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Ind. Eng. Chem. Res. 2008, 47, 9108–9114
Emulsion Texture and Stability: Role of Surfactant Micellar Interactions in the Presence of Proteins Youngsun Kong, Alex Nikolov, Darsh Wasan,* and Akihiro Ogawa† Illinois Institute of Technology, Chicago, Illinois
This article presents the results of our recent research on the texture and stability of oil-in-water emulsions containing sucrose ester and proteins. We used both the direct microscopic imaging and nondestructive backlight scattering (Kossel diffraction) techniques to evaluate the emulsion texture and the energy barrier between droplets for two different emulsifier compositions with and without the proteins present. The microinterferometric method employing our capillary force balance was used to study the stability of the confined thin film (containing surfactant micelles and proteins) between two droplets. In addition to the film stability, we also measured the second virial coefficient of the micellar solutions with and without protein and assessed the intermicellar interaction and related it to the stabilities of the emulsions prepared using two different emulsifier compositions. The effect of protein on the oil-in-water emulsion stability was also assessed and was found to lead to the depletion attraction between droplets, resulting in a less stable emulsion. The results offer new insight into the understanding of how the micellar interactions in the presence of proteins affect emulsion texture and stability. 1. Introduction Many food beverages, such as “coffee milk”, are oil-in-water emulsions. Emulsions are thermodynamically unstable systems containing oil (or fat) and water. The two phases separate over time because of their density differences. The primary modes of destabilization of emulsions are creaming, flocculation, and the coalescence of droplets.1,2 Emulsifiers (surface active agents) are normally used to lower the interfacial tension; this improves the emulsion stability by reducing the average oil drop size and preventing the oil droplets from flocculating and coalescing. Over the past two decades, we have conducted research in our laboratory on the mechanisms of foam and emulsion stability in a variety of industrial systems using surfactants, micelles, proteins, and fine particles as stabilizers.3,4 We have also used statistical mechanical theories and computer simulations to rationalize the experimental findings. Our present article particularly highlights the role of surfactant micellar interactionss with and without protein presentsin affecting emulsion texture and stability.
two solutions were mixed at 60 °C, after which the soybean oil was added to the blended solution. The blended solution was mixed well using a homomixer at 10 000 rpm for 10 min, resulting in a coarse emulsion. Homogenization was conducted, yielding an average oil drop size of 1.3 µm. The emulsion was then sterilized at 121 °C for 40 min. The prepared emulsion sample was placed into a glass vial 10 cm in height, and the average oil drop size and drop size distribution were observed over time both in the bulk and inside the creaming layer (ring) formed near the air-emulsion interface.
2. Experiments The model beverage system of an oil-in-water emulsion was prepared using soybean oil and sucrose ester as emulsifiers. Two types of sucrose esters, palmitric acid (Ryoto Sugar Ester P1670) and steric acid (Ryoto Sugar Ester S570), were used. The structure of the emulsifier and the emulsion composition are depicted in Figure 1. The critical micelle concentration (CMC) of the sucrose ester emulsifier was about 10-5 mol/L (∼0.0006 wt %), determined from interfacial tension measurements. The density of the soybean oil was 0.92 g/cm3 at 20 °C. The emulsion contained 0.7 wt % soybean oil, 0.06 wt % sugar ester (about 100 times its CMC), 0.54 wt % caseinate, 0.16 wt % whey protein isolate, 0.13 wt % sodium bicarbonate (final pH of 7.0), and water. The micellar solution containing sucrose ester and a protein solution were each first dissolved in water at 60 °C. Then, the * To whom the correspondence should be addressed: email:
[email protected] † Mitsubishi Chemical Ltd., Yokohama, Japan.
Figure 1. Emulsifier structure and composition of the two emulsions.
10.1021/ie8001815 CCC: $40.75 2008 American Chemical Society Published on Web 10/11/2008
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Figure 3. Oil drop size distributions inside the ring. Figure 2. Oil drop size distributions in the bulk for the two different emulsifier compositions.
The interfacial tension between the oil and water phases in the presence of the emulsifiers was measured using drop shape analysis.5 The oil droplet was formed in the micellar solution. The oil droplet volume was increased, and the image was captured using a goniometer (Kernco Instruments Co., Inc.). As the oil droplet volume increased, the oil droplet eventually reached the maximum height, at which point the hydrostatic pressure balances the capillary pressure. The interfacial tension was calculated using the Laplace equation. The nondestructive light back-scattering technique (also known as Kossel diffraction) was used to obtain quantitative information on the collective droplet interactions inside the emulsion system. The technique involves shining a monochromatic laser beam on an emulsion sample in a glass cell and measuring the back-scattered light with a vertically polarized charged-complex digital camera. The diffraction pattern was transformed into an intensity profile and analyzed to give the structure factor that reveals the emulsion’s microstructure. More information about this technique is in our previous article.6 We used a capillary force balance in conjunction with common interference microscopy to measure the film-thinning process, the film thickness, and the film stability between a pair of approaching emulsion droplets in the presence of micellar solutions and proteins.7 We also used a refractometer to determine the second virial coefficient of the micellar solutions both with and without the protein present by measuring the turbidity and the refractive index at a temperature of 25 °C as a function of the emulsifier concentration. We calculated the osmotic pressure using the second virial coefficient. 3. Results and Discussion 3.1. Drop Size Distribution. The stability of the oil-in-water emulsion was evaluated by monitoring the drop size distributions both in the bulk and inside the creaming layer (the ring formed near the air-emulsion interface). Figure 2 shows plots of the percent oil volume in the bulk versus the droplet diameter (in microns) over a period of 30 days for two different emulsifier compositions. The photographs depict the microscopic images of the droplets in the emulsion samples at 25 °C after homogenization and after 30 days. The average oil drop size distribution in the bulk changed from 1.3 ( 0.24 to 1.4 ( 0.29 µm for the combined high- and low-HLB system, whereas the
average oil droplet size changed from 1.3 ( 0.24 to 1.6 ( 0.35 µm for the high-HLB (P1670) sample. Figure 3 shows plots of the oil drop size distributions over time inside the ring. Over time, the differences in the drop size become more significant. For the high-HLB emulsifier, the drop size increased to 2.5 ( 0.64 µm, from an initial value of 1.3 µm, whereas it was 2.2 ( 0.54 µm for the combined system. The content of large droplets (4-5 µm) was 20 vol % for the high-HLB system compared to about 10 vol % for the combined system. Therefore, one would expect a more stable emulsion for the combined-emulsifier system. Our interfacial tension data indicated that the interfacial tension was lower (∼1.8 mN/m2) for the combined system than for the high-HLB emulsifier system alone (∼2.3 mN/m2), suggesting that the combined system would result in a more stable emulsion sample with smaller drop sizes. This is indeed what we observed experimentally, as indicated by the data in Figures 2 and 3. 3.2. Competitive Adsorption. To understand the role of the surfactant in a mixed emulsion system containing both protein and surfactant, we estimated the surface coverage of surfactant around oil droplets using the Gibbs adsorption isotherm. The area per molecule for sucrose ester is 0.58 nm2/molecule. For an emulsion system containing 0.7 wt % soybean oil with an average droplet diameter of 1.3 µm, the total surface area of oil droplets is 3.23 × 105 cm2/L. Therefore, the total surface area occupied by the sucrose ester surfactant molecules is 5.80 × 10-15 cm2. Consequently, the concentration of the surfactant covering 100% of the surface area of the droplets is 9.25 × 10-5 mol/L. Because the initial concentration of sucrose ester in the bulk aqueous phase is 1.0 × 10-3 mol/L (i.e., 100 times its CMC), the bulk concentration is diminished by only 9.07 × 10-4 mol/L as a result of surface adsorption. Hence, after adsorption of the surfactant around all oil droplets, there is still 90% of the surfactant left in the micellar form in the bulk aqueous phase. Therefore, the presence of both the surfactant micelles and proteins needs to be considered in affecting emulsion stability. 3.3. Interdroplet Interactions inside the Ring. The emulsion ring forms because of the increase in the oil volume fraction in the creaming layer, and the size of the ring increases over time. We used the Kossel diffraction technique to obtain information on the development of the microstructure as quantified by the structure factor inside the ring, which depends on the interdroplet interactions. The radial distribution function, which describes the droplet distribution, was calculated from the measured structure factor by a Fourier transform. The
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Figure 4. Radial distribution functions and interaction energy.
effective pair potential (i.e., potential of the mean force), which is the potential (free) energy between two droplets in the presence of other droplets (i.e., the collective interaction), was then calculated from the radial distribution function using the Boltzmann energy equation.6 Figure 4b,c depicts the radial distribution function and the effective pair potential for the P1670 and P1670/S570 systems, respectively. Figure 4a also shows the photographs of the Kossel diffraction patterns after 30 days. The energy barriers for the two emulsifier systems are marked in Figure 4c. The structural energy barrier between the droplets (difference in the energy between the first maximum and the second minimum) of the combined emulsifier system is about 2.2kT, whereas it is only 1.5kT for the single-emulsifier system. Both energy barriers are small, but the value for the combined system is higher than that for the single micellar sample, which suggests that the combined high- and low-HLB emulsifier should result in a more stable emulsion system. In fact, the drop size distribution was found to be narrower for the combined-emulsifier system than for the high-HLB system alone (Figure 3). The increase in the average oil drop size was also less for the combined system, which resulted in a more stable emulsion. 3.4. Effect of Protein on Emulsion Stability. The effects of 0.7 wt % protein (0.54 wt % caseinate plus 0.16 wt % whey protein isolate) combined with high- and low-HLB emulsifiers (0.06 wt %/0.02 wt %) on the emulsion stability, such as oil drop size distribution and the degree of clustering, were studied. First, we studied the effect of protein on emulsion stability using image analysis for the coarse emulsions. The average oil drop sizes with and without protein after the high-speed mixing were 3.0 ( 1.3 and 3.1 ( 1.2 µm, respectively. The coarse emulsions
with and without protein had similar average oil drop sizes after the high-speed mixing. However, we observed more clusters in the presence of protein, as shown in Figure 5b. The degree of clustering (calculated by examining more than 1200 droplets) of the coarse emulsion containing protein is higher (13%) than that (7%) of the coarse emulsion without protein. At a 0.7 wt % protein concentration, the presence of protein enhances the droplet depletion attraction between droplets, which results in clustering of droplets. The coarse emulsions with and without protein were homogenized to produce a narrowly distributed droplet size. The average oil drop size of the combined high- and low-HLB emulsifier composition after homogenization decreased from 3.1 ( 1.2 to 1.2 ( 0.23 µm without protein and from 3.0 ( 1.3 to 1.3 ( 0.24 µm with protein (compare Figures 2 and 5). Initially, the average oil drop size after homogenization was about the same (1.2 vs 1.3 µm). However, over time, the oil droplets in the emulsion creamed to the top and formed a concentrated layer (ring). We observed the change in the oil drop size distribution inside the ring after 30 days (Figure 6). The average oil drop size increased from 1.2 ( 0.23 to 1.5 ( 0.40 µm after 30 days without the presence of proteins. The average oil drop size with protein increased more (from 1.3 ( 0.24 to 2.2 ( 0.54 µm) after 30 days. The presence of 0.7 wt % protein induced the depletion attraction between oil droplets, leading to flocculation and an increase in the average oil drop size. The percent oil volume of large droplets (4-5 µm) inside the ring increased after 30 days, as seen in Figure 3. In general, interdroplet coalescence makes the oil drop size distribution broader with one maximum peak over time. However, our results show that the oil drop size distribution curve inside the ring has two maxima: one is a main broader peak, and the other is a small bump in the higher size range (Figure 3). The appearance of the second peak for large droplets suggests that the Ostwald ripening phenomenon might be occurring inside the ring after 30 days. We plan to explore this possibility in a future study. 3.5. Intermicellar Interactions. To better understand the differences in emulsion stability using the high-HLB and the combined high- and low-HLB micellar systems, we examined the role of intermicellar interactions and protein using the liquidfilm-thinning approach.8 We also determined the second virial coefficients of the micellar solutions with and without the protein present. Liquid Film Stability. The stability of an emulsion system is related to the thinning of the liquid film between two approaching oil drops. We employed a capillary force balance using light interference microscopy to study the film-thinning process and the film thickness stability. Figure 7 shows a sketch of the capillary force balance and photomicrographs displaying the micellar-layering phenomenon inside the emulsion film for the two emulsifier systems with and without protein. The film formed from the high-HLB micellar solution thinned from 75 to about 10 nm in a stepwise manner, and black spots (i.e., areas containing no micelles) appeared in the middle of the film when the size of the film increased (Figure 7b). The interferogram revealed the effective size of the micelle was about 55 ( 5 nm in diameter. However, this film was unstable, so the drops tended to flocculate. However, the film formed from the combined micellar solution of P1670/S570 thinned in a different manner (Figure 7c). This film thinned to about 75 nm and remained stable at this thickness; it did not thin any further when its size was increased or decreased.
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Figure 5. Microscopic images and degree of clustering after the high-speed mixing of the combined P1670/S570 emulsifier composition without and with protein.
Figure 6. Effect of protein on the oil drop size distribution inside the ring after 30 days for the combined high- and low-HLB emulsifier compositions.
The liquid film formed from the combined micellar solution of P1670/S570 with protein had one stepwise thinning of 40nm thickness (Figure 7d). This is because the protein molecules
(i.e., submicelles of about 20-nm diameter) stay inside the film, causing depletion attraction leading to droplet flocculation (Figure 5b). The liquid film containing 0.7 wt % protein is less
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Figure 7. (a) Schematic of the microinterferometric method and (b-d) stepwise film-thinning processes of the (b) high-HLB emulsifier (P1670), (c) combined high- and low-HLB emulsifier (P1670/S570), and (d) combined high- and low-HLB emulsifier with protein.
stable (thinner film, 40 nm) than the film with no protein (thicker film, 75 nm). Second Virial Coefficient. To understand why the film formed from the combined-emulsifier system remained stable, we conducted measurements to determine the second virial coefficients of the micellar solution with and without protein. Both turbidimetric and refractive index measurements were made. The data are shown as Debye plots in Figure 8. The value of Hc/τ was plotted against the micellar concentration (c), where τ is the turbidity of the micellar solution. The values of H were obtained from the equation9 H)
32π3n02
( dndc )
3NAλ4
2
(1)
where n0 is the refractive index of the solvent, λ is the wavelength of light at which the measurements were made, NA is Avogadro’s number, and dn/dc is the refractive index gradient. The latter was determined from measurements of the refractive index as a function of the emulsifier concentration. According to the Debye equation Hc 1 ) + 2Bc τ M
(2)
where M is the molecular weight of the micelle and B is the second virial coefficient. The latter was evaluated from the slope of the Debye plots (Figure 8). The second virial coefficient of a micellar solution characterizes the micellar interactions. The combined high- and lowHLB emulsifier system (P1670/S570) had a higher overall
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Figure 8. Debye plots for the high-HLB and combined high- and low-HLB emulsifier compositions and for the combined-emulsifier system with protein.
positive value for the second virial coefficient (B ) 7.5 × 10-3 mol cm3/g2) than did the high-HLB emulsifier alone (B ) 4.9 × 10-3 mol cm3/g2). The higher positive value of the second virial coefficient for the combined-emulsifier system signifies a higher repulsive interaction between the micelles, implying a greater emulsion film stability. In fact, our experimental observations of film thickness stability confirmed the predicted outcome. The second virial coefficient of micellar solution containing protein was also studied. The second virial coefficient of the combined high- and low-HLB emulsifier system containing protein (Figure 8b) was 1.7 × 10-3 mol cm3/g2, which is lower than that without protein (B ) 7.5 × 10-3 mol cm3/g2). We can conclude that the presence of 0.7 wt % protein reduces the second virial coefficient through the depletion attraction among protein submicelles. Consequently, the emulsion is less stable. The second virial coefficient of the high- HLB emulsifier system alone containing protein was 1.2 × 10-3 mol cm3/g2. Osmotic Pressure. We calculated the osmotic pressure from the second virial coefficient using the van’t Hoff equation Πosm 1 ) + Bc (3) RTc M where Πosm is the osmotic pressure, c is the concentration, R is the gas constant, and T is the temperature. The combined high- and low-HLB emulsifier (with the higher second virial coefficient) with protein had a greater osmotic
pressure of 1500 dyn/cm2 compared to the value of 940 dyn/ cm2 for the high-HLB emulsifier with protein. Micellar Film Interaction Energy. We calculated the film interaction energy using the osmotic pressure obtained from the measurement of the second virial coefficient and the equation proposed by Trokhymchuk et al.10 Trokhymchuk et al. 10 derived a simple analytic expression for the film interaction energy as a function of the film thickness and volume fraction of the micelles. The equation for the film interaction energy per unit area is given by W(H) ) -P(d - H) - 2σ
0
