Article pubs.acs.org/JAFC
Effect of Salts on Formation and Stability of Vitamin E‑Enriched Miniemulsions Produced by Spontaneous Emulsification Amir Hossein Saberi,† Yuan Fang,§ and David Julian McClements*,† †
Biopolymers and Colloids Laboratory, Department of Food Science, University of MassachusettsAmherst, Amherst, Massachusetts 01003, United States § PepsiCo Global R&D, 100 Stevens Avenue, Valhalla, New York 10595, United States ABSTRACT: Emulsion-based delivery systems are being utilized to incorporate lipophilic bioactive components into various food, personal care, and pharmaceutical products. This study examined the influence of inorganic salts (NaCl and CaCl2) on the formation, stability, and properties of vitamin E-enriched emulsions prepared by spontaneous emulsification. These emulsions were simply formed by titration of a mixture of vitamin E acetate (VE), carrier oil (MCT), and nonionic surfactant (Tween 80) into an aqueous salt solution with continuous stirring. Salt type and concentration (0−1 N NaCl or 0−0.5 N CaCl2) did not have a significant influence on the initial droplet size of the emulsions. On the other hand, the isothermal and thermal stabilities of the emulsions depended strongly on salt levels. The cloud point of the emulsions decreased with increasing salt concentration, which was attributed to accelerated droplet coalescence in the presence of salts. Dilution (2−6 times) of the emulsions with water appreciably improved their thermal stability by increasing their cloud point, which was mainly attributed to the decrease in aqueous phase salt levels. The isothermal storage stability of the emulsions also depended on salt concentration; however, increasing the salt concentration decreased the rate of droplet growth, which was the opposite of its effect on thermal stability. Potential physicochemical mechanisms for these effects are discussed in terms of the influence of salt ions on van der Waals and electrostatic interactions. This study provides important information about the effect of inorganic salts on the formation and stability of vitamin E emulsions suitable for use in food, personal care, and pharmaceutical products. KEYWORDS: emulsions, nanoemulsions, vitamin E, salts, stability, cloud point, spontaneous emulsification
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INTRODUCTION Emulsion-based delivery systems are finding increasing utilization within food, personal care, and pharmaceutical products to encapsulate, protect, and release lipophilic active ingredients. This category of delivery system includes various kinds of colloidal suspension that utilize lipid droplets as building blocks, such as emulsions, solid lipid nanoparticles, multilayer emulsions, multiple emulsions, and filled hydrogel particles.1,2 Emulsions are the simplest type of emulsion-based delivery system consisting of lipid droplets suspended in an aqueous phase, with each lipid droplet being coated by a layer of emulsifier molecules. Nevertheless, the functional performance of emulsions can still be manipulated by altering oil phase composition, emulsifier type, and particle size distribution. For example, reducing the particle size can lead to higher optical clarity, greater stability to gravitational separation and droplet aggregation, and higher oral bioavailability.3−5 Emulsions containing very small droplets (d < 200 nm) are often referred to as nanoemulsions, mini-emulsions, or ultrafine emulsions. In this paper, we will simply refer to them as emulsions for the sake of convenience. Emulsions can be prepared using several approaches, which are usually categorized as either high-energy or low-energy methods.6,7 High-energy methods use mechanical devices to generate intense forces that intermingle and break up the oil and water phases, such as high-pressure homogenizers, microfluidizers, and ultrasonic generators.3,8 Low-energy methods rely on the spontaneous formation of ultrafine droplets within surfactant−oil−water (SOW) mixtures when © 2014 American Chemical Society
specific changes in composition and/or environmental conditions occur.3,8 A number of low-energy methods have been developed to form ultrafine oil droplets including spontaneous emulsification (SE), phase inversion temperature (PIT), phase inversion composition (PIC), and emulsion inversion point (EIP) methods.5,8−10 Among these methods, spontaneous emulsification is one of the easiest and most costeffective to implement because it simply involves metering an oil−surfactant mixture into an aqueous solution with constant agitation at a fixed temperature. Commercial food and beverage products typically contain a variety of ingredients that may influence the formation and stability of emulsions, including sugars, cosolvents, and salts. Salts are incorporated into foods for a number of purposes, for example, to control flavor, water activity, stability, and nutritional profile. Salts have been reported to influence various physicochemical properties of nonionic surfactants, including solubility and cloud point,11−15 critical micelle concentration,16,17 emulsion phase inversion temperature,14,15,18,19 and emulsion stability.11,19,20 Mei et al.19 suggested that salt may facilitate the formation of emulsions using the PIT method. Salts may alter the interlayer spacing of the liquid crystalline phases formed during the intermediate steps of low-energy emulsification, thereby leading to the formation of ultrafine Received: Revised: Accepted: Published: 11246
August 10, 2014 October 22, 2014 October 24, 2014 October 24, 2014 dx.doi.org/10.1021/jf503862u | J. Agric. Food Chem. 2014, 62, 11246−11253
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droplets.21 Furthermore, it has been reported that the storage stability of emulsions stabilized by nonionic surfactants is strongly dependent upon their PIT,22−24 which depends on salt concentration. Many previous studies on the impact of salt on nanoemulsion formation have utilized simple organic solvents or liquids as the oil phase. In this study, we utilized an oil phase that is more representative of those that might be used in the food industry, that is, vitamin E acetate dissolved in mediumchain triglycerides (MCT). Recently, we demonstrated that stable vitamin E-enriched emulsions containing very fine droplets (d < 60 nm) could be produced by spontaneous emulsification.25 We also showed that addition of cosolvents to the aqueous phase of these emulsions had an appreciable influence on their formation and stability.26,27 The purpose of the present study was to investigate the influence of two salts commonly used in industrial applications (NaCl and CaCl2) on the formation and stability of vitamin E emulsions produced using spontaneous emulsification. The knowledge gained from this study will be useful for the successful incorporation of emulsion-based delivery systems into commercial products.
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Statistical Analysis. All experiments were carried out two or three times using two freshly prepared samples, and the results are reported as the calculated mean and standard deviation of these measurements. Differences between group means were determined by analysis of variance (ANOVA) using statistical software (version 12.0; SPSS, Inc., Chicago, IL, USA). Means were subjected to Duncan’s test, and a P value of 1), and changes the optimum curvature so that it favors the formation of water-in-oil emulsions (Figure 1b). The water droplets in the water-in-oil emulsions formed above the PIT are relatively large and therefore scatter light strongly, leading to increased turbidity.3,29 The impact of salt on the turbidity−temperature profiles of the emulsions can be attributed to the fact that salt decreases the PIT of the emulsions. In the absence of salt, the PIT is relatively high, and therefore we observed only the initial droplet coalescence regimen. In the presence of sufficient salt, the PIT moved into the experimental temperature range, and we could observe droplet coalescence, microemulsion formation, and phase inversion. A similar behavior was observed in the turbidity−temperature profiles of vitamin E-enriched emulsions in the absence and presence of glycerol; that is, glycerol reduced the PIT.26 The turbidity versus temperature profiles were used to determine the cloud points of the emulsions so that we could establish the influence of salt type and concentration on their thermal behavior. The cloud point of the emulsions decreased from around 78 to 65 °C when the NaCl concentration was increased from 0 to 1 M and decreased from around 78 to 70 °C when the CaCl2 concentration was increased from 0 to 0.5 N (Figure 1c). The ability of NaCl14,15,20,37 and CaCl214,15,37 to decrease the cloud points of nonionic surfactant has been reported previously. Our results are also in agreement with previous studies that have reported that the cloud point and/or PIT of nonionic surfactants is a linear function of their salt concentration.15,38,39 The decrease in thermal stability (cloud point) of emulsions in the presence of NaCl and CaCl2 can be attributed to the effect of these salts on the solubility and packing characteristics of nonionic surfactants. Specific anionic19,40,41 and cationic12,16,19,40 salt ions may either increase (salting in) or decrease (salting out) the solubility of nonionic surfactants due to their effects on the structural organization of water molecules. Ions can be classified as either “structure-makers” (salting out) or “structure-breakers” (salting in) according to their position in the Hofmeister series.42,43 The presence of mineral ions in an aqueous solution may alter the PIT of a nonionic surfactant by altering the solubility and packing characteristics of the surfactant molecules. In particular, salts may affect the optimum curvature of the surfactants by altering the hydration of their
Figure 2. Influence of dilution on the cloud point of vitamin Eenriched mini-emulsion produced using 10% oil phase (vitamin E/ MCT, 8:2), 10% surfactant phase (Tween 80), and 80% aqueous phase (0.5 M salt). Oil phase concentrations were 10, 5, 3.33, 2.5, and 1.67, and salt concentrations were 0.4, 0.2, 0.13, 0.1, 0.08, and 0.067 N for the emulsion diluted with dilution factors of 1, 2, 3, 4, 5, and 6, respectively.
influence on the dehydration of the hydrophilic headgroups at different temperatures. Emulsion formation was carried out at low (room) temperature, and therefore the surfactant molecules would have been highly hydrated. Presumably, headgroup hydration was still sufficient in the presence of salt to generate a strong steric repulsion between the droplets and to have a high surfactant curvature. However, once the emulsions were heated, the effects of salts became more pronounced because the surfactants were already partially dehydrated, and any further change due to salt addition was sufficient to promote instability. Influence of Dilution on Thermal Stability. Emulsions are diluted when they are incorporated into functional food and beverage products. Our previous studies have shown that emulsion dilution may have an appreciable influence on emulsion thermal stability.26,27,45 We therefore examined the influence of dilution with buffer solution on the thermal stability of vitamin E emulsions initially containing 0.5 N NaCl or CaCl2 in the aqueous phase. Qualitatively, all of the diluted 11249
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emulsions had similar turbidity−temperature profiles as the undiluted emulsions (Figure 1a): upon increasing the temperature from 20 to 95 °C, the turbidity initially remained low, then steeply increased, then sharply decreased to a minimum, then steeply increased again, and then remained high (data not shown). The cloud points determined from these profiles increased from 73 to 81 °C for NaCl and from 70 to 81 °C CaCl2 as the emulsion dilution factor (DF) increased from 1 to 6 (Figure 2). As these samples were diluted with buffer solution, the observed results may have been due to a decrease in salt concentration in the aqueous phase or due to a decrease in oil droplet concentration. We therefore carried out additional experiments in which the emulsions were diluted with various salt solutions so as to obtain samples where (i) the droplet concentration was kept constant, but the salt concentration varied (Figure 3) or (ii) the
Figure 4. (a) Influence of oil phase concentration on the cloud point of diluted vitamin E-enriched mini-emulsions containing 0.4 N salts. The initial emulsion was produced using 10% oil phase (vitamin E/ MCT, 8:2), 10% surfactant phase (Tween 80), and 0.4 N NaCl. (b) Influence of temperature on the turbidity of vitamin E-enriched miniemulsions containing 0.4 N CaCl2 and different oil phase concentrations. The initial emulsion was produced using 10% oil phase (vitamin E/MCT, 8/2), 10% surfactant phase (Tween 80), and 0.4 N CaCl2.
Figure 3. Influence of salt concentration on the cloud point of vitamin E-enriched mini-emulsions containing 5% oil phase. The initial emulsion was produced using 10% oil phase (vitamin E/MCT, 8:2), 10% surfactant phase (Tween 80), and 0.4 N salt.
droplet concentration varied, but the salt concentration was kept constant (Figure 4). In the diluted emulsions with fixed droplet concentration, the cloud point increased from around 73 to 77 °C for NaCl and from around 72 to 77 °C for CaCl2 as the salt content decreased from 0.4 to 0.2 N, respectively (Figure 3). These results suggest that at least part of the increase in cloud point observed upon emulsion dilution (Figure 2) was related to a reduction in salt concentration in the aqueous phase. In the diluted emulsions with fixed salt concentration, the cloud point initially increased and then decreased as the droplet content was reduced from 10 to 1% (Figure 4a). This effect may have been an artifact of the cloud point measurement at high droplet concentration. As the droplet concentration increased, there was an increase in the overall turbidity of the samples due to an increase in light scattering by the droplets (Figure 4b). In the emulsions with the highest droplet concentrations, the turbidity was so great (>3 cm−1) that it was not possible to observe the entire region where rapid droplet coalescence occurred. Consequently, it was not possible to accurately measure the cloud points for these samples. In the more dilute emulsions (≤3% oil), the cloud point decreased with increasing dilution (decreasing droplet concentration), which may have been due to changes in the nature of the microemulsion phase formed around the PIT when the SOW ratio was changed. Taken together, these results
suggest that the increase in cloud point observed when saltcontaining emulsions were diluted with buffer solution was primarily due to the reduced salt concentration in the aqueous phase, rather than the reduced droplet concentration. Influence of Salts and Dilution on Isothermal Stability. Physical stability of emulsion-based delivery systems during storage, transport, and utilization is required for most commercial applications. Emulsions may become unstable through a number of different physicochemical processes, including flocculation, coalescence, Ostwald ripening, and gravitational separation.10,33 The mechanism and rate of breakdown depend on emulsion composition and microstructure, as well as the environmental conditions experienced during its lifetime. We therefore examined the influence of salt addition and dilution on the storage stability of vitamin E emulsions. A series of emulsions was fabricated with different droplet concentrations (2.5 or 10 wt %) and salt concentrations (0, 0.2, and 0.4 N NaCl or CaCl2) by diluting an initial emulsion with different types and amounts of salt solution. After preparation, the resulting emulsions were stored for 1 11250
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decreased their thermal stability by reducing the cloud point (Figure 2). This independence of thermal and isothermal stability of vitamin E emulsions has also been reported upon posthomogenization addition of cosurfactants.46 There are a number of possible mechanisms that might be responsible for the observed instability of the emulsions during storage: flocculation, coalescence, and Ostwald ripening.4,33 We do not believe that flocculation played an important role in emulsion instability as we did not observe any flocs using an optical microscope. We therefore postulated that the observed instability was due to either coalescence or Oswald ripening. It is often difficult to distinguish between these two mechanisms because they both lead to a net increase in mean droplet diameter over time.33,47 An increase in droplet growth rate with increasing temperature could be attributed to the effects of temperature on coalescence and/or Ostwald ripening. Increasing temperature can dehydrate the surfactant headgroups and thereby increase droplet coalescence by (i) decreasing the hydration repulsion between droplets and (ii) by changing the optimum curvature of the surfactant monolayer toward unity.3,31,48 A temperature rise may also increase the rate of Ostwald ripening by increasing the diffusion rate or solubility of the oil molecules in the aqueous phase.49 Inorganic salts might influence the isothermal storage stability of oil-in-water emulsions stabilized by nonionic surfactant through a number of mechanisms: Effect of Salts on Surfactant Headgroup Dehydration. Previous studies have reported that salts that decrease the cloud point of emulsions stabilized by nonionic surfactant also increase droplet coalescence.11,20 This effect was attributed to the ability of the salts to promote dehydration of the surfactant headgroup, thereby reducing the hydration repulsion between droplets and altering the surfactant monolayer curvature. However, the fact that we actually observed a decrease in droplet growth during storage in the presence of NaCl and CaCl2 suggested that this mechanism was not prominent under the conditions used. Effect of Salts on van der Waals Interactions. van der Waals interactions are attractive forces that almost always act between oil droplets suspended in water and, therefore, promote droplet aggregation. The addition of salts to an aqueous solution appreciably decreases the magnitude of the van der Waals interaction due to an electrostatic screening effect.33 This mechanism may therefore account for the increased storage stability of the emulsions in the presence of salt, because there would have been a reduction in the attractive forces acting between the oil droplets. It should be noted that salt can also decrease the electrostatic interaction between the droplets through electrostatic screening, thereby increasing droplet coalescence. Effect of Salts on Oil Solubility. One of the major factors influencing the rate of Ostwald ripening in oil-in-water emulsions is the solubility of the oil phase in the aqueous phase.33 The addition of salts to the aqueous phase changes the structural organization of the water molecules, which will alter the equilibrium solubility of oil molecules. Salt addition causes an increase in interfacial tension (Table 1), which would increase the free energy associated with introducing a nonpolar molecule into water, thereby decreasing its water solubility. Consequently, addition of salt to the emulsions may have reduced the rate of Ostwald ripening, which is consistent with the reduction in droplet growth rate observed in the presence of salt observed in this study.
month at three storage temperatures representing refrigeration conditions (5 °C), ambient storage in mild climates (20 °C), and ambient storage in hot climates (37 °C). The initial emulsion formed by spontaneous emulsification contained 0% salt and 10% oil and was therefore designated “0/ 10”. The extent of droplet growth in this emulsion after 1 month of storage was appreciable and increased with increasing storage temperature: ∼15% at 5 °C, 19% at 20 °C, and 54% at 37 °C (Figure 5). The droplet size distribution of these
Figure 5. (a) Influence of storage time and temperature on the particle growth (%) of vitamin E-enriched mini-emulsions containing different ratios of NaCl (N)/oil phase. The initial emulsions were produced using 10% oil phase (vitamin E/MCT, 8:2), 10% surfactant phase (Tween 80), and different NaCl concentrations (0, 0.4, and 0.8 N). Means within different letters and the same number of stars are significantly (P < 0.05) different. (b) Influence of storage time and temperature on the particle growth (%) of vitamin E-enriched miniemulsions containing different ratios of CaCl2 (M)/oil phase. The initial emulsions were produced using 10% oil phase (vitamin E/MCT, 8:2), 10% surfactant phase (Tween 80), and different CaCl2 concentrations (0, 0.2, and 0.4 N). Means within different letters and the same number of stars are significantly (P < 0.05) different.
emulsions remained monomodal throughout storage at all temperatures (data not shown). The stability of the emulsions to droplet growth improved significantly (P < 0.05) as the level of NaCl or CaCl2 added was increased, especially at the elevated temperatures (Figure 5). In addition, reducing the droplet concentration from 10 to 2.5% also led to a significant (P < 0.05) inhibition of droplet growth for emulsions containing both types of salt (Figure 5). Overall, these results suggest that the addition of NaCl or CaCl2 improved the isothermal storage stability of the emulsions, even though it 11251
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(3) McClements, D. J.; Rao, J. Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity. Crit. Rev. Food Sci. Nutr. 2011, 51, 285−330. (4) Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C. Formation and stability of nano-emulsions. Adv. Colloid Interface Sci. 2004, 108−109, 303−318. (5) McClements, D. J. Nanoemulsion-based oral delivery systems for lipophilic bioactive components: nutraceuticals and pharmaceuticals. Ther. Delivery 2013, 4, 841−57. (6) Anton, N.; Benoit, J. P.; Saulnier, P. Design and production of nanoparticles formulated from nano-emulsion templates − a review. J. Controlled Release 2008, 128, 185−199. (7) Solans, C.; Esquena, J.; Forgiarini, A. M.; Uson, N.; Morales, D.; Izquierdo, P.; Azemar, N.; Garcia-Celma, M. J. Nano-emulsions: formation, properties, and applications. In Adsorption and Aggregation of Surfactants in Solution; Mittal, K. L., Shah, D. O., Eds.; CRC Press: Boca Raton, FL, USA, 2003; Vol. 109, pp 525−554. (8) Solans, C.; Solé, I. Nano-emulsions: formation by low-energy methods. Curr. Opin. Colloid Interface Sci. 2012, 17, 246−254. (9) Anton, N.; Benoit, J.-P.; Saulnier, P. Design and production of nanoparticles formulated from nano-emulsion templates − a review. J. Controlled Release 2008, 128, 185−199. (10) McClements, D. J. Edible nanoemulsions: fabrication, properties, and functional performance. Soft Matter 2011, 7, 2297−2316. (11) Schott, H.; Royce, A. E. Effect of inorganic additives on solutions of non-ionic surfactants V: emulsion stability. J. Pharm. Sci. 1983, 72, 1427−1436. (12) Schott, H. Salting in of nonionic surfactants by complexation with inorganic salts. J. Colloid Interface Sci. 1973, 43, 150−155. (13) Schott, H.; Han, S. K. Effect of inorganic additives on solutions of nonionic surfactants. 2. J. Pharm. Sci. 1975, 64, 658−664. (14) Shinoda, K.; Takeda, H. The effect of added salts in water on the hydrophile-lipophile balance of nonionic surfactants: the effect of added salts on the phase inversion temperature of emulsions. J. Colloid Interface Sci. 1970, 32, 642−646. (15) Sunderland, V. B.; Enever, R. P. The influence of formulation variables on phase inversion temperatures of emulsions as determined by a programmed viscometric technique. J. Pharm. Pharmacol. 1972, 24, 804−814. (16) Schott, H.; Han, S. K. Interaction of inorganic additives with solutions of nonionic surfactants. 3. Effect on critical micelle concentrations and krafft points. Abstr. Papers Am. Chem. Soc. 1975, 26−26. (17) Schott, H.; Han, S. K. Effect of inorganic additives on solutions of nonionic surfactants. 3. Cmcs and surface properties. J. Pharm. Sci. 1976, 65, 975−978. (18) Anton, N.; Saulnier, P.; Béduneau, A.; Benoit, J. P. Salting-out effect induced by temperature cycling on a water/nonionic surfactant/ oil system. J. Phys. Chem. B 2007, 111, 3651−3657. (19) Mei, Z.; Xu, J.; Sun, D. O/W nano-emulsions with tunable PIT induced by inorganic salts. Colloids Surf., A 2011, 375, 102−108. (20) Florence, A. T.; Madsen, F.; Puisieux, F. Emulsion stabilization by nonionic surfactants − relevance of surfactant cloud point. J. Pharm. Pharmacol. 1975, 27, 385−394. (21) Iwanaga, T.; Suzuki, M.; Kunieda, H. Effect of added salts or polyols on the liquid crystalline structures of polyoxyethylene-type nonionic surfactants. Langmuir 1998, 14, 5775−5781. (22) Shinoda, K.; Saito, H. Stability of O/W type emulsions as functions of temperature and HLB of emulsifiers − emulsification by PIT-method. J. Colloid Interface Sci. 1969, 30, 258−263. (23) Rao, J.; McClements, D. J. Stabilization of phase inversion temperature nanoemulsions by surfactant displacement. J. Agric. Food Chem. 2010, 58, 7059−7066. (24) Ee, S. L.; Duan, X.; Liew, J.; Nguyen, Q. D. Droplet size and stability of nano-emulsions produced by the temperature phase inversion method. Chem. Eng. J. 2008, 140, 626−631. (25) Saberi, A. H.; Fang, Y.; McClements, D. J. Fabrication of vitamin E-enriched nanoemulsions: factors affecting particle size using spontaneous emulsification. J. Colloid Interface Sci. 2013, 391, 95−102.
In summary, the addition of salt may have improved the storage stability of the emulsions by decreasing the attractive interactions between them (thereby decreasing coalescence) or by reducing the water solubility of the oil phase (thereby decreasing Ostwald ripening). Further studies are required to establish the relative importance of these two different physicochemical mechanisms. The improved stability of the emulsions to droplet growth when the droplet concentration was reduced can simply be attributed to the reduction in droplet collision frequency in the more dilute system. Conclusions. In this study, we examined the influence of salts on the formation, properties, and stability of vitamin Eenriched emulsions prepared by spontaneous emulsification. We have shown that the addition of salts (NaCl and CaCl2) to the aqueous phase of the emulsions did not change the initial droplet size produced. However, the ability of salts to modulate surfactant properties had an appreciable effect on their thermal stability: the cloud point decreased with increasing salt concentration. This effect was attributed to the ability of salts to promote dehydration of the surfactant headgroup, which alters the optimum curvature of the surfactant monolayer. The isothermal stability of the emulsions was influenced by storage temperature, with the droplet growth rate increasing with increasing temperature. This effect was attributed to the impact of temperature on the rate of droplet coalescence and Ostwald ripening. Addition of salts to the emulsions decreased the rate of droplet growth during isothermal storage. This effect was attributed to the ability of salts to reduce the attractive van der Waals interactions between droplets (thereby reducing coalescence) or to reduce the solubility of the oil molecules in water (thereby reducing Ostwald ripening). These results have important implications for the development of emulsionbased delivery systems for application in products containing salts.
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AUTHOR INFORMATION
Corresponding Author
*(D.J.M.) Phone: (413) 545-1019. Fax: (413) 545-1262. Email: [email protected]. Funding
This material is based upon work supported by the Cooperative State Research, Extension, Education Service, U.S. Department of Agriculture, Massachusetts Agricultural Experiment Station (Project 831), and by the U.S. Department of Agriculture, NRI Grants (2011-03539 and 2013-03795). Notes
The opinions expressed in this paper are those of the authors and do not represent statements of position, intention, or strategy of PepsiCo Inc. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank William Mutilangi of PepsiCo for useful advice and discussions on this research. REFERENCES
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Effect of Salts on Formation and Stability of Vitamin E‑Enriched Miniemulsions Produced by Spontaneous Emulsification Amir Hossein Saberi,† Yuan Fang,§ and David Julian McClements*,† †
Biopolymers and Colloids Laboratory, Department of Food Science, University of MassachusettsAmherst, Amherst, Massachusetts 01003, United States § PepsiCo Global R&D, 100 Stevens Avenue, Valhalla, New York 10595, United States ABSTRACT: Emulsion-based delivery systems are being utilized to incorporate lipophilic bioactive components into various food, personal care, and pharmaceutical products. This study examined the influence of inorganic salts (NaCl and CaCl2) on the formation, stability, and properties of vitamin E-enriched emulsions prepared by spontaneous emulsification. These emulsions were simply formed by titration of a mixture of vitamin E acetate (VE), carrier oil (MCT), and nonionic surfactant (Tween 80) into an aqueous salt solution with continuous stirring. Salt type and concentration (0−1 N NaCl or 0−0.5 N CaCl2) did not have a significant influence on the initial droplet size of the emulsions. On the other hand, the isothermal and thermal stabilities of the emulsions depended strongly on salt levels. The cloud point of the emulsions decreased with increasing salt concentration, which was attributed to accelerated droplet coalescence in the presence of salts. Dilution (2−6 times) of the emulsions with water appreciably improved their thermal stability by increasing their cloud point, which was mainly attributed to the decrease in aqueous phase salt levels. The isothermal storage stability of the emulsions also depended on salt concentration; however, increasing the salt concentration decreased the rate of droplet growth, which was the opposite of its effect on thermal stability. Potential physicochemical mechanisms for these effects are discussed in terms of the influence of salt ions on van der Waals and electrostatic interactions. This study provides important information about the effect of inorganic salts on the formation and stability of vitamin E emulsions suitable for use in food, personal care, and pharmaceutical products. KEYWORDS: emulsions, nanoemulsions, vitamin E, salts, stability, cloud point, spontaneous emulsification
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INTRODUCTION Emulsion-based delivery systems are finding increasing utilization within food, personal care, and pharmaceutical products to encapsulate, protect, and release lipophilic active ingredients. This category of delivery system includes various kinds of colloidal suspension that utilize lipid droplets as building blocks, such as emulsions, solid lipid nanoparticles, multilayer emulsions, multiple emulsions, and filled hydrogel particles.1,2 Emulsions are the simplest type of emulsion-based delivery system consisting of lipid droplets suspended in an aqueous phase, with each lipid droplet being coated by a layer of emulsifier molecules. Nevertheless, the functional performance of emulsions can still be manipulated by altering oil phase composition, emulsifier type, and particle size distribution. For example, reducing the particle size can lead to higher optical clarity, greater stability to gravitational separation and droplet aggregation, and higher oral bioavailability.3−5 Emulsions containing very small droplets (d < 200 nm) are often referred to as nanoemulsions, mini-emulsions, or ultrafine emulsions. In this paper, we will simply refer to them as emulsions for the sake of convenience. Emulsions can be prepared using several approaches, which are usually categorized as either high-energy or low-energy methods.6,7 High-energy methods use mechanical devices to generate intense forces that intermingle and break up the oil and water phases, such as high-pressure homogenizers, microfluidizers, and ultrasonic generators.3,8 Low-energy methods rely on the spontaneous formation of ultrafine droplets within surfactant−oil−water (SOW) mixtures when © 2014 American Chemical Society
specific changes in composition and/or environmental conditions occur.3,8 A number of low-energy methods have been developed to form ultrafine oil droplets including spontaneous emulsification (SE), phase inversion temperature (PIT), phase inversion composition (PIC), and emulsion inversion point (EIP) methods.5,8−10 Among these methods, spontaneous emulsification is one of the easiest and most costeffective to implement because it simply involves metering an oil−surfactant mixture into an aqueous solution with constant agitation at a fixed temperature. Commercial food and beverage products typically contain a variety of ingredients that may influence the formation and stability of emulsions, including sugars, cosolvents, and salts. Salts are incorporated into foods for a number of purposes, for example, to control flavor, water activity, stability, and nutritional profile. Salts have been reported to influence various physicochemical properties of nonionic surfactants, including solubility and cloud point,11−15 critical micelle concentration,16,17 emulsion phase inversion temperature,14,15,18,19 and emulsion stability.11,19,20 Mei et al.19 suggested that salt may facilitate the formation of emulsions using the PIT method. Salts may alter the interlayer spacing of the liquid crystalline phases formed during the intermediate steps of low-energy emulsification, thereby leading to the formation of ultrafine Received: Revised: Accepted: Published: 11246
August 10, 2014 October 22, 2014 October 24, 2014 October 24, 2014 dx.doi.org/10.1021/jf503862u | J. Agric. Food Chem. 2014, 62, 11246−11253
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droplets.21 Furthermore, it has been reported that the storage stability of emulsions stabilized by nonionic surfactants is strongly dependent upon their PIT,22−24 which depends on salt concentration. Many previous studies on the impact of salt on nanoemulsion formation have utilized simple organic solvents or liquids as the oil phase. In this study, we utilized an oil phase that is more representative of those that might be used in the food industry, that is, vitamin E acetate dissolved in mediumchain triglycerides (MCT). Recently, we demonstrated that stable vitamin E-enriched emulsions containing very fine droplets (d < 60 nm) could be produced by spontaneous emulsification.25 We also showed that addition of cosolvents to the aqueous phase of these emulsions had an appreciable influence on their formation and stability.26,27 The purpose of the present study was to investigate the influence of two salts commonly used in industrial applications (NaCl and CaCl2) on the formation and stability of vitamin E emulsions produced using spontaneous emulsification. The knowledge gained from this study will be useful for the successful incorporation of emulsion-based delivery systems into commercial products.
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Statistical Analysis. All experiments were carried out two or three times using two freshly prepared samples, and the results are reported as the calculated mean and standard deviation of these measurements. Differences between group means were determined by analysis of variance (ANOVA) using statistical software (version 12.0; SPSS, Inc., Chicago, IL, USA). Means were subjected to Duncan’s test, and a P value of 1), and changes the optimum curvature so that it favors the formation of water-in-oil emulsions (Figure 1b). The water droplets in the water-in-oil emulsions formed above the PIT are relatively large and therefore scatter light strongly, leading to increased turbidity.3,29 The impact of salt on the turbidity−temperature profiles of the emulsions can be attributed to the fact that salt decreases the PIT of the emulsions. In the absence of salt, the PIT is relatively high, and therefore we observed only the initial droplet coalescence regimen. In the presence of sufficient salt, the PIT moved into the experimental temperature range, and we could observe droplet coalescence, microemulsion formation, and phase inversion. A similar behavior was observed in the turbidity−temperature profiles of vitamin E-enriched emulsions in the absence and presence of glycerol; that is, glycerol reduced the PIT.26 The turbidity versus temperature profiles were used to determine the cloud points of the emulsions so that we could establish the influence of salt type and concentration on their thermal behavior. The cloud point of the emulsions decreased from around 78 to 65 °C when the NaCl concentration was increased from 0 to 1 M and decreased from around 78 to 70 °C when the CaCl2 concentration was increased from 0 to 0.5 N (Figure 1c). The ability of NaCl14,15,20,37 and CaCl214,15,37 to decrease the cloud points of nonionic surfactant has been reported previously. Our results are also in agreement with previous studies that have reported that the cloud point and/or PIT of nonionic surfactants is a linear function of their salt concentration.15,38,39 The decrease in thermal stability (cloud point) of emulsions in the presence of NaCl and CaCl2 can be attributed to the effect of these salts on the solubility and packing characteristics of nonionic surfactants. Specific anionic19,40,41 and cationic12,16,19,40 salt ions may either increase (salting in) or decrease (salting out) the solubility of nonionic surfactants due to their effects on the structural organization of water molecules. Ions can be classified as either “structure-makers” (salting out) or “structure-breakers” (salting in) according to their position in the Hofmeister series.42,43 The presence of mineral ions in an aqueous solution may alter the PIT of a nonionic surfactant by altering the solubility and packing characteristics of the surfactant molecules. In particular, salts may affect the optimum curvature of the surfactants by altering the hydration of their
Figure 2. Influence of dilution on the cloud point of vitamin Eenriched mini-emulsion produced using 10% oil phase (vitamin E/ MCT, 8:2), 10% surfactant phase (Tween 80), and 80% aqueous phase (0.5 M salt). Oil phase concentrations were 10, 5, 3.33, 2.5, and 1.67, and salt concentrations were 0.4, 0.2, 0.13, 0.1, 0.08, and 0.067 N for the emulsion diluted with dilution factors of 1, 2, 3, 4, 5, and 6, respectively.
influence on the dehydration of the hydrophilic headgroups at different temperatures. Emulsion formation was carried out at low (room) temperature, and therefore the surfactant molecules would have been highly hydrated. Presumably, headgroup hydration was still sufficient in the presence of salt to generate a strong steric repulsion between the droplets and to have a high surfactant curvature. However, once the emulsions were heated, the effects of salts became more pronounced because the surfactants were already partially dehydrated, and any further change due to salt addition was sufficient to promote instability. Influence of Dilution on Thermal Stability. Emulsions are diluted when they are incorporated into functional food and beverage products. Our previous studies have shown that emulsion dilution may have an appreciable influence on emulsion thermal stability.26,27,45 We therefore examined the influence of dilution with buffer solution on the thermal stability of vitamin E emulsions initially containing 0.5 N NaCl or CaCl2 in the aqueous phase. Qualitatively, all of the diluted 11249
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emulsions had similar turbidity−temperature profiles as the undiluted emulsions (Figure 1a): upon increasing the temperature from 20 to 95 °C, the turbidity initially remained low, then steeply increased, then sharply decreased to a minimum, then steeply increased again, and then remained high (data not shown). The cloud points determined from these profiles increased from 73 to 81 °C for NaCl and from 70 to 81 °C CaCl2 as the emulsion dilution factor (DF) increased from 1 to 6 (Figure 2). As these samples were diluted with buffer solution, the observed results may have been due to a decrease in salt concentration in the aqueous phase or due to a decrease in oil droplet concentration. We therefore carried out additional experiments in which the emulsions were diluted with various salt solutions so as to obtain samples where (i) the droplet concentration was kept constant, but the salt concentration varied (Figure 3) or (ii) the
Figure 4. (a) Influence of oil phase concentration on the cloud point of diluted vitamin E-enriched mini-emulsions containing 0.4 N salts. The initial emulsion was produced using 10% oil phase (vitamin E/ MCT, 8:2), 10% surfactant phase (Tween 80), and 0.4 N NaCl. (b) Influence of temperature on the turbidity of vitamin E-enriched miniemulsions containing 0.4 N CaCl2 and different oil phase concentrations. The initial emulsion was produced using 10% oil phase (vitamin E/MCT, 8/2), 10% surfactant phase (Tween 80), and 0.4 N CaCl2.
Figure 3. Influence of salt concentration on the cloud point of vitamin E-enriched mini-emulsions containing 5% oil phase. The initial emulsion was produced using 10% oil phase (vitamin E/MCT, 8:2), 10% surfactant phase (Tween 80), and 0.4 N salt.
droplet concentration varied, but the salt concentration was kept constant (Figure 4). In the diluted emulsions with fixed droplet concentration, the cloud point increased from around 73 to 77 °C for NaCl and from around 72 to 77 °C for CaCl2 as the salt content decreased from 0.4 to 0.2 N, respectively (Figure 3). These results suggest that at least part of the increase in cloud point observed upon emulsion dilution (Figure 2) was related to a reduction in salt concentration in the aqueous phase. In the diluted emulsions with fixed salt concentration, the cloud point initially increased and then decreased as the droplet content was reduced from 10 to 1% (Figure 4a). This effect may have been an artifact of the cloud point measurement at high droplet concentration. As the droplet concentration increased, there was an increase in the overall turbidity of the samples due to an increase in light scattering by the droplets (Figure 4b). In the emulsions with the highest droplet concentrations, the turbidity was so great (>3 cm−1) that it was not possible to observe the entire region where rapid droplet coalescence occurred. Consequently, it was not possible to accurately measure the cloud points for these samples. In the more dilute emulsions (≤3% oil), the cloud point decreased with increasing dilution (decreasing droplet concentration), which may have been due to changes in the nature of the microemulsion phase formed around the PIT when the SOW ratio was changed. Taken together, these results
suggest that the increase in cloud point observed when saltcontaining emulsions were diluted with buffer solution was primarily due to the reduced salt concentration in the aqueous phase, rather than the reduced droplet concentration. Influence of Salts and Dilution on Isothermal Stability. Physical stability of emulsion-based delivery systems during storage, transport, and utilization is required for most commercial applications. Emulsions may become unstable through a number of different physicochemical processes, including flocculation, coalescence, Ostwald ripening, and gravitational separation.10,33 The mechanism and rate of breakdown depend on emulsion composition and microstructure, as well as the environmental conditions experienced during its lifetime. We therefore examined the influence of salt addition and dilution on the storage stability of vitamin E emulsions. A series of emulsions was fabricated with different droplet concentrations (2.5 or 10 wt %) and salt concentrations (0, 0.2, and 0.4 N NaCl or CaCl2) by diluting an initial emulsion with different types and amounts of salt solution. After preparation, the resulting emulsions were stored for 1 11250
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decreased their thermal stability by reducing the cloud point (Figure 2). This independence of thermal and isothermal stability of vitamin E emulsions has also been reported upon posthomogenization addition of cosurfactants.46 There are a number of possible mechanisms that might be responsible for the observed instability of the emulsions during storage: flocculation, coalescence, and Ostwald ripening.4,33 We do not believe that flocculation played an important role in emulsion instability as we did not observe any flocs using an optical microscope. We therefore postulated that the observed instability was due to either coalescence or Oswald ripening. It is often difficult to distinguish between these two mechanisms because they both lead to a net increase in mean droplet diameter over time.33,47 An increase in droplet growth rate with increasing temperature could be attributed to the effects of temperature on coalescence and/or Ostwald ripening. Increasing temperature can dehydrate the surfactant headgroups and thereby increase droplet coalescence by (i) decreasing the hydration repulsion between droplets and (ii) by changing the optimum curvature of the surfactant monolayer toward unity.3,31,48 A temperature rise may also increase the rate of Ostwald ripening by increasing the diffusion rate or solubility of the oil molecules in the aqueous phase.49 Inorganic salts might influence the isothermal storage stability of oil-in-water emulsions stabilized by nonionic surfactant through a number of mechanisms: Effect of Salts on Surfactant Headgroup Dehydration. Previous studies have reported that salts that decrease the cloud point of emulsions stabilized by nonionic surfactant also increase droplet coalescence.11,20 This effect was attributed to the ability of the salts to promote dehydration of the surfactant headgroup, thereby reducing the hydration repulsion between droplets and altering the surfactant monolayer curvature. However, the fact that we actually observed a decrease in droplet growth during storage in the presence of NaCl and CaCl2 suggested that this mechanism was not prominent under the conditions used. Effect of Salts on van der Waals Interactions. van der Waals interactions are attractive forces that almost always act between oil droplets suspended in water and, therefore, promote droplet aggregation. The addition of salts to an aqueous solution appreciably decreases the magnitude of the van der Waals interaction due to an electrostatic screening effect.33 This mechanism may therefore account for the increased storage stability of the emulsions in the presence of salt, because there would have been a reduction in the attractive forces acting between the oil droplets. It should be noted that salt can also decrease the electrostatic interaction between the droplets through electrostatic screening, thereby increasing droplet coalescence. Effect of Salts on Oil Solubility. One of the major factors influencing the rate of Ostwald ripening in oil-in-water emulsions is the solubility of the oil phase in the aqueous phase.33 The addition of salts to the aqueous phase changes the structural organization of the water molecules, which will alter the equilibrium solubility of oil molecules. Salt addition causes an increase in interfacial tension (Table 1), which would increase the free energy associated with introducing a nonpolar molecule into water, thereby decreasing its water solubility. Consequently, addition of salt to the emulsions may have reduced the rate of Ostwald ripening, which is consistent with the reduction in droplet growth rate observed in the presence of salt observed in this study.
month at three storage temperatures representing refrigeration conditions (5 °C), ambient storage in mild climates (20 °C), and ambient storage in hot climates (37 °C). The initial emulsion formed by spontaneous emulsification contained 0% salt and 10% oil and was therefore designated “0/ 10”. The extent of droplet growth in this emulsion after 1 month of storage was appreciable and increased with increasing storage temperature: ∼15% at 5 °C, 19% at 20 °C, and 54% at 37 °C (Figure 5). The droplet size distribution of these
Figure 5. (a) Influence of storage time and temperature on the particle growth (%) of vitamin E-enriched mini-emulsions containing different ratios of NaCl (N)/oil phase. The initial emulsions were produced using 10% oil phase (vitamin E/MCT, 8:2), 10% surfactant phase (Tween 80), and different NaCl concentrations (0, 0.4, and 0.8 N). Means within different letters and the same number of stars are significantly (P < 0.05) different. (b) Influence of storage time and temperature on the particle growth (%) of vitamin E-enriched miniemulsions containing different ratios of CaCl2 (M)/oil phase. The initial emulsions were produced using 10% oil phase (vitamin E/MCT, 8:2), 10% surfactant phase (Tween 80), and different CaCl2 concentrations (0, 0.2, and 0.4 N). Means within different letters and the same number of stars are significantly (P < 0.05) different.
emulsions remained monomodal throughout storage at all temperatures (data not shown). The stability of the emulsions to droplet growth improved significantly (P < 0.05) as the level of NaCl or CaCl2 added was increased, especially at the elevated temperatures (Figure 5). In addition, reducing the droplet concentration from 10 to 2.5% also led to a significant (P < 0.05) inhibition of droplet growth for emulsions containing both types of salt (Figure 5). Overall, these results suggest that the addition of NaCl or CaCl2 improved the isothermal storage stability of the emulsions, even though it 11251
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(3) McClements, D. J.; Rao, J. Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity. Crit. Rev. Food Sci. Nutr. 2011, 51, 285−330. (4) Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C. Formation and stability of nano-emulsions. Adv. Colloid Interface Sci. 2004, 108−109, 303−318. (5) McClements, D. J. Nanoemulsion-based oral delivery systems for lipophilic bioactive components: nutraceuticals and pharmaceuticals. Ther. Delivery 2013, 4, 841−57. (6) Anton, N.; Benoit, J. P.; Saulnier, P. Design and production of nanoparticles formulated from nano-emulsion templates − a review. J. Controlled Release 2008, 128, 185−199. (7) Solans, C.; Esquena, J.; Forgiarini, A. M.; Uson, N.; Morales, D.; Izquierdo, P.; Azemar, N.; Garcia-Celma, M. J. Nano-emulsions: formation, properties, and applications. In Adsorption and Aggregation of Surfactants in Solution; Mittal, K. L., Shah, D. O., Eds.; CRC Press: Boca Raton, FL, USA, 2003; Vol. 109, pp 525−554. (8) Solans, C.; Solé, I. Nano-emulsions: formation by low-energy methods. Curr. Opin. Colloid Interface Sci. 2012, 17, 246−254. (9) Anton, N.; Benoit, J.-P.; Saulnier, P. Design and production of nanoparticles formulated from nano-emulsion templates − a review. J. Controlled Release 2008, 128, 185−199. (10) McClements, D. J. Edible nanoemulsions: fabrication, properties, and functional performance. Soft Matter 2011, 7, 2297−2316. (11) Schott, H.; Royce, A. E. Effect of inorganic additives on solutions of non-ionic surfactants V: emulsion stability. J. Pharm. Sci. 1983, 72, 1427−1436. (12) Schott, H. Salting in of nonionic surfactants by complexation with inorganic salts. J. Colloid Interface Sci. 1973, 43, 150−155. (13) Schott, H.; Han, S. K. Effect of inorganic additives on solutions of nonionic surfactants. 2. J. Pharm. Sci. 1975, 64, 658−664. (14) Shinoda, K.; Takeda, H. The effect of added salts in water on the hydrophile-lipophile balance of nonionic surfactants: the effect of added salts on the phase inversion temperature of emulsions. J. Colloid Interface Sci. 1970, 32, 642−646. (15) Sunderland, V. B.; Enever, R. P. The influence of formulation variables on phase inversion temperatures of emulsions as determined by a programmed viscometric technique. J. Pharm. Pharmacol. 1972, 24, 804−814. (16) Schott, H.; Han, S. K. Interaction of inorganic additives with solutions of nonionic surfactants. 3. Effect on critical micelle concentrations and krafft points. Abstr. Papers Am. Chem. Soc. 1975, 26−26. (17) Schott, H.; Han, S. K. Effect of inorganic additives on solutions of nonionic surfactants. 3. Cmcs and surface properties. J. Pharm. Sci. 1976, 65, 975−978. (18) Anton, N.; Saulnier, P.; Béduneau, A.; Benoit, J. P. Salting-out effect induced by temperature cycling on a water/nonionic surfactant/ oil system. J. Phys. Chem. B 2007, 111, 3651−3657. (19) Mei, Z.; Xu, J.; Sun, D. O/W nano-emulsions with tunable PIT induced by inorganic salts. Colloids Surf., A 2011, 375, 102−108. (20) Florence, A. T.; Madsen, F.; Puisieux, F. Emulsion stabilization by nonionic surfactants − relevance of surfactant cloud point. J. Pharm. Pharmacol. 1975, 27, 385−394. (21) Iwanaga, T.; Suzuki, M.; Kunieda, H. Effect of added salts or polyols on the liquid crystalline structures of polyoxyethylene-type nonionic surfactants. Langmuir 1998, 14, 5775−5781. (22) Shinoda, K.; Saito, H. Stability of O/W type emulsions as functions of temperature and HLB of emulsifiers − emulsification by PIT-method. J. Colloid Interface Sci. 1969, 30, 258−263. (23) Rao, J.; McClements, D. J. Stabilization of phase inversion temperature nanoemulsions by surfactant displacement. J. Agric. Food Chem. 2010, 58, 7059−7066. (24) Ee, S. L.; Duan, X.; Liew, J.; Nguyen, Q. D. Droplet size and stability of nano-emulsions produced by the temperature phase inversion method. Chem. Eng. J. 2008, 140, 626−631. (25) Saberi, A. H.; Fang, Y.; McClements, D. J. Fabrication of vitamin E-enriched nanoemulsions: factors affecting particle size using spontaneous emulsification. J. Colloid Interface Sci. 2013, 391, 95−102.
In summary, the addition of salt may have improved the storage stability of the emulsions by decreasing the attractive interactions between them (thereby decreasing coalescence) or by reducing the water solubility of the oil phase (thereby decreasing Ostwald ripening). Further studies are required to establish the relative importance of these two different physicochemical mechanisms. The improved stability of the emulsions to droplet growth when the droplet concentration was reduced can simply be attributed to the reduction in droplet collision frequency in the more dilute system. Conclusions. In this study, we examined the influence of salts on the formation, properties, and stability of vitamin Eenriched emulsions prepared by spontaneous emulsification. We have shown that the addition of salts (NaCl and CaCl2) to the aqueous phase of the emulsions did not change the initial droplet size produced. However, the ability of salts to modulate surfactant properties had an appreciable effect on their thermal stability: the cloud point decreased with increasing salt concentration. This effect was attributed to the ability of salts to promote dehydration of the surfactant headgroup, which alters the optimum curvature of the surfactant monolayer. The isothermal stability of the emulsions was influenced by storage temperature, with the droplet growth rate increasing with increasing temperature. This effect was attributed to the impact of temperature on the rate of droplet coalescence and Ostwald ripening. Addition of salts to the emulsions decreased the rate of droplet growth during isothermal storage. This effect was attributed to the ability of salts to reduce the attractive van der Waals interactions between droplets (thereby reducing coalescence) or to reduce the solubility of the oil molecules in water (thereby reducing Ostwald ripening). These results have important implications for the development of emulsionbased delivery systems for application in products containing salts.
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AUTHOR INFORMATION
Corresponding Author
*(D.J.M.) Phone: (413) 545-1019. Fax: (413) 545-1262. Email: [email protected]. Funding
This material is based upon work supported by the Cooperative State Research, Extension, Education Service, U.S. Department of Agriculture, Massachusetts Agricultural Experiment Station (Project 831), and by the U.S. Department of Agriculture, NRI Grants (2011-03539 and 2013-03795). Notes
The opinions expressed in this paper are those of the authors and do not represent statements of position, intention, or strategy of PepsiCo Inc. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank William Mutilangi of PepsiCo for useful advice and discussions on this research. REFERENCES
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