Making Food Protein Gels via an Arrested Spinodal Decomposition

Adolphe Merkle Institute, University of Fribourg, Route de l'ancienne Papeterie 1, Marly, Switzerland. ‡ Physical Chemistry, Lund University, Geting...
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Making Food Protein Gels via an Arrested Spinodal Decomposition Najet Mahmoudi†,‡ and Anna Stradner*,‡ †

Adolphe Merkle Institute, University of Fribourg, Route de l’ancienne Papeterie 1, Marly, Switzerland Physical Chemistry, Lund University, Getingevägen 60, Lund, Sweden

‡

S Supporting Information *

ABSTRACT: We report an investigation of the structural and dynamic properties of mixtures of food colloid casein micelles and low molecular weight poly(ethylene oxide). A combination of visual observations, confocal laser scanning microscopy, diffusing wave spectroscopy, and oscillatory shear rheometry is used to characterize the state diagram of the mixtures and describe the structural and dynamic properties of the resulting fluid and solidlike structures. We demonstrate the formation of gel-like structures through an arrested spinodal decomposition mechanism. We discuss our observations in view of previous experimental and theoretical studies with synthetic and food colloids, and comment on the potential of such a route toward gels for food processing.

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attraction, however, is much less consensual.10 Several mechanisms were proposed including mode-coupling theory (MCT) for attractive glasses,16 cluster MCT,17 viscoelastic phase-separation,18 and arrested spinodal decomposition.19−21 The latter has become recently favored in both experimental investigations on model colloids interacting via short-range attractions, including colloids with depletion attraction19,20 and lysozyme with temperature-dependent attractions,21 and numerical studies.22,23 Moreover, the incorporation of a screened Coulomb repulsion in such systems leads to the formation of equilibrium clusters.24 Such phases have been shown to form in protein solutions as well as in colloid−polymer mixtures.25,26 A schematic state diagram depicting the fluid-to-solid boundary in colloidal systems with mixed potentials has recently been drawn.27 Such a diagram has already been used to rationalize the behavior of casein particle gels and casein−xanthan mixtures.3 Controlling the mechanical properties of food gels represents a prominent challenge in food technology because they define the texture and sensory properties of many food products. Since most food gels are made of proteins alone or in mixtures with other biopolymers, solid-like states encountered in model colloidal systems offer interesting alternatives to the costly classical gelation processes.1 In particular, the ability of short-range attractive colloids to form elastic networks at intermediate ϕ, as a result of arrested spinodal decomposition, appears very attractive.4,19−21,28−32 This unconventional route to gelation leads to the formation of amorphous protein networks with elastic properties that depend remarkably strongly on changes in ϕ.33 It could thus offer

INTRODUCTION Gels are ubiquitous in biological and industrial applications such as food, pharmaceuticals, and cosmetics. There are two types of food gels, polymer gels, and particle gels. The archetypical polymer gel is made of gelatin, a polydisperse biopolymer obtained from collagen hydrolysis, and is quite uniformly structured and rubberlike. In contrast, particle food gels are soft solid-like materials with often heterogeneous network structures, formed, for example, by acidification or enzymatic coagulation of casein micelles1 or temperature-induced formation of whey protein gels.2 The production of these types of gels involves complex formulation such as addition of enzymes, bacteria, and/or other chemicals to induce the required pH changes, and/or energy-demanding processing such as heattreatment. Hence, food technologists need to develop alternative routes to gels by exploiting recent experimental and theoretical developments in fluid-gel transitions in model colloidal suspensions.3 For instance, studying simple model colloid− polymer mixtures has helped to rationalize and understand the behavior of complex systems such as emulsion droplets and biopolymers,4,5 silica particles and biopolymers,6 and proteins and polysaccharides.7,8 Investigations of disordered solid-like states in colloidal suspensions have shown that dynamic arrest is strongly dependent on the colloidal volume fraction, ϕ, and the strength and nature of the interactions described by a potential of mean force U.9,10 Solid-like structures such as colloidal glasses or particle gels form under either attractive or repulsive interactions.10−12 For short-range attraction, it is well established now that gelation is driven by the diffusion-limited cluster aggregation (DLCA) of fractal clusters of colloidal particles.13,14 For hard spheres and weakly attractive colloids, dynamical arrest occurs through a glass or jamming transition at high ϕ.15 The mechanism of gel or glass formation at intermediate strength of © 2015 American Chemical Society

Received: September 11, 2015 Revised: November 18, 2015 Published: November 23, 2015 15522

DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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The Journal of Physical Chemistry B

39. We have used the latter value for all further calculations. Stock solutions of PEO were prepared by dissolving PEO powder in Milli-Q water with 100 mM NaCl and 2 mM NaN3 under gentle stirring over 3 days. The solutions were filtered using a serum Acrodisc filter (37 mm of diameter, GF/0.2 μm Supor Membrane) and their solid content determined using a moisture analyzer (MA35, Sartorius). The calculated overlap concentration c* using the relationship c* = 3Mw/4πNAR3g , where NA is Avogadro’s number, is 10 mg mL−1 compared to the one determined from microrheology, 13.7 mg mL−1. The latter was used. The size ratio between the polymer and casein is δ ∼ 0.31.40 Casein−PEO mixtures were prepared from stock solutions in 1.5 mL diffusing-wave spectroscopy cuvettes and vigorously mixed for 1 min using a minishaker (MS1 minishaker, IKA). An aliquot of 400 μL was labeled with rhodamine B for structural characterization by confocal laser scanning microscopy. The remaining mixture was studied in terms of its dynamics by diffusing-wave spectroscopy. Phase behavior was determined by direct visual observation over 2 weeks at 22 °C. Mixtures are defined in terms of their total casein volume fraction ϕ and their PEO concentration expressed in mg mL−1 of the total mixture and normalized by the overlap concentration, c* = 13.7 mg mL−1. Methods. The microstructure of caseins dispersed in PEO was visualized by confocal laser scanning microscopy (CLSM) (Leica TCS SP5) on an inverted microscope (Leica DMI 6000 B) and motorized stage using a 63× glycerol−water immersion objective. Caseins were noncovalently labeled with a rhodamine B solution at 0.02 wt % (excitation at 561 nm). Twenty microliters of mixed sample was loaded on a microscope slide and fixed with a spacer (Secure-Seal imaging spacer, Sigma, 120 μm thickness, window diameter 13 mm). Images of 25 × 25 to 250 × 250 μm2 at 512 × 512 pixel resolution were acquired over a depth of approximately 30−40 μm, which is within the bottom phase of sedimenting samples. To probe the microscopic dynamics of casein micelles in PEO solutions, diffusing-wave spectroscopy (DWS), a multiple scattering technique that probes the fast dynamics in turbid systems, was used. We utilized a commercial DWS instrument (Rheolab, LS Instruments, Switzerland) operating at a laser wavelength of 632.8 nm and with the two-cell scheme in transmission geometry as described in the literature.41 Typical measurement times were in the range of 6−10 min. Samples were measured at 22 °C in standard plastic cuvettes (Sigma) with a path length L of 4 mm and a width of 10 mm. We use a reference sample (0.5 wt % polystyrenesulfonate particles (Polysciences Inc.) in water; diameter 457 nm) with known photon transport mean free path, l*, to determine l* of casein solutions and casein−PEO mixtures from the ratio of transmitted intensities of the reference particles and the casein sample according to the following equation:42

a mechanism that would allow us to design protein-based food gels with tailored mechanical and structural properties. Van Gruijthuijsen et al.34 tested the feasibility of such an alternative route to gelation in longer-ranged attractive systems using casein micelles as colloids in combination with xanthan, where xanthan induces a long-range depletion interaction. They have shown that the system indeed macroscopically arrests but that its mechanical properties are fully determined by the continuous xanthan-rich phase and not by an “arrested” casein-rich phase. In this article, we present a proof of concept that gelation through arrested spinodal decomposition could provide an alternative route to food gels with controllable structural and elastic properties. We now use an alternative polymer, poly(ethylene oxide) (PEO), to induce a medium-range depletion attraction. We indeed find that casein micelles at intermediate ϕ exhibit a fluid to gel transition as a result of an arrested spinodal decomposition. Our results show that the elastic properties of arrested casein gels exhibit a strong dependence on ϕ, and we compare them with short-range attraction lysozyme gels.

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EXPERIMENTAL SECTION Experimental System. We used pure casein micelles (ref MPI-85 MC), free from whey proteins and lactose, which were a gift from the Hungarian Institute of Dairy Research. Their size was measured using small angle neutron scattering at the Paul Scherrer Institute. The size distribution is broad with a numberaverage radius, R, of 55 nm and a polydispersity, p = σ/R, with σ2 the variance of the distribution, of 0.40 using a Schulz distribution. The gravitational Péclet number35 of the system, which compares the relative rates of sedimentation to Brownian motion, Peg = 4πΔρgR4/3kBT, with Δρ the density mismatch between casein micelles (casein micelle density ≈1.07 g/cm3 determined through density measurements36) and solvent background as a function of the polymer concentration in the free volume (solvent density between 1.004 and 1.01 g/cm3 from the lowest to the highest PEO concentration in the gel region, respectively) ranging from 0.06 to 0.066 g/cm3, is ≈6 × 10−6 and thus more than 3 orders of magnitude smaller than the threshold value Pecrit g ≈ 0.01 beyond which gravity is shown to impact gelation.37 Stock solutions of casein micelles were prepared by slowly adding casein powder to stirred Milli-Q water in 100 mM NaCl to screen electrostatic repulsions and containing 2 mM NaN3 to prevent microbial growth, at 50 °C to a final concentration of ∼6 wt %. Solutions were then centrifuged at 7750g for 30 min to sediment the nondissolved material, filtered with 5 and 1.2-μm syringe filters (Pall Life Sciences, Acrodisc 32 mm, Supor Membrane), and finally concentrated using a Vivaflow 50 ultrafiltration unit (Sartorius AG) with a 50-kDa cut off. The casein volume fraction ϕ was determined from ϕ = ccρυ, with cc the casein concentration in g per g determined using a moisture analyzer (MA35, Sartorius), ρ the density of the casein solution (in g mL−1) determined with a density and sound analyzer (DSA500, Anton Paar), and υ the voluminosity of casein (4.4 mL g−1).38 As a depletant polymer, we used PEO from Polymer Source Inc. with a weight-average molecular weight Mw of 121.1 kg/mol and a polydispersity index PDI = Mw/Mn = 1.05, where Mn is the number-average molecular weight, determined using size exclusion chromatography. The radius of gyration stated by the manufacturer is Rg = 15.86 nm compared to a value of 17 nm calculated using the relationship Rg = 0.99 M0.6 w , as described in ref

It 5l* /3L = 4l* I0 1 + 3L

(1)

where I0 is the intensity of the light source and It the transmitted intensity through the sample/reference. From the DWS intensity autocorrelation function g2(t) = 1 + βg21(t), the ensemble averaged particle mean square displacement, ⟨Δr2(t)⟩, was obtained assuming the diameter of the illuminating extended beam d > L (d = 6 mm), the autocorrelation time t ≪ τ, and L/l* ≫ 1:43,44 15523

DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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The Journal of Physical Chemistry B

g1(t ) =

( lL* + 43 ) (1 + 38τt )sin h⎡⎣ lL*

⎤ k 02⟨Δr 2(t )⟩ ⎦ +

where the characteristic decay time τ = (Dk20)−1 with D the particle diffusion coefficient and k0 = 2πn/λ the wave vector of light, n the refractive index of the solvent (1.33), and λ the wavelength of the laser. Measurements were done in the middle of the sample volume for homogeneous mixtures and in the lower phase for sedimenting samples. The macroscopic mechanical behavior of gels of casein−PEO mixtures was characterized using a stress-controlled rheometer (ARG2, TA Instruments) in double concentric cylinders geometry with a solvent trap to reduce evaporation. We performed oscillatory measurements at 22 °C. After loading and prior to any measurement, we presheared the casein−PEO mixture at 103 s−1 for 1 min to ensure the same starting history for all samples. The building-up of the gel elasticity was then monitored by recording its viscoelastic response to a 1%-strain oscillatory measurement at 1 rad/s as a function of time. After 24 h, the sample was shown to have reached a steady state. The steady state viscoelastic response was then characterized by performing a frequency sweep at 1%-strain amplitude. The strain amplitude was chosen to be within the linear regime, which we determined by measuring the strain dependence of G′ and G″ at three frequencies (0.1, 1, and 10 rad/s).

k 02⟨Δr 2(t )⟩ 4 3

⎡L ⎤ k 02⟨Δr 2(t )⟩ cos h⎣ l* k 02⟨Δr 2(t )⟩ ⎦

(2)

casein volume fraction leads to arrest/gelation, the mixture not flowing upon tube inversion. Three types of gels were identified based on their stability: gels that collapse within hours termed rapidly collapsing gels (△); gels that are stable for days to weeks before suddenly collapsing called delayed collapsing gels (▲); and gels that are stable for over three months termed stable gels (■). Casein−PEO mixtures were systematically investigated in terms of structure by CLSM and dynamics by DWS. In Figure 2, we show typical confocal images of casein−PEO mixtures at ϕ = 0.25 and increasing c/c* (left panel), and c/c* = 0.51 and increasing ϕ (right panel). For mixtures at ϕ = 0.25, the casein− micelle structure shifts from clusters in a homogeneous fluid (point A) to that of a concentrated attractive fluid with large density fluctuations (point B), with increasing c/c* from 0.36 to 0.51. Quenching mixtures deeper within the two-phase region, we found that the structure is a bicontinuous network with protein-rich and protein-poor domains for both rapidly collapsing (point C) and delayed collapsing gels (point D). Such large length-scale structures in samples C and D appear immediately after mixing and appear static over the duration of CLSM measurements, suggesting that arrested gels are formed. The emergence of such large length scale structures is reminiscent of colloidal systems undergoing spinodal decomposition.30,45 In contrast to classical spinodal decomposition where we distinguish between three characteristic regimes (Gibaud and Shurtenberger45 and references therein), here the occurrence of such a process is too fast to be monitored by CLSM, and the structure is invariant in time. Along a ϕ-cut at c/c* = 0.51, the casein−micelle structure evolves from that of large clusters for the homogeneous fluid at ϕ = 0.15 (point E) to that of a concentrated attractive fluid with large density fluctuations at ϕ = 0.2 (point F). Stable gels at 0.3 ≤ ϕ ≤ 0.35 (points G and H) show a space-spanning network but with significantly smaller mesh sizes than the rapidly collapsing gels. The appearance of space-spanning networks with increasing attraction is accompanied by a pronounced change in the microscopic dynamics of the individual particles, averaged over all particles, as probed by DWS at length scales ranging from a few nanometers up to the particle radius. Indeed, when the structure of casein−PEO mixtures at ϕ = 0.25 transitions from a cluster fluid to an arrested space-spanning network, the dynamics correspondingly shift from a fully decaying g1(t) for fluids (open circles in Figure 3a) to a slowly relaxing but still fully decaying g1(t) for fluid−fluid separated mixtures (plus signs in Figure 3a) and to an only partially decaying g1(t) for interconnected systems with a nearly time-independent plateau value (dotted and dashed lines in Figure 3a); this is consistent with dynamic arrest of the network over the time scales of the experiment. Furthermore, the mixture with the highest c/c* (dotted line in Figure 3a) shows an instantaneous nonergodic behavior, while that with lower c/c* (dashed line) shifts from ergodic to nonergodic dynamics upon the collapse of the gel. Along a ϕ-cut at c/c* ∼ 0.5, fluid−fluid phase-separated samples show a fluid-like behavior (crosses in Figure 3a and b), the measurement being done in the bottom

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RESULTS AND DISCUSSION State Diagram: Macroscopic, Structural, and Dynamic Hallmarks. The state diagram in Figure 1 shows the macroscopic state of casein−PEO mixtures. At low PEO concentration and casein volume fraction, homogeneous fluids (○) are observed. Upon increasing PEO concentration, mixtures phase-separate (+) into a casein-rich lower phase and caseindepleted upper phase with a clear interface between the two phases. An additional increase of PEO concentration and/or

Figure 1. Experimental state diagram of casein/poly(ethylene oxide) mixtures in H2O containing 100 mM NaCl with the PEO concentration in units of its overlap concentration on the y-axis and the casein volume fraction on the x-axis. The PEO/casein size ratio is δ ∼ 0.31. Circles, crosses, and open triangles correspond to homogeneous fluid, fluid− fluid phase separation, and rapidly collapsing gels. Filled triangles and squares correspond to delayed collapsing gels and stable gels. The dotted line corresponds to the coexistence curve. Data from samples A− D along a c/c*-cut (vertical solid line) and E−H along a ϕ-cut (horizontal solid line) are shown in Figures 2 and 3. 15524

DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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The Journal of Physical Chemistry B

the homogeneous fluid boundary the system undergoes a fluid− fluid phase separation. Upon slightly increasing the attraction, the system forms a rapidly collapsing gel that then results in a coexistence of fluid and gel phases. At even higher attraction, the system forms a more stable gel. The occurrence of three types of gels with a distinct time evolution of the collapse under their own weight (gravitational stress) demonstrates the importance of gravity for the long-time stability of colloidal gels even in the current case with only a small density mismatch between the gel network and the suspending fluid. Indeed, Secchi et al. reported on the appearance of three well-defined regions of restructuring and collapse of gels with short-range depletion attractions consisting of spherical particles of MFA, a copolymer of tetrafluoroethylene and perfluoromethylvinylether, and a nonionic surfactant Triton X100, which forms micelles serving as the depletant.46 This colloidal system has, however, a much higher density mismatch, Δρ ≈ 1.1 g/cm3, compared to that of the casein micelle-PEO system, where Δρ ≈ 0.063 g/cm3. An important result in our system is that we can produce macroscopically stable gels at volume fractions higher than 0.3 and for deep quenches into the metastable region due to the low density mismatch between casein micelles and the solvent. We compared our experimental phase diagram with calculations from generalized free volume theory (GFVT)40 of the binodal and spinodal lines for mixtures of hard spheres with excluded-volume polymers with a size ratio of δ = 0.31. The almost vertical low-ϕ branch (ϕ < 0.1) of the calculated spinodal line roughly reproduces the fluid−solid transition. This agrees with the scenario expected for a system showing arrested spinodal decomposition as, for example, seen with lysozyme,21 where sufficiently deep quenches into the spinodal region result in the formation of a macroscopically arrested system. The calculated binodal as well as the spinodal at higher ϕ lie within the experimental one-phase region, indicating that the GFVT calculations seem to overestimate the effect of adding PEO compared to our experimental observations. However, it is important to mention here that casein micelles have a significantly high degree of polydispersity (40%) that is not included in the theoretical calculations. In addition, this offset between experimental and theoretical coexistence curves might partially also originate from the fact that casein micelles have an uneven surface48 and that depletion attraction is reduced in particles with a topographically rough surface relative to the smooth hard sphere case.49 Another possible reason for this discrepancy could come from the partial or complete adsorption of PEO on the casein micelle surface. Polyethylene glycol (PEG), PEO with Mw < 20 000 Da, is the most common hydrophilic nonionic polymer to induce protein separation or crystallization, and its extensive use as a precipitating agent for proteins generated fundamental studies that aimed at understanding protein−protein and protein−PEG interactions.50−52 This interaction is often described in terms of a depletion attraction, which proceeds with the exclusion of polymer molecules from the region between adjacent proteins.50,51 However, a departure from pure entropic depletion was reported and attributed to an energetic attraction between lysozyme and PEG,53 to a penetration of PEG into the cavities on α-Crystallin’s surface,51 and to a flattening of PEG coils near the γD-Crystallin surface.54 Since investigating PEO adsorption on casein micelles is experimentally extremely challenging because of the broad size distribution of the caseins combined with a relatively small PEO/casein size ratio, we tested the effect of PEO adsorption on the state diagram by replotting it assuming that

Figure 2. CLSM micrographs of casein−PEO mixtures obtained at ϕ = 0.25 and various c/c* (A−D), and at c/c* = 0.51 and various ϕ (E−H) as depicted in the state diagram of Figure 1. The images were taken 1 to 4 days after mixing. The caseins are dyed with rhodamine B and appear red in the images. The scale bar shown in g is 15 μm and is the same for all images.

casein-rich phase. At higher ϕ, stable gels (samples G and H) show nonergodic dynamics (dashed and dotted line in Figure 3b). The combined use of visual observations, DWS, and CLSM allowed us to refine the state diagram of the transition from a homogeneous ergodic fluid of clusters at low c/c* to a nonequilibrium nonergodic gel at higher c/c* and/or ϕ (Figure 4). Note that because of the high polydispersity of casein micelles the equilibrium fluid−crystal phase transition vanishes. The gelation proceeds directly from the homogeneous fluid phase at ϕ ≥ 0.3, in contrast to lower ϕ where more complex phase behavior is observed. Indeed, at ϕ ≤ 0.25 and immediately above 15525

DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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The Journal of Physical Chemistry B

Figure 3. DWS field autocorrelation functions measured for casein−PEO mixtures along a c/c*-cut at ϕ = 0.25 (a) and a ϕ-cut at c/c* = 0.51 (b). Measurements were done after the same waiting time as for the CLSM. Circles and plus signs in a correspond to points A and B in Figure 2, respectively. Dashed and dotted lines denote points C and D, respectively. The solid line corresponds to the casein solution without added polymer. Plus signs and dashed and dotted lines in b correspond to points F, G, and H, respectively. The variation in the background viscosity, due to the presence of the polymer along the c/c*-cut, is accounted for by rescaling the lag-time axis with the ratio of the solvent viscosity to the background viscosity, η0/ηPEO. To account for the ϕ dependence of l* along the ϕ-cut, the lag-time axis is normalized with (L/l*)2.

Connecting Structure and Elasticity. One important issue in arrested spinodal decomposition gels is the connection between gel structure and its bulk mechanical properties. In casein−PEO gels, the elasticity of the gel increases over a period of time (Figure 5b), while the structure arrests within minutes of mixing cessation as shown in a sequence of confocal images taken at various ages of the gel in Figure 5a. The temporal evolution of G′ is very similar to that of short-range attraction colloidal gels such as thermo-reversible silica gels,60 charge-stabilized particle gels,61 and arrested spinodal decomposition protein gels.45 The structural arrest seen here was also reported for other systems with short-to-intermediate range attraction where the gel structure arrests completely at some time after gelation.10,21,62−64 The gelation thus occurs as the spinodal decomposition process is interrupted, and the bicontinuous structure subsequently arrests into an elastic self-bearing network with the colloid-rich regions undergoing dynamical arrest as their concentration crosses the arrest line. In Figure 5, we show the mechanical properties of three casein−PEO gels with various casein ϕ and “equal” depletion potential, U, calculated using generalized free volume theory (equation S3 in Supporting Information).40 The temporal evolution of their rigidity (Figure 5b) can be divided into three stages: a short latency period (≲200s) in which the mixture is fluid (G″/G′ > 1), followed by a rapid rise in G′ and G″ indicating gel formation. The G′ increase in this regime is approximated by an exponential growth. At longer times, G′ continues increasing but does so slowly (approximation with a weak power law). Along with the increase in G′, G″/G′ decreases to an asymptotic value around 0.15−0.2. The presence of these three regimes was also observed in thermo-reversible silica gels.62 Guo et al. suggested that the crossover from an exponential to a weak power law growth of G′ along with the saturation of G″/G′ to a time-invariant value corresponds to a separation in the formation of the gel at short times and its aging at longer times.62 While the temporal evolution of elasticity is similar between the three gels of almost equal depletion strength and structure, differences between the gels appear in the length of the latency and the gel formation stages and the strength of the gel. Indeed, the gel elasticity increases with ϕ, while the latency and gel formation period decrease with increasing ϕ. The frequency dependence of G′ and G″ in the linear regime (Figure 5c) shows similar trends for the three gels: a weak increase of G′ with ω and a minimum in G″ characteristic of colloidal gels and glasses.29,65,66 Again, the gel elasticity increases with ϕ.

Figure 4. Experimental state diagram compared to theoretical binodal and spinodal lines. Symbols and axes are the same as those in Figure 1, and X corresponds to the theoretical critical point. The blue solid and dashed lines are generalized free volume theory binodal and spinodal, respectively, calculated at δ = 0.31. The dot-dashed line denotes the structural arrest of pure casein micelles (ϕ = 0.69).47

PEO adsorbs on casein micelle surfaces. We find that even for the extreme case of a full adsorption of PEO on casein, we only see a relatively small shift of the binodal at higher casein concentrations. The macroscopic state diagram remains qualitatively the same, as demonstrated in Figure S2 in Supporting Information, and PEO adsorption would thus not change the conclusions drawn from our experiments nor account for the discrepancies between experiments and the calculated theoretical curves. We also compared our results with other colloid−polymer systems reported in the literature for a better understanding of our state diagram and especially the gelation transition. The formation of stable or long-lived clusters is consistent with findings in model colloid−polymer (PMMA/PS) mixtures over a wide range of attraction δ ∼ 0.02−0.37, in the absence of longrange electrostatic repulsions.30,55−57 Our finding of a narrow fluid−fluid phase separation region is consistent with an observable gas−liquid coexistence beyond the threshold value of δ ∼ 0.25 in experimental systems58 and 0.3 theoretically.40,59 The transition from a single-phase fluid to fluid−fluid coexistence to gelation with increasing c/c* for ϕ ≤ 0.25 is consistent also with findings for model colloid−polymer mixtures, namely, silica/polydimethylsiloxane (PDMS) at δ = 0.2532 and polystyrene colloids/PEO at δ = 0.24−0.68.39 15526

DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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Figure 5. (a) Typical time sequence of confocal images showing the arrest of gel structure within minutes of mixing cessation (sample at ϕ = 0.15 and c/ c* = 1.31). The scale bar is 25 μm for all images. The casein network appears in red. (b) Mechanical properties of casein−PEO gels of “equal” attraction strength (deep quench at −U ∼ 9 ± 0.5 kBT): elastic modulus G′ (full symbols) increases as a function of time, tw, elapsed after preshear. Triangles denote casein at ϕ = 0.35 and PEO at c/c* = 0.71; diamonds, casein at ϕ = 0.25 and PEO at c/c* = 1.02; and circles, casein at ϕ = 0.15 and PEO at c/c* = 1.31. The plot shows the long length of time needed for a steady-state modulus value to be obtained. This was consistent for all the deeply quenched gel samples studied. For clarity, we show G″/G′ for only one system (ϕ = 0.25 and PEO at c/c* = 1.02, open diamonds). (c) Linear viscoelastic moduli, elastic, G′ (full symbols), and viscous, G″ (open symbols), as a function of frequency in a dynamic oscillatory shear measurement. The measurement is made after a steady-state modulus is reached. The solid lines in b are results of exponential fits; see text. The dashed line in b indicates the asymptotic value of G″/G′ at long tw.

Compared to our results, lysozyme gels are stronger, and their pore-geometry prefactor is ∼5 times larger. The differences observed are likely to be due to the higher size ratio between strand thickness and particle size of lysozyme gels (∼103 for lysozyme versus ∼20−50 for casein−PEO).33 However, the range of volume fractions covered in our experiments is not sufficient to unambiguously distinguish between such an exponential ϕ-dependence and a power law G0 ∝ ϕn often described for depletion and other colloidal gels.65−70

In Figure 6, we show that the low-frequency elastic modulus G0 (taken at ω = 10−1 rad/s) increases with ϕ for a given quench

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CONCLUSIONS We have studied the phase behavior of a moderately concentrated food protein system with a medium-range attraction induced by the addition of a nonadsorbing polymer. In this biocolloid/polymer system, phase transitions have been investigated in terms of structure, dynamics, and mechanical properties. Under nonbuoyancy-matched and screened-repulsion conditions, depletion attraction between casein micelles leads to a variety of structures such as clusters and arrested gels. The transition from fluid clusters to gels is direct and coincides with the phase separation line at high volume fraction, ϕ ≥ 0.3. Conversely, at lower volume fraction, ϕ ≤ 0.25, a continuous crossover from phase separation to gelation is observed. We have also shown that casein−PEO gels form via an arrested spinodal decomposition mechanism. Using confocal imaging, we observe the immediate formation of a bicontinuous network that remains unchanged with age. This structural arrest contrasts with the temporal evolution of the gel elasticity. A strong increase of the elastic shear modulus with the attraction strength and the volume fraction is observed. Our work provides a proof of concept that food protein based gels with tunable structural and dynamical properties can indeed

Figure 6. Low-frequency elastic modulus G0 as a function of casein volume fraction for depletion gels at different attraction potentials −U = 9 ± 0.5 kBT (circles) and 13.3 ± 0.5 kBT (triangles). The solid lines are exponential fits of the data with coefficients decreasing from 19.2 to 11.6 with increasing U. The inset shows G0 versus the volume fraction for the casein with the deepest quench data compared to data on lysozyme gels from Gibaud et al.33

(U). In a recent paper on arrested spinodal decomposition lysozyme gels, Gibaud et al. found that their elasticity increases exponentially with ϕ.33 This unusual strong dependence, reminiscent of porous media, was attributed to the large difference between the particle and strand size.33 As shown in Figure 6, our data appear indeed consistent with such a behavior. 15527

DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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The Journal of Physical Chemistry B

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be produced through an arrested spinodal decomposition mechanism. This requires the use of a small nonadsorbing biopolymer, i.e., utilizing depletion interactions in the colloid rather than in the protein limit previously attempted. While the current study has used a synthetic nonfood polymer, it nevertheless clearly illustrates the enormous potential of such an approach, where protein gels with tailor-made properties can be obtained by simple mixing without additional complex and/or expensive processing steps. In a next step, this pathway to gelation could be investigated using food-grade depletants like neutral polysaccharides, such as dextran,71 or anionic polysaccharides, such as alginates,72 both meeting the criteria of being nonadsorbing on casein micelle surfaces and providing a wide range of radii of gyration encompassing the range needed to induce short-to-intermediate-range depletion attraction.

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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.jpcb.5b08864. Description of the GFVT calculations and effect of polymer adsorption on experimental state diagram and theoretical GFVT lines (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 46 222 8214. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kitty van Gruijthuijsen for the GFVT calculations and Yuki Umehara for her help in the laboratory. We thank the Hungarian Institute of Dairy Research for providing us with the pure casein micelles. We gratefully acknowledge financial support from the Nestlé Research Center, Lausanne, Switzerland, the University of Fribourg, Switzerland, the Adolphe Merkle Foundation, the Science Faculty at Lund University, and the Swedish Research Council (grants 621-2012-2422 and 20096794).

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DOI: 10.1021/acs.jpcb.5b08864 J. Phys. Chem. B 2015, 119, 15522−15529

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