we noted that the viscosity of fibril dispersions is likely related to both the volume fraction of fibrils and their degree of entanglement. Thioflavin T fluorescence assay results5 indicate that the volume fraction of fibrils would continue to increase beyond 3 h, and yet the viscosity did not increase correspondingly. This suggests that the degree of entanglement may have been more important than volume fraction. In Figure 11, we have attempted a conceptual explanation of the mechanisms by which CaCl2 affects fibril morphology, fibril entanglements, and network percolation and thereby affects bulk rheological properties. Earlier TEM results5 showed that increasing the amount of CaCl2 increased the proportion of curly, twisted, β-lactoglobulin fibrils (presumably highly

CaCl2 was removed from WPI fibril samples by dialysing, to see whether the presence of Ca2+ was causing precipitation or whether it was due to an inherent property of the short, curled fibrils. Fibril dispersions were dialyzed at pH 2, 4, or 6.7, and dispersions dialyzed at pH 2 were subsequently adjusted to pH 4 and pH 6.7. The visual appearance of samples (Figure 7) shows that dialysis at pH 4 or pH 6.7 did not reverse precipitation, probably because salt ions were bound to proteins. However, samples dialyzed at pH 2 did not precipitate at pH 4 or pH 6.7, suggesting that the salts removed by dialysis were responsible for pH-related precipitation. The effect on fibril morphology of dialysing then adjusting pH was examined with TEM (Figure 8). The morphology seen in the pH 2 control (Figure 8A) was apparently unchanged by dialysis at pH 2 (Figure 8B) and subsequent adjustment to pH 4 (Figure 8C). However, dialyzed fibrils at pH 6.7 (Figure 8D) were shorter and more irregular than under other conditions, 3784


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Figure 9. Effect of pH and dialysis on the viscosity of fibril dispersions made with 0, 40, or 120 mM CaCl2. Control samples were heated 3 h, and samples with CaCl2 were heated for 16 h. Vertical bars are the maximum and minimum from two replicates.

influences macroscopic properties like dynamic moduli, as described by classical polymer theory.26 The slight decrease in viscosity of heated WPI fibrils between 0 and 40 mM CaCl2 was surprising, given earlier evidence of a monotonic increase in the rate of self-assembly of β-lactoglobulin with increasing CaCl2.5 The lower viscosity of WPI fibrils with 40 mM CaCl2 may be related to the fact that no semiflexible fibrils were present (Figure 2B). Semiflexible fibrils can be up to 10 μm long, and they could link flocs of curly fibrils into larger mesoscopic networks (Figure 11, panel B). The larger networks would increase bulk viscosity more effectively than dispersed flocs. The increase in viscosity between 40 and 120 mM CaCl2 might be explained by an increasing amount of fibrils, but ThT results10 and measurements of nonaggregated protein4 suggest that NaCl and CaCl2 have a relatively small impact on fibril yield. The internal structure of fibrils could also be altered by CaCl2, resulting in a higher elastic modulus. Fibrils formed at ionic strength of up to 255 mM10 do not look dramatically different to those formed at moderate CaCl2 content (Figure 2), but there may still be differences in fine structure that are not clear in TEM images. A further possibility is suggested in panels C and D of Figure 11. The strong concentration-dependent effect of calcium on the lag phase suggests that it catalyzes nucleation, e.g., via cation−π interactions.5 The number of nucleation events per unit volume and unit time would affect the balance between intrafibril and interfibril entanglements. With low nucleation density, fibrils would not readily encounter other

Figure 8. Effects of dialysis followed by pH adjustment on fibril morphology. Fibrils were prepared as for Figure 7.

flexible) and decreased the proportion of long semiflexible fibrils. Molecular models of polysaccharides indicate that highly flexible molecules (i.e., short persistence length) are able to “hyperentangle” better than stiffer molecules,25 so the curly fibrils may entangle better than semiflexible fibrils (Figure 11, panels A and B). The number density of entanglements 3785


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Figure 10. Whey protein fibrils created by heating 2% w/w WPI at pH 2 and 80 °C for 16 h (A) and the same fibrils with pH adjusted to 6.7 (B).

Figure 11. Proposed mechanism for the effect of CaCl2 on the rheological properties of whey protein fibril dispersions.

fibrils around them, and so they would become tangled up in themselves because of their curly, twisted shape. When nucleation events happen closer together, growing fibrils would encounter each other more frequently and would form interfibril entanglements that effectively compete with intrafibril entanglements. The result of high nucleation density is more linkages between fibrils and a stronger network. Higher nucleation density would produce more individual fibrils with shorter lengths. Perhaps a large number of shorter fibrils entangle more effectively than fewer very long fibrils. At 10% WPI the self-assembly reaction was much faster than at 2% WPI, probably because of a higher number of nucleation events per unit volume and a higher concentration of assemblycompetent building blocks. CaCl2 shortened tgel for 10% WPI (Table 1) in the same way it shortened the lag time, tlag, for selfassembly in 1% β-lactoglobulin,5 i.e., a large decrease in tgel or tlag with the addition of a little CaCl2 and a diminishing additional effect with further CaCl2. The G′ of the sample without added CaCl2 followed eq 1 right from the onset of the G′ increase. However, G′ for the 40 and 80 mM samples fell below the fit prediction line for a short period immediately after the onset of gelation. This may be related to the change in fibril morphology (Figure 2). The radius of gyration per unit fibril length will be much smaller for curly fibrils than for long semiflexible fibrils, so network percolation throughout a sample would occur at a higher volume fraction for curly fibrils. This is supported by simulation of dispersions of rod-like or coil-like particles.27 During the

period between tgel and when G′ meets the fit line, there may be local networks of curly fibrils that have not yet percolated throughout the sample. The 120 mM CaCl2 sample also followed eq 1 from the onset of the G′ increase. Percolation may have occurred quite quickly because of the rapid nucleation throughout the sample. The three methods for finding tgel showed the same trends, and on the face of it the choice does not appear to make much difference to the conclusions. However, manually designating a time point as tgel is not very rigorous. Given the difficulty of accurately measuring G′ of very low-viscosity samples with our equipment, the method of selecting the first point at which G′ exceeds an arbitrary threshold was also problematic. A more sensitive geometry such as a concentric cylinder might fare better than our 40 mm cone in this respect, but this would be harder to fabricate from chloride-resistant 316 stainless steel and would take longer to heat or cool. G′inf was higher with 80 mM CaCl2 than at any other level. This may be because the rate of fibril nucleation with 80 mM CaCl2 was optimal for the formation of a strong elastic network. The good superposition of time course data into a master curve (Figure 5) suggested that CaCl2 did not alter the gel formation mechanism,28 even though fibril morphology was affected by CaCl2 (Figure 2). Given that most amino acids with charged side chains would be protonated at pH 2 and would repel cations, we expected that Ca2+ influenced self-assembly via its effect on the ionic environment and/or its ability to 3786


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it provides information on the kinetics of mesostructure development in a self-assembling system.

temporarily bridge between two electron-rich side chains, rather than fulfilling an ongoing structural role in fibrils or fibril gels. To investigate this, we tested the effect of heating WPI with CaCl2 and then removing Ca2+ by dialysis against water adjusted to pH 2 with HCl. If Ca2+ ions were an integral, ongoing part of the fibril structure and/or the gel structure, then their removal would result in gross changes to fibril morphology and/or bulk rheological properties, respectively. However, dialysing at pH 2 had no noticeable effect on fibril morphology (Figure 8) and had little effect on bulk viscosity (Figure 9). These findings were in line with earlier work29 in which β-lactoglobulin fibrils formed with or without NaCl were diluted in solutions of different ionic strength. They confirmed our expectation that Ca2+ did not play an ongoing role in either fibril structure or gel structure at pH 2. Raising the pH above 2 would shift the dissociation equilibria of charged amino acid side chains away from the protonated state, thus eliminating positive charges and facilitating the interaction of cations with negatively charged and electron-rich side chains. Divalent Ca2+ ions can bridge between residues on two different peptides, thus promoting aggregation. We believed that such “salt bridging” was behind the precipitation seen at pH 3−4 and pH 6−7 with CaCl2 fibrils (Figure 6), but it was also possible that intrinsic properties of fibrils with CaCl2 made them more prone to electrostatic aggregation than those made without CaCl2. Removing Ca2+ by dialysis at pH 2 prevented precipitation at pH 4 (Figure 7) without altering fibril morphology (Figure 8), so salt bridging was probably the cause of precipitation at that pH. Precipitates which formed on raising the pH of CaCl2-containing samples to 4 were not dispersed by subsequent dialysis at pH 4 (Figure 7), which indicated a strong interaction in precipitated material between Ca2+ and proteins at pH 4. Removing Ca2+ at pH 2 prevented precipitation when the pH was subsequently raised to 6.7 (Figure 7), but dialysing and then neutralizing also disrupted fibrils into shorter pieces (Figure 8). Fibrils made without CaCl2 were also partly disrupted at pH 6.7 (Figure 10), and the viscosity of these fibrils fell slightly (Figure 9). The fall in viscosity was much larger for fibrils made with 40 mM CaCl2 than for those made without CaCl2, perhaps because fibrils were assembled faster with 40 mM CaCl25 and thus had a more irregular, disorganized structure, which was more fragile. At pH 6.7, the carboxyl groups on most aspartic acid and glutamic acid residues will be negatively charged, creating significant electrostatic repulsions within a fibril. The fact that some fibrils remained intact while others fractured or disintegrated indicates that not all fibrils are created equal. The source of this diversity in fibril structure is not clear, but there are unlikely to be different building blocks in different fibrils, since the protein/peptide composition should be homogeneous throughout a sample, provided temperature gradients are avoided.

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ASSOCIATED CONTENT S Supporting Information * The analytical solution for (dG′/dt)max. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *Fax: (+64) 6 3505655. E-mail: [email protected].

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ACKNOWLEDGMENTS We are grateful for the skillful assistance of Doug Hopcroft at the Manawatu Microscopy and Imaging Centre, IMBS, Massey University. This work was funded by Fonterra Cooperative Ltd. and the New Zealand Ministry of Science and Innovation, Contract DRIX0701.

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REFERENCES

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CONCLUSIONS This study has examined the effects of CaCl2 on the rheological properties and microstructure of WPI fibrils, in complement to earlier work examining its effect on the kinetics of βlactoglobulin self-assembly. The in situ heating method has proven to be a good way of investigating self-assembly kinetics at higher protein concentrations, where gelation precludes the use of fluorimetric assays. The in situ method does not directly measure volume fraction or length concentration of fibrils, but 3787


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Effect of Calcium on the Morphology and Functionality of Whey Protein

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