Impact of Sodium Caseinate Concentration and Location on

pubs.acs.org/Langmuir © 2010 American Chemical Society

Impact of Sodium Caseinate Concentration and Location on Magnesium Release from Multiple W/O/W Emulsions Marie Bonnet,† Maud Cansell,*,† Fr
ed
eric Placin,† Marc Anton,‡ and Fernando Leal-Calderon† †

Universit
e Bordeaux 1, TREFLE UMR CNRS 8508, ENSCBP, 16 avenue Pey Berland, 33607 Pessac, France, and ‡UR1268 Biopolym eres Interactions Assemblages, Equipe Interfaces et Syst emes Dispers
es, INRA, 44360 Nantes Cedex 3, France Received January 7, 2010. Revised Manuscript Received March 1, 2010 Water-in-oil-in-water (W/O/W) double emulsions were prepared and the rate of release of magnesium ions from the internal to the external aqueous phase was followed. Sodium caseinate was used not only as a hydrophilic surface-active species but also as a chelating agent able to bind magnesium ions. The release occurred without film rupturing (no coalescence). The kinetics of the release process depended on the location (in only one or in both aqueous compartments) and on the concentration of sodium caseinate. The rate of release increased with the concentration of sodium caseinate in the external phase and decreased when sodium caseinate was present in the inner droplets. The experiments were interpreted within the frame of a mean-field model based on diffusion, integrating the effect of ion binding. The data could be adequately fitted by considering a time-dependent permeation coefficient of the magnesium ions across the oil phase. Our results suggested that ion permeability was influenced by the state of the protein interfacial layers which itself depended on the extent of magnesium binding.

1. Introduction Water-in-oil-in-water emulsions (W/O/W) are compartmented systems comprising aqueous droplets dispersed in oil globules, these latter being dispersed in an aqueous continuous phase.1-3 Such materials have potential applications for the encapsulation and controlled release of hydrosoluble compounds3-8 in various domains such as pharmaceutics, cosmetics and food. The goal of encapsulation is to isolate substances from the environment and/ or to retain their activity until required. Double emulsions are metastable systems stabilized by two emulsifiers of opposite solubility, i.e., a water-soluble emulsifier and an oil-soluble one following the well-known Bancroft rule. Both emulsifiers mix at the interfaces and the kinetic evolution of double emulsions is mainly governed by the composition of the binary mixture. The evolution of double emulsions results from a complex interplay between coalescence and diffusion phenomena. Coalescence involves film rupturing and may occur at several levels:9 (i) between the small inner aqueous droplets, (ii) between the large oil globules, and (iii) between the globule and the small droplets dispersed within it. This latter mechanism leads to the release of the whole droplet content into the external aqueous phase. Alternatively, the globules are permeable to many different chemical species which can migrate from the internal phase to *To whom correspondence should be addressed. Telephone: 33 (0)5 40 00 38 19. E-mail: [email protected]. (1) Florence, A. T.; Whitehill, D. Int. J. Pharm. 1982, 11, 277–308. (2) Garti, N.; Aserin, A. Adv. Colloid Interface Sci. 1996, 65, 37–69. (3) Benichou, A.; Aserin, A.; Garti, N. Adv. Colloid Interface Sci. 2004, 108-109, 29–41. (4) Sela, Y.; Magdassi, S.; Garti, N. J. Controlled Release 1995, 33, 1–12. (5) Tedajo, G. M.; Bouttier, S.; Fourniat, J.; Grossiord, J. L.; Marty, J. P.; Seiller, M. Int. J. Pharm. 2005, 288, 63–72. (6) Shima, M.; Kobayashi, Y.; Kimura, Y.; Adachi, S.; Matsuno, R. Colloids Surf., A 2004, 238, 83–90. (7) Shima, M.; Morita, Y.; Yamashita, M.; Adachi, S. Food Hydrocolloids 2006, 20, 1164–1169. (8) O’Regan, J.; Mulvihill, D. Food Hydrocolloids 2009, 23, 2339–2345. (9) Pays, K.; Giermanska-Kahn, J.; Pouligny, B.; Bibette, J.; Leal-Calderon, F. J. Controlled Release 2002, 79, 193–205.

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the external one and vice versa, without film coalescence. The diffusion processes is driven by concentration gradients involving by the whole set of molecules (surfactant, electrolytes, osmotic regulators and encapsulated compounds). Several possible mechanisms have been proposed to account for the diffusive transport of the encapsulated compounds: (i) direct solubilization of the entrapped species in the oil phase (for neutral molecules), (ii) transport via the hydrophilic surfactant polar headgroup in the case of water,10,11 (iii) transport through the oil phase into reverse micelles,1,3,4,12-15 and (iv) formation of thermally activated transient holes in the thin liquid films separating the internal droplets and the globule surface.9 Achieving sufficient storage stability for commercial applications is a common issue that arises when considering potential uses of double emulsions. In practice, a large set of parameters may influence the stability of the compartmented structure as well as the release kinetics of the encapsulated compounds: the type of the surface-active species (monomeric or polymeric) and their concentrations,4,9,13,16 the osmotic pressure mismatch between the aqueous compartments,10,11,17,18 the average diameter and volume fractions of the inner droplets and of the oil globules,1,9,19 and the oil chemical nature.9,20-22 Among all the parameters, the (10) Wen, L.; Papadopoulos, K. D. Colloids Surf., A 2000, 174, 159–167. (11) Wen, L.; Papadopoulos, K. D. J. Colloid Interface Sci. 2001, 235, 398–404. (12) Garti, N. Colloids Surf., A 1997, 123-124, 233–246. (13) Garti, N. Lebensm. Wiss. Technol. 1997, 30, 222–235. (14) Benichou, A.; Aserin, A.; Garti, N. Colloids Surf., A 2007, 294, 20–32. (15) Cheng, J.; Chen, J. F.; Zhao, M.; Luo, Q.; Wen, L. X.; Papadopoulos, K. D. J. Colloid Interface Sci. 2007, 305, 175–182. (16) Jager-Lezer, N.; Terrisse, I.; Bruneau, F.; Tokgoz, S.; Ferreira, L.; Clausse, D.; Seiller, M.; Grossiord, J. L. J. Controlled Release 1997, 45, 1–13. (17) Mezzenga, R.; Folmer, B. M.; Hughes, E. Langmuir 2004, 20, 3574–3582. (18) Guery, J.; Baudry, J.; Weitz, D. A.; Chaikin, P. M.; Bibette, J. Phys. Rev. 2009, E 79, 060402R. (19) Leadi Cole, M.; Whateley, T. L. J. Controlled Release 1997, 49, 51–58. (20) Weiss, J.; Scherze, I.; Muschiolik, G. Food Hydrocolloids 2005, 19, 605–615. (21) Knoth, A.; Scherze, I.; Muschiolik, G. Eur. J. Lipid Sci. Technol. 2005, 107, 857–863. (22) Bonnet, M.; Cansell, M.; Berkaoui, A.; Ropers, M. H.; Anton, M.; Leal-Calderon, F. Food Hydrocolloids 2009, 23, 92–101.

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surfactant type plays a major role. Pioneering studies on double emulsions were performed in presence of low-molecular weight surfactants.1,9 It was found that the fast destabilization and release of double emulsions based on short surfactants was incompatible with commercial applications. However, recent studies in presence of amphiphilic polymers, proteins, and solid colloidal particles2-4,19,22-25 reveal that coalescence can be inhibited and that diffusive transport may be extremely slow, thus motivating renewed interest for such materials. In recent studies, we have studied the kinetics of release of magnesium ions in W/O/W double emulsions based on triglyceride oils. Magnesium (Mg2þ) was used not only as a model species to probe the release mechanism but also as a compound suitable for food supplementation. Indeed, magnesium is a mineral trace element that is involved in a wide range of fundamental, enzymatic cellular reactions and in protein synthesis. Because of a change in the nutrition habits, the daily intake in magnesium is lower than the recommended value. Thus, magnesium supplementation of food could be an alternative to prevent magnesium deficiency and its associated clinical disorders (hypertension, cardiovascular diseases, muscular weakness, diarrhea). However, magnesium addition in foods can induce chemical degradations and protein aggregation and generate an unpleasant taste. These drawbacks could be avoided or at least reduced by encapsulation. Magnesium ions were initially dissolved in the internal droplets and the kinetics of release into the external aqueous phase was followed using flame spectroscopy. Magnesium leakage occurred without film rupturing through entropically driven diffusion/ permeation phenomena. The characteristic retention time varied between weeks and months depending on the oil chemical nature.22 When the emulsions were exposed to pancreatic lipase, the triglycerides composing the oil phase were rapidly hydrolyzed (within minutes) by the enzyme suggesting that magnesium could be available at the intestinal site. These results indicated a possible use of W/O/W emulsions loaded with magnesium ions in food or pharmaceutical applications. In the present study, ion leakage was studied over a month, at 25 and/or 4
C, for emulsions containing either miglyol or olive oil. Miglyol is a synthetic oil only based on medium chain triacylglycerols. In contrast, olive oil is naturally occurring and comprises a larger range of fatty acids, oleic acid (18:1) being the most abundant (more than 77%). Following the preliminary work of Bonnet et al.,22 we could obtain systems where the release was mainly due to diffusion phenomena. Double emulsions were all formulated using sodium caseinate as the hydrophilic surfaceactive species. This protein was chosen because of its specific properties: adsorption at the oil/water interface, binding capacity with magnesium ions, and large molecular size, which precludes permeation of the protein across the oil phase. The main objective of the present study was to deepen the knowledge about passive permeation phenomena of ionic encapsulated species in order to improve retention in W/O/W emulsions. Our study was focused on the impact of caseinate location and concentration. These parameters should influence the rate of delivery because of the possible coupling between the diffusion process, ion chelation and the interfacial properties of caseinate. The influence of caseinate was probed when present: (i) in the external aqueous phase only, and (ii) in both the internal and (23) Michaut, F.; H
ebraud, P.; Perrin, P. Polym. Int. 2003, 52, 594–601. (24) Aveyard, R.; Binks, B. P.; Clint, J. H. Adv. Colloid Interface Sci. 2003, 100-102, 503–546. (25) Garti, N.; Lutz, R. In: Emulsions: Structure, stability and interactions; Petsev, D. N., Ed.; Elsevier, Academic Press: San Diego, CA, 2004, Vol. 14, pp 557-605.

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external aqueous compartments. A theoretical model based on Fick’s law and on the binding capacity of casein was proposed to analyze the experimental results. By means of this model, we could assess the impact of several formulation parameters and we could derive a quantitative estimate of the characteristic permeation coefficient of magnesium ions across the oil phase.

2. Materials and Methods 2.1. Materials. The oil phase used for the formulation of W/O/W emulsions was either miglyol or olive oil. Miglyol (from St
earinerie Dubois Fils, France) is a mixture of triglycerides with C8/C10 fatty acids chains in the mass ratio 55/45. Olive oil (from Lesieur, extra virgin) is essentially composed of triglyceride molecules with the following main fatty acid chain composition: 77% of oleic acid (18:1), 11% of palmitic acid (16:0), 7% of linoleic acid (18:2), 3% of stearic acid (18:0). The refractive index of the oil at 20
C under white light was 1.466 and the viscosity was equal to 80 mPa.s at 25
C. The lipophilic surface-active species, polyglycerol polyricinoleate (PGPR) (esters of polyglycerol and polyricinoleate fatty acids, Mw ≈ 1 766 g 3 mol-1), was purchased from Palsgraad (France) and the hydrophilic one, sodium caseinate (SC) (Mw ≈ 20 000 g 3 mol-1), was obtained from Lactoprot (Germany). Magnesium chloride (hexahydrate 99%) was from Acros Organics (Belgium) and sodium azide from Merck (Germany). Lactose and sorbitan monooleate (Span 80) were purchased from Sigma-Aldrich (Germany). All the species were used as received. The water used in the experiments was deionized with a resistivity close to 15 MΩ 3 cm at 20
C.

2.2. W/O/W Emulsion Formulation and Preparation.

W/O/W emulsions were prepared at room temperature by using a two-step emulsification process, as previously described.22 Various aqueous phases hereafter quoted WI, WII and WIII, were prepared at different magnesium chloride, SC and lactose concentrations (Table 1). They were manually dispersed at 80 wt % into the oil phase (miglyol or olive oil) containing PGPR (30 wt %). The obtained crude W/O emulsions were then submitted to a strong shear using a Couette’s cell (concentric cylinders geometry, Ademtech SA, France), with a gap of 200 μm. The shear rates were adapted to each formulation, depending on the viscosity ratio of the dispersed to the continuous phase and on the oil/aqueous phase interfacial tension,26 to obtain approximately the same average droplet size (Table 2). Once fragmented, the emulsions were diluted with oil, at a droplet content of 40 wt %. In the second step, the W/O emulsions were incorporated into an aqueous phase, up to 70 wt %. The external aqueous phase contained lactose, 12 wt % of SC and 0.08 wt % of sodium azide (bactericide agent). Lactose was introduced as an osmotic pressure matching agent in the internal and/or external aqueous phases in order to limit water transfer.16,17,22 The W/O/W emulsions were fragmented in the Couette cell at different shear rates depending on the formulation (Table 2), and diluted with solutions containing lactose and SC to set the final globule concentrations at 10 wt % and to vary SC concentrations in the external aqueous phase from 1.9 to 12 wt %. In the final state, the internal aqueous phase represented 4% of the total weight and PGPR was present at 5 wt % in the oil phase. The maximum surface concentration of SC has been measured at various water/triglyceride interfaces27,28 and is of the order of 3.5  10-3 g.m-2. Considering the average globule size (∼10 μm, see below) in our emulsions, the amount of SC in the external aqueous phase required to fully cover the globule surfaces was thus 0.25 wt %. The final compositions of all formulations are reported in Table 1. The pH of both internal and external aqueous phases (26) Goubault, C.; Pays, K.; Olea, D.; Bibette, J.; Schmitt, V.; Leal-Calderon, F. Langmuir 2001, 17, 5184–5188. (27) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437–471. (28) 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 2003, 31, 219–229.

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Bonnet et al. Table 1. Composition of the W/O/W Emulsions globules (10 wt %) oil phase (6 wt %)

internal aqueous droplets (4 wt %)

miglyol or olive oil (95 wt %) PGPR (5 wt %)

SC (3.4; 7.0; 12.0 wt %)/ lactose (0.3 M)/ sodium azide (0.08 wt %)

WI MgCl2 (0.1 M) WII MgCl2 (0.014 M)/lactose (0.2 M) WIII MgCl2 (0.014 M)/lactose (0.2 M)/SC (1.9 wt %)

was always close to 6.5. The emulsions were stored at 4 and/or 25
C for 1 month. The globules tended to cream after a few hours of settling. To maintain homogeneity, instead of applying a continuous mechanical stirring that could accelerate the release process because of convective effects, the samples were turned upside down at regular time intervals (5 h). 2.3. W/O/W Emulsion Characterization. The structural evolution of the double emulsions was followed by means of an Olympus BX51 microscope equipped with a phase-contrast device, an oil immersion objective (100/1.3, Zeiss, Germany), and a video camera. In parallel, droplet sizing was carried out on both the primary W/O and multiple W/O/W emulsions. Water droplet size distribution of the primary W/O emulsions was measured by static light-scattering, using a Coulter LS 230 apparatus. To avoid multiple scattering, the W/O emulsion samples were diluted with a dodecane solution containing 0.5 wt % of sorbitan monooleate (Span 80). This oil-soluble surfactant was used because of its total miscibility with dodecane, and in order to avoid destabilization of the droplets due to the strong dilution with oil. The measuring cell was filled with the dodecane solution, and a small volume of the sample was introduced under stirring. Measurements were performed at room temperature. The volume weighted average diameter d4,3 was obtained from Mie’s theory. The particle-size distribution of oil globules in the W/O/W emulsions was also measured using static light-scattering. To dilute the emulsions, we used NaCl aqueous solutions with the same osmotic pressure as that of the inner droplets in order to avoid water transfer phenomena. In this case, d4,3 was obtained using Fraunhofer’s model. In principle, this model applies to optically homogeneous spheres with diameters equal to or greater than 10 μm. In our case, although the globules were not homogeneous due to the presence of the inner water droplets, the measured values were in agreement with the microscopic observations. For each type of formulation, three samples of the W/O and W/O/W emulsions were analyzed right after preparation and after one-month storage at 4 and/or 25
C.

2.4. Quantification of Magnesium Release from W/O/W Emulsions. Magnesium release was measured right after preparation and during 1-month storage. A small amount of the double-emulsion was collected from the stock volume at regular time intervals for further titration of the continuous phase. The collected sample was centrifuged at 1100 g for 30 min (Jouan CR 1000) in order to separate the globules from the external aqueous Table 2. Shear Rates (s-1) Applied To Prepare the W/O and W/O/W emulsions shear rate (s-1) W/O emulsion internal aqueous phasea

miglyol

olive oil

W/O/W emulsion miglyol

olive oil

WI 2625 1575 6300 8400 WII 1575 2625 WII 2100 2625 a The composition of WI, WII, and WII phases is reported in Table 1.

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external aqueous phase (90 wt %)

SC (1.9 wt %)/ lactose (0.2 M)/ sodium azide (0.08 wt %)

phase. We checked that the applied centrifugation did not lead to coalescence phenomena that could produce further release of Mg2þ. The cream was redispersed again after centrifugation and the globules were observed under the microscope. Both the internal droplet size and the droplet concentration within the globules remained apparently invariant. Moreover, droplet sizing experiments based on static light-scattering (see above) were carried out and we checked that the average globule diameter before and after centrifugation were the same. Magnesium in the subnatant aqueous phase was titrated by flame atomic absorption spectroscopy (Perkin-Elmer AAnalyst 100) as previously described.22 Magnesium release was measured for 1 month for emulsions stored at 25
C. Three samples from each type of W/O/W emulsions were analyzed. Magnesium was hardly detectable right after emulsion fabrication (t = 0) and the encapsulation yield was close to 100%. We can conclude from the measurements at t = 0 that the experimental procedure used for the quantification of magnesium release did not generate any artifact, e.g., droplet/globule coalescence that would produce further release of Mg2þ. The percentage of magnesium released from the internal (subscript “1”) to the external (subscript “2”) aqueous phase was calculated according to the following formula: %Mg2þ ðtÞ ¼ 100 

N2, Mg2þ ðtÞ N10, Mg2þ

ð1Þ

0 In eq 1, N1,Mg 2þ was the initial encapsulated magnesium in the internal droplets (in mole), and N2,Mg2þ was the total amount of magnesium present in the external aqueous phase at time t and given by:

N2, Mg2þ ðtÞ ¼ C2, Mg2þ ðtÞ  V2

ð2Þ

where C2,Mg2þ(t) was the concentration measured by flame spectroscopy at time t and V2 the volume of the external aqueous phase. Volume V2 was assumed to remain constant over time since it was very large compared with the internal volume V1 (V2/V1 ≈ 22 under the initial conditions). Thus, any instability (coalescence) or moderate osmotic mismatch would have a negligible effect on V2.

3. Experimental Results and Discussion 3.1. Characterization of W/O/W Emulsions and Structural Evolution. Parts a and b of Figure 1 are typical microscope images taken right after fabrication and after 30 days. The compartmented structure remained apparently unchanged during storage. Indeed, we could not visualize any significant variation neither in size nor in concentration of the internal droplets under the microscope. The mean diameters of the internal droplets, dd, in the primary W/O emulsion and of the oil globules, dg, in W/O/W emulsions measured by static light-scattering were in the same range for all the prepared emulsions, dd = 1.2 ( 0.4 μm and dg = 10.2 ( 2.5 μm. Within experimental uncertainty ((0.2 μm), both diameters remained invariant over the whole storage period for Langmuir 2010, 26(12), 9250–9260

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Figure 1. Optical microscopy images of a W/O/W emulsion based on miglyol with 0.1 M of magnesium chloride in the internal aqueous phase, 12 wt % of sodium caseinate and 0.3 M of lactose in the external aqueous phase (a) just after preparation; (b) after a 30 day-storage period at 25
C. The scale bar represents 10 μm.

each emulsion indicating the absence of external (globuleglobule) and internal (droplet-droplet) coalescence phenomena. As indicated in section 2.4, the emulsions were submitted to centrifugation in order to collect the subnatant phase prior to magnesium titration. The thickness of the cream measured after centrifugation remained constant over 1 month, suggesting that the internal droplet fraction was preserved over time. To improve accuracy, measurements were also made with W/O/W double emulsions containing 50 wt % of globules and it was identically found that the thickness of the corresponding cream did not vary after one month. From the whole set of results, we deduced that coalescence was insignificant and that magnesium leakage occurred principally under the effect of diffusion phenomena. Because of its comparatively large volume, the external water phase can be considered as a reservoir that fixes the osmotic pressure. Because of the transfer at different rates of the various solutes from one compartment to the other, water permanently migrates and modifies the internal droplet size in order to set the osmotic pressure at the same level as in the external reservoir. The apparent invariance of the internal droplets size indicated that the osmotic balance did not significantly evolve over time. However, it should be underlined that only very large volume variations would be discernible since a k-fold variation in the volume only induces a k1/3-fold variation in the droplet size. Nevertheless, because the structural parameters of the double emulsions were almost identical irrespectively of the composition of the water compartments, the rates of magnesium release could be straightforwardly compared for all the W/O/W emulsions. Langmuir 2010, 26(12), 9250–9260

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3.2. Magnesium Release as a Function of SC Concentration in the External Aqueous Phase. Magnesium release was measured as a function of SC concentration in the external aqueous phase at 25
C. Magnesium chloride was initially encapsulated at 0.1 M in the internal droplets (WI solution, Table 1) and the concentration of SC in the external aqueous phase ranged between 1.9 and 12 wt %. In Figure 2, the experimental curves show that the higher the SC concentration, the higher the proportion of magnesium released at any time t. Pays et al.9 identically observed that the release was accelerated upon increasing the concentration of the hydrophilic surfaceactive species. However, in their case, release was due to coalescence of the inner droplets on the globule surface and the surfaceactive species were low-molecular weight surfactants. Our results revealed the same tendency when the release process was diffusion-controlled and the hydrophilic surface-active species was a macromolecule (protein). At constant temperature and assuming ideal behavior of the solutes, the osmotic pressure is simply proportional to the total concentration of dissolved species (vant’t Hoff equation). In the experimental conditions, MgCl2 is totally dissociated in water and thus 0.1 M of this electrolyte generates 0.3 M of dissolved species. This concentration is osmotically compensated by lactose dissolved in the external compartment at 0.3 M. Caseinate molecules have a negligible contribution to the osmotic pressure because of their very large molar mass. However, each protein yields on average 25 Naþ counterions (SC contains 3 wt % of sodium) whose contribution is significant (for the sake of simplicity, we assume that SC is fully dissociated). For instance, for the more concentrated sample containing 12 wt % of SC, Naþ concentration is about 0.15 M, representing an osmotic pressure increment of nearly 50%. Thus, the external osmotic pressure was initially larger than the internal one and the mismatch increased linearly with SC concentration. As a consequence, it is likely that water migrated from the droplets to the external aqueous phase until osmotic equilibration was achieved. Osmotic equilibration by water transfer across the oil phase being a relatively fast process,10 the resulting reduction of the droplet volume caused a rapid jump of the internal magnesium concentration. According to Fick’s law, the rate of magnesium release is proportional to the concentration difference between the two aqueous compartments. By varying SC concentration from 1.9 to 12 wt %, the initial rate of release should exhibit a nearly 30% increment. In comparison, the average rates of release deduced from the initial slopes in Figure 2 exhibited at least a 2-fold increase (more than 100% increment). Thus, the osmotic pressure difference resulting from the presence of SC could not quantitatively explain the experimental observations. It is well-known that casein can bind divalent ions (Ca2þ, Mg2þ, etc.) mostly because of the presence of phosphoryl groups.29-31 In the experimental conditions probed here, we assumed that each protein could bind on average 5 magnesium ions.30 For instance, with 12 wt % SC in the external aqueous phase, the amount of available protein sites for chelation was about 7 times larger than the total number of encapsulated Mg2þ ions. Thus, caseinate acted as a chemical “pump” that lowered the concentration of free Mg2þ ions in the external aqueous phase. Since the diffusion rate between the internal and external aqueous phases was determined by the concentration difference of free ions, chelation should accelerate the process and the effect should (29) Parker, T. G.; Dalgleish, D. G. J. Dairy Res. 1981, 48, 71–76. (30) Baumy, J. J.; Brul
e, G. Le Lait 1988, 68, 409–418. (31) Gaucheron, F. In Min
eraux et produits laitiers; Tec and Doc Eds., Europe Media Duplication: Lassay-les-Ch^ateaux, France, 2003; Vol. 4, pp 81-112.

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Figure 2. Magnesium release for W/O/W emulsions based on olive oil (a) and miglyol (b), in the presence of 3.4 wt % (9), 7.0 wt % (2), and 12.0 wt % (b) of sodium caseinate in the external aqueous phase, at 25
C. Magnesium chloride at 0.1 M was initially encapsulated in the internal droplets. The solid lines are theoretical calculations using the model developed in section 4 (see text for details; the following 0 0 0 0 0 parameters were adopted: K(25
C) = 1200 M-1; s = 5; C1,Mg 2þ = 0.1 M; C2,lact = 0.3 M; dg = 10 μm; V1 = 0.040 L; V2 = 0.890 L; Voil = 0.07 L; 0 the values of V1 after osmotic equilibrium, PMg 2þ and k are provided in Table 3). Inset: kinetic evolution of PMg2þ.

be as more pronounced as SC concentration was larger. Although being satisfactory on a qualitative point of view, this explanation was also discarded for quantitative reasons. Because of the large volume ratio of the internal to external aqueous compartments (V2/V1 ≈ 22), Mg2þ ions underwent a strong dilution after being released. In such conditions, chelation weakly modified the concentration difference, and the diffusion rate between the two aqueous compartments remained essentially unchanged. In the early stages of the process, the relative difference in the diffusion rate between the two limiting situations, i.e., no chelation at all and total chelation of the released ions, was estimated to be(V2/V1 ≈ 4.5%. Such a difference was negligible compared with the experimental variations observed in Figures 2. More precise calculations are performed in section 4.2 to definitely consolidate our conclusion. The data of Figure 2 suggested some possible relation between the surface-active species at interfaces and the diffusion/permeation process across the oil globules. Caseinate is a surface-active protein whose adsorption determines both static and dynamic interfacial properties of the globule/water interface. It has been established that the interfacial properties are strongly modified when divalent cations are present in the aqueous phase.32,33 We thus hypothesized that ion permeability was influenced by the binding state of the proteins characterized by the proportion of ligand sites occupied by magnesium ions. This proportion tended to decrease as the concentration of caseinate in the external phase increased. Following this hypothesis, the rate of the permeation (32) Husband, F. A.; Wilde, P. J. J. Colloid Interface Sci. 1998, 205, 316–322. (33) Belhomme, C.; David-Briand, E.; Gu
erin-Dubiard, C.; Vi
e, V.; Anton, M. Colloids Surf., B 2008, 63, 12–20.

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process should be time-dependent and its kinetic evolution should depend on caseinate concentration. This hypothesis and its impact on the rate of release will be quantitatively assessed in section 4. 3.3. Magnesium Release as a Function of SC Location. The influence of SC location was probed in a different set of experiments. The osmotic pressure was mainly fixed by lactose, initially dissolved in both aqueous phases at the same concentration (0.2 M), well above all other possible contributions. The strategy consisted in comparing the kinetics of magnesium leakage when SC was present: (i) in the external aqueous phase only, and (ii) in both the internal and external aqueous compartments at the same concentration. The initial concentration of Mg2þ ions in the internal droplets was equal to 0.014 M (WII and WIII solutions, Table 1). When present, SC concentration in the corresponding phase was equal to 1.9 wt % (Table 1). We attempted to increase the internal concentration of SC above 1.9 wt % but the resulting W/O emulsions were prone to coalescence. Hereafter, each double emulsion were identified by a simple code “x/y”, where x represented the composition of SC in the internal droplets and y that in the external phase (in wt %). The evolutions of magnesium release during 1-month storage at 25
C in miglyol-based emulsions are reported in Figure 3a. The amount of magnesium released at any time t was smaller for the “1.9/1.9” system compared with the “0/1.9” system. Caseinate molecules in the internal droplets could bind up to 30% of the magnesium ions initially encapsulated. Chelation of magnesium by casein proteins decreased the internal concentration of free (unbound) ions, which certainly contributed to slow down the release in the “1.9/1.9” system. Langmuir 2010, 26(12), 9250–9260

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Figure 3. Magnesium release for W/O/W emulsions based on miglyol, as a function of sodium caseinate concentrations in the internal and in the external aqueous phases (internal (wt %)/external (wt %)): 1.9/1.9 (9) and 0/1.9 ((). Emulsions were stored at 25
C (a) and at 4
C (b). Magnesium chloride at 0.014 M was initially encapsulated into the internal droplets. The solid lines are theoretical calculations using the model developed in section 4 (see text for details; the following parameters were adopted: K(25
C) = 1200 M-1; K(4
C) = 860 M-1; s = 5; 0 -4 0 0 0 0 C1,Mg 2þ = 0.014 M; C1,chel = 0 or 9.5  10 M;C2,chel = 9.5  10-4 M; C1,lact = C2,lact = 0.2 M; dg0 = 10 μm; V10 = 0.040 L; V 02 = 0.890 L; Voil = 0.070 L; the values of V1 after osmotic equilibrium, PMg2þ0 and k are provided in Table 3). Inset: kinetic evolution of PMg2þ.

The same qualitative evolution was observed when the double emulsions were stored at 4
C (Figure 3b). However, the rates of release were much slower at this temperature, thus confirming the activated nature of the process, as already discussed.9,22

4. Theoretical Approach 4.1. Basic Principles. A theoretical approach is now proposed to account for the transfer of magnesium ions by diffusion. The model proposed by Pays et al.9 was adopted and extended to take into account osmotic pressure differences and the possible chelation induced by ligand molecules. These latter are large hydrophilic species (like proteins), hereafter denoted P, with s potential sites for magnesium binding and a total net charge zP ( V10), magnesium ions are diluted in the internal droplets and consequently the release is slowed down in comparison with iso-osmotic conditions (triangles) (V1 = V10). Conversely, the release is accelerated when the droplet volume decreases (V1