Article pubs.acs.org/JAFC
Impact of Environmental Stresses on Orange Oil-in-Water Emulsions Stabilized by Sucrose Monopalmitate and Lysolecithin David Julian McClements,† Eric Andrew Decker,† and Seung Jun Choi*,§ †
Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States Department of Food Science and Technology and Department of Interdisciplinary Bio IT Materials, Seoul National University of Science and Technology, Seoul 139-743, Republic of Korea
§
ABSTRACT: The food and beverage industry is trying to replace synthetic functional ingredients with more “label-friendly” ingredients in many commercial products. This study therefore examined the influence of environmental stresses on the stability of emulsions stabilized by a combination of lysolecithin and sucrose monopalmitate (SMP). Orange oil-in-water emulsions (5% (w/w) oil) stabilized by SMP (1%) and lysolecithin (0−0.5%) were prepared using high-pressure homogenization (pH 3). In the absence of lysolecithin, all emulsions were highly unstable to droplet aggregation, which was attributed to low droplet charge (weak electrostatic repulsion) and small SMP headgroup size (weak steric repulsion). Incorporation of 0.1−0.5% lysolecithin into the emulsions greatly improved their stability to droplet aggregation, which was mainly attributed to the increase in negative charge on the droplets (strong electrostatic repulsion). The addition of high levels of salt (NaCl) to the emulsions promoted droplet aggregation and creaming. Emulsions containing 0.5% lysolecithin were stable to heating (30−90 °C) in the absence of salt, but exhibited droplet aggregation and creaming when held at high (>50 °C) temperatures in the presence of 300 mM salt. This study has implications for the development of emulsion-based delivery systems for use in food and beverage products. KEYWORDS: emulsions, environmental stress, lecithin, orange oil, sucrose monopalmitate, beverages, stability, sucrose esters
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beverage industries.11 However, at present there is a relatively poor understanding of the influence of environmental stresses that beverage products might experience during their production, transport, storage, and utilization on the properties of emulsions stabilized by sucrose monoesters. The objective of the present study was therefore to systematically study the ability of sucrose monopalmitate (SMP) to form and stabilize orange oil-in-water emulsions and to examine the influence of environmental stresses (salt and temperature) and cosurfactants (lysolecithin) on their stability. The information obtained from this study should be useful for designing food and beverage emulsions with improved properties and consumer acceptance.
INTRODUCTION Oils extracted from orange peel are widely used as flavoring ingredients in food and beverage products.1 Some studies suggest they may also contain phytochemicals that have healthpromoting effects, such as anticarcinogenic and anti-inflammatory activities.2,3 There is therefore considerable interest in incorporating orange oils into food and beverages as flavoring agents or bioactive ingredients. The majority of chemical constituents of orange oils are hydrophobic compounds that do not readily dissolve in water4 and therefore have to be incorporated into foods in the form of colloidal dispersions, such as microemulsions, nanoemulsions, or emulsions.5,6 The beverage industry is under increasing pressure to create products that are perceived as being healthy.5 Consequently, they are attempting to reformulate many of their products to remove synthetic ingredients that consumers perceive as being undesirable. Synthetic surfactants (such as “Tweens” or “polysorbates”) are commonly used commercially to fabricate emulsion-based beverage products. According to market research, the food emulsifier market will globally grow from around U.S. $2109 million in 2012 to around U.S. $2859 million by 2018 with synthetic emulsifiers accounting for about two-thirds of the total market and natural emulsifiers the rest.7 Synthetic surfactants are highly effective at forming and stabilizing emulsions, and therefore it is often difficult to find more label-friendly alternatives. Sucrose monoesters are surface-active materials produced from natural products, such as sucrose and vegetable oils.8 They have been widely used in commercial cosmetic and pharmaceutical products to stabilize emulsions due to their low toxicity, good biocompatibility, and high ecological acceptability.9,10 Sucrose monoesters are also good candidates for application as surfactants in the food and © 2014 American Chemical Society
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MATERIALS AND METHODS
Materials. Sucrose monopalmitate and 4-fold orange oil were supplied by International Flavors and Fragrances (Union Beach, NJ, USA), and an enzyme-modified soy lecithin was supplied by Solae (SOLEC K-EML, St. Louis, MO, USA). All other chemicals were of reagent grade or purer and were obtained from Fisher Scientific (Pittsburgh, PA, USA). Emulsion Preparation. Orange oil-in-water emulsions were prepared by mixing 5% (w/w) oil phase containing different concentrations of lysolecithin with 95% (w/w) aqueous phase (1% SMP, 10 mM phosphate buffer, pH 7.0) following the method described by Choi et al.12 A coarse emulsion premix was prepared by homogenizing oil and aqueous phases using a high-speed blender (Biohomogenizer, Biospec Products, Bartlesville, OK, USA) for 2 min Received: Revised: Accepted: Published: 3257
November 6, 2013 March 10, 2014 March 10, 2014 March 10, 2014 dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
Article
Figure 1. Evolution of the particle size distribution of SMP- or SMP/lysolecithin-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) stored at 20 °C: (A) SMP-stabilized emulsion stored at pH 7; (B) SMP-stabilized emulsion stored at pH 3; (C) SMP/0.1% lysolecithinstabilized emulsion at pH 3; (D) SMP/0.5% lysolecithin-stabilized emulsion at pH 3. at 10000 rpm at room temperature. For reduction of droplet size, the premixed emulsions were homogenized by five passes through a microfluidizer (M-100L, Microfluidics, Newton, MA, USA) at 48.3 MPa. Then, the emulsion was adjusted to pH 3.0 using 1.0 and 0.1 N hydrochloric acid solutions. The final concentrations of lysolecithin in the whole emulsions varied from 0 to 0.5% (w/w), and the final concentration of SMP in the whole emulsions was 1% (w/w). To avoid the effect of light, all samples were stored in the dark before analysis. Stability to Environmental Stresses. To determine the stability of SMP/lysolecithin-stabilized emulsions against heat treatment, emulsions were placed in glass test tubes and then incubated in a water bath for 15 min at different temperatures (30−90 °C). The emulsions were then allowed to cool to room temperature and held overnight prior to subsequent analysis. To evaluate the stability of SMP/lysolecithin-stabilized emulsions against the change in the electrostatic interaction, a series of 2.5 wt % emulsions with different NaCl concentrations was prepared by diluting the concentrated emulsions with 10 mM phosphate buffer (pH 3.0) containing different concentrations of sodium chloride. Then, the emulsions were kept at room temperature overnight prior to subsequent analysis. Droplet Size Measurements. Mean droplet diameters of emulsions were measured using static light scattering (laser diffraction). To avoid multiple scattering effects, all emulsion samples were diluted to a droplet concentration of approximately ≈0.005% (w/ w) using buffer solution at the pH of the sample and stirred continuously throughout the measurements to ensure they were homogeneous. The droplet size distribution of the emulsions was then measured using a commercial static light scattering instrument (Mastersizer 2000, Malvern Instruments, Worcestershire, UK).
Droplet size data are reported as either the volume-surface mean diameter, d32 = (∑nidi3/∑nid2i ), or the volume-weighted mean diameter, d43 = (∑nid4i /∑nid3i ), where ni is the number of droplets with diameter di. If an emulsion sample to be analyzed had phaseseparated due to droplet creaming, then it was gently mixed to evenly disperse the droplets prior to collection of an aliquot for making the particle size measurements. Electrical Charges of Oil-in-Water Emulsions. The ζ-potential of the emulsion droplets was measured using a microelectrophoresis instrument (ZetaSizer Nano, Malvern Instruments). The ζ-potential was calculated from measurement of the electrophoretic mobility of droplets in an applied oscillating electric field using laser Doppler velocimetry. Samples were prepared by diluting emulsions 1:500 with buffer solution at the pH of the sample and placing the diluted emulsions into disposable capillary cells (DTS1060, Malvern Instruments). If an emulsion sample to be analyzed had phase-separated due to creaming, then it was evenly dispersed by gentle stirring before collection of an aliquot for analysis. Statistical Analysis. Experiments were repeated two or three times using freshly prepared samples, and the mean and standard deviations were calculated from these measurements.
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RESULTS AND DISCUSSION Influence of Lysolecithin. Previously, we examined the influence of pH on the stability of SMP-stabilized orange oil emulsions.12 Stable orange oil emulsions with small mean droplet diameters (d43 < 0.15 μm) could be formed at neutral pH. However, when the emulsion was stored under acidic conditions (pH 3−5), a large increase in mean droplet diameter 3258
dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
Article
Figure 5. Influence of NaCl concentration on the electrical charge of SMP-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) at pH 3, stored at 20 °C.
Figure 2. Influence of lysolecithin concentration on the ζ-potential of SMP-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) at pH 3, stored at 20 °C.
droplets have a high negative charge at high pH values, which stabilizes them from aggregation due to electrostatic repulsion. On the other hand, they have a low net charge at low pH and are therefore susceptible to aggregation due to the dominance of attractive colloidal interactions (such as van der Waals or hydrophobic attractions). We therefore examined the impact of adding an anionic cosurfactant (lysolecithin) on the aggregation stability of orange oil-in-water emulsions under acid conditions (Figure 1). We hypothesized that this cosurfactant would improve the physical stability of the emulsions by increasing the electrostatic repulsion between the droplets. Emulsions were initially prepared under neutral pH conditions to ensure that they were stable. The initial mean droplet diameter at pH 7 was around 0.125 μm for all of the emulsions prepared (Figure 1A), independent of the lysolecithin concentration present, which suggested that the initial particle size was primarily governed by the homogenization conditions or the SMP. All of the emulsions were then adjusted to pH 3 and stored for 15 days. In the absence of lysolecithin, the emulsions were highly unstable to droplet growth at pH 3, which was attributed to droplet coalescence during storage (Figure 1B). Conversely, in the presence of lysolecithin (0.1−0.5%), all of the emulsions were stable to droplet growth throughout 15 days of storage (Figures 1C,D). These results indicated that lysolecithin was an effective cosurfactant for improving the stability of SMP-coated droplets to growth, which can be attributed to the increased electrostatic repulsion between the droplets in the presence of this anionic surface active substance. Figure 2 shows the ζ-potential of the SMP-stabilized orange oil emulsions with various lysolecithin concentrations. The ζpotential of the oil droplets was close to zero (−1.7 mV) in the absence of lysolecithin, but it became increasingly negative as the amount of lecithin incorporated into the system increased. The ζ-potential reached a value of around −35 mV at 0.5% lysolecithin, which should be large enough to provide good stability through electrostatic repulsion. Theoretically, sucrose esters containing SMP are nonionic surfactants,8 but they may have a negative charge at high pH because of the presence of ionized impurities, such as palmitic acid.12 As described above, palmitic acid should have little charge at acid pH, which would account for the low net charge on the emulsions in the absence
Figure 3. Photographs of SMP-stabilized orange oil-in-water emulsions (0.5% SMP, 2.5% orange oil, and 20 °C) with different NaCl concentrations.
Figure 4. Influence of NaCl concentration on the mean droplet diameter of SMP-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) at pH 3, stored at 20 °C.
was observed during storage. The reason for this phenomenon was attributed to the presence of small amounts of impurities (such as palmitic acid) in the SMP ingredient used to prepare the emulsions.12 Because most of the palmitic acid molecules are ionized at pH values above the pKa (≈4.9),13 they are negatively charged. However, the predominant form of palmitic acid is neutral at pH values below the pKa, indicating a low or almost zero net charge on the oil droplets. Therefore, the oil 3259
dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
Article
Figure 6. Photographs of 2.5 wt % orange oil-in-water emulsions stabilized by SMP and different amounts of lysolecithin after heat treatment in the presence of 0 or 300 mM NaCl.
visible phase separation at the highest salt concentrations (400 and 500 mM). Interestingly, the particle size measurements suggested that the emulsions containing 0.1% lysolecithin remained relatively small across the entire salt range (Figure 4). We hypothesize that droplet flocculation occurred in these emulsions because they exhibited rapid creaming (indicative of an increase in particle size), but still had relatively small particle sizes when measured by static light scattering. This effect can be attributed to dissociation of flocs due to dilution and stirring within the measurement chamber of the light-scattering instrument. For the emulsions containing 0.5% lysolecithin, the mean particle diameter remained relatively small for low salt concentrations (0−300 mM NaCl), but increased steeply at higher salt levels. This result suggests that phase separation occurred due to an increase in droplet diameter (coalescence), followed by droplet creaming. Droplet aggregation can be attributed to screening of the electrostatic repulsion between negatively charged emulsion droplets at increasing ionic strength, leading to droplet flocculation and/or coalescence.16 Emulsions containing 0.1% lysolecithin had a lower net charge than those containing 0.5% lysolecithin at all salt concentrations (Figure 5), which would account for the fact that they were less stable to salt addition. A low droplet charge leads to a lower energy barrier that has to be overcome before droplets can come into close proximity; therefore, less salt is needed to promote aggregation. However, the screening of the electrostatic repulsion between negatively charged emulsion droplets could not explain the difference in the origin of destabilization of emulsions stabilized with 0.1% (mainly flocculation) and 0.5% (mainly coalescence) lysolecithin. This effect may have been due to differences in interfacial composition: emulsions containing higher levels of lysolecithin are presumably more sensitive to electrolyte addition because of their charge characteristics. Electrolytes could change the hydrophilic−lipophilic balance and hydration conditions of the surfactants by depletion of the hydration shell around the polar head groups of surfactant molecules.17 Because of the greater amount of lysolecithin, which is charged negatively over a broader range of pH values, the interfacial layer of emulsions containing 0.5% lysolecithin could be much more sensitive to these alterations in the physical properties of the surfactant molecules. Even if oil droplets are able to come into close
Figure 7. Influence of heat treatment on the mean droplet diameter of SMP-stabilized orange oil-in-water emulsions (0.5% SMMP, 2.5% orange oil, and 300 mM NaCl) containing lysolecithin at pH 3.
of lysolecithin. Lysolecithin has been reported to have a negative charge over a wide range of pH values.14 Because the pKa value of the phosphate groups on lysolecithin is typically around pH 1.5,15 lysolecithin should still be negatively charged at pH 3.0. The presence of lysolecithin at the oil droplet surfaces would therefore account for the good stability of the orange oil emulsions to droplet growth at acidic conditions. Influence of Ionic Strength. Commercial food and beverage products often contain different types and amounts of mineral ions, and therefore it is important to understand how ionic strength influences their stability. We therefore examined the influence of salt addition on the stability of orange oil-inwater emulsions stabilized by a combination of SMP and lysolecithin. Visual observations of orange oil emulsions containing different salt levels were made after they were stored at room temperature for 8 h. In the absence of added salts, the emulsions were visibly stable to phase separation, but in the presence of salt many of them separated into two distinct layers: a cream layer at the top and a serum layer at the bottom (Figure 3). Emulsions containing 0.1% lysolecithin were unstable to phase separation from 50 to 500 mM, whereas emulsions containing 0.5% lysolecithin were only unstable to 3260
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proximity due to attractive interactions, the presence of more nonpolar surfactants (SMP) within the interfacial layer of emulsions containing 0.1% lysolecithin may give better resistance against coalescence. Influence of Temperature. Commercial food and beverage emulsions are commonly subjected to thermal processing, and so it is important to understand how heating influences their physical stability. In the absence of salt, all of the emulsions appeared to be visibly stable to phase separation after they were heated (Figure 6). In the presence of salt (300 mM NaCl), emulsions containing 0.1% lysolecithin were highly unstable to phase separation at all holding temperatures (from 30 to 90 °C). On the other hand, emulsions containing 0.5% lysolecithin were stable to phase separation at relatively low temperatures (30 and 40 °C), but unstable at higher temperatures. Previous studies have reported that SMPstabilized emulsions were also unstable to droplet growth when they were stored at 40 °C for prolonged periods.18 A possible reason for the destabilization of SMP-coated droplets at elevated temperatures is dehydration of the hydrophilic surfactant head groups. Dehydration leads to an alteration in the optimum curvature of the surfactant monolayer and to reduced steric repulsion between droplets.15 The fact that the emulsions were stable to droplet aggregation in the absence of salt suggests that electrostatic repulsion was strong enough to overcome any attractive interactions in these systems. In the presence of 0.5% lecithin and 300 mM NaCl, the emulsions may have become unstable to aggregation at elevated temperatures due to the combined influence of a reduction in electrostatic repulsion and in steric repulsion. Particle size analysis of emulsions exposed to different holding temperatures was also carried out (Figure 7). The mean droplet diameter remained relatively small (
Impact of Environmental Stresses on Orange Oil-in-Water Emulsions Stabilized by Sucrose Monopalmitate and Lysolecithin David Julian McClements,† Eric Andrew Decker,† and Seung Jun Choi*,§ †
Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States Department of Food Science and Technology and Department of Interdisciplinary Bio IT Materials, Seoul National University of Science and Technology, Seoul 139-743, Republic of Korea
§
ABSTRACT: The food and beverage industry is trying to replace synthetic functional ingredients with more “label-friendly” ingredients in many commercial products. This study therefore examined the influence of environmental stresses on the stability of emulsions stabilized by a combination of lysolecithin and sucrose monopalmitate (SMP). Orange oil-in-water emulsions (5% (w/w) oil) stabilized by SMP (1%) and lysolecithin (0−0.5%) were prepared using high-pressure homogenization (pH 3). In the absence of lysolecithin, all emulsions were highly unstable to droplet aggregation, which was attributed to low droplet charge (weak electrostatic repulsion) and small SMP headgroup size (weak steric repulsion). Incorporation of 0.1−0.5% lysolecithin into the emulsions greatly improved their stability to droplet aggregation, which was mainly attributed to the increase in negative charge on the droplets (strong electrostatic repulsion). The addition of high levels of salt (NaCl) to the emulsions promoted droplet aggregation and creaming. Emulsions containing 0.5% lysolecithin were stable to heating (30−90 °C) in the absence of salt, but exhibited droplet aggregation and creaming when held at high (>50 °C) temperatures in the presence of 300 mM salt. This study has implications for the development of emulsion-based delivery systems for use in food and beverage products. KEYWORDS: emulsions, environmental stress, lecithin, orange oil, sucrose monopalmitate, beverages, stability, sucrose esters
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beverage industries.11 However, at present there is a relatively poor understanding of the influence of environmental stresses that beverage products might experience during their production, transport, storage, and utilization on the properties of emulsions stabilized by sucrose monoesters. The objective of the present study was therefore to systematically study the ability of sucrose monopalmitate (SMP) to form and stabilize orange oil-in-water emulsions and to examine the influence of environmental stresses (salt and temperature) and cosurfactants (lysolecithin) on their stability. The information obtained from this study should be useful for designing food and beverage emulsions with improved properties and consumer acceptance.
INTRODUCTION Oils extracted from orange peel are widely used as flavoring ingredients in food and beverage products.1 Some studies suggest they may also contain phytochemicals that have healthpromoting effects, such as anticarcinogenic and anti-inflammatory activities.2,3 There is therefore considerable interest in incorporating orange oils into food and beverages as flavoring agents or bioactive ingredients. The majority of chemical constituents of orange oils are hydrophobic compounds that do not readily dissolve in water4 and therefore have to be incorporated into foods in the form of colloidal dispersions, such as microemulsions, nanoemulsions, or emulsions.5,6 The beverage industry is under increasing pressure to create products that are perceived as being healthy.5 Consequently, they are attempting to reformulate many of their products to remove synthetic ingredients that consumers perceive as being undesirable. Synthetic surfactants (such as “Tweens” or “polysorbates”) are commonly used commercially to fabricate emulsion-based beverage products. According to market research, the food emulsifier market will globally grow from around U.S. $2109 million in 2012 to around U.S. $2859 million by 2018 with synthetic emulsifiers accounting for about two-thirds of the total market and natural emulsifiers the rest.7 Synthetic surfactants are highly effective at forming and stabilizing emulsions, and therefore it is often difficult to find more label-friendly alternatives. Sucrose monoesters are surface-active materials produced from natural products, such as sucrose and vegetable oils.8 They have been widely used in commercial cosmetic and pharmaceutical products to stabilize emulsions due to their low toxicity, good biocompatibility, and high ecological acceptability.9,10 Sucrose monoesters are also good candidates for application as surfactants in the food and © 2014 American Chemical Society
■
MATERIALS AND METHODS
Materials. Sucrose monopalmitate and 4-fold orange oil were supplied by International Flavors and Fragrances (Union Beach, NJ, USA), and an enzyme-modified soy lecithin was supplied by Solae (SOLEC K-EML, St. Louis, MO, USA). All other chemicals were of reagent grade or purer and were obtained from Fisher Scientific (Pittsburgh, PA, USA). Emulsion Preparation. Orange oil-in-water emulsions were prepared by mixing 5% (w/w) oil phase containing different concentrations of lysolecithin with 95% (w/w) aqueous phase (1% SMP, 10 mM phosphate buffer, pH 7.0) following the method described by Choi et al.12 A coarse emulsion premix was prepared by homogenizing oil and aqueous phases using a high-speed blender (Biohomogenizer, Biospec Products, Bartlesville, OK, USA) for 2 min Received: Revised: Accepted: Published: 3257
November 6, 2013 March 10, 2014 March 10, 2014 March 10, 2014 dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
Article
Figure 1. Evolution of the particle size distribution of SMP- or SMP/lysolecithin-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) stored at 20 °C: (A) SMP-stabilized emulsion stored at pH 7; (B) SMP-stabilized emulsion stored at pH 3; (C) SMP/0.1% lysolecithinstabilized emulsion at pH 3; (D) SMP/0.5% lysolecithin-stabilized emulsion at pH 3. at 10000 rpm at room temperature. For reduction of droplet size, the premixed emulsions were homogenized by five passes through a microfluidizer (M-100L, Microfluidics, Newton, MA, USA) at 48.3 MPa. Then, the emulsion was adjusted to pH 3.0 using 1.0 and 0.1 N hydrochloric acid solutions. The final concentrations of lysolecithin in the whole emulsions varied from 0 to 0.5% (w/w), and the final concentration of SMP in the whole emulsions was 1% (w/w). To avoid the effect of light, all samples were stored in the dark before analysis. Stability to Environmental Stresses. To determine the stability of SMP/lysolecithin-stabilized emulsions against heat treatment, emulsions were placed in glass test tubes and then incubated in a water bath for 15 min at different temperatures (30−90 °C). The emulsions were then allowed to cool to room temperature and held overnight prior to subsequent analysis. To evaluate the stability of SMP/lysolecithin-stabilized emulsions against the change in the electrostatic interaction, a series of 2.5 wt % emulsions with different NaCl concentrations was prepared by diluting the concentrated emulsions with 10 mM phosphate buffer (pH 3.0) containing different concentrations of sodium chloride. Then, the emulsions were kept at room temperature overnight prior to subsequent analysis. Droplet Size Measurements. Mean droplet diameters of emulsions were measured using static light scattering (laser diffraction). To avoid multiple scattering effects, all emulsion samples were diluted to a droplet concentration of approximately ≈0.005% (w/ w) using buffer solution at the pH of the sample and stirred continuously throughout the measurements to ensure they were homogeneous. The droplet size distribution of the emulsions was then measured using a commercial static light scattering instrument (Mastersizer 2000, Malvern Instruments, Worcestershire, UK).
Droplet size data are reported as either the volume-surface mean diameter, d32 = (∑nidi3/∑nid2i ), or the volume-weighted mean diameter, d43 = (∑nid4i /∑nid3i ), where ni is the number of droplets with diameter di. If an emulsion sample to be analyzed had phaseseparated due to droplet creaming, then it was gently mixed to evenly disperse the droplets prior to collection of an aliquot for making the particle size measurements. Electrical Charges of Oil-in-Water Emulsions. The ζ-potential of the emulsion droplets was measured using a microelectrophoresis instrument (ZetaSizer Nano, Malvern Instruments). The ζ-potential was calculated from measurement of the electrophoretic mobility of droplets in an applied oscillating electric field using laser Doppler velocimetry. Samples were prepared by diluting emulsions 1:500 with buffer solution at the pH of the sample and placing the diluted emulsions into disposable capillary cells (DTS1060, Malvern Instruments). If an emulsion sample to be analyzed had phase-separated due to creaming, then it was evenly dispersed by gentle stirring before collection of an aliquot for analysis. Statistical Analysis. Experiments were repeated two or three times using freshly prepared samples, and the mean and standard deviations were calculated from these measurements.
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RESULTS AND DISCUSSION Influence of Lysolecithin. Previously, we examined the influence of pH on the stability of SMP-stabilized orange oil emulsions.12 Stable orange oil emulsions with small mean droplet diameters (d43 < 0.15 μm) could be formed at neutral pH. However, when the emulsion was stored under acidic conditions (pH 3−5), a large increase in mean droplet diameter 3258
dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
Article
Figure 5. Influence of NaCl concentration on the electrical charge of SMP-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) at pH 3, stored at 20 °C.
Figure 2. Influence of lysolecithin concentration on the ζ-potential of SMP-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) at pH 3, stored at 20 °C.
droplets have a high negative charge at high pH values, which stabilizes them from aggregation due to electrostatic repulsion. On the other hand, they have a low net charge at low pH and are therefore susceptible to aggregation due to the dominance of attractive colloidal interactions (such as van der Waals or hydrophobic attractions). We therefore examined the impact of adding an anionic cosurfactant (lysolecithin) on the aggregation stability of orange oil-in-water emulsions under acid conditions (Figure 1). We hypothesized that this cosurfactant would improve the physical stability of the emulsions by increasing the electrostatic repulsion between the droplets. Emulsions were initially prepared under neutral pH conditions to ensure that they were stable. The initial mean droplet diameter at pH 7 was around 0.125 μm for all of the emulsions prepared (Figure 1A), independent of the lysolecithin concentration present, which suggested that the initial particle size was primarily governed by the homogenization conditions or the SMP. All of the emulsions were then adjusted to pH 3 and stored for 15 days. In the absence of lysolecithin, the emulsions were highly unstable to droplet growth at pH 3, which was attributed to droplet coalescence during storage (Figure 1B). Conversely, in the presence of lysolecithin (0.1−0.5%), all of the emulsions were stable to droplet growth throughout 15 days of storage (Figures 1C,D). These results indicated that lysolecithin was an effective cosurfactant for improving the stability of SMP-coated droplets to growth, which can be attributed to the increased electrostatic repulsion between the droplets in the presence of this anionic surface active substance. Figure 2 shows the ζ-potential of the SMP-stabilized orange oil emulsions with various lysolecithin concentrations. The ζpotential of the oil droplets was close to zero (−1.7 mV) in the absence of lysolecithin, but it became increasingly negative as the amount of lecithin incorporated into the system increased. The ζ-potential reached a value of around −35 mV at 0.5% lysolecithin, which should be large enough to provide good stability through electrostatic repulsion. Theoretically, sucrose esters containing SMP are nonionic surfactants,8 but they may have a negative charge at high pH because of the presence of ionized impurities, such as palmitic acid.12 As described above, palmitic acid should have little charge at acid pH, which would account for the low net charge on the emulsions in the absence
Figure 3. Photographs of SMP-stabilized orange oil-in-water emulsions (0.5% SMP, 2.5% orange oil, and 20 °C) with different NaCl concentrations.
Figure 4. Influence of NaCl concentration on the mean droplet diameter of SMP-stabilized orange oil-in-water emulsions (1% SMP and 5% orange oil) at pH 3, stored at 20 °C.
was observed during storage. The reason for this phenomenon was attributed to the presence of small amounts of impurities (such as palmitic acid) in the SMP ingredient used to prepare the emulsions.12 Because most of the palmitic acid molecules are ionized at pH values above the pKa (≈4.9),13 they are negatively charged. However, the predominant form of palmitic acid is neutral at pH values below the pKa, indicating a low or almost zero net charge on the oil droplets. Therefore, the oil 3259
dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
Article
Figure 6. Photographs of 2.5 wt % orange oil-in-water emulsions stabilized by SMP and different amounts of lysolecithin after heat treatment in the presence of 0 or 300 mM NaCl.
visible phase separation at the highest salt concentrations (400 and 500 mM). Interestingly, the particle size measurements suggested that the emulsions containing 0.1% lysolecithin remained relatively small across the entire salt range (Figure 4). We hypothesize that droplet flocculation occurred in these emulsions because they exhibited rapid creaming (indicative of an increase in particle size), but still had relatively small particle sizes when measured by static light scattering. This effect can be attributed to dissociation of flocs due to dilution and stirring within the measurement chamber of the light-scattering instrument. For the emulsions containing 0.5% lysolecithin, the mean particle diameter remained relatively small for low salt concentrations (0−300 mM NaCl), but increased steeply at higher salt levels. This result suggests that phase separation occurred due to an increase in droplet diameter (coalescence), followed by droplet creaming. Droplet aggregation can be attributed to screening of the electrostatic repulsion between negatively charged emulsion droplets at increasing ionic strength, leading to droplet flocculation and/or coalescence.16 Emulsions containing 0.1% lysolecithin had a lower net charge than those containing 0.5% lysolecithin at all salt concentrations (Figure 5), which would account for the fact that they were less stable to salt addition. A low droplet charge leads to a lower energy barrier that has to be overcome before droplets can come into close proximity; therefore, less salt is needed to promote aggregation. However, the screening of the electrostatic repulsion between negatively charged emulsion droplets could not explain the difference in the origin of destabilization of emulsions stabilized with 0.1% (mainly flocculation) and 0.5% (mainly coalescence) lysolecithin. This effect may have been due to differences in interfacial composition: emulsions containing higher levels of lysolecithin are presumably more sensitive to electrolyte addition because of their charge characteristics. Electrolytes could change the hydrophilic−lipophilic balance and hydration conditions of the surfactants by depletion of the hydration shell around the polar head groups of surfactant molecules.17 Because of the greater amount of lysolecithin, which is charged negatively over a broader range of pH values, the interfacial layer of emulsions containing 0.5% lysolecithin could be much more sensitive to these alterations in the physical properties of the surfactant molecules. Even if oil droplets are able to come into close
Figure 7. Influence of heat treatment on the mean droplet diameter of SMP-stabilized orange oil-in-water emulsions (0.5% SMMP, 2.5% orange oil, and 300 mM NaCl) containing lysolecithin at pH 3.
of lysolecithin. Lysolecithin has been reported to have a negative charge over a wide range of pH values.14 Because the pKa value of the phosphate groups on lysolecithin is typically around pH 1.5,15 lysolecithin should still be negatively charged at pH 3.0. The presence of lysolecithin at the oil droplet surfaces would therefore account for the good stability of the orange oil emulsions to droplet growth at acidic conditions. Influence of Ionic Strength. Commercial food and beverage products often contain different types and amounts of mineral ions, and therefore it is important to understand how ionic strength influences their stability. We therefore examined the influence of salt addition on the stability of orange oil-inwater emulsions stabilized by a combination of SMP and lysolecithin. Visual observations of orange oil emulsions containing different salt levels were made after they were stored at room temperature for 8 h. In the absence of added salts, the emulsions were visibly stable to phase separation, but in the presence of salt many of them separated into two distinct layers: a cream layer at the top and a serum layer at the bottom (Figure 3). Emulsions containing 0.1% lysolecithin were unstable to phase separation from 50 to 500 mM, whereas emulsions containing 0.5% lysolecithin were only unstable to 3260
dx.doi.org/10.1021/jf404983p | J. Agric. Food Chem. 2014, 62, 3257−3261
Journal of Agricultural and Food Chemistry
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proximity due to attractive interactions, the presence of more nonpolar surfactants (SMP) within the interfacial layer of emulsions containing 0.1% lysolecithin may give better resistance against coalescence. Influence of Temperature. Commercial food and beverage emulsions are commonly subjected to thermal processing, and so it is important to understand how heating influences their physical stability. In the absence of salt, all of the emulsions appeared to be visibly stable to phase separation after they were heated (Figure 6). In the presence of salt (300 mM NaCl), emulsions containing 0.1% lysolecithin were highly unstable to phase separation at all holding temperatures (from 30 to 90 °C). On the other hand, emulsions containing 0.5% lysolecithin were stable to phase separation at relatively low temperatures (30 and 40 °C), but unstable at higher temperatures. Previous studies have reported that SMPstabilized emulsions were also unstable to droplet growth when they were stored at 40 °C for prolonged periods.18 A possible reason for the destabilization of SMP-coated droplets at elevated temperatures is dehydration of the hydrophilic surfactant head groups. Dehydration leads to an alteration in the optimum curvature of the surfactant monolayer and to reduced steric repulsion between droplets.15 The fact that the emulsions were stable to droplet aggregation in the absence of salt suggests that electrostatic repulsion was strong enough to overcome any attractive interactions in these systems. In the presence of 0.5% lecithin and 300 mM NaCl, the emulsions may have become unstable to aggregation at elevated temperatures due to the combined influence of a reduction in electrostatic repulsion and in steric repulsion. Particle size analysis of emulsions exposed to different holding temperatures was also carried out (Figure 7). The mean droplet diameter remained relatively small (
