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Langmuir 2008, 24, 13364-13375
Thermally Induced Gelling of Oil-in-Water Emulsions Comprising Partially Crystallized Droplets: The Impact of Interfacial Crystals Florence Thivilliers,† Eric Laurichesse,† Hassan Saadaoui,† Fernando Leal-Calderon,*,‡ and Ve´ronique Schmitt*,† Centre de Recherche Paul Pascal, UniVersite´ Bordeaux 1, CNRS, AVenue du Dr. Albert Schweitzer, 33600 Pessac, France, and Laboratoire TREFLE, UniVersite´ Bordeaux 1, CNRS, AVenue des Faculte´s, 33405 Talence, France ReceiVed August 5, 2008. ReVised Manuscript ReceiVed September 9, 2008 We produced triglyceride-in-water emulsions comprising partially crystallized droplets, stabilized by a mixture of protein and low molecular weight surfactant. The emulsions were emulsified in the melted state of the oil phase and stored at low temperature (4 °C) right after fabrication to induce oil crystallization. The systems were then warmed to room temperature for a short period of time and cooled again to 4 °C. Owing to this treatment referred to as temperature cycling or “tempering”, the initially fluid emulsions turned into hard gels. We followed the bulk rheological properties of the materials during and after tempering. The storage modulus, G′, exhibited a dramatic increase when tempering was applied. We showed that the systems evolved following two distinct regimes that depend on the average droplet size and on the surfactant-to-protein molar ratio. Gelling may involve partial coalescence of the droplets, i.e., film rupturing with no further shape relaxation because of the solid nature of the droplets. Alternatively, gelling may occur without film rupturing, and is reminiscent of a jamming transition induced by surface roughness. We discussed the origin of these two mechanisms in terms of the properties (size and protuberance) of the interfacial oil crystals.
Introduction Emulsions are metastable materials with a widespread range of applications, the most important ones including cosmetics, foods, detergency, adhesives, coatings, paints, surface treatment, road surfacing, and pharmaceutics. Some emulsions may evolve quite rapidly under the effect of coalescence and Ostwald ripening,1 while others may become trapped in deep metastable states, exhibiting considerable robustness to changes in thermodynamic conditions. These latter materials are particularly suitable for applications requiring a long period of storage. In general, the final properties of emulsions are governed not only by the thermodynamic conditions but also by the entire process history. Such versatility can be exploited to produce a very large set of materials in terms of rheological properties, from low viscosity fluids to highly elastic pastes.1,2 The main purpose of this paper is to examine a thermally induced gelling process occurring in emulsions whose dispersed phase is composed of crystallizable oil. The initial spherical and smooth shape of the warm dispersed droplets is controlled by surface tension. Upon cooling, the surface becomes rough and rippled as a result of the formation of irregularly shaped/oriented crystals, which can protrude into the continuous phase. It is wellknown that when such crystals are present within the thin film separating two droplets, they may pierce the film and bridge the surfaces, causing the droplets to coalesce.3-18 If the crystallized fraction within the droplets is sufficient, the intrinsic rigidity * Corresponding authors. E-mail:
[email protected] (V.S.);
[email protected] (F.L.-C.). † Centre de Recherche Paul Pascal. ‡ Laboratoire TREFLE. (1) Bibette, J.; Leal-Calderon, F.; Schmitt, V.; Poulin, P. Springer Tracts in Modern Physics: Emulsion Science. Basic Principles. An OVerView; SpringerVerlag: Berlin, 2002. (2) Encyclopedia of Emulsion Technology; Becher, P., Ed.; Dekker: New York, 1996; Vol 4. (3) van Boekel, M. Thesis, University of Wageningen, Netherlands, 1980. (4) Melsen, J. P. The stability of recombined milk fat globules. Ph.D. Thesis, University of Wageningen, Netherlands, 1987. (5) Boode, K.; Walstra, P. Colloids Surf., A 1993, 81, 121.
inhibits relaxation to the spherical shape driven by surface tension after each coalescence event. Large clusters appear and grow by the accretion of any other primary droplet or cluster until a rigid network made of partially coalesced droplets is formed. For instance, the fabrication of viscoelastic aerated food emulsions such as whipped creams or ice creams is based on the application of intense mechanical agitation, which promotes formation of the fat droplet network.10 Emulsions made of crystallizable oils exhibit variable internal dynamics and mechanical properties. In this class of colloids, the transition from the fluid to the gelled state is frequent and may occur either by application of an intense shear9-12 or in quiescent storage conditions upon thermal cycling.13-15 Whatever the kinetic pathway followed to attain the final gelled state, interfacial crystals play a major role.5,6,13 However, is it not yet very clear how some parameters such as the thermal history, the nature of the surface active species, the droplet size, and the dispersed phase volume fraction impact the interfacial properties and the gel structure. Following the pioneering work of Thivilliers et al.,13 Golemanov et al.16 and Giermanska et al.,17 we have (6) Boode, K.; Walstra, P.; de Groot-Mosert, A. E. A. Colloids Surf., A 1993, 81, 139. (7) Govin, R.; Leeder, J. G. J. Food Sci. 1971, 36, 718. (8) Goff, H. D.; Jordan, W. K. J. Dairy Sci. 1989, 72, 18. (9) Davies, E.; Dickinson, E.; Bee, R. Food Hydrocolloids 2000, 14, 145. (10) van Aken, G. A. Colloids Surf., A 2001, 190, 333. (11) Giermanska-Kahn, J.; Laine, V.; Arditty, S.; Schmitt, V.; Leal-Calderon, F. Langmuir 2005, 21, 4316. (12) Vanapalli, S. A.; Coupland, J. N. Food Hydrocolloids 2001, 15, 507. (13) Thivilliers, F.; Drelon, N.; Schmitt, V.; Leal-Calderon, F. Europhys. Lett. 2006, 76, 332. (14) Coupland, J. N.; Dickinson, E.; McClements, D. J.; Povey, M. J. W.; de Rancourt de Mimmerand, C. In Food Colloids and Polymers: Stability and Mechanical Properties; Dickinson, E., Waltra, P., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1993; p 243. (15) Boode, K.; Bisperink, C.; Walstra, P. Colloids Surf. 1991, 61, 55. (16) Golemanov, K.; Tcholakova, S.; Denkov, N. D.; Gurkov, T. Langmuir 2006, 22, 3560. (17) Giermanska, J.; Thivilliers, F.; Backov, R.; Schmitt, V.; Drelon, N.; LealCalderon, F. Langmuir 2007, 23, 4792. (18) Vanapalli, S. A.; Palanuwech, J.; Coupland, J. N. Colloids Surf., A 2002, 204, 227.
10.1021/la802521f CCC: $40.75 2008 American Chemical Society Published on Web 10/29/2008
Impact of Oil Interfacial Crystals on O/W Emulsions
Figure 1. Proportion of solid of the oil phase as a function of temperaturer.
investigated two possible routes to produce emulsion gels in quiescent conditions, both of them exploiting the crystallized state of the droplets. One is due to partial coalescence, whereas the other one occurs without film rupturing and is provoked by a restriction of the internal dynamics due to the change in droplet surface roughness. Triglyceride-in-water emulsions stabilized by a mixture of proteins and low molecular weight surfactants were initially fabricated at high temperature and quenched at 4 °C. After a period of storage sufficient to achieve oil crystallization, the systems were warmed at room temperature for a short delay to produce partial melting of the oil, and cooled again at 4 °C. Such treatment referred to as temperature cycling or “tempering” induced a spectacular transition from a fluid emulsion to a strong gel. We followed the bulk rheological properties of the materials during and after tempering. The origin of the gelling process was deduced from macroscopic observations, measurements of the droplet size distribution, and AFM images of the oil/water interface. We show that the gelling mechanism is controlled by the average droplet size and by the surfactantto-protein molar ratio, both of them influencing the size and protrusion of the interfacial oil crystals.
Materials and Methods Emulsion Preparation. The emulsions were of the oil-in-water type. The dispersed phase was partially crystallized at room temperature. We used natural anhydrous milk fat provided by Fle´chard (France) (density ∼ 0.9 g · cm-3), which is composed of a complex mixture of triglycerides with a wide range of melting temperatures from -40 °C to +40 °C (Figure 1). We adopted that oil because the crystallized fraction can be tuned easily and continuously with temperature, the material always remaining homogeneous.19-21 The emulsions were stabilized by sodium caseinate (Mw ≈ 23 300 g · mol-1), purchased from Aldrich and/or by a nonionic surfactant, Tween 20 (Mw ) 1228 g · mol-1, critical micellar concentration (cmc) ≈ 8 × 10-5 mol · L-1 ≈ 10-2 wt % at T ) 25 °C), from Fluka. We first prepared crude polydisperse emulsions stabilized by sodium caseinate alone, by progressively incorporating the liquefied oil into the aqueous phase at T ) 45 °C. Quasi-monodisperse emulsions were obtained by shearing the polydisperse ones within a narrow gap (100 µm) in a Couette cell (Ademtech) at T ) 45 °C.22 The final droplet size was fixed by the shear rate, with the interfacial tension and the viscosity ratio between the dispersed and continuous phases.22 Thus, the shear rates and initial formulations were adapted to obtain (19) Lavigne, F.; Ollivon, M. Ol., Corps Gras, Lipides 1997, 4, 212. (20) Lopez, C.; Bourgaux, C.; Lavigne, F.; Lesieur, P.; Ollivon, M. J. Dairy Sci. 2001, 84, 756. (21) Lopez, C.; Lavigne, F.; Lesieur, P.; Bourgaux, C.; Ollivon, M. J. Dairy Sci. 2001, 84, 2402. (22) Mabille, C.; Leal Calderon, F.; Bibette, J.; Schmitt, V. Europhys. Lett. 2003, 61, 708.
Langmuir, Vol. 24, No. 23, 2008 13365 the targeted average droplet size between 2 and 50 µm. All details concerning the composition and average diameter of the mother emulsions are provided in Table 1. In the particular case of emulsions stabilized by Tween 20 alone, the viscosity ratio was not adequate, so we had to dissolve 1 wt % of sodium alginate (nonadsorbing polymer, HF 120 L from PRONOVA Biopolymers, Mw ) 54 000 g · mol-1) as a thickening agent in the aqueous phase (Table 2). One of the formulated emulsions was obtained with an Ultra-Turrax T25 mixer (Janke & Kunkel equipped with a S25 KV-25F rotor head) operating at 13 500 rpm for 50 s (Table 2). The final systems were obtained by diluting the mother sheared emulsions with an aqueous solution containing Tween 20 and/or sodium caseinate at T ) 45 °C. That way we could control the overall concentrations of protein and surfactant, the mass fraction of dispersed oil, φ, and the average drop size, D. Unless otherwise specified, φ was equal to 45 wt %. The mixture of surface-active species was characterized in terms of the surfactant-to-protein molar ratio, Rm. All the prepared emulsions contained 0.3 wt % of sodium azide (NaN3 from Aldrich) in the aqueous phase as a bactericide agent. Crystallization. The emulsions were always cooled down to 4 °C in a thermostatically controlled chamber in order to induce partial crystallization of the oil droplets. Crystallization of milk fat is a slow and complex process involving variable polymorphic forms. Metastable soft crystals first appear at short times. Then, they progressively evolve toward stable and rigid crystalline forms over a characteristic time scale of 10 h.20,21 This is why our emulsions were always stored at low temperature for 15 h. At T ) 4 °C, the proportion of solid oil at the end of the crystallization process is of the order of (57 ( 3)% as deduced from NMR measurements.13,23 In that state, the emulsions could be destabilized under the effect of shear or creaming. Consequently, they were not stored for more than 48 h, and, to avoid creaming, they were attached to a wheel rotating at 15 rpm. For that purpose, 6 mL tubes were completely filled with the emulsions to avoid the presence of air bubbles that could induce shear instability. In such conditions, the emulsions remained stable during storage (no evolution of the droplet size distribution), and the experiments were reproducible. Differential Scanning Calorimetry (DSC) Experiments. Thermal analyses were conducted on a differential scanning calorimeter (Perkin-Elmer, Pyris 1) in 50 µL hermetically sealed aluminum pans. An empty, hermetically sealed aluminum pan was used as a reference. DSC experiments were carried out in the temperature range from 4 to 50 °C (melting curves), in order to check the crystallization state of the emulsion drops. Optical Microscopy. The emulsions were directly observed with an inverted optical microscope (Zeiss Axiovert X100, resolution of 200 nm) equipped with a digital camera (Sony) and a homemade Peltier module for observations at 4 °C. Crossed analyzers were used to reveal the presence of birefringent oil crystals. Fluorescence Microscopy. Some observations were performed with a fluorescent microscope Leica (DM IRE2) equipped with a digital camera and with a bloc filter characterized by the excitation and emission wavelengths of 560 and 580 nm, respectively. For such observations, 0.02 wt % of a fluorescent red hydrophobic dye (referred to as FY131SC) was dissolved in the oil prior to emulsification. Emulsion Characterization. The emulsions were characterized by their volume-averaged diameter D and polydispersity index P, defined as D ) (∑iNiDi4)/(∑iNiDi3) and P ) 1/D[(∑iNiDi3|D - Di|)/ (∑iNiDi3)], where Ni is the total number of droplets with diameter j , is the value for which the cumulative Di. The median diameter, D undersized volume fraction is equal to 50%. Both D and P were deduced from static light-scattering measurements performed at 45 °C with a Mastersizer 2000 Hydro SM device (Malvern) using Mie theory. Partial coalescence produced irreversible tenuous aggregates that could be easily disrupted by the agitation imposed in the measurement cell. Hence, the samples were first diluted at T ) 45 °C under very low agitation to provoke melting of the crystals and subsequent shape relaxation of the aggregates into single drops. The (23) Drelon, N.; Gravier, E.; Boisserie, L.; Omari, A.; Leal-Calderon, F. Int. Dairy J. 2006, 16, 1454.
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Table 1. Formulation and Process Parameters for the Preparation of Mother Emulsions Stabilized by Sodium Caseinate average diameter D ((0.5 µm) caseinate concentration in the aqueous phase (wt %) oil fraction (wt %) shear rate (103 s-1) shearing time (s)
2 12 85 7 10
5 10 85 12 10
7 10 85 10.5 10
Table 2. Formulation and Process Parameters for the Preparation of Mother Emulsions Stabilized by Tween 20 Alonea average diameter 2.5 5 7 10 14 18 21 D ((0.5 µm) Tween 20 in the aqueous 5 5 5 5 5 5 5 phase (wt %) alginate concentration in 0.5 1 1 1 1 1 1 the aqueous phase (wt %) oil fraction (wt %) 45 85 70 70 70 70 65 shear rate (103 s-1) b 8.7 12 8.7 3.5 2.1 2.1 shearing time (s) 50 10 10 10 10 10 10 a A non-adsorbing polymer, sodium alginate, was added in order to increase the aqueous phase viscosity. b In this particular case, agitation was imposed by an UltraTurrax T25 mixer (Janke & Kunkel, equipped with an S25 KV25F rotor head) operating at 13 500 rpm for 50 s.
aqueous phase used for dilution contained sodium dodecyl sulfate (SDS) at the cmc (cmc ) 8 × 10-3 mol · L-1). This surfactant was used because of its ability to dissociate protein aggregates that could reversibly bridge emulsion drops.23 Rheology. Oscillatory rheological measurements were performed with a strain-controlled rheometer (ARES-LS, TA Instruments) equipped with a parallel-plate geometry (1 mm gap), thermostatically controlled by a Peltier module. Despite the fact that the applied strain was not constant over the whole sheared volume, parallelplate geometry was preferred in order to avoid problems related to confinement. The surfaces of the plates were made rough in order to avoid wall slipping, and the cell was equipped with an antievaporating device. An oscillatory shear strain characterized by its amplitude γ0 and its pulsation ω was applied to the sample, and the resulting shear stress σ was measured. As long as the strain amplitude belongs to the linear regime, the measured stress is sinusoidal and characterized by its amplitude σ0 and by the same pulsation ω with a phase shift δ with respect to the strain. In the linear regime, the elastic G′ and loss G′′ moduli defined as G′ ) σ0/γ0 cos(δ) and G′′ ) σ0/γ0 sin(δ) are representative of the stored and dissipated parts of the energy. These two parameters fully characterize the sample viscoelasticity. We checked that G′ and G′′ were very weakly dependent on the pulsation (for 10-2 rad · s-1 < ω < 20 rad · s-1). All the rheological measurements reported in this paper were obtained at the same pulsation ω ) 1 rad · s-1. Tempering Procedure and Parameters. Tempering was applied immediately after loading the emulsions in the rheometer’s cell. The main parameters characterizing the tempering process were as follows: - the initial temperature Ti ) 4 °C. - the warming rate, from Ti to the tempering plateau fixed at +5 °C · min-1. - the maximum temperature (or tempering plateau), Tp: 15 °C < Tp < 30 °C. - the holding time tp at the tempering plateau: 15 min < tp < 300 min. - the cooling rate from Tp to Ti was fixed at a constant value of -5 °C · min-1. Atomic Force Microscopy (AFM). In order to determine the influence of the aqueous phase composition on the interface topography, we performed AFM experiments. A liquid oil drop (approximate volume 4 µL) at T ) 45 °C was deposited on a graphite substrate, then turned upside down and put in contact with the aqueous phase. Graphite was preferred to mica because of its better oil wettability. Highly ordered pyrolitic graphite was freshly cleaved before each experiment. Then the sample was cooled at 4 °C and stored at this temperature for 15 h to induce oil crystallization.
8 10 85 8.7 10
9 10 85 7 10
10 10 85 5.2 10
11 10 80 7 10
12 10 80 5.2 10
15 10 70 12 10
18 10 70 8.7 10
20 10 70 7 10
25 10 70 5.2 10
35 10 70 3.5 10
50 10 70 3.5 1
The samples were observed by means of a commercial Nanoscope III multimode AFM (from DI-Veeco, Santa Barbara, CA, USA) equipped with a 140 µm scanner (J-scanner), introduced in a thermostatically controlled chamber at T ) 4 °C. The samples were imaged using tapping-mode phase imaging and a standard silicon cantilever (∼40 N/m) to provide topographic and corresponding phase images. Typical imaging scan rates varied between 0.02 and 0.5 Hz, and proportional and integral gains between 0.8 and 1 were used. It was not possible to observe the samples at the tempering plateau (15 °C < Tp < 30 °C) because of the insufficient hardness of the samples in this temperature range. All the experiments were performed in triplicate, and representative images of the interface are reported hereafter.
Results and Discusion 1. Description of the Phenomenon. In the following experiments, the emulsion was characterized by an oil mass fraction φ ) 45 wt %, a mean diameter D ) 15 ( 0.5 µm and a polydispersity P ) 0.24 ( 0.05 (Figure 2). The overall contents of the surface-active species relative to the aqueous phase were 0.5 wt % for Tween 20 and 3.5 wt % for sodium caseinate, corresponding to Rm ) 2.7. After crystallization, the drops surface became rough, and the presence of crystals was revealed by the birefringence between crossed analyzers (Figure 3). At 4 °C, the emulsion remained fluid, and its size distribution did not evolve under storage conditions. Surprisingly, when heated at ambient temperature (T ) 25 °C) for approximately 30 min, the fluid emulsion became much thicker and, when cooled again at T ) 4 °C, it did not flow anymore under its own weight. To characterize the evolution at T ) 25 °C, we measured the size distribution at different times (Figure 4). The initial monomodal distribution centered around 15 µm transformed into a bimodal one with large drops from 70 to 500 µm over a very short period of time (less than 5 min). Considering the protocol used for size measurements, this evolution definitely proves that gelling is due to partial coalescence. We also directly observed the formation of irreversible links between the oil drops. For that purpose, we mixed two coarse emulsions that were fabricated separately at 45 °C, one being labeled with a fluorescent hydrophobic dye (Figure 5). The mixed system was stored for 15 h at 4 °C and then observed under the microscope. The emulsion remained stable at 4 °C and was composed of individual drops, as can be deduced from the images in Figure 5A,A′. By adopting the
Figure 2. Size distributions of the emulsion before and after crystallization; the two curves perfectly superimpose showing that crystallization does not destabilize the emulsion.
Impact of Oil Interfacial Crystals on O/W Emulsions
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Figure 3. Microscope images at 4 °C of the same emulsion as in Figure 2: (a) illuminated by white light and (b) between crossed analyzers. The scale bar represents 50 µm.
Figure 4. Evolution of the size distribution as a function of time at the tempering plateau (Tp ) 25 °C): b tp ) 0, 9 tp ) 5 min, [ tp ) 6 min, and 2 tp ) 8 min.
fluorescent configuration of the microscope, dark spheres in contact with bright labeled ones could be distinguished, proving that fluorochrom molecules were confined inside the drops and that coalescence did not occur (see drops (a) and (b) in Figure 5A′). When the sample was heated at T ) 25 °C, partial coalescence was revealed by the diffusion of the fluorochrom molecules (Figure 5B,B′). For example the initially unlabeled drop (b) became fluorescent, while the fluorescence intensity in drop (a) decreased. The previous observations strongly suggest that the drop connectivity progressively increased at the tempering plateau. A macroscopic sample was tempered at T ) 25 °C for 60 min and then immediately heated at 45 °C to allow total shape relaxation of the aggregates formed by partial coalescence. A macroscopic layer sitting at the top of the sample and containing almost all the oil phase (Figure 6a) was finally obtained. We thus conclude that after a sufficient delay, the network of partially coalesced droplets is almost continuous. We also deposited a water drop containing a hydrophilic dye (methyl blue at 0.5 wt %) at T ) 25 °C. The dye diffused over the whole sample after less than 1 h (Figure 6b) revealing the existence of a continuous water path. Hence, it is likely that the gel structure resulting from tempering is composed of entangled networks of oil and water.
To quantitatively characterize the effect of tempering, the emulsions were loaded at Ti ) 4 °C in the rheometer’s gap, and oscillatory rheological measurements were performed in the linear regime. Tempering was then applied, and the kinetic evolutions of G′ and G′′ were followed (Figure 7, adapted from ref 13). Both moduli exhibited low but measurable values at Ti ) 4 °C, mainly reflecting the flocculated state of the droplets due the attractive depletion interaction induced by excess proteins and surfactant in the continuous phase.24,25 A substantial increase of both moduli was observed upon warming and during the tempering plateau. An even sharper raise occurred as temperature was lowered from Tp to Ti as a result of oil crystallization, until the final asymptotic values, G′f and G′′f, were achieved. The comparatively large values of G′ revealed the essentially elastic nature of the materials and the presence of stress-bearing paths in the samples. The influence of the tempering plateau Tp and holding time tp on the final elasticity were examined in ref 13. The evolution of the elasticity as a function of Tp was not monotonous, with G′ being relatively small for Tp < 15 °C and Tp > 35 °C and exhibiting a maximum value around 25 °C. At this temperature, the proportion of solidified oil in bulk samples is on the order of 10-15%.13 It was concluded that partial coalescence requires the simultaneous presence of solid and liquid oil within the droplets. An excess of solid or liquid oil inhibits coalescence since gelling did not occur neither at temperatures exceeding the upper limit of the oil melting domain (40 °C) nor at low temperatures, such as 4 °C, where the solid content is important (∼57%). The kinetic evolution of the bulk elastic modulus G′ during the thermal treatment was interpreted within the frame of percolation theory.13 The experimental data supported the conclusion that coalescence involved irreversible bridging between crystallized and melted patches in the thin liquid films. In the remainder, we propose a detailed examination of some key parameters that control the kinetics of the process and the final strength of the gel. Experiments were all conducted at the optimum temperature, Tp ) 25 °C, corresponding to the fastest rate of partial coalescence.13 However, the tempering delay, tp, (24) Dickinson, E.; Golding, M.; Povey, M. J. W. J. Colloid Interface Sci. 1997, 185, 515. (25) Dickinson, E.; Ritzoulis, C.; Povey, M. J. W. J. Colloid Interface Sci. 1999, 212, 466.
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Figure 5. Microscope images of a mixture of two emulsions, one being labeled with a fluorescent dye. (A,B) Sample viewed under conventional white light. (A′,B′) Sample viewed under fluorescent conditions. Images A and A′ were taken at 4 °C, and images B and B′ at 25 °C. Scale bar ) 50 µm.
Figure 6. (a) Oil released after tempering for 60 min at 25 °C and melting the sample at 45 °C. (b) Diffusion of a hydrophilic dye showing the existence of a continuous aqueous path in the tempered system.
was adapted to each particular system to obtain reliable results and experimental trends well beyond the intrinsic experimental uncertainty.
2. Influence of the Interfacial Composition. The interfacial composition was modified, all other parameters being constant (D ) 15 ( 0.5 µm, Tp ) 25 °C, tp ) 30 min, φ ) 45 wt %).
Impact of Oil Interfacial Crystals on O/W Emulsions
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Figure 7. Evolutions of the elastic, G′, and loss, G′′, moduli during a tempering cycle (D ) 15 µm, Rm ) 2.7), adapted from ref 13. The images show the macroscopic state of the samples at the beginning and at the end of the tempering cycle.
Figure 8. Final elasticity at 4 °C after tempering (Tp ) 25 °C; tp ) 30 min) as a function of the surfactant-to-protein molar ratio, Rm, and at a constant average drop size, D ) 15 µm. The line is a guide for the eyes.
For that purpose, the surfactant-to-protein molar ratio, Rm, was varied from 0 (protein alone) to infinity (surfactant alone). Except for Rm ) ∞, this was achieved by maintaining the overall mass fraction of protein with respect to the aqueous phase equal to 3.5 wt % and by varying the amount of Tween 20. After the crystallization step, the emulsions were loaded in the rheometer, and the tempering cycle was applied. The final elastic modulus, G′f, plotted as a function of Rm on Figure 8, exhibits a sharp increase for Rm ≈ 0.3. Below this threshold value, tempering had no appreciable effect, and the variation of the droplet size was negligible. However, for Rm values larger than 0.3, a dramatic jump in the elastic modulus was observed, the asymptotic value being close to 4 × 104 Pa. We checked that, in this regime, gelling was due to partial coalescence: a macroscopic oil layer was released as the sample was heated at T ) 45 °C after tempering. Our results are in agreement with previous studies where it was shown that emulsions stabilized by sodium caseinate remained unaltered after repeated temperature cycling but underwent fast destabilization when a sufficient amount of a small molecule surfactant was added.26 To interpret the data, we first consider the competitive adsorption of Tween 20 and casein molecules at the oil-water interface. Casein layers are generally viscoelastic and almost (26) Palanuwech, J.; Coupland, J. N. Colloids Surf., A 2003, 223, 251.
always tangentially immobile.27-29 This property, combined with the large electrosteric hindrance existing between casein layers,30 efficiently protects the droplets against coalescence. The nonionic surfactant Tween 20 is known to weakly interact with casein in the aqueous phase.31,32 Even at very low concentration, it preferentially adsorbs at the oil-water interface and locates in the defects of the protein film.33 As Rm increases, surfactant adsorption induces a progressive displacement of casein.32,34,35 It is also well-known that the interactions between proteins are weakened by surfactant adsorption.36 The interface becomes more fluid, allowing the diffusion of the adsorbed species, even if the protein is not totally displaced.37 Moreover, from interferometry,37 it has been shown that air-water-air films stabilized with pure casein are much thicker than those stabilized by casein-surfactant mixtures. Another effect has to be taken in account. The presence of hydrophilic surfactant in water is known to promote the transfer of lipid crystals from the oil to the water phase.38 This phenomenon is applied for industrial lipid purification and is known as the Lanza process39 after the name of its inventor in 1905. Although we did not observe the presence of crystals in the aqueous phase, it is likely that Tween 20 favors protrusion of crystals at the interface. To verify this hypothesis, we examined the topography of the interface between the aqueous and oil phases at T ) 4 °C by AFM in the two limiting situations: aqueous phase containing protein alone (Rm ) 0; 3.5 wt % in the water phase) and surfactant alone (Rm ) ∞; 5 wt % in the water phase). The images reported in Figure 9 reveal a significant difference between the two samples. In the presence of sodium caseinate, the surface exhibits (27) Bressy, L.; He´braud, P.; Schmitt, V.; Bibette, J. Langmuir 2003, 19, 598. (28) Dickinson, E.; Radford, S. J.; Golding, M. Food Hydrocolloids 2003, 17, 211. (29) Dickinson, E. Colloids Surf. B 2001, 20, 197. (30) Dimitrova, T.; Leal-Calderon, F. Langmuir 2001, 17, 3235. (31) Dickinson, E.; Woskett, C. Competitive adsorption between proteins and small-molecule surfactants in food emulsions. In Food Colloids; Royal Society of Chemistry: Cambridge, U.K., 1989; pp 74-96. (32) Dickinson, E.; Tanai, S. J. Agric. Food Chem. 1992, 40, 179. (33) Mackie, A.; Gunning, A.; Wilde, P.; Morris, V. Langmuir 2000, 16, 2242. (34) Courthaudon, J. L.; Dickinson, E.; Dalgleish, D. J. Colloid Interface Sci. 1991, 145, 390. (35) Girardet, J.; Humbert, G.; Creusot, N.; Chardot, V.; Campagna, S.; Courthaudon, J.; Gaillard, J. J. Colloid Interface Sci. 2001, 243, 515. (36) Chen, J.; Dickinson, E. Food Hydrocolloids 1995, 9, 35. (37) Kra¨gel, J.; Wu¨stneck, R.; Husband, F.; Wilde, P.; Makievski, A.; Grigoriev, D.; Li, J. Colloids and Surfaces B 1999, 12, 399. (38) Spicer, P. T.; Hartel, R. W. Aust. J. Chem. 2005, 58, 655. (39) Lanza, F. German Patent 191 238, 1905.
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Figure 9. AFM topography of the oil surface maintained at T ) 4 °C for 15 h in the presence of an aqueous phase containing (a) 3.5 wt % of sodium caseinate and (b) 5 wt % of Tween 20 + 0.5 wt % of non adsorbing polymer (sodium alginate).
undulations due to the formation of irregularly shaped crystals, and the characteristic roughness does not exceed 500 nm. In the presence of surfactant, the same undulations are present but, in addition, large protrusions are visible. It should be noticed that the real topography is deformed by the pyramidal shape of the AFM tip. This is due to the fact that the tip cannot scan nearly vertical surfaces because of the sterical constraints. Despite this technical limitation, it can be stated that large crystals are really present at the interface. We are also aware that the scans have been obtained on a macroscopic interface and that the characteristic roughness in emulsion drops is certainly less pronounced because of curvature and finite size effects. However, the same qualitative behavior is expected in emulsions, with crystal protrusion progressing as Rm increases. Above some critical surfactant concentration, the characteristic protrusion length of the crystals should become sufficient to pierce the thin liquid films separating the drops. 3. Influence of the Oil Mass Fraction. By diluting the same mother emulsion, it was possible to vary the oil fraction from 5 wt % to 60 wt %, keeping all the other parameters constant (D ) 15 ( 0.5 µm, 3.5 wt % of sodium caseinate, and 0.5 wt % of Tween 20 corresponding to Rm ) 2.7). Rheological
measurements were performed only for φ g 40 wt % since the most dilute samples tended to cream within the time scale of the experiments. For 5 wt % e φ e 45 wt %, the influence of the oil mass fraction was assessed by visual inspection of macroscopic samples (6 mL tubes) placed on a wheel rotating at 15 rpm to avoid buoyancy-driven phase separation during all the successive steps: drop crystallization, tempering (tp ) 300 min, Tp ) 25 °C), and shape relaxation at 45 °C. Once at rest, some samples underwent rapid phase separation into three phases: an oil layer (top), a creamed emulsion (middle), and a clear aqueous solution (bottom). The rate of phase separation and the amount of oil released were representative of the extent of partial coalescence. Figure 10 shows the final aspect of the samples as a function of the oil mass fraction, after shape relaxation in quiescent conditions for 30 min at T ) 45 °C. By combining rheology (Figure 11), droplet size measurements, and the previous macroscopic observations, we could identify three different regimes: (i) for 5 wt % < φ e 25 wt %, the droplet size distribution evolved marginally after tempering, and the emulsions remained homogeneous after shape relaxation; (ii) for 30 wt % e φ < 50 wt %, partial coalescence was significant as revealed by the macroscopic oil layer formed at the top of the samples after
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Figure 10. Final aspect of the emulsions as a function of the oil mass fraction (D ) 15 µm, Rm ) 2.7). After crystallization at 4 °C for 15 h, tempering (tp ) 300 min; Tp ) 25 °C) was applied, and then the emulsions were heated at 45 °C to allow shape relaxation and subsequent phase separation. The oil released at the top of the sample is representative of the extent of partial coalescence. The dashed line is a guide for the eyes that delimitates the released oil from the remaining creamed emulsion; the solid line delimitates the creamed emulsion from the clear subnatant water phase.
Figure 11. Influence of the oil mass fraction on the final elasticity at 4 °C after tempering (Tp ) 25 °C; tp ) 300 min); D ) 15 µm; Rm ) 2.7
shape relaxation (Figure 10); (iii) for more concentrated emulsions (φ g 50 wt %), a gradual thickening of the emulsions was observed even during the crystallization step 4 °C prior to tempering. Concomitantly, the droplet size distribution evolved because of partial coalescence. Despite this initial destabilization, the elasticity after tempering was about 105 times larger than before. From the previous experiments, we deduce that the kinetics of partial coalescence is influenced by the drop fraction. For coalescence to occur, drops must be in contact, and we guess that, below a critical drop fraction, the number of contacts is insufficient for the phenomenon to be detectable after the tempering delay tp. As the drop fraction increases, the connectivity rises in accord with the evolution of the elasticity (Figure 11). Moreover, the local constraints imposed by droplet confinement at high droplet fraction may favor film piercing.16 4. Influence of the Drop Size. a. Rm ) 2.7. The drop size was varied from 2 to 50 µm at a constant composition (φ ) 45 wt %, 3.5 wt % of sodium caseinate, 0.5 wt % of Tween 20, Rm)2.7). After crystallization, the emulsions were tempered in the rheometer for tp ) 30 min at Tp ) 25 °C. Figure 12 shows the evolution of the final elasticity as a function of the drop diameter. A sharp transition occurs near 10 µm: the elasticity rises over almost 4 decades between 9 and 15 µm. The same tendency was observed for a longer tempering duration of 300
Figure 12. Influence of the drop size on the final elasticity measured at 4 °C after tempering at Tp ) 25 °C for tp ) 30 min (full symbols) and tp ) 300 min (open symbols); φ ) 45 wt %, Rm ) 2.7. The lines are guides for the eyes.
min. In order to avoid any possible artifact due to creaming, the experiments were reproduced in 6 mL tubes fixed on a rotating wheel for 300 min at 25 °C. The samples were then heated at 45 °C to allow shape relaxation (Figure 13). Between 9 and 12 µm, the quantity of oil released, representative of the extent of coalescence, exhibits a sharp variation, in close agreement with the rheological data. This experiment confirms the existence of a transition between a regime where gelling by partial coalescence is slow (D < 9 µm) and a regime where partial coalescence is fast and leads to a rigid gel over a very short period of time (D g 12 µm). It is well-known that supercooling effects become increasingly pronounced as the average droplet size decreases: the smaller the droplets, the lower the probability for homogeneous or heterogeneous crystal nucleation and, consequently, the longer the characteristic time to obtain the first nuclei.40,41 The weak gels obtained at low average droplet size could be due to incomplete (delayed) crystallization of the droplets. To probe this hypothesis, DSC measurements were performed upon heating (40) Coupland, J. N. Curr. Opin. Colloid Interface Sci. 2002, 7, 445. (41) Lopez, C.; Bourgaux, C.; Lesieur, P.; Bernadou, S.; Keller, G.; Ollivon, M. J. Colloid Interface Sci. 2002, 254, 64.
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Figure 13. Final aspect of the emulsions as a function of the droplet size (φ ) 45 wt %; D )15 µm, Rm ) 2.7). After crystallization at 4 °C for 15 h, tempering (Tp ) 25 °C; tp ) 300 min) was applied, and then the emulsions were heated at 45 °C to allow shape relaxation and subsequent phase separation. The oil released at the top of the sample is representative of the extent of partial coalescence. The dashed line is a guide for the eyes that delimitates the released oil from the remaining creamed emulsion; the solid line delimitates the creamed emulsion from the clear subnatant water phase. Table 3. Comparative Data at the Onsets of Transitions in Figures 8 and 12
Figure 8 Figure 12 a
D (µm)
Rm
S (m2/g oil)
maximum fraction of protein adsorbed (%)a
total amount of surfactant/total surface area of the droplets (10-6 mol/m2)
15 9-12
0.3-0.6 2.7
0.44 0.44-0.74
3.6 3.6-6.1
1.2-2.5 6.7-9.1
Assuming a maximum coverage of 3.5 × 10-3 g · m-2 of the protein at the oil/water interface.
the emulsions from 4 to 50 °C at a constant rate of +1 °C · min–1 (the systems were held at 4 °C for 15 h before DSC recording). The signals were integrated, and a constant value of about 67 ( 3 J per gram of dispersed phase was found whatever the droplet size. Hence, it can be concluded that droplet crystallization was achieved for all the systems in the experimental conditions. To interpret the data of Figure 12, it can be argued that the surface contact area between aggregated drops is an increasing function of the drop size.1 This could explain why the kinetics of coalescence is faster for bigger drops, but it does not explain at all the sharpness of the transition with respect to the drop diameter. An alternative explanation would be the increase in relative Tween 20 surface concentration because the overall surface area of the emulsion is reduced as D increases. The sharp transition observed in Figure 12 could thus have the same origin as the one in Figure 8: the displacement of the adsorbed protein by surfactant. The surface area per unit mass of dispersed phase is inversely related to the mean diameter of the droplets:
S)
6 FDs
where F is the density of the oil phase, and Ds is the surfacediameter assumed to be equal to the volume-averaged value, D, since our emulsions are monodisperse. The onset of the transition occurs for 0.3 < Rm < 0.6 in Figure 8, and for 9 µm < D < 12 µm in Figure 12. The comparative data are available on Table 3. The maximum surface concentration of sodium caseinate alone has been measured at various water/triglyceride interfaces,43,44 and is on the order of 3.5 × 10-3 g · m-2. At the transition onset, the total amount of protein is large enough to fully cover the
interfaces in both cases, and we can estimate that at least 94% of protein remains free (nonadsorbed) in the continuous phase at a concentration close to 3.5 wt %. In Table 3, we have also reported the ratio of the total amount of surfactant to the total interfacial area of the droplets. The ratios are clearly not in the same range, the amount of surfactant available to displace the protein being much larger in Figure 12 than in Figure 8. If gelling was linked to the interfacial composition only, the onset in Figure 12 would be expected at diameters much lower than 9 µm. Thus, in our experiments, the droplet size can be considered as a key control variable independent of the interfacial composition. It can also be argued that the characteristic crystal size is an increasing function of the drop size. Since partial coalescence requires protrusion of crystals over distances larger than the film thickness, gelling should occur above a critical drop size, as observed experimentally. By combining optical microscope observation between crossed analyzers and DSC measurements coupled to X-ray diffraction (XRD), Lopez et al.41 demonstrated that the emulsion drop size affects the crystal size and/or the structure of the crystal network in milk fat-in-water emulsions. The formation of crystalline structures was revealed by the presence of diffractions peaks. The decrease in the average fat drop size induced a decrease in small-angle XRD peaks maximum intensity correlated with an increase in peaks width. This was interpreted as resulting from defaults in the organization of (42) Sonoda, T.; Takata, Y.; Ueno, S.; Sato, K. Cryst. Growth Des. 2006, 6, 306. (43) Bos, M. A.; van Vliet, T. AdV. Colloid Interface Sci. 2001, 91, 437. (44) Sliwinski, E. L.; Lavrijsen, B. W. M.; Vollenbroek, H. J.; van der Stege, H. J.; van Boekel, M. A. J. S.; Wouters, J. T. M. Colloids Surf., B: Biointerfaces 2003, 31, 219.
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Figure 14. Microscope images under crossed analyzers at 4 °C of an emulsion made of (a) 5 µm drops and of (b) 15 µm drops. The scale bar represents 25 µm.
triglyceride molecules in the crystals, either directly due to the curvature of the oil/water interface from which crystals are supposed to grow, or indirectly due to the faster relaxation that can induce the formation of crystals of smaller size. Indeed, the decrease of droplet size induces a faster supercooling relaxation, and consequently a higher disorder and/or a smaller size of triglyceride crystals within the emulsion droplets.40,41 Similar results were obtained by Sonoda et al.42 for oil-in-water emulsions comprising triglycerides of different types. We observed two crystallized emulsions with average diameter of 5 and 15 µm by optical microscopy under crossed analyzers after a storage period of 15 h at T ) 4 °C (Figure 14). Drops with D ) 5 µm were poorly birefringent compared to D ) 15 µm, most probably reflecting the much smaller crystal size. We also probed the influence of crystal size by adding 2 × 10-3 wt % of fumed 16 nm silica beads hydrophobically functionalized by dimethyl dichlorosilane (Aerosil R972 purchased from Evonik) in the oil phase of an emulsion with D ) 15 ( 0.5 µm prior to emulsification. The beads act as hydrophobic substrates (on average, 7000 beads per drop) that favor heterogeneous nucleation. After crystallization, birefringence could be hardly observed between crossed analyzers, revealing the much reduced size of the crystals. Tempering (tp ) 30 min, Tp ) 25 °C) was then applied to the emulsion in the rheometer, and the final elasticity was only equal to 103 Pa compared to 2 × 104 Pa without silica. This experiment qualitatively confirms that the kinetics of partial coalescence is influenced by the crystal size. b. Rm ) ∞ The influence of the drop size was also investigated for a pure surfactant-stabilized interface corresponding to Rm ) ∞ (Figure 15). The drop size was varied from 3 to 47 µm, and the composition was the same for all emulsions (φ ) 45 wt %, 5 wt % of Tween 20, 0.5 wt % of sodium alginate in the aqueous phase after dilution). We did not observe any evolution of the size distribution after droplet crystallization at 4 °C for 15 h. The emulsions were submitted to tempering in the rheometer (tp ) 30 min, Tp ) 25 °C). Again, they turned to a hard gel, and the final elasticity versus drop size is reported in Figure 15. The elasticity increases with the drop size and saturates at a value close to 105 Pa, the variation being smoother than that for Rm ) 2.7. In order to determine the structure of the tempered emulsions, they were examined under a microscope at 4 °C (Figure 16), and droplet size measurements were performed. Emulsions
Figure 15. Influence of the drop size on the final elasticity measured at 4 °C after tempering (Tp ) 25 °C; tp ) 30 min) for emulsions at φ ) 45 wt %, stabilized by a mixture of surfactant and sodium caseinate (Rm ) 2.7; filled circles) or by pure surfactant (Rm ) ∞; open triangles).
with D e 10 µm were made of individual drops easily dispersible with exactly the same size distribution as the initial one. In contrast, irreversible aggregates were observed in emulsions with D g 15 µm, and their presence was confirmed by drop size measurements. We thus conclude that the elasticity jump induced by tempering has different origins depending on the average droplet size. For D e 10 µm, we guess that elasticity results from a jamming transition induced by surface roughness. Jamming was first observed in suspensions of solid spherical particles submitted to an intense shear for which lubrication between spheres was inoperative.45-48 This phenomenon was extended to colloidal systems where attractive interactions hinder the exploration of the accessible configurations.49,50 A general fluid-jammed diagram that rationalizes different phenomena such as glass transition, aggregation, and gelling has been proposed, where the system state is determined by particle volume fraction, thermal (45) Melrose, J.; Ball, R. Europhys. Lett. 1995, 32, 535. (46) Farr, R.; Melrose, J.; Ball, R. Phys. ReV. E 1997, 55, 7203. (47) Cates, M. E.; Wittmer, J. P.; Bouchaud, J. P.; Claudin, P. Phys. ReV. Lett. 1998, 81, 1841. (48) Liu, A.; Nagel, S. Nature 1998, 396, 21. (49) Holmes, C.; Fuchs, M.; Cates, M. E. Europhys. Lett. 2003, 63, 240. (50) Bertrand, E.; Bibette, J.; Schmitt, V. Phys. ReV. E 2002, 66, 060401.
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Figure 16. Microscope images showing the state of the emulsion after a tempering cycle (Tp ) 25 °C; tp ) 30 min): (a) D ) 10 µm and (b) D ) 47 µm. The emulsions were diluted to facilitate observation. The scale bar represents 50 µm.
energy, and applied stress.51 In our case, it is likely that the attractive depletion interactions induced by excess surfactant24 combined with surface roughness of the drops provoke a restriction of the internal dynamics characteristic of a jammed state. As discussed earlier, the hydrophilic surfactant promotes crystal location at the oil/water interface and protrusion toward the water phase (Figure 9). Boode et al.15 identically argued that tempering changed the crystal size and position, without changing any further system properties: after tempering, the crystals became bigger, and more of them were present at the oil/water interface. Partial coalescence did not take place in our fine emulsions most probably because the average crystal size in small drops remained insufficient to pierce the films. For emulsions with large droplet size (D g 15 µm), gelling was due to a combination of jamming and partial coalescence. As explained above, the occurrence of partial coalescence involves the formation of interfacial crystals whose characteristic protrusion size is large enough to pierce the films. We verified that the gel (51) Trappe, V.; Prasad, V.; Cipelletti, L.; Segre, P.; Weitz, D. Nature 2001, 411, 772.
elasticity decreased in the presence of smaller crystals by introducing 2 × 10-3 wt % of hydrophobic fumed silica beads in the oil phase of an emulsion with D ) 15 ( 0.5 µm, as previously. Tempering (tp ) 30 min, Tp ) 25 °C) was then applied to the emulsion in the rheometer, and the final elasticity was only equal to 5 × 103 Pa compared to 105 Pa without silica. In addition, we observed by AFM the topography of an interface between oil containing the same amount of silica and a 5 wt % Tween 20 aqueous solution. The image in Figure 17 has to be compared with that in Figure 9b: the presence of silica has the effect to smooth down the interface because more crystals with smaller average size have been formed.
Conclusion In this paper, we have explored the gelation of semisolid oilin-water emulsions triggered by thermal treatment. The two limiting mechanisms at the origin of the transition are partial coalescence and jamming. Partial coalescence involves the formation of irreversible links between drops when an optimal proportion of interfacial crystals is attained. In the jamming
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Figure 17. AFM topography of the oil surface maintained at T ) 4 °C for 15 h in the presence of an aqueous phase containing 5 wt % of Tween 20 and 0.5 wt % of a nonadsorbing polymer (sodium alginate). The oil phase contained 2 × 10-3 wt % of silica beads acting as crystallization substrates.
Figure 18. Schematic diagram of the different states obtained after tempering as a function of drop size, D, and the surfactant-to-protein molar ratio in the continuous phase, Rm. Strong gels are preferentially formed in the shaded zones.
mechanism, the integrity of the drops is preserved, but they are flocculated and rearrangements are hindered because of surface roughness. We have demonstrated that the behavior depends in a critical way on the crystal size and/or protrusion at the oil/ water interface, both properties being controlled by the average droplet size, D, and by the protein-to-surfactant molar ratio, Rm, which sets the interfacial composition. In Figure 18, we propose a graph that qualitatively summarizes the whole set of observations, with the different accessible states after tempering: weakly aggregated droplets, strong gels made of partially coalesced droplets, and strong gels made of aggregated jammed droplets. The position of the boundaries depends on the emulsion components and on the tempering conditions. For small Rm values, sodium caseinate forms a rigid layer at the drop surface, reducing crystals protrusion and avoiding both partial coalescence and
jamming. When the interface is enriched with surfactant, lateral protein interactions are weakened and interfacial crystals can protrude toward the water phase. Interfacial protrusions may be large enough to pierce the films. In smaller droplets, jamming occurs because the crystal size is sufficient to induce surface roughness, but insufficient to pierce the films. We have observed similar trends with other triglyceride oils such as cocoa butter, and we guess that the diagram reported in Figure 18 accounts for the general phenomenology of emulsions made of partially solidified oil drops comprising interfacial crystals. Acknowledgment. We are very grateful to Le Conseil Re´gional d’Aquitaine for financial support. LA802521F
