Article pubs.acs.org/Langmuir
Contribution of Long Fibrils and Peptides to Surface and Foaming Behavior of Soy Protein Fibril System Zhili Wan,†,‡ Xiaoquan Yang,† and Leonard M. C. Sagis*,‡,§ †
Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China ‡ Laboratory of Physics and Physical Chemistry of Foods, Wageningen University, Bornse Weilanden 9, 6708WG Wageningen, The Netherlands § Department of Materials, Polymer Physics, ETH Zürich, Leopold-Ruzicka-Weg 4, 8093 Zurich, Switzerland S Supporting Information *
ABSTRACT: When soy glycinin (11S) is heated for a prolonged time at pH 2 (20 h at 85 °C), a mixture is formed consisting of long semiflexible 11S fibrils and small peptides. The surface and foaming properties of this mixture were investigated at different pHs, and compared to the behavior of pure fibrils and pure peptides, to determine the individual contributions of these two factions to the behavior of the mixture. The adsorption of these three systems at air−water interfaces and the resulting surface rheological properties were studied by combining drop shape analysis tensiometry, ellipsometry, and surface large amplitude oscillatory dilatational (LAOD) rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. Our results show that the adsorption kinetics, dilatational rheological properties, and the foaming behavior of the mixture were mainly dominated by the small peptides in the fibril system. Compared to pH 2, the fibril mixture at pH 5 and 7 provides much better foam stability and appears to be a very promising protein material to make stable foams, even at low protein concentration (0.1 wt %). The presence of fibril clusters and peptide aggregates at pH 5 and 7 contributed to foam stability of the mixture. In contrast, pure fibril formed an interface with a highly pH-responsive adsorption and rheological behavior, and the foamability and foam stability of the pure fibrils were very poor.
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INTRODUCTION Protein adsorption at the interface plays a crucial role in many industrial processes, for example, in the formation and stabilization of multiphase food systems.1−3 In systems such as foams, upon adsorption, globular food proteins are known to form highly elastic networks around air bubbles, which appear to be able to stabilize foams against drainage, bubble coalescence, and coarsening.2−6 Studies on the adsorption of globular proteins at the air−liquid interface have been carried out extensively and suggest that the changes in molecular structure of proteins could strongly affect their adsorption behavior and thereby the foaming properties.7−14 For example, several papers have reported that even a slight modification of protein molecules by a physical or chemical treatment can enhance their adsorption kinetics.9−13 These treatments can lead to more exposed hydrophobicity or lower net charge of proteins, which can decrease the energy barrier to adsorption at the interface, and thus lead to faster adsorption kinetics.9,10,15,16 The faster adsorption process generally contributes positively to the foaming performance of globular food proteins. In food processing, the heat treatment of many food proteins, like whey proteins and soy proteins, at different © 2016 American Chemical Society
pHs and ionic strengths can induce structural changes and lead to the formation of supramolecular aggregates with various structures, such as fibrillar structures.17−19 The long, semiflexible protein fibrillar aggregates obtained by heat treatment (80−90 °C) at pH 2 and low ionic strength have attracted increasing interest as functional ingredients in food products due to their high aspect ratio, which could make them ideal as thickening or gelling agents.20 Such fibrils, like their source proteins, have also been shown to be efficient stabilizers for foams or emulsions.21,22 Oboroceanu et al. reported that fibrillized whey protein isolate (WPI) dispersion had better foaming capacity and foam stability than the native protein, and the fibril length (long and short) did not significantly affect the foaming properties.23 They also found that WPI fibril dispersions at pH 7 had better foaming properties than at pH 2. It is well-known that the formation of fibrils, through the heat denaturation of proteins at pH 2, consists of a two-step process.17,24 First, the proteins are hydrolyzed into small Received: April 20, 2016 Revised: June 30, 2016 Published: July 25, 2016 8092
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Langmuir peptides, and then part of the peptides is assembled into fibrils. Thus, the obtained fibril dispersions actually contain a mixture of pure fibrils and unconverted peptides. The small peptides are expected to contribute to the foaming properties of fibril systems due to their amphiphilic structure. So far, no studies have appeared quantifying the contributions of these peptides to the foaming behavior of fibril systems. Recently, the interfacial properties of air−water and oil− water interfaces stabilized by fibrillar aggregates from various proteins have been studied.21,22,25−28 Jung et al. showed that the adsorption kinetics of β-lactoglobulin fibril systems at both air−water and oil−water interfaces were faster than those of native protein, and the adsorption process of pure fibrils seemed to be independent of their length or flexibility.21 Based on surface shear and dilatational rheology measurements, these fibrillar aggregates were found to be able to form a highly elastic interface with solid-like behavior, and long semiflexible fibrils exhibited the highest modulus. Similar findings have been obtained in the case of lysozyme fibrils.22 Through the use of subphase exchange interfacial rheology, Rühs et al. studied the influence of pH on the interfacial rheological properties of long β-lactoglobulin fibrils and found the highest interfacial moduli were observed at pH values close to the isoelectric point (pH 5−6).25 In addition, the influence of unconverted molecules (peptides and protein monomers) on the adsorption kinetics and interfacial viscoelasticity of the fibril system were also investigated.21 From the results, it was shown that these unconverted molecules could increase the rate of adsorption and interfacial modulus of the fibril system. These studies suggest that the microstructure and rheological properties of interfaces stabilized by protein fibrillar aggregates are modified not only by the length and flexibility but also by their polydispersity.21,28 As a result of the high specific surface area, it is known that the macroscopic dynamic behavior of foams can be affected strongly by surface properties, such as surface tension and surface rheological parameters.29 However, studies on the link between surface behavior and foam properties of protein fibrillar aggregates have not been carried out to date. From an industrial point of view, the whole protein fibril mixture is a more relevant material to be used as a foaming agent, since separation of peptides and fibrils can be costly. However, it is important to have knowledge about the surface and foaming properties of pure long fibrils and unconverted peptides to understand their individual contributions to the complex fibril system. Therefore, in this work, we first characterized the surface properties of pure fibrils, unconverted peptides, and the whole fibril system in terms of adsorption kinetics and dilatational rheology at the air−water interface, and then we further investigated the foaming properties of all three systems to assess the links between surface behavior and foam properties. We have chosen to use soy proteins (soy glycinin, 11S) to produce long semiflexible fibrillar aggregates at pH 2, since they are widely used in different research fields, relatively low cost, and biocompatible. Previous studies have shown that most properties of soy glycinin fibrils are similar to βlactoglobulin and whey protein isolate fibrils.19 Since most food products have a pH between 4 and 7, we investigated the influence of pH changes on the surface and foaming properties of the fibril systems. The adsorption kinetics at the air−water interface were determined by drop shape analysis tensiometry and ellipsometry. Subsequently, we performed a detailed study of the frequency and amplitude dependence of the surface
dilatational modulus to obtain information about the microstructure of interfaces formed by pure fibrils, peptides, and the whole fibril system. We used the Lissajous plots to quantify the degree of nonlinearity in the rheological response of interfaces. Finally, we evaluated foam formation and stability of these three systems and correlated foam properties with the results of adsorption and surface dilatational rheology.
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EXPERIMENTAL SECTION
Materials. Defatted soy flour was provided by Shandong Yuwang Industrial and Commercial Co., Ltd., China. The protein content of soy flour was 55.10% (determined by micro-Kjeldahl method, N × 6.25, dry basis). Soy glycinin (11S) was prepared according to a previously method by Yuan et al.30 The protein content of 11S was 97.98%, determined by using Dumas analysis (N × 6.25) using a Flash EA 1112 NC analyzer (Thermo Fisher Scientific Inc., Waltham, MA). Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDSPAGE) analysis showed that the purity of isolated 11S was above 94.5% (data not shown). All other chemicals used were of analytical grade. Preparation of 11S Fibril System. Preparation of the 11S fibril system was carried out according to a previously described protocol.19 Briefly, protein stock solutions of 11S were made by dissolving the protein in Millipore water. The pH of the solution was adjusted to pH 2.0 with 6 M HCl solution, followed by centrifugation (10000g, 30 min, 4 °C) to remove undissolved materials (around 3.5% of the protein sample). The protein concentration of solutions was determined using Dumas analysis (N × 6.25). The obtained 11S solution (2 wt %) was placed in small glass vials (20 mL) and then incubated at 85 °C for 20 h under continuous stirring at 300 rpm by using a metal heating and stirring plate. After heat treatment, samples were immediately cooled in an ice bath and then stored at 4 °C for further use, typically within 1 week. The formed 11S fibrils are long and semiflexible with a thickness of a few nanometers, a persistence length of about 2.3 μm, and a contour length of about 1 μm.19 Separation of Fibrils and Unconverted Peptides. Fibrils and unconverted peptides were separated according to a previously described method.19,31 The fibril solution was first diluted to a protein concentration of 0.2 wt % with HCl solution of pH 2. The solution was divided into centrifugal filters (Amicon Ultra 100 K-15 centrifugal filters) and centrifuged at 3000g and 15 °C for 30 min (Allegra X-22R centrifuge, Beckman Coulter). The retentate (containing the fibrils) was washed twice with fresh pH 2 HCl solution after each centrifugation to remove unconverted protein left in the retentate. The filtrate was recovered after each washing step, and after the last washing step, the retentate was recovered. The protein concentration in the three filtrates was measured using Dumas analysis, and in the third filtrate no protein was detected anymore. Further analysis was also done on the recovered retentate solution. The difference between the total amount of protein and the amount of protein in the retentate was used to calculate the amount of protein converted into fibrils. On the basis of this method, we found that the prepared 11S fibril system consists of approximately 20% fibrils and 80% unconverted peptides. Surface Tension and Surface Dilatational Rheology. The surface tension and surface dilatational modulus of the air−water interface were determined using a profile analysis tensiometer (PAT1M, Sinterface Technologies, Germany). A drop of sample solution was formed in a rectangular glass cuvette and monitored with a video camera. The surface tension (γ) was calculated from the shape analysis of a pendent drop according to the Gauss−Laplace equation. The dynamic surface tension of all the solutions was monitored for 180 min at a constant area of 25 mm2 of the drop. The surface tension data were converted into surface pressure by using the relation Π(t) = γ(water) − γ(t), where γ(water) = 72 mN m−1 is the surface tension value of pure water. All measurements were performed at a bulk protein concentration of 0.1 wt %. After 180 min of adsorption, quasi-equilibrium conditions of interface were obtained, and then the following dilatational rheology measurements were performed: (1) A frequency sweep was performed 8093
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Langmuir with frequencies between 0.005 and 1 Hz, at a constant amplitude of 10%. The slope of a double-logarithmic plot of complex surface dilatational modulus versus frequency was determined using linear regression. For higher frequencies, the fit used to establish the firstharmonic Fourier moduli was not always of sufficient quality, which reduced the accuracy of values for the moduli. Therefore, the regression fit was based on the frequency range from 0.005 to 0.1 Hz. (2) To investigate the rheological response as a function of dilatational amplitude, amplitude sweeps were performed with deformations ranging from 1.5% to 30%, at a frequency of 0.1 Hz. The complex modulus was calculated from the intensity and phase of the first harmonic of a Fourier transform of the oscillatory surface pressure signal. All experiments were performed at 20 °C and at a bulk protein concentration of 0.1 wt %. Reported values represent the average of 3−7 measurements. Lissajous Plots. The large amplitude dilatational measurements were also analyzed with a method developed by Van Kempen et al.32 Lissajous plots were constructed of the surface pressure (Π = γ − γ0) versus deformation (δA/A0), where δA = A − A0, γ and A are the surface tension and area of the deformed interface, and γ0 and A0 are the surface tension and area of the nondeformed interface. For the analysis of the curves, we defined the following four factors: EL,E, defined as the large strain modulus in extension, EM,E, defined as the minimum strain modulus in extension, EL,C, defined as the large strain modulus in compression, and EM,C, defined as the minimum strain modulus in compression. The method for determining these factors has been discussed in detail in a previous study.32 On the basis of these moduli, we further defined two nonlinearity parameters: one for the extension part of the cycle, Sexp = (EL,E − EM,E)/EL,E, and one for the compression part, Scom= (EL,C − EM,C)/EL,C. For both factors, S = 0 may be interpreted as a linear elastic response, S > 0 indicates strain hardening behavior in the interface, and S < 0 corresponds to intracycle strain softening. With these factors, we could quantitatively analyze the degree of nonlinearity in the response of interfaces during extension and compression. Ellipsometry. The adsorbed amount and the adsorption layer thickness after 3 h were measured by null ellipsometry using a Multiskop instrument (Optrel GBR, Sinzing, Germany). The details of this apparatus and the procedure to calculate adsorbed amount and layer thickness have been given elsewhere.33 The light source was a He−Ne laser with a wavelength of 632.8 nm, and the incidence angle of the light was set at 50°. The changes of ellipsometric angles (Δ and Ψ) due to the adsorption of protein at the air−water interface were measured and fitted using a three-layer model. This model assumes a single homogeneous adsorption layer characterized by the average refractive index (nad ) and the thickness (δad) between two homogeneous phases (water and air). Subsequently, the adsorbed amount (Γ, mg/m2) can be calculated according to the following equation:33
Γ=
the surface of foam tube. The time for the foam to drain 50% of its initial liquid content is defined as the foam liquid stability (s). The time required for the foam volume to reduce to half of its initial volume (half-life, t1/2) was used as an indication of foam stability. Reported values are averages of at least three individual measurements. All experiments were performed at 20 °C and at a bulk protein concentration of 0.1 wt %.
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RESULTS AND DISCUSSION In this work, the adsorption and dilatational rheological properties of pure fibrils, unconverted peptides, and the whole mixed fibril system at the air−water interface, as well as the foaming properties of these three systems, were investigated to determine the individual contributions of fibrils and unconverted peptides to surface and foaming properties of the complex fibril system. To study the influence of pH changes on these properties, the pHs of all three systems were adjusted to pH 2, 5, and 7, respectively. All systems had the same protein concentration of 0.1 wt %. As shown in Figure 1S (see Supporting Information), these three systems were stable and transparent at pH 2 due to the high electrostatic repulsive force (Table 1). The fibril system and pure peptide became turbid at Table 1. Zeta-Potential (mV) of the Fibril System, Pure Fibril, and Pure Peptide at pH 2, 5, and 7a zeta-potential (mV)
a
sample
pH 2
pH 5
pH 7
fibril system pure fibril pure peptide
36.2 ± 2.4 36.4 ± 2.2 25.1 ± 0.1
4.3 ± 1.0 −2.6 ± 0.1 4.6 ± 1.1
−14.4 ± 1.6 −24.2 ± 2.9 −9.3 ± 0.8
Protein concentrations are 0.1 wt %.
pH 5 and 7, especially at pH 5, since their net charge is close to zero at pH 5 (Table 1). However, the pure fibrils stayed relatively transparent at pH 5 and 7. Previous studies have shown that the turbidity of β-lactoglobulin fibril systems around pH 5 could be decreased drastically by removing the unconverted peptides from the system and thus confirmed that the unconverted peptides are the main cause for the turbidity.31 Despite the relatively transparent appearance, pure fibrils actually formed dilute clusters around pH 5 since the surface charge of the fibrils was also close to zero at this pH (Table 1).31 We conclude that at pH 5 the 11S fibril system predominantly consists of dilute fibril clusters and peptide aggregates, and at pH 7 free peptides, peptide aggregates, dilute fibril clusters, and individual fibrils coexist. These pronounced changes on the structure and net charge of all the systems as a function of pH are expected to have a significant influence on their adsorption kinetics and thus foaming properties, and the results are described in the following sections. Adsorption Behavior of 11S Fibril System, Pure Fibrils, and Unconverted Peptides. Adsorption Kinetics. Figure 1 shows the time evolution of surface pressure (Π) of the fibril system, pure fibrils, and pure peptide at the air−water interface. As can be seen, at all investigated pHs, the Π curves for the fibril system and pure peptide almost completely overlapped, and they differed significantly from those for pure fibrils (especially at pH 5 and 7). As mentioned above, the prepared fibril system consists of approximately 20% pure fibrils and 80% unconverted peptides; therefore, the adsorption process of the 11S fibril system at the air−water interface was seen to be mainly dominated by the high proportion of
(nad − n water)δad dn/dc
where nwater is the refractive index of water (1.333) and dn/dc is the refractive index increment of the protein in water (0.185 mL g−1). The measurements were performed at a bulk protein concentration of 0.1 wt % and performed in triplicate. Foamability and Foam Stability. The foaming properties of protein fibrillar aggregates were measured using a Foamscan device (Teclis IT-Concept, Longessaigne, France). An initial volume of 60 mL of solutions was foamed by sparging nitrogen at a constant gas flow rate of 400 mL/min through a metal frit (60 mm diameter, pore size 27 ± 2 μm, 100 μm distance between centers of pores, square lattice). Bubbling was stopped after a volume of 400 cm3 of foam was obtained. The foam formation, the drainage of liquid from the foam, and foam stability were monitored by a combination of optical (CCD camera) and conductivity measurements. Series of images of the foam were taken every 20 s by a CCD camera to observe the changes of bubbles. The bubble size was measured at the middle of the foam column using a CCD camera and a prism and lighting arrangement at 8094
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mixed interface where both species are adsorbed at the interface together.28,34 As expected, the adsorption of all three systems at the air− water interface was affected by pH changes. The effect of pH on the fibril system and pure peptide was different from that observed in pure fibril (Figure 1). At both pH 5 and 7, the fibril system and pure peptide exhibited similar adsorption kinetics and reached nearly identical values for the surface pressure after 3 h (about 25 mN/m). At pH 2 these systems had much slower adsorption and reached only a surface pressure of 18 mN/m. Pure fibrils showed a markedly different effect of pH on adsorption kinetics, especially in the initial adsorption stage. The initial adsorption rate for pure fibrils was faster at pH 7 than at both pH 2 and 5. At pH 5, there was an obvious lag time of around 10 s for pure fibrils (Figure 1B). However, the subsequent increase in Π was faster than the increase observed at pH 2 and 7. It is known that the initial adsorption kinetics of proteins is generally limited by their diffusion from the bulk to the interface. The diffusion of proteins to air−water interfaces can be affected by a subtle balance between molecular size, shape, surface charge, and surface hydrophobicity.9,10,35 As previously mentioned, the fibril system and pure peptides are highly positively charged at pH 2 (Table 1), and therefore the electrostatic repulsion is strong, which would lead to a higher electrostatic adsorption barrier. This is probably the main reason that the fibril system and pure peptides showed a faster initial adsorption and higher surface pressure values at both pH 5 and 7 than at pH 2. This analysis is supported by previous studies, which showed that around the isoelectric point (pI) proteins appear to be more surface active and have faster adsorption kinetics.16,36,37 However, for pure fibrils, the observed Π curve at pH 5 seems to contradict these earlier observations. As can be seen from Figure 1B, almost no adsorption (increase in Π) was observed in the initial 10 s (lag time), although the surface charge of the fibrils was close to zero at pH 5 (Table 1). Since the fibrils formed dilute clusters at pH 5, we attributed the presence of the so-called lag phase to the large size of these fibril clusters, which hindered their migration to the interface. Once they have diffused to the interface, a very fast increase in Π was observed since the electrostatic repulsion energy barrier for adsorption is low (Figure 1B). The fact that the fibrils exhibited faster initial adsorption at pH 7 than at pH 2 can be attributed to the lower energy barrier to adsorption at the former pH, since at pH 2 the fibrils have a higher charge. It is described extensively that the adsorption kinetics, especially the initial adsorption, is well correlated to the foam formation of proteins.2,16 Therefore, we expect these observed differences in adsorption kinetics would affect the foaming properties. Ellipsometry. Figure 2 shows the adsorbed amount (Γ) and thickness (δad) of adsorbed layers formed from all systems after 3 h of adsorption. As can be seen, the fibril system and pure peptides showed similar trends in changes of Γ and δad at all three pHs. This can be explained by their similar adsorption processes at the air−water interface (Figure 1). The fibril system and pure peptides exhibited similar values for Γ (around 3.0 mg/m2) and δad (around 8 nm) at both pH 5 and 7, which differed from the values at pH 2 (Γ ∼ 2.0 mg/m2, δad ∼ 10−12 nm). This indicates that a higher packing density of adsorbed proteins at the interface was obtained at pH 5 and 7. As previously discussed, there was a lower electrostatic adsorption barrier and thus a faster adsorption for proteins at pH 5 and 7 (Figure 1). Also, there was less electrostatic repulsion between
Figure 1. Surface pressure (Π) as a function of time for the fibril system, pure fibril, and pure peptide at pH 2 (A), 5 (B), and 7 (C). All systems have the same protein concentration of 0.1 wt %.
unconverted peptides. This is not surprising since, compared to large fibrils (long fibrils at pH 2 and fibril clusters at pH 5 and 7), these small peptide materials (free peptides and/or peptide aggregates) should have faster mass transport rate toward the interface, decreasing the surface tension more rapidly, thus dominating the adsorption kinetics (especially the initial adsorption) of the fibril system. However, it should be noted that at pH 2, although pure fibril had a lower Π during the initial phase of adsorption, pure fibril and the fibril system showed similar final Π, which was slightly higher than that of pure peptide, implying that pure fibril may contribute to the equilibrium surface properties of the fibril system. A similar observation was reported for β-lactoglobulin fibrils at pH 2, a system which also consists of a mix of fibrils and small peptides, and for which an interfacial structure was proposed which consists either of a multilayer with the peptides directly at the interface and the fibrils attached below this primary layer or a 8095
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more accurately characterize the dilatational behavior, we instead used Lissajous plots of dynamic surface pressure versus strain to help interpret the dilatational behavior during amplitude sweeps. Previous studies demonstrated that Lissajous plots are very useful in analyzing nonlinear dilatational behavior.26,32,40 The Lissajous plots of the amplitude sweeps for all systems at pH 2 and 7 are given in Figures 3A and 3B, respectively (additional plots at intermediate amplitude values between 1.5 and 30% are given in the Supporting Information). While the amplitude sweep plots unveiled comparable dilatational moduli for all systems at pH 2 (Figure 3S-A), the Lissajous plots showed significant differences for the fibril system, pure fibrils, and pure peptides, especially at higher amplitudes (7.5−30%) (Figure 3 and Figure 4S). For all systems at pH 2, at amplitudes up to 5% the Lissajous plots were characterized by a mostly elastic response, without pronounced asymmetries. At higher amplitudes of 15% and 30%, the plots for the fibril system and pure peptides became increasingly asymmetric, indicating different responses in extension and compression. It can be seen that the maximum surface pressure observed in compression was much higher than that observed in extension, and the plot also became narrower toward maximum compression, which indicates that the surface is strain hardening upon compression. A widening of the plots was observed in extension (compared to the compression phase), implying that the surface has a more viscous response (strain softening) in extension. Compared to the fibril system and pure peptides, for pure fibrils the shape of the Lissajous plots were less asymmetric, and the response was still mostly elastic when the amplitude was increased. Relatively pronounced nonlinearities only appeared at higher amplitudes (15−30%), where the extension phase of the plots became narrower, implying a relatively small strain hardening upon extension. In addition, it can be seen that the shapes of the Lissajous plots for the fibril system and pure peptides at pH 7 were similar to those obtained at pH 2 (Figure 3, Figures 4S and 5S), but at pH 7 the nonlinearities appeared even at the smallest amplitude (1.5%) and the nonlinear response seemed to be more pronounced when the amplitude was increased from 7.5% to 30%. Additionally, it is worth noting that compared to pure fibrils at pH 2, pure fibrils at pH 7 showed an obvious widening of the Lissajous plots at small amplitudes (1.5−7.5%), indicating a relative increase in the viscous components during deformation. At higher amplitudes (15−30%), the asymmetries became more pronounced, with strain hardening both in extension and compression. This is consistent with the results from the amplitude sweeps (Figure 3S), which show that at smaller amplitudes (1.5−7.5%) the surface layer stabilized by pure fibrils had much higher dilatational modulus at pH 2 than at pH 7 (Figure 3S). The pH may have affected the in-plane interactions between the fibrils, which could have resulted in a decrease in in-plane cohesion of the interfacial microstructure, thus leading to changes in strain softening or hardening behavior of the interface. Quantitative Analysis of Lissajous Plots. To further quantify the degree of nonlinearity, the nonlinearity parameters Sext and Scom for both phases of the cycle were determined, as shown in Figure 4. It can be seen that at pH 2 the absence of nonlinearity at amplitudes up to 5% for all the systems was confirmed (Figures 4A,D). At 7.5 and 10% amplitude, the fibril system and pure peptides showed pronounced nonlinear responses, with strain hardening during compression and strain
Figure 2. (A) Adsorbed amount (Γ, mg/m2) and (B) thickness (δad, nm) of adsorption layers after 3 h formed from the fibril system, pure fibril, and pure peptide at pH 2, 5, and 7. All systems have the same protein concentration of 0.1 wt %.
adsorbed proteins, which can increase their packing density at the interface.10,16 A higher density of the adsorbed layer could provide stronger steric stabilization, thus contributing to the foam stability. It may also enhance the surface shear and dilatational moduli. For pure fibrils, the changes of Γ and δad as a function of pH differed from those observed for pure peptide. As can be seen from Figure 2A, for pure fibrils the adsorbed amount and layer thickness are less sensitive to changes in pH: these showed a small decrease in Γ upon increasing the pH from 2 to pH 5 and pH 7, with no significant change in layer thickness (within the margin of error of the measurement), suggesting a slightly higher packing density of adsorbed fibrils at pH 2. This result is consistent with the surface pressure measurements, since pure fibril at all three pHs showed similar final surface pressures (Figure 1). Nonlinear Dilatational Rheological Properties. To gain more information about the microstructure and mechanical properties of interfaces stabilized by these systems, surface dilatational rheology measurements including frequency and amplitude sweeps were performed. A detailed discussion of these sweeps is given in the Supporting Information. In the strain sweeps we observed that even at small deformation amplitudes the response of the interfaces stabilized by all systems was in the nonlinear regime (Figure 3S, Supporting Information). The moduli shown in these sweeps were determined from a Fourier transform of the oscillatory surface pressure signal, in particular from the intensity and phase of the first harmonic of the Fourier spectrum. As pointed out by Ewoldt et al., any nonlinearity that could be present in the raw signal will be disregarded when using this first-harmonic based Fourier analysis.39 Therefore, the values of the first harmonic moduli presented in Figure 3S are bound to be inaccurate. To 8096
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Figure 3. Lissajous plots obtained during amplitude sweeps (1.5, 15, and 30%) of air−water interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2 (A) and 7 (B). Protein concentrations are 0.1 wt %.
the latter is also dominated by the peptides, which is supported by the similarities in adsorption kinetics (Figure 1), the similarities in adsorbed amounts of protein (Figure 2), and the nearly identical frequency response of both types of interfaces (Figure 2S). This analysis needs to be further confirmed by structural characterization methods, such as Brewster angle microscopy (BAM) and particle tracking observed by microscopy.41 At pH 5 and 7, for interfaces stabilized by peptides or the fibril system, the degree of nonlinearity during extension and compression showed a similar tend to the one observed for pH 2, but with significantly higher values for the S factors over the whole range of amplitudes (Figures 4B,E and Figures 4C,F), indicating the interface had a stronger response to deformation. This may be attributed to the enhanced interactions among components at the interface at both pH 5 and 7 due to the reduced electrostatic repulsion. For pure fibrils, at pH 2, at high amplitudes (15−30%), the interfaces showed a mild strain hardening during extension and a near linear elastic response during compression (Figures 4A,D). A possible explanation for the different responses of the
softening during extension. When the amplitude was further increased from 15% to 30%, the strain hardening in compression became increasingly larger, but the degree of strain softening in extension was reduced, and finally, at 30% amplitude, the response in extension became strain hardening. Combined with the low frequency dependence and high dilatational modulus during frequency and amplitude sweeps (Figures 2S and 3S), the most likely structure of the air−water interfaces formed by the peptides at pH 2 is a heterogeneous structure, in which areas of densely packed peptides coexist with areas with a more dilute composition. Upon compression a shift occurs in the fractions of dense and dilute domains, toward a higher fraction of dense domains, which is responsible for the observed strain hardening in compression. Upon expansion the relative fraction of dilute domains increases leading to strain softening. The decrease of the softening effect at higher amplitudes (with even a mild strain hardening at 30% deformation, Figure 4A) suggests that the dense domains are weakly connected. In view of the similarity between the largeamplitude response of pure peptides stabilized interfaces and those stabilized by the complete fibril system, the structure of 8097
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Figure 4. S factor during extension (above the dotted line) and during compression (below the dotted line), determined during amplitude sweeps (1.5−30%) of air−water interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2 (A, D), 5 (B, E), and 7 (C, F). All systems have the same protein concentration of 0.1 wt %.
other foams. For the fibril system and (especially) pure peptides, at pH 5, the initial bubbles had a smaller bubble size with a more uniform size distribution (Figure 5B) as compared to the two systems at pH 2 and 7 (Figures 5A,C). For pure fibrils at pH 5, it should be noted that it is difficult to produce foams with enough volume for measurements at the present fibril concentration (0.1 wt %). For all systems, it can be seen that with increasing time the bubble size gradually increased and the bubble shapes started to transform from spherical to polyhedral due to the destabilization processes, such as liquid drainage, coarsening, and coalescence. The foam stability of all systems was evaluated by measuring the half-life time (t1/2) and liquid stability of foams, as shown in Figures 6A and 6B, respectively. It can be clearly seen from Figure 6A that at pH 2 the foam stability of pure peptides was significantly higher than the fibril system and pure fibrils. Furthermore, at pH 5 and 7, both the fibril system and pure peptides could form more stable foams with comparable foam stability. The t1/2 values for foams were around 10 h, which were much higher than that of pure fibril, i.e., roughly 5 times higher (Figure 6A). Similar observations were also reported by Oboroceanu et al., who found that a whey protein fibril system at pH 7 had better foaming properties than at pH 2.23 For pure fibrils, the foam stability at pH 7 was also shown to be much higher than that at pH 2, but pure fibrils appeared to be unable to make foams effectively at pH 5. Delayed liquid drainage generally can contribute to foam stability. Therefore, the foam liquid stability was calculated to assess the liquid drainage rate (Figure 6B). A high foam liquid stability indicates a low liquid drainage rate. As can be seen, the pure fibrils had a much higher liquid drainage rate than foams prepared from pure peptides and the full fibril system. Although the formed foams were not very stable (Figure 6A), pure fibrils at pH 2 and 7 did show a capacity to delay liquid drainage (Figure 6B). On the basis of these results, it can be concluded that the foaming properties of the fibril system appeared to be mainly determined by the high proportion of unconverted peptides in the system, especially at pH 5 and 7 (Figure 6). This observation is in good agreement
pure fibrils may be due to a structural transition in the interface induced by the deformation. Previous studies by Humblet-Hua et al.22 and others27,28 have shown that at relatively high bulk protein concentration (0.1 wt %) the long, semiflexible protein fibrils form a structure after adsorption to the interface in which nematic domains and disordered domains coexist at the interface. Upon compression, the surface fraction of nematic domains grows at the expense of the surface fraction of disordered domains, but if at the maximum degree of compression we are still in the coexistence regime and do not cross into a fully nematic structure, we would expect a near linear response with only a minor degree of hardening. Upon extension the interfacial structure changes gradually into a more and more disordered state, which in view of the relatively high electrostatic repulsion has only limited connectivity between the fibrils, which results into a moderate degree of strain hardening during extension. At pH 5 and 7, the strain hardening in compression and extension is clearly more pronounced, especially at high amplitudes (20 and 30%) (Figures 4B,E and Figures 4C,F). The strain hardening in both extension and compression indicates that a more disordered gel-like interfacial structure was formed by pure fibril at these two pHs, which could be explained by the fact that the fibrils could more easily aggregate and build a stronger network at the interface due to the reduced electrostatic repulsive forces.25,42 Foaming Properties. In this section, we examine the relation between the obtained surface properties of all protein systems to their macroscopic foaming behavior. Here, we use the initial bubble size to assess the foamability of all systems as a function of pH. As can be seen from Figure 5, at all investigated pHs, the bubbles formed from the fibril system and pure peptides showed comparable initial bubble size (at 0 s), which was much smaller than that observed in pure fibrils. This indicates that the fibril system and pure peptides had a better foamability. Additionally, the initial bubbles of the fibril system and pure peptides were spherical, but the bubbles from pure fibrils appeared to be polyhedral, implying the liquid drainage in foams formed from pure fibrils is much faster than in the 8098
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Figure 6. (A) Half-life time (t1/2, h) and (B) liquid stability (s) of foams generated from the fibril system, pure fibril, and pure peptide at pH 2, 5, and 7. All systems have the same protein concentrations of 0.1 wt %.
electrostatic adsorption barrier. In addition, compared to pure peptides, pure fibrils showed a much slower adsorption kinetics due to the large size of fibril or fibril clusters. The results from surface dilatational rheology showed that, again, the unconverted peptides mostly dominated the dilatational rheological properties of surface layers stabilized by the fibril system. At all three pHs, the fibril system and pure peptide formed interfaces with similarly high dilatational moduli, an identical lowfrequency dependence of the modulus, and produced Lissajous plots with strain hardening during compression and strain softening during extension. We argue that the rheological properties of interface are most likely the result of the formation of a heterogeneous structure by the peptides, in which dense and dilute regimes coexist. The presence of fibrils in this structure could be neither confirmed nor excluded, solely based on the surface rheological results. The interface stabilized by pure fibrils had a highly pH-responsive response behavior and showed different rheological properties and interfacial microstructures as a function of pH. Not only the surface properties of the fibril system are dominated by peptides, it appears that the foaming properties of the fibril system were also dominated by the unconverted peptides and not by the protein fibrils as previously thought. Compared to pH 2, the fibril system at pH 5 and 7 seems to be a more promising protein material to make stable foams, even at low protein concentration (0.1 wt %), which is mainly due to the faster adsorption kinetics and the formation of highly elastic surface layer by the peptide materials. Additionally, the presence of fibril clusters and peptide aggregates at pH 5 and 7 may also contribute to the foam stability of fibril system. For pure fibrils, the foaming properties appeared to be mostly dependent on their adsorption kinetics, and both the foamability and foam stability of pure fibrils were very poor.
Figure 5. Time evolution of air bubbles within foams formed by the fibril system, pure fibril, and pure peptide at pH 2 (A), 5 (B), and 7 (C). Protein concentrations are 0.1 wt %. The magnification is same for all images (1 cm × 1 cm).
with the results from adsorption and dilatational rheology measurements (Figures 1 and 2, Figures 2S and 3S).
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CONCLUSIONS In this work, we have studied the surface and foaming properties of an 11S soy protein fibril system, consisting of a mix of long semiflexible protein fibrils (20%) and unconverted peptides (80%). We also study the purified 11S fibrils and purified peptides of this system, with the aim to understand the individual contributions of these two factions to the complex fibril system. We examined the influence of pH changes on these properties of all three systems. From the surface pressure and ellipsometry measurements at the air−water interface, we were able to shed light on two major observations. First, the adsorption kinetics of the fibril system appeared to be mainly dominated by its high proportion of unconverted peptides, especially in the initial adsorption, which could be explained by the faster adsorption of smaller peptide materials at the interface. Second, the fibril system and pure peptides showed faster adsorption kinetics and higher packing density at the interface at pH 5 and 7 than at initial pH 2, due to the reduced 8099
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01511. Visual observations of the fibril system, pure fibril, and pure peptide at pH 2, 5, and 7; complex surface dilatational modulus as a function of frequency for interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2, 5, and 7; slope of a doublelogarithmic plot of the modulus versus frequency; complex surface dilatational modulus as a function of amplitude for air−water interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2, 5, and 7; Lissajous plots obtained during amplitude sweeps (1.5− 30%) of air−water interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2; Lissajous plots obtained during amplitude sweeps (1.5−30%) of air−water interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 7 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(L.M.C.S.) E-mail
[email protected]; Fax +31 317 483669; Tel +31 317 485023. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank M. A. H. de Beus, M. Chen, and H. Baptist (Food Physics Group, Wageningen University) for their help with the pendant drop tensiometer measurements and protein fibril characterization and R. J. B. M. Delahaije and F. J. Lech (Food Chemistry Group, Wageningen University) for their help with the ellipsometry and foamscan experiments. We also thank the China Scholarship Council (CSC) research program for providing funding for Zhili Wan.
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