O Emulsification and its Impact on

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Quantification of spontaneous W/O emulsification and its impact on the swelling kinetics of multiple W/O/W emulsions Jana Bahtz, Deniz Zeynel Gunes, Axel Syrbe, Nicola Mosca, Peter Fischer, and Erich J. Windhab Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00425 • Publication Date (Web): 19 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

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Quantification of spontaneous W/O emulsification and its impact on the swelling kinetics of multiple W/O/W emulsions Jana Bahtz*#ǂ, Deniz Z. Gunes*||, Axel Syrbe||, Nicola Mosca#, Peter Fischer*#, Erich J. Windhab# #

ETH Zurich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 9, 8092 Zurich, Switzerland Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland ǂ Nestlé Product Technology Centre, Lange Str. 21, 78224 Singen, Germany ||

KEYWORDS: multiple emulsion, spontaneous emulsification, polyglycerol polyricinoleate, osmotic swelling, controlled release

ABSTRACT: An osmotic imbalance between the two water phases of multiple water-in-oil-in-water (W1/O/W2) emulsions results in either emulsion swelling or shrinking due to water migration across the oil layer. A controlled mass transport is not only of importance for the emulsion stability, it also allows a transient emulsion thickening or a controlled release of encapsulated substances, such as nutriments or simply salt. Our prior work has shown that the mass transport follows two sequential stages. In a first stage the oil phase structure is changed in a way, which allows a rapid osmotically driven water transport in the second, osmotically dominated stage. These structural changes in the oil layer are strongly facilitated by the spontaneous formation of tiny water droplets in the oil phase, induced by the oil-soluble surfactant, i.e., polyglycerol polyricinoleate (PGPR). This study provides a simple method based on microscopy image analysis, allowing detailed investigation of spontaneous W/O emulsification. It quantitatively describes the volume of droplets generated and rate of droplet creation. Moreover, it describes the effect of spontaneous W/O emulsification on the swelling kinetics of microfluidic processed W1/O/W2 emulsions. Two different concentration regimes of the oilsoluble surfactant are identified: below a critical concentration the overall water transport rate increases, while above the critical concentration the water transport stagnates due to a maximized structure formation.

INTRODUCTION Multiple water-in-oil-in-water (W1/O/W2) emulsions are oil-in-water emulsions (O/W2) with entrapped internal water droplets (W1). In general, they have potential in applications such as fat reduction, encapsulation, and controlled delivery of hydrophilic drugs or other active components in food [1,2], pharmaceutical [3-6], cosmetic [7], or agricultural products [8]. An osmotic pressure gradient ∆Π between both aqueous phases caused by different electrolyte concentrations results in water transport across the oil layer. The direction of the water transport depends on the direction of the osmotic pressure imbalance ∆Π. In the case of ∆Π being directed toward the inner water phase W1, water migrates into the inner droplets resulting in emulsion swelling. In the opposite case, water migrates to the outer aqueous phase W2 and the droplets shrink. In both cases, water transport strongly degrades the emulsion stability. Nevertheless, one may also take advantage of the water transport providing that it is precisely controlled. Emulsion swelling can be used for the production of viscous firm emulsions with high internal water loadings [9,10]. On the other hand, precise tailoring of the water transport in direction of the outer aqueous phase W2 is required for controlled delivery of encapsulated sub-

stances, such as aroma or drug components in the presence of water. Control of the water migration across the oil layer is only possible if the underlying transport mechanisms are understood in detail. In a prior study we investigated the water transport in osmotically imbalanced W1/O/W2 emulsions stabilized by PGPR in the oil phase. The emulsions contained one internal water droplet W1, which was separated from the outer water phase W2 by an oil layer of a certain thickness [9]. Thus, the oil layer initially established a border between both aqueous phases W1 and W2. The study demonstrated that in such emulsion systems the swelling kinetics follows two sequential stages. The first stage is the lag stage, in which water migrates spontaneously, but slowly into the oil from both aqueous phases. It is assumed that the lag stage is required for structural changes of the oil layer, i.e., the formation of a water transport structure. This allows for the osmotic pressure gradient to act and the second, osmotically dominated stage starts. It is characterized by a fast and initial linear droplet volume change with time, which is called swelling rate in the following. The osmotic pressure imbalance ∆Π between W1 and W2 is the driving force for the water transport in the osmotically dominated stage. With time ∆Π is reduced and

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the swelling rate decreases as the system tends toward the osmotic equilibrium. The prerequisite of a lag stage before the osmotically dominated stage implicates a contribution of other emulsion components, such as the oil-soluble surfactant to the water transport. Most authors state a supporting role of the oil-soluble surfactant to the water transport by increasing the water permeation rate [11-14]. Others observed an inhibiting effect [15]. Yan et al. [16] and Colinart et al. [17] described a maximum value of the water transport rate at a certain surfactant concentration. Both authors supposed the oil-soluble surfactant to act as a carrier during the water transport. They explained the initial increase of the water transport rate by decreasing interfacial tensions [16] and increasing polarity of the oil phase [17] with increasing concentration of the oilsoluble surfactant. Above a certain concentration the increased viscosity of the oil phase would most likely dominate the interfacial tension effect and the water transport rate decreases. Nevertheless, they emphasized that this is one possible explanation without knowing the exact mechanism. In fact, the potential functionality of the oil-soluble surfactant during the water transport seems to be more complex. It is assumed to act as hydrated carrier molecule, to form inverse carrier micelles, or to stabilize transient membrane pores [18]. Thus, it contributes to structure formation mechanisms in the oil phase. Our investigations on the swelling kinetics in single dropin-drop emulsions provided evidence that the oil-soluble surfactant contributes to the water transport by stabilizing spontaneously formed tiny water droplets in the oil [9]. It has been shown that such spontaneous W/O emulsification accelerates the structural changes of the oil layer during the lag stage. Spontaneous emulsification in oil has already been observed for a variety of different oil-soluble surfactants [19-22] or amphiphilic block-copolymers [23], but without an indication either of the importance or consequences of the phenomenon. A detailed overview of the non-equilibrium phenomena leading to spontaneous emulsification is given by LopezMontilla et al. [24]. Widely suggested mechanisms of spontaneous emulsification are i) an interfacial growth caused by notional negative interfacial tensions [25-30], ii) interfacial turbulences [30,31] and interfacial tension fluctuations due to local concentration or temperature gradients, iii) diffusion and stranding [24,30-32] of a cosolvent, which are all based on non-equilibrium effects, or iv) simply an impurity effect [23]. Mechanisms ii) and iii) are often related to a sudden change of the spontaneous surfactant curvature resulting in an entrapment of water in reverse micelles [33], which turn into spherical droplets above a certain degree of swelling. [34] However, published studies contain neither enough of qualitative information nor a quantitative description of the similar phenomena observed for double-emulsions.[19-21]

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Figure 1. Detection of PGPR induced spontaneous W/O emulsification (a) above a planar O/W interface with (W) the bulk water phase, and (b) in oil droplets (O) surrounded by a continuous water phase (W).

For example, the dependency of the spontaneous emulsification rate or related droplet size on the surfactant concentration is generally not predicted or quantified. It is also not known if and how the phenomenon of spontaneous W/O emulsification is coupled to other driving forces, such as the osmotic pressure gradient between both water phases. Moreover, lack of quantitative description of the spontaneous water uptake prevented quantification of the osmotic contribution to the swelling rate and of the lag phase duration preceding the swelling. In this study the influence of spontaneous W/O emulsification induced by the oil-soluble surfactant polyglycerol polyricinoleate (PGPR, average Mw of around 2000 g/mol) is quantified as a function of time and concentration. PGPR can be considered as a block copolymer with low polymerization degree and thus might be prone to self-assembly [35,36]. It has to be noted that the purpose of the study does not include investigations of very rapid initial steps of the spontaneous structural organization at molecular level leading to W/O emulsification. PGPR was chosen as oil-soluble surfactant as it has shown to be one of the most powerful surfactants to stabilize W/O emulsions [37-39]. Thus, PGPR is extensively used in food industry [2,40,41]. Nevertheless, it is of synthetic origin and has a low limit of acceptable daily intake [40,41]. Hence, its concentration in food products should be reduced when possible, e.g. by replacement by natural substitutes. However, substitutes can only be found when the complex functionality of PGPR in W1/O/W2 emulsions is clarified. Up to now, knowledge about the interfacial behavior of PGPR, and more general of any food-grade surfactant stabilizing W/O interfaces and of its interaction with other additives is only limited [2,42,43]. Our study combines investigations of the interfacial stabilization by PGPR in combination with water-soluble surfactants and the spontaneous water entrapment in the oil phase. It aims at quantifying the kinetics of spontaneous emulsification. Based on this, the task of PGPR during the different stages of water transport is clarified dependent on the concentration regime.

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Figure 2. Microscopy images of PGPR induced spontaneous W/O emulsification above a planar W/O interface with (a) 0.5 %, (b) 1 %, (c) 1.5 %, and (d) 4 % PGPR in MCT after 90 (scale bar = 100 ).

EXPERIMENTAL SECTION Microscopy monitoring of spontaneous W/O emulsification. Spontaneous W/O emulsification was monitored in two different systems illustrated in Figure 1. First, in a specifically designed microscopy observation chamber [44] MilliQ water was covered by an oil layer (Figure 1a), which contained medium chain triglyceride (MCT Delios V from Impag AG, Switzerland) and various concentrations of polyglycerol polyricinoleate (Grinsted PGPR 90 Kosher from Danisco, Switzerland). The droplet formation in the oil phase directly above the O/W interface was monitored using microscopy at a certain focus level using a Diaphot-TMD inverted microscope and a DS-Fi1 high-definition colour camera head (Nikon Corporation, Japan). Images were taken automatically every 10 for 120 using the software NISElements D3.0. The droplet number in a defined image area was determined in quintuplicate. The maximum detectable droplet number was 50 10 / . Above this a distinction between single droplets was no longer possible. The volume-weighted median droplet diameter , was detected in triplicate using the in-house software Bubble Detect. It has to be noted, that droplets with a diameter below 2 were too small to be detected. Second, O/W emulsions were prepared using a rotorstator device (Polytron PT6000, Kinematica AG, Switzerland) with the dispersing aggregate PT-DA 20/EC. The oil phase contained pure MCT or MCT with 2 % PGPR (Figure 1b). It was slowly added to the water phase, which consisted of MilliQ water and 2 % whey protein isolate (WPI, BiPRO from Davisco) at 4000 for 4 . Then the rotational speed was increased to 10000 for 20 . Microscopy images of the emulsions were taken directly after the emulsification step. Pendant drop tensiometry. The interfacial tension of an oil droplet surrounded by a continuous water phase was measured by pendant drop tensiometry as described by Gunde et al. [45]. The oil droplet contained MCT with varied concentration of PGPR. The water phase

contained MilliQ water with either polyethylene glycol sorbitanmonolaurate (Tween 20 from Sigma Aldrich, Switzerland), whey protein isolate (WPI, BiPRO from Davisco), or casein sodium salt from bovine milk (NaCas from Sigma Aldrich, Switzerland). The oil droplet was hanging at the tip of an upwards capillary with a radius of 0.737 . The capillary was connected to a 2.5 gastight syringe (Hamilton, Switzerland) embedded in a thermostat-controlled syringe cell. A glass cuvette, which contained the continuous water phase was inserted into a thermostat-controlled cuvette-cell. The temperature was set to 20°!. A DFW-V 500 camera (Sony, Japan) was used as optical detector, taking an image every ten seconds over 125 . The drop profiles were fitted with an in-house Matlab algorithm. The interfacial tensions were determined based on the change of drop curvature over time. Interfacial shear rheology. The interfacial storage and loss moduli "#$ and "#$$ of a water/oil interface were measured using a Physica MCR 501 rheometer (Anton Paar GmbH, Austria) with a biconical disk BIC 68-5 (radius % = 34.1 , cone angle ' = 5°). The detailed experimental setup is described in Erni et al. [46]. The temperature was set to 20°! with a Peltier element. Time sweep measurements were performed with a strain of ( = 0.1% and a frequency of ) = 1 */. "#$ and "#$$ of a WPI (2 %) /MCT system were measured for 60 before 2 % PGPR was added to the oil phase. Microscopy characterization of swelling kinetics. The microscopy characterization of the swelling kinetics of single drop-in-drop emulsion produced by microfluidics is described elsewhere [9]. A microfluidic glass chip purchased from Dolomite Centre Ltd (30x15x4 mm) with two flow-focusing junctions aligned in series was used for the production of the single drop-in-drop emulsions. The oil phase contained MCT with different concentrations of PGPR. In addition 10 % sucrose acetate isobutyrate (SAIB from Eastman, Switzerland) was added in order to

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Figure 3. Quantitative description of the evolution of number and size of PGPR induced, time dependent spontaneously formed water droplets in the oil phase close to a planar W/MCT interface. (a) Droplet number increase ∆, = ,- . , as a function of time dependent on the PGPR concentration, (b) Rate of droplet number increase ∆,/ as a function of PGPR concentration, (c) Median droplet diameter , as a function of PGPR concentration at different times; (d) Total water uptake expressed by total droplet volume /-0- as a function of PGPR concentration at different times (lines are drawn to guide the eye).

increase the oil density to 0.960 1/2 . Both aqueous phases contained MilliQ water and tetramethylammonium chloride (TMACl from Fluka, Switzerland). An osmotic pressure difference of ∆Π = 13.1 3* was created between W1 and W2 by adding 0.1 and 3 % TMACl to the outer W2 and the inner W1 phase, respectively. The inner water phase W1 contained additionally 1 % sodium dodecyl sulfate (SDS from Sigma Aldrich, Switzerland). The outer water phase W2 contained additionally 2 % Tween 20. The change of droplet size with time was monitored using microscopy. The diameters of five inner and outer droplets were measured by help of the program NIS-Elements D3.0. The refractive effects derived from the refractive index between the outer water phase W2 and the oil phase O were eliminated by correcting the diameters as described in Bahtz et al. [9].

RESULTS AND DISCUSSION Spontaneous W/O emulsification induced by PGPR. In the presence of PGPR in the oil phase and in contact with water tiny water droplets are spontaneously formed either above a planar W/O interface (Figure 1a) or inside O/W droplets (Figure 1b), i.e., no external energy such as stirring or shaking has to be applied. This phenomenon is depicted in Figure 2, which shows microscopic images of the formation of water droplets in the oil phase above a planar W/O interface after 90 as a function of the PGPR concentration in oil. Without any PGPR in the oil phase, water droplet formation has not been observed, independent of the presence of a water-soluble surfactant in the water phase (not shown in Figure 2). Spontaneous droplet formation has only been observed in the oil, but not in the water phase. With 0.5 % PGPR (Figure 2a) only a few water droplets were formed after 90 . Higher PGPR concentrations resulted in a strong increase of the droplet number. At 4 % PGPR

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the W/O emulsion droplets were densely packed after the same time elapsed: the microscopic image appears very dark (Figure 2d). Thus, the results indicate in contrast to the study by Davies & Haydon [47] that spontaneous emulsification strongly depends on the surfactant concentration, which is in agreement with Wen & Papadopoulos [14, 20] and Yan & Pal [16]. In addition, the droplet size increased with an increasing PGPR concentration up to 1 . 1.5 % (Figure 2b). Figure 3a shows the increase of the W/O emulsion droplet number ∆, = ,- . , as a function of time , with ,and , the droplet number at time and time = 0, respectively. The droplet number increase ∆, was larger for an increased concentration of PGPR and correlates linearly with time . In general, the water droplets may originate from the water bulk phase (Figure 1a; i) from water droplets which were formed before (Figure 1a; ii), or both. Droplet generation from the water bulk phase would result in a linear increase of the droplet number with time. While generation mainly from already present droplets would not change the total volume of W1 drops: it would result in an increase of the droplet number faster than linear, since the surface area created would increase with time. Thus, the linear behavior shown in Figure 3a can have multiple causes, namely (i) a strong dominance of water droplets originating from the water bulk phase, (ii) a reduction of the droplet number due to Ostwald ripening or coalescence, or (iii) the limited detection of very small droplets (see above). The slopes of the linear fits, denoting the rates of the droplet number increase ∆,/ are shown in Figure 3b as a function of the PGPR concentration. The curve fit demonstrates an exponential dependency for the investigated concentration range. Comparable to the droplet number also the median diameter of the spontaneously formed W/O emulsion droplets , depend on concentration and time (Figure 3c). In general, droplet size increases with time either due to droplet coalescence, Ostwald ripening, and/or swelling due to ongoing water uptake from the water bulk phase. However, Figure 3c shows not only a strong dependency of the median droplet size on time but also on the PGPR concentration. The PGPR concentration had an increasing effect on , up to a critical value of approximately 1.5 % . Above this concentration, which is close to the critical micellar concentration (cmc, see below) the droplet diameter decreased with increasing PGPR concentration. In contrast to this, the emulsion stability against coalescence and Ostwald ripening is expected to increase strongly with an increasing PGPR concentration up to the cmc and slightly for concentrations above the cmc. Moreover, our experiments showed on average a continuous growth of the spontaneously formed droplets with time (Figure 3c) and thus a continuous increase of the total interfacial area. Thus, we exclude dropletdroplet coalescence and Ostwald ripening to be the only reasons for the observed droplet growth. The amount of water entrapped by spontaneous emulsification results from the product of droplet number , and single droplet volume /, defined as total droplet volume

/-0- . Figure 3d shows the total droplet volume /-0- as a function of time. At = 10 no significant difference of /-0- between different PGPR concentrations was detected. With time more water was taken up and /-0- increased significantly, which excludes again Ostwald ripening and coalescence being the main mechanisms of spontaneous emulsification. This trend was observed up to a concentration of 1.5 % PGPR. Above 1.5 % of PGPR the total droplet volume /-0- did no longer change significantly. Discussion on the mechanism of spontaneous W/O emulsification. The spontaneous formation of new droplets and the average droplet size increase over time results in a strong increase of the total interfacial area. On the first glance, this seems to contradict thermodynamic principles. It has to be assumed that the formation of tiny water droplets releases more energy than required for the increase of the interfacial area. Thus, ∆"-0- = ∆"4 + ∆"6

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follows, with "-0- the total Gibb’s free energy, "4 the interfacial free energy including the spontaneous curvature of PGPR stabilized W/O interfaces and "6 the free energy of other contributions related to the composition and structure in the bulk phases including e.g. PGPR in the bulk and the salt concentration in water. As shown in the following paragraph, in all of our emulsion systems the measured interfacial tension values were very low, but positive. Thus, ∆"4 must possess a positive value for an increasing total interface. Moreover, at 2 % Tween 20 in the water phase (Figure 4b: no PGPR in the oil phase) a comparable interfacial tension to systems with high PGPR concentrations in the oil phase was reached (Figure 4a). However, neither spontaneous W/O nor O/W emulsification was observed without PGPR. Thus, in our systems the reason for spontaneous emulsification cannot be a pure interfacial tension effect. Moreover, neither of the additional mechanisms cited in literature, i.e. interfacial turbulences [24], ‘diffusion and stranding’ [24,30], or impurities are believed to be the cause of the continuous surface area creation in our system, as we did not observe convective motion, did not add ‘cosolvent’ molecules and obtained a very high reproducibility under various conditions. We assume that there are two other potential mechanisms of spontaneous water droplet formation in the oil phase: i) Water diffuses into the oil phase until the saturated concentration is reached, form clusters, which nucleate to tiny droplets. ii) The droplet formation occurs directly at the interface by entrapment of water from the bulk phase into PGPR micelles. As the solubility of water in oil and thus the saturated concentration are very low [9], the contribution of mechanism (i) is expected to be limited. In both assumed mechanisms the spontaneously formed water droplets are stabilized by PGPR molecules. Wen & Papadopoulos [20] showed already that the water transport rate in osmotically imbalanced

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W1/O/W2 emulsions is controlled by the rates of interfacial processes (under their experimental conditions), however without further specifications. We assume spontaneous W/O emulsification to be a consequence of the spontaneous curvature of PGPR stabilized W/O interfaces due to the structural properties of the PGPR molecules after the adsorption to the interface. PGPR molecules are not fully symmetric and rather large compared to conventional surfactant molecule. Ziegler [48] described the interfacial adsorption of PGPR as follows: in contrast to many other surfactants, the polar hydroxyl and carbonyl groups of PGPR do not form a polar “head”. They are in fact distributed throughout the branched surfactant molecule. As a result, PGPR molecules have a high affinity to water and adsorb almost evenly at an interface. Consequently, we assume that PGPR molecules prefer slightly negatively curved interfaces, i.e., of the spontaneously formed W/O emulsion droplets compared to a planar O/W interface or strongly curved pure micellar interfaces. In contact with an O/W interface, the PGPR molecules will tend to their optimal curvature, an effect which depends on the PGPR concentration, the available amount of water and O/W interface. The optimal droplet size distribution will arise with time. We assume these structural changes make the main contribution to ∆"4 . Thus, they have to be thermodynamically favorable. For concentrations below the cmc (c < cmc) the droplet size increased with increasing PGPR concentration (Figure 3c). In this concentration regime all surfactant molecules have access to the initial O/W interface. Thus, they are rapidly in contact with water and tiny water droplets started to be formed. With an increasing concentration of PGPR, more surfactant molecules were available and the kinetics of droplet formation was increased. The spontaneously formed droplets grew in number and size. Consequently, the total amount of entrapped water /-0was increased. At high PGPR concentrations (c > cmc) an excessive amount of PGPR molecules is present in the oil phase, forming micellar structures and thus increasing the oil phase viscosity. Due to the limited accessibility of the O/W interface to the excessive PGPR molecules and the increased viscosity the kinetics of droplet formation and thus the amount of entrapped water are restricted. In contrast to a few large water droplets, a high number of small droplets strongly increased the total W/O interface (at a constant volume of entrapped water) and thus enable most PGPR molecules to be in contact with water, even in the case of a limited amount of entrapped water in the oil. It has to be noted, that the above assumption still requires an experimental proof of the nano-scale mechanism and the corresponding driving force. In order to show that the effect of PGPR on the spontaneous W/O structure formation (demonstrated in Figure 2,3) was not limited to O/W systems with a planar interface, it was also detected in oil droplets surrounded by a continuous water phase. Figure 4 shows microscopy images of simple O/W emulsions prepared by using a

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Figure 4. Microscopy images of O/W emulsions stabilized by (a) 2 wt% WPI in the water phase and (b) 2 wt% WPI in the water phase and 2 wt% PGPR in the oil phase. The emulsions were prepared using a Polytron PT 6000. Structure formation in the oil droplets corresponds to water entrapment. (scale bar = 50 )

rotor-stator device. The emulsions were stabilized by WPI with or without PGPR in the oil phase. The droplets without PGPR appeared clear (Figure 4a). An internal W/O-structure formation occurred only in the presence of PGPR (Figure 4b). Thus, spontaneous emulsification is not limited to planar interfaces, but occurs also in O/W droplets (curved interface). This qualitatively agrees with previous observations [14,20]. Interfacial composition. As demonstrated above, the PGPR dependent spontaneous emulsification requires access of the PGPR molecules to the W/O interface. Thus, transferring the conclusions of spontaneous emulsification above one W/O interface to multiple W1/O/W2 emulsions needs first to consider the accessibility and composition of both interfaces, the outer O/W2 and the inner W1/O interface. In W1/O/W2 emulsions watersoluble surfactants, e.g. low molecular weight Tween 20, or proteins, such as NaCas or WPI are added to the outer water phase W2 to stabilize the outer O/W2 interface. Compared to this, the oil-soluble surfactant PGPR is primarily added to the oil phase to stabilize the inner W1/O interface. Nevertheless, PGPR has a very high interfacial activity, which is demonstrated in the following. Figure 5a shows the interfacial tension of (MCT+ PGPR)/W systems versus time (no water-soluble surfactant in the W-phase). A PGPR concentration of 2 = 0.001 % had no effect on the interfacial tension. The measured values were comparable to pure MCT/W system, which had an interfacial tension of ( = 21.0 ,/. Increasing the PGPR concentration resulted in a fast adsorption to the interface and a rapid decrease of the interfacial tension. The cmc of PGPR was measured to be 1.8 %. Figure 5b shows the interfacial tensions under presence of water-soluble surfactants in the W-phase, i.e., (MCT+ PGPR)/(W+2 % water-soluble surfactant).

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Figure 5. Interfacial properties resulting from the competitive adsorption of PGPR and water-soluble surfactants at the O/W interface. (a) Interfacial tension of an MCT/W system as a function of time with a varying concentration of PGPR in MCT. (b) Interfacial tension as a function of PGPR concentration in MCT without any water-soluble surfactant or in combination with NaCas, WPI, and Tween 20 in water. (c) Influence of PGPR addition to MCT on the interfacial loss and storage moduli of an established WPI network. Both surfactants were used in a concentration of 2 % in oil and water, respectively. The measurements were performed at 20°!.

The data show the interfacial tension values reached after 100 min as a function of the PGPR concentration. Without PGPR (0 %) all water-soluble surfactants, i.e.,

Tween 20, WPI, NaCas were less effective in reducing the interfacial tension compared to 2 % PGPR. However, the interfacial tensions decreased with increasing PGPR concentrations, already at very low concentrations of PGPR and independent of the type of watersoluble surfactant. The interfacial tension values of the combined surfactant systems (PGPR + water-soluble surfactant) were lower than using only PGPR (Figure 5a,b black symbols). In combination of 2 % PGPR with 2 % Tween 20 the interfacial tension was even reduced to 9 = 1 ,/. This indicates that in multiple W1/O/W2 emulsions both surfactant types, the oil and the water-soluble surfactant have access to the outer O/W2 interface and adsorb simultaneously, especially at high PGPR concentrations. In the case that PGPR and the water-soluble surfactant do not adsorb simultaneously, but sequentially to the O/W2 interface (Figure 5b), PGPR can displace already adsorbed water-soluble surfactants from the interface. Such impact of PGPR is evidenced more strongly by measuring shear interfacial viscoelastic properties of a WPI network at the MCT/W interface while adding PGPR to the oil phase. WPI was chosen for these experiments since low molecular weight surfactants such as Tween 20 do not form networks [49], and the viscoelasticity of NaCas covered interfaces is considerably smaller than those of WPI [50]. Figure 5c illustrates the interfacial storage "#$ and loss moduli "#$$ of the WPI covered MCT/W interface versus time, initially without PGPR in the oil phase. As the aqueous solution had a pH of 7.2, the WPI molecules were negatively charged (isoelectric point: 4.8). During the time sweep WPI was allowed to adsorb to the interface and form a network, before PGPR was added to the MCT phase after 60 . After a lag time of approximately 15 "#$ and "#$$ decreased rapidly below the detection limit. If both surfactants, PGPR and WPI were added to the MCT and W-phase right from the beginning no viscoelastic response was detected. Consequently, PGPR interferes with the WPI network, leading to a loss of the protein film integrity. The complete loss of elasticity of a previously formed interface by PGPR indicates a segregated interfacial structure [51]. Further studies using Brewster angle microscopy or large amplitude oscillatory dilatation measurements could provide evidence for this. The observed effect indicates, without being experimentally proven, that PGPR displaced WPI at least partially from the interface, but confirms the presence of PGPR at the outer O/W2 interface shown by the interfacial tension measurements (Figure 5b). The phenomenon is not limited to WPI networks. So Guelseren & Corredig [52] showed that in combination with NaCas and β-lactoglobulin the interfacial tension of a PGPR stabilized W/O interface is lowered. In summary, in multiple emulsions PGPR does not only stabilize the inner W1/O interface. It has also a strong contribution to the composition of the outer O/W2 interface. Thus, it can be concluded that in presence of PGPR in the oil phase in W1/O/W2 emulsions water

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The size increase of the droplets was monitored using microscopy, resulting in characteristic two-stage kinetics curves [9] (Figure 6a), including a lag stage (I), and an osmotically dominated stage (II). Figure 6b shows the lag time :;< , the water migration rate in the lag stage, and the swelling rate in the osmotically dominated stage as a function of the PGPR concentration. Based on the above results the mass balance equation can be written as: = = ># ?@#,#A . @#,0B- C + >0 ?@0,#A . @0,0B- C

Figure 6. Influence of PGPR on swelling kinetics of single drop-in-drop W1/O/W2 emulsions. (a) Exemplary two-stage swelling kinetics with I the lag stage and II the osmotically dominated stage. b) Lag time, normalized migration rate (both lag stage), and normalized swelling rate (osmotically dominated stage) as a function of PGPR concentration. The oil phase contained MCT, PGPR and 10 % SAIB. The W1 phase contained 1 % SDS and 3 % TMACl. The W2 phase contained 2 % Tween 20 and 0.1 % TMACl.

droplets are spontaneously formed originating from both water phases, W1 and W2. This conclusion has to be considered while interpreting the swelling mechanism of osmotically imbalanced W1/O/W2 emulsions. Swelling kinetics of single drop-in drop W1/O/W2 emulsions. In general, in osmotically imbalanced W1/O/W2 emulsions water migrates across the oil layer in direction of the osmotic pressure gradient. In our prior study we already provided evidence of the existence of a lag stage, during which structure formation in the oil phase occurs. The structure formation appeared to be supported by spontaneous W/O emulsification. Moreover, the lag stage has shown to be a prerequisite to osmotically driven swelling [9]. In the second part of this study the influence of the PGPR concentration and thus the spontaneous emulsification on the swelling kinetics of microfluidics processed single drop-in-drop emulsions was investigated.

(2)

with = the total mass flow rate of water into the oil phase, ># and >0 the area of the inner and outer interface, respectively, @#A a mass transport factor describing the water uptake into the oil, and @0B- a mass transport factor describing the transport of water from the oil phase back to either water phase W1 or W2. The transport of water into the oil phase is determined by the rate of spontaneous W/O emulsification, which depends on the composition of the water phases, e.g. the salt concentration [53]. In contrast to the water transport into the oil, the transport of water from the oil phase back to the aqueous phases is mainly caused by coalescence of the spontaneously formed water droplets with either the W1 or the W2 phase, and/or Ostwald ripening. It is reasonable and widely accepted that the back-transfer of water and solutes entrapped in reverse micelles and spontaneously formed droplets is governed by an interfacial process and by coalescence at the oil-water interface [54]. For both, the water uptake and the release we assume the contribution of diffusion of single water molecules to be only small. In the case where i) the compositions of the inner and the outer water phases are identical, and ii) the curvature difference between the inner water droplet W1 and the oil droplet O is only marginal (which is true for the very large droplet sizes in our experiments, @#,#A = @0,#A and @#,0B- = @0,0B- ) eq 1 becomes = = D># + >0 ) D@#A . @0B- )

(3)

Eq. 3 neglects droplets generated from already formed droplets. Consequently, the total water migration rate in the lag stage was calculated by = + |F | =

∆DGH /4H ) I |∆DGJ /4J )| -

(4)

with F, the water migration rate from direction of the inner and the outer aqueous phase, and /0,# and >0,# the volume and the surface area of the outer and inner droplet, respectively. The swelling rate correlates to the linear slopes of the initial part of the swelling curve of the inner droplet in the osmotically dominated stage. Thus, is defined as:

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=

Langmuir

∆DGJ /4J ) -

(5)

In the lag stage the water migration rate (Figure 6b, grey symbols) increased up to a critical concentration of 1 % PGPR (dotted line). Above this concentration the water migration rate decreased slightly. Moreover, the curve implies that in the lag stage there is no net flux of water migration ( = 0 /) into the oil without PGPR in the oil phase. Two conclusions can be drawn from this observation: 1) PGPR is required for the spontaneous formation of W/O emulsion droplets as part of the water transport structure permitting the swelling; 2) Spontaneous emulsification, and not osmotically driven transport, is the major mechanism of water transport in the lag stage of the swelling. Comparable to the migration rate , also the spontaneous emulsification increased with PGPR up to a critical concentration. Corresponding to the water migration rate , the lag time :;< decreased up to 1 % PGPR, confirming the correlation between the kinetics of spontaneous water droplet formation with the formation of the water transport structure during the lag stage, as prerequisite to the osmotically dominated stage. At higher PGPR concentrations the lag time :;< increased, probably caused by an increase in oil phase viscosity, which results in a slight decrease of the migration rate in combination with steric effects: at a high PGPR concentration a very high number of small water droplets are formed in the oil phase (see above). Such concentration increases significantly the effective viscosity of the spontaneously formed W/O emulsion und thus increases the lag time :;< . [9] In the osmotically dominated stage the swelling rate (black symbols) increased strongly up to 1 % PGPR. Consequently, the total swelling process was accelerated in this concentration range. Based on the curve, it can be assumed that without PGPR in the oil phase, the swelling rate would be very small. Such emulsion systems were not investigated, as droplet formation and stabilization without PGPR is not possible. Above 1 % PGPR the swelling rate leveled off. This shows a strong effect of PGPR not only on the structural changes of the oil in the lag stage (permitting the osmotic contact between W1 and W2), but also on the water transport in the osmotically dominated stage. Once both water phases are osmotically connected, an osmotically driven exchange of water between W1 and W2 was possible. The swelling rate was limited by the water migration rate . Thus, it was determined by oil bulk and interfacial structures given by PGPR, which reach a stationary state above the cmc. Comparing the above results to the study of Wen & Papadopoulos [14], who investigated the influence of the Span 80 concentration (from 0.05 to to 0.5 mol/l n-Hexadecane) on the water transport rate through spontaneous emulsification, we find strong similarities in the regime of lower oil-soluble surfactant concentration. They detected a linear increase of the overall water transport rate with the Span 80 concentration, which we find below 1% PGPR. Moreover, they also showed that the spontaneous droplet formation is the

controlling step for the overall water transport rate [53]. This indicates the similarity of our PGPR stabilized emulsions to other surfactant systems, at least for low concentration ranges (< cmc). CONCLUSIONS In the current study the mechanism of spontaneous W/O emulsification induced by PGPR is discussed beyond the existing explanations. The droplet formation rate is quantified and conclusions to the water transport in multiple W/O/W emulsions are drawn. We combined already established properties of the oil-soluble surfactant PGPR in multiple W1/O/W2 emulsions with its complex role during the osmotically dominated water transport. The functionality of PGPR in W1/O/W2 emulsions is far in excess of stabilizing the internal W1/O interface, which is its primary task. It has been shown that PGPR occupies also the outer O/W1 interface and is even able to interact with other additives, such as water-soluble surfactants and to replace them from the interface. Consequently, it has a major contribution to the overall interfacial stabilization during production, storage, and droplet swelling. In addition, PGPR induces the spontaneous formation of tiny W/O emulsion droplets. The droplet population coarsens with time into a wide distribution of sizes. We provided evidence that the spontaneous water droplet formation cannot be a pure interfacial tension or turbulence effect, as could be assumed for our system from published literature. We discussed the structural properties of PGPR and the correlated spontaneous curvature of the surfactant or surfactant aggregates at the W/O interface to cause spontaneous W/O emulsification. The dependency of the spontaneous emulsification rate and related droplet size on the surfactant concentration has been quantified. It has been shown that the functionality of PGPR to stabilize the W1/O and O/W2 interface, as well as spontaneously formed W/O emulsion droplets highly impacted the different stages of the swelling kinetics in osmotically imbalanced W1/O/W2 emulsions. The quantitative description of the spontaneous water uptake allowed the quantification of the osmotic contribution to the swelling rate, and of the lag phase duration preceding the swelling [9]. Two different concentration regimes of the contribution of PGPR to the water transport were evidenced.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected] [email protected]

Funding Sources The authors gratefully acknowledge the financial support provided by the Nestlé Research Center Lausanne.

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(19) Koroleva, M.Y.; Yurtov, E.V. Water mass transfer in W/O emulsions. J. Colloid Interface Sci. 2006, 297(2), 778-784. (20) Wen, L.X.; Papadopoulos, K.D. Visualization of water transport in W1/O/W2 emulsions. Colloids Surf. A, 2000, 174(1-2), 159-167. (21) Yan, J.; Pal, R. Effects of aqueous-phase acidity and salinity on isotonic swelling of W/O/W emulsion liquid membranes under agitation conditions. J. Membrane Sci. 2004, 244(1-2), 193-203. (22) Aronson, M.P.; Petko, M.F. Highly concentrated waterin-oil emulsions: Influence of electrolyte on their properties and stability. J. Colloid Interface Sci. 1993, 159(1), 134-149. (23) Bae, J.; Russell, T.P.; Hayward, R.C. Osmotically driven formation of double emulsion stabilized amphiphilic block copolymers. Angewandte Chemie Internationale Edition 2014, 53(31), 8240-8245. (24) Lopez-Montilla, J.C.; Herrera-Morales, P.E.; Pandey, S.; Shah, D.O. Spontaneous emulsification: Mechanisms, physicochemical aspects, modeling, and applications. J. Dispersion Sci. Techn. 2001, 23(1-3), 219-268. (25) Okazawa, T.; Bron, J. On thermodynamically stable emulsions. J. Colloid Interface Sci. 1979, 69, 86-96. (26) Schulman, J.H.; Stoeckenius, W.; Prince, L.M. Mechanism of formation and structure of micro emulsions by electron microscopy. J. Phys. Chem. 1959, 63(10), 1677-1680. (27) Shah, D.O.; Tamjeedi, A.; Falco, J.W.; Walker, R.D. Interfacial instability and spontaneous formation of microemulsions. AIChE J. 1972, 18, 1116. (28) Prince, L.M. A theory of aqueous emulsions I. Negative interfacial tension at the oil/water interface. J. Colloid Interface Sci. 1967, 23, 165. (29) Granek, R.; Ball, R.C.; Cates, M.E. Dynamics of spontaneous emulsification. J. Phys. II 1993, 3(6), 829-849. (30) Davies, J.T.; Haydon, D.A., Proc. Int. Congr. Surface active. 2nd 1957, 1, 417. (31) Leal-Calderon, F.; Schmitt, V.; Bibette, J. Emulsion Science: Basic Principles. Springer, 2007. (32) Miller, C.A. Spontaneous emulsification produced by diffusion – A review. Colloids and Surfaces, 1988, 29(1), 89-102. (33) Granek, R. Spontaneous curvature-induced Rayleighlike instability in swollen cylindrical micelles. Langmuir, 1996, 12, 5022-5027. (34) Eastoe, J. Small-angle neutron-scattering from dilute didodecyldimethylammonium bromide water-in-oil microemulsions – Evidence for polymer-like aggregates. Langmuir, 1992, 8, 1503-1506. (35) Zhu, J.; Hayward, R.C. Spontaneous generation of amphiphilic block copolymer micelles with multiple morphologies through interfacial instabilities. J. Am. Chem. Soc., 2008, 130, 7496-7502. (36) Nikova, A.T.; Gordon, V.D.; Cristobal, G.; Talingting, M.R.; Bell, D.C.; Evans, C.; Joanicot, M.; Zasadziniski, J.A.; Weitz, D.A. Swollen vesicles and multiple emulsions from block copolymers. Macromolecules, 2004, 37, 2215-2218. (37) Muschiolik, G. Multiple emulsions for food use. Current Opinion Colloid & Interface Sci. 2007, 12(4-5), 213-220. (38) Garti, N.; Binyamin, H.; Aserin, A. Stabilization of waterin-oil emulsions by submicrocrystalline alpha-form fat particles. J. Am. Oil Chem. Soc. 1998, 75(12), 18251831. (39) Surh, J.; Vladisavljevic, G.T.; Mun, S.; McClements, D.J. Preparation and characterization of water/oil and water/oil/water emulsions containing biopolymer-gelled wa-

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(40)

(41) (42)

(43)

(44)

(45)

(46)

(47) (48)

(49) (50)

(51) (52)

Langmuir ter droplets. J. Agricultural Food Chem. 2007, 55(1), 175-184. Wilson, R.; van Schie, B.J.; Howes, D. Overview of the preparation, use and biological studies on polyglycerol polyricinoleate (PGPR). Food Chem. Toxicol. 1998, 36(9-10), 711-718. Dickinson, E. Double emulsions stabilized by food biopolymers. Food Biophysics, 2011, 6(1), 1-11. Dedinaite, A.; Campbell, B. Interactions between mica surfaces across triglyceride solution containing phospholipid and polyglycerol polyricinoleate. Langmuir, 2000, 16, 2248-2253. Middendorf, D.; Juadjur, A.; Bindrich, U.; Mischnick, P. AFM approach to study the function of PGPR’s emulsifying properties in cocoa butter based suspensions. Food Structure, 2015, 4, 16-26. Rühs, P.A.; Böcker, L.; Inglis, R.F.; Fischer, P. Studying bacterial hydrophobicity and biofilm formation at liquidliquid interfaces through interfacial rheology and pendent drop tensiometry. Colloids Surfaces B: Biointerfaces. 2014, 117, 174-184. Gunde, R.; Kumar, A.; Lehnert-Batar, S.; Mäder, R.; Windhab, E.J. Measurement of the surface and interfacial tension from maximum volume of a pendant drop. J. Colloid Interface Sci. 2001, 1, 113-122. Erni, P.; Fischer, P.; Windhab, E.J.; Kusnezov, V.; Stettin, H.; Läuger, J. Stress- and strain-controlled measurements of interfacial shear viscosity and viscoelasticity at liquid/liquid and gas/liquid interfaces. Rev. Sci. Instruments, 2003, 74(11), 4916-4924. Davies, J.T.; Rideal, E.K. Interfacial phenomena, Academic Press, New York, N.Y., 1961. Ziegler, G.R.; Garbolino, C.; Coupland, J.N. The influence of surfactants and moisture on the colloidal and rheological properties of model chocolate dispersions. 3rd International Symposium on Food Rheology and Structure, 2003, 335-339. Bos, M.A.; van Vliet, T. Interfacial rheological properties of adsorbed protein layers and surfactants: a review. Adva. Colloid Interface Sci. 2001, 91(3), 437-471. Mezzenga, R.; Fischer, P. The self-assembly, aggregation and phase transitions of food protein systems in one, two and three dimensions. Rep. Prog. Phys. 2013, 76(4), 046601. Patino, J.M.R.; Pilosof. A.M.R. Proteune-polysaccaride interactions at fluid interfaces. Food Hydrocolloids, 2011, 25,1925-1937.. Guelseren, I.; Corredig, M. Interactions at the interface between hydrophobic and hydrophilic emulsifiers: Poly-

glycerol polyricinoleate (PGPR) and milk proteins, studied by drop shape tensiometry. Food Hydrocolloids. 2012, 29, 193-198. (53) Wen, L.; Papadopulos, K.D. Effects of osmotic pressure on water transport in W1/O/W2 emulsions. J. Colloid Interface Sci., 2001, 235, 398-404. (54) Ono, T.; Goto, M. Bioseparation through liquid-liquid interfaces. Interfacial Nanochemistry, 2005, 287-302.

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Table of Content

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Langmuir

Detection of PGPR induced spontaneous W/O emulsifica-tion (a) above a planar O/W interface with (W) the bulk water phase, and (b) in oil droplets (O) surrounded by a continuous water phase (W). 64x36mm (150 x 150 DPI)


Langmuir

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Microscopy images of PGPR induced spontaneous W/O emulsification above a planar W/O interface with (a) 0.5 wt%, (b) 1 wt%, (c) 1.5 wt%, and (d) 4 wt% PGPR in MCT after 90 min (scale bar = 100 µm). 170x41mm (150 x 150 DPI)


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Langmuir

Quantitative description of the evolution of number and size of PGPR induced, time dependent spontaneously formed water droplets in the oil phase close to a planar W/MCT interface. (a) Droplet number increase ∆N= N_t- N_0 as a function of time t dependent on the PGPR concentration, (b) Rate of droplet number increase ∆N/t as a function of PGPR concentration, (c) Median droplet diameter d_50,3 as a function of PGPR concentration at different times; (d) Total water uptake expressed by total droplet volume V_tot as a function of PGPR concentration at different times (lines are drawn to guide the eye). 362x243mm (150 x 150 DPI)


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Microscopy images of O/W emulsions stabilized by (a) 2 wt% WPI in the water phase and (b) 2 wt% WPI in the water phase and 2 wt% PGPR in the oil phase. The emulsions were prepared using a Polytron PT 6000. Structure formation in the oil droplets corresponds to water entrapment. (scale bar = 50 µm) 75x37mm (150 x 150 DPI)


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Langmuir

Interfacial properties resulting from the competitive adsorption of PGPR and water-soluble surfactants at the O/W interface. (a) Interfacial tension of an MCT/W system as a function of time with a varying concentration of PGPR in MCT. (b) Interfacial tension as a function of PGPR concentration in MCT without any water-soluble surfactant or in combination with NaCas, WPI, and Tween 20 in water. (c) Influence of PGPR addition to MCT on the interfacial loss and storage moduli of an established WPI network. Both surfactants were used in a concentration of 2 wt% in oil and water, respectively. The measurements were performed at 20°C. 128x254mm (150 x 150 DPI)


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Influence of PGPR on swelling kinetics of single drop-in-drop W1/O/W2 emulsions. (a) Exemplary two-stage swelling kinetics with I the lag stage and II the osmotically dominated stage. b) Lag time, normalized migration rate (both lag stage), and normalized swelling rate (osmotically dominated stage) as a function of PGPR concentration. The oil phase contained MCT, PGPR and 10 %wt SAIB. The W1 phase contained 1 %wt SDS and 3 %wt TMACl. The W2 phase contained 2 %wt Tween 20 and 0.1 %wt TMACl. 183x247mm (150 x 150 DPI)


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Langmuir

169x87mm (150 x 150 DPI)


O Emulsification and its Impact on

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