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Comparison of Droplet Flocculation in Hexadecane Oil-in-Water Emulsions Stabilized by β-Lactoglobulin at pH 3 and 7 H.-J. Kim, E. A. Decker, and D. J. McClements* Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003 Received May 4, 2004 The influence of surface and thermal denaturation of adsorbed β-lactoglobulin (β-Lg) on the flocculation of hydrocarbon oil droplets was measured at pH 3 and compared with that at pH 7. Oil-in-water emulsions (5 wt % n-hexadecane, 0.5 wt % β-Lg, pH 3.0) were prepared that contained different levels of salt (0-150 mM NaCl) added immediately after homogenization. The mean particle diameter (d43) and particle size distribution of diluted emulsions were measured by laser diffraction when they were either (i) stored at 30 °C for 48 h or (ii) subjected to different thermal treatments (30-95 °C for 20 min). In the absence of salt, little droplet flocculation was observed at pH 3 or 7 because of the strong electrostatic repulsion between the droplets. In the presence of 150 mM NaCl, a progressive increase in mean particle size with time was observed in pH 7 emulsions during storage at 30 °C, but no significant change in mean particle diameter with time (d43 ∼ 1.4 ( 0.2 µm) was observed in the pH 3 emulsions. Droplet aggregation became more extensive in pH 7 emulsions containing salt (added before thermal processing) when they were heated above 70 °C, which was attributed to thermal denaturation of adsorbed β-Lg leading to interdroplet disulfide bond formation. In contrast, the mean particle size decreased and the creaming stability improved when pH 3 emulsions were heated above 70 °C. These results suggest that the droplets in the pH 3 emulsions were weakly flocculated at temperatures below the thermal denaturation temperature of β-Lg (T < 70 °C) but that flocs did not form so readily above this temperature, which was attributed to a reduction in droplet surface hydrophobicity due to protein conformational changes. The most likely explanation for the difference in behavior of the emulsions is that disulfide bond formation occurs much more readily at pH 7 than at pH 3.
Introduction Many globular proteins are used as emulsifiers because of their ability to facilitate the formation and improve the stability of oil-in-water emulsions.1-6 Proteins rapidly adsorb to the surface of oil droplets formed by mechanical agitation of an oil-water-protein mixture using a homogenizer, where they facilitate further droplet disruption by lowering the interfacial tension and retard droplet coalescence within the homogenizer by forming protective membranes around the droplets.2,3,7,8 The ability of proteins to generate repulsive interactions (e.g., steric and electrostatic) between oil droplets also plays an important role in stabilizing the droplets against coalescence and flocculation after homogenization.7-10 There has therefore been a considerable research effort to understand the factors that influence the interfacial behavior of adsorbed * Corresponding author. Tel: +1-413-545-1019. Fax: +1-413545-1262. E-mail:
[email protected]. (1) Dickinson, E. Introduction to Food Colloids; Oxford University Press: Oxford, 1992. (2) Damodaran, S. In Food Chemistry, 3rd ed.; Fennema, O. R., Ed.; Marcel Dekker: New York, 1996; p 321. (3) Dalgleish, D. G. In Emulsions and Emulsion Stability; Sjoblom, J., Ed.; Marcel Dekker: New York, 1996. (4) Nakai, S.; Modler, H. W. Food Proteins: Properties and Characterization; VCH: New York, 1996. (5) Euston, S. R.; Hirst, R. L. J. Food Sci. 2000, 65, 934-940. (6) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176-181. (7) Dickinson, E. Colloids Surf., B 1999, 15, 161-176. (8) McClements, D. J. Food Emulsions: Principles, Practice and Techniques; CRC Press: Boca Raton, FL, 1999. (9) Bergensthanl, B. A.; Claesson, P. M. In Food Emulsions, 3rd ed.; Friberg, S. E., Larsson, K., Eds.; Marcel Dekker: New York, 1997; pp 57-109. (10) Walstra, P. In Physical Chemistry of Foods; Marcel Dekker: New York, 2003.
proteins and to elucidate the relationship between interfacial characteristics and bulk physicochemical properties of emulsions.11-16 In previous studies, we have examined the influence of surface and thermal denaturation of adsorbed β-lactoglobulin (β-Lg) on the stability and physicochemical properties of oil-in-water emulsions at neutral pH.14,15 In the absence of added salt, the protein-stabilized emulsions exhibited no droplet flocculation when stored at room temperature (30 °C for 48 h) or when heated (from 30 to 95 °C for 20 min). On the other hand, in the presence of 150 mM NaCl, appreciable droplet flocculation was observed in the emulsions during storage at room temperature, and a much greater degree of droplet flocculation was observed when the emulsions were heated above the thermal denaturation temperature of the adsorbed proteins (Tm ∼ 70 °C). Interestingly, there was little evidence of droplet flocculation in emulsions that were heated above Tm in the absence of salt, and then 150 mM NaCl was added after they had been cooled to room temperature.15 The origin of droplet flocculation in β-Lg-stabilized emulsions can be attributed to the influence of protein (11) Fang, Y.; Dalgleish, D. G. J. Colloid Interface Sci. 1997, 196, 292-298. (12) Mackie, A. R.; Husband, F. A.; Holt, C.; Wilde, P. J. Int. J. Food Sci. Technol. 1999, 34, 509-516. (13) Euston, S. R.; Finningan, S. R.; Hirst, R. L. Food Hydrocolloids 2000, 14, 155-161. (14) Kim, H.-J.; Decker, E. A.; McClements, D. J. J. Agric. Food Chem. 2002, 50, 7131-7137. (15) Kim, H.-J.; Decker, E. A.; McClements, D. J. Langmuir 2002, 18, 7577-7583. (16) Norde, W. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 27-54.
10.1021/la048899b CCC: $27.50 © 2004 American Chemical Society Published on Web 06/11/2004
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conformation and salt on the colloidal and chemical reactions between the oil droplets. β-Lg undergoes conformational changes after adsorption to the surfaces of oil droplets because of the alteration in its molecular environment.16 These conformational changes lead to exposure of reactive amino acid residues originally located in the hydrophobic interior of the native protein, for example, nonpolar groups or sulfur-containing groups.17,18 Exposure of nonpolar groups leads to increased hydrophobic interactions between surface-denatured protein molecules,19 whereas exposure of sulfur-containing groups leads to disulfide bond formation or disulfide interchange reactions at neutral pH.17,20 In the absence of salt, the increased hydrophobic attraction between the emulsion droplets is insufficient to promote flocculation because of the relatively strong electrostatic repulsion. On the other hand, in the presence of sufficient salt the electrostatic repulsion is screened and the hydrophobic attraction is large enough to promote droplet flocculation. Once the droplets come into close proximity, then disulfide bonds may form between protein molecules adsorbed onto different droplets, which strengthens the bonds holding the droplets in the flocs together. In the present study, we compare the influence of surface and thermal denaturation of adsorbed β-Lg on the stability of oil-in-water emulsions at pH 3 and 7 to establish if the protein behaves differently under dissimilar solution conditions. Emulsion stability was studied at pH 3 and 7 because these values are sufficiently far below or above the isoelectric point of the protein to ensure that the droplet charge is large enough to prevent flocculation (in the absence of electrostatic screening effects). In addition, there are many commercially important protein-stabilized emulsions that have pH values close to these values, for example, beverages, infant formula, and dairy products. We postulated that the behavior of the proteins in the emulsions would be different at these two pH values because the conformation, stability, and association of β-Lg is pH dependent21,22 and because β-Lg is able to form disulfide bonds much more readily at higher pH values.23 The information obtained from this study will have important implications for the formulation and production of oil-in-water emulsions stabilized by globular proteins. Materials and Methods Materials. Analytical grade sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium azide (NaN3), Tween 20, 2-mercaptoethanol, and n-hexadecane were purchased from the Sigma Chemical Co. (St. Louis, MO). Powdered β-lactoglobulin was obtained from Davisco Foods International (lot no. JE 001-1-922, Le Sueur, MN). As stated by the manufacturer, the β-Lg content of the powder determined by electrophoresis was 98% (the remainder being mostly globulins). The decrease in mass of the protein powder upon drying was 2.6%, and the nitrogen content of the powder was 15.6%. Distilled and deionized water was used for the preparation of all solutions. Solution Preparation. Emulsifier solutions were prepared by dispersing 1 wt % of powdered β-Lg into 5 mM imidazole/ (17) Dickinson, E.; Matsumura, Y. Int. J. Biol. Macromol. 1991, 13, 26-32. (18) Monahan, F. J.; McClements, D. J.; Kinsella, J. E. J. Agric. Food Chem. 1993, 41, 1826-1831. (19) McClements, D. J.; Monahan, F. J.; Kinsella, J. E. J. Food Sci. 1993, 58, 1036-1039. (20) Apenten, R. K. O.; Galani, D. J. Sci. Food Agric. 2000, 80, 447452. (21) Tanford, C.; De, P. K. J. Biol Chem. 1961, 236, 1711-1961. (22) Kinsella, J. E.; Whitehead, D. M. Adv. Food Nutr. Res. 1989, 33, 343-438. (23) Monahan, F. J.; McClements, D. J.; German, J. B. J. Food Sci. 1996, 61, 504-510.
Kim et al. acetate buffer (pH 3.0) containing 0.04 wt % NaN3 (as an antimicrobial agent) and stirring for at least 2 h to ensure complete dispersion. Solutions containing different NaCl concentrations were prepared by dispersing weighed amounts of the powdered material into the same buffer (pH 3.0). Emulsion Preparation. An oil-in-water emulsion was prepared by homogenizing 10 wt % n-hexadecane oil and 90 wt % emulsifier solution (1 wt % β-Lg in 5 mM imidazole/acetate buffer) at room temperature. The oil and emulsifier solution were blended using a high-speed blender for 2 min (model 33BL79, Waring Inc., New Hartford, CT) and then passed through a high-pressure valve homogenizer five times at 7500 psi (Rannie High Pressure, APV-Gaulin, model Mini-Lab 8.30H, Wilmington, MA). The pH of this emulsion was readjusted to 3.0 using HCl solution (pH Meter 320, Corning Inc., Corning, NY). The emulsions were then diluted to 5 wt % oil with 5 mM imidazole/acetate buffer (pH 3) containing salt either before or after heat treatment to obtain a final NaCl concentration of either 0 or 150 mM. In the isothermal storage experiments, emulsions were kept in a temperaturecontrolled water bath at 30 °C with constant swirling, and samples were selected periodically for analysis. In the heat-treatment experiments, the emulsions were placed in glass test tubes and stored at fixed temperatures ranging from 30 to 95 °C for 20 min. The test tubes were then stored in a temperature-controlled water bath at 30 °C for 24 h prior to emulsion analysis. Particle Size Determination. The particle size distribution of the emulsions was measured using a laser diffraction instrument (LS230, Coulter Corp., FL). This instrument measures the angular dependence of the intensity of light scattered from a stirred dilute emulsion and then indicates the particle size distribution that gives the closest fit between theoretical calculations (Mie theory) and experimental measurements. A refractive index ratio of 1.08 was used in the particle size calculations. To avoid multiple scattering effects, the emulsions were diluted with pH-adjusted distilled water (pH 3) prior to making the measurements. Dilution and stirring may have partially disrupted weakly flocculated droplets, although it is unlikely that they will have disrupted any strongly flocculated droplets. To examine this effect, we monitored changes in mean particle size with time immediately after the emulsions were placed in the measurement chamber. The theory used to calculate the particle size distribution assumes that the particles are spherical and homogeneous, and therefore the data obtained on emulsions that contained flocs should be treated with caution because they are nonspherical and nonhomogeneous. Particle size measurements are reported as either full particle size distributions or as weight-average mean diameters, d43 ()Σnidi4/ Σnidi3, where ni is the number of particles with diameter di). Mean particle diameters were calculated as the average and standard deviation of measurements made on at least two freshly prepared samples. Creaming Stability Measurements. Ten grams of emulsion was transferred into a test tube (internal diameter, 16 mm; height, 160 mm) and then stored for 24 h at room temperature. After storage, a number of emulsions separated into a “cream” layer at the top and a transparent “serum” layer at the bottom. The total height of the emulsions (HE) and the height of the serum layer (HS) were measured. The extent of creaming was characterized by a creaming index ) 100 × HS/HE. The creaming index provided indirect information about the extent of droplet aggregation in an emulsion: the more aggregation, the larger the particles and the faster the creaming.
Results and Discussion In previous studies, we have used laser diffraction to measure the extent of droplet flocculation in β-Lgstabilized oil-in-water emulsions at pH 7. This technique was used to quantify the influence of temperature, storage time, NaCl, and sucrose on the susceptibility of proteinstabilized emulsions to droplet flocculation.14,15,24 In the present study, we used the same technique to investigate the factors that influence droplet flocculation in β-Lg(24) Kim, H.-J.; Decker, E. A.; McClements, D. J. J. Agric. Food Chem. 2003, 51, 766-772.
Droplet Flocculation in Stabilized Emulsions
Figure 1. Laser diffraction study of the influence of pH (3 or 7) and NaCl (0 or 150 mM) on the evolution of mean particle diameter of diluted 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-Lg) stored at 30 °C.
stabilized oil-in-water emulsions at pH 3 and compare the data with results from our earlier studies carried out at pH 7. Droplet Aggregation during Isothermal Storage at 30 °C. Initially, the influence of pH on droplet growth during isothermal storage in β-Lg-stabilized emulsions was investigated. In this study, 5 wt % oil-in-water emulsions (pH 3) were stored in glass test tubes at 30 °C for 48 h with constant swirling. Small aliquots of emulsion were periodically removed from the test tubes and placed in the measurement chamber of the laser diffraction instrument. Inside the measurement chamber, they were diluted with water adjusted to pH 3 and stirred for 5 min before making a measurement. The evolution of the mean particle diameter of emulsions at pH 3 and 7 is compared in Figure 1. In the absence of salt, the pH 7 emulsion showed no evidence of particle growth (d43 ∼ 0.46 ( 0.01 µm), which has been attributed to the relatively strong electrostatic repulsion between the droplets.14 In the presence of 150 mM NaCl, the pH 7 emulsion exhibited steady growth in mean particle diameter with time, until a relatively constant value was reached after about 24 h. This increase has been attributed to the screening of the electrostatic repulsion between the droplets, combined with increased hydrophobic attraction and disulfide bond formation associated with surface denaturation of the adsorbed globular proteins.14,19,25,26 In the absence of salt, there was little change in the mean particle diameter of the pH 3 emulsions with time (d43 ∼ 1.3 ( 0.2 µm), as was observed in the pH 7 emulsions. However, in the presence of 150 mM NaCl the behavior of the emulsions was very different at pH 3 and 7. At pH 3, the initial mean particle diameter was significantly higher than in the pH 7 emulsions and there was little change in mean particle diameter with time (d43 ∼ 1.6 ( 0.2 µm). When 1 wt % Tween 20 was mixed with the pH 3 emulsions prior to carrying out the laser diffraction experiments, we found that the mean particle diameter decreased to 0.59 ( 0.02 µm, which indicated that there was some flocculation in the original emulsions. The addition of mercaptoethanol to the emulsions did not produce any further decrease in mean particle diameter, which suggests that the proteins at the droplet interface (25) Damodaran, S.; Anand, K. J. Agric. Food Chem. 1997, 45, 38133820. (26) Dufour, E.; Dalgalarrondo, M.; Adam, L. J. Colloid Interface Sci. 1998, 207, 264-272.
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were not extensively cross-linked by disulfide bonds.14,15 The fact that the diameter of the individual droplets was larger at pH 3 than at pH 7 when similar homogenization conditions were used suggests that the β-Lg is not as efficient at emulsion formation at low pH. This may be because differences in molecular structure or self-association at different pH values cause differences in the surface activity or kinetics of emulsifier adsorption during homogenization.8 Droplet Aggregation Due to Heat Treatment. In this series of experiments, we examined the influence of pH (3 or 7), heat treatment (30-95 °C, 20 min), and salt content (0 or 150 mM NaCl) on the flocculation stability of β-Lg-stabilized oil-in-water emulsions. We also examined the influence of adding the salt before or after heat treatment, since previous studies have shown that the order of addition of salt relative to heating has a strong influence on droplet flocculation in β-Lg-stabilized emulsions.15 The measurements made at pH 3 in this study were compared with measurements made at pH 7 in an earlier study.15 In the absence of salt, there was no evidence of droplet aggregation at any holding temperature in the pH 7 emulsions. In the absence of salt, we also observed no significant change in the mean particle diameter of the pH 3 emulsions with holding temperature (d43 ∼ 1.04 ( 0.05 µm). These results can be attributed to the relatively strong electrostatic repulsion between the droplets at low salt concentrations, which effectively prevents droplet flocculation. In the presence of 150 mM NaCl, the extent of droplet aggregation in the emulsions measured by laser diffraction was highly dependent on holding temperature, pH, and the order of salt addition relative to the thermal processing step. NaCl Added before Heating. The influence of pH on the mean particle diameters of heat-treated β-Lgstabilized oil-in-water emulsions to which 150 mM NaCl was added before thermal treatment is shown in Figure 2a. Using these conditions, there was no strong electrostatic repulsion between the droplets when they were heated because these interactions were effectively screened by salt; therefore we would expect that the droplets could come into fairly close contact during thermal processing (providing the attractive interactions were strong enough). In the pH 7 emulsions, an appreciable amount of droplet aggregation was observed when they were held at temperatures ranging from 30 to 65 °C, which has been attributed to surface denaturation of the adsorbed β-Lg.15 At higher temperatures (70-95 °C), the extent of droplet aggregation in the pH 7 emulsions increased appreciably, which can be attributed to thermal denaturation of the adsorbed β-Lg.15,27 Surface or thermal denaturation of proteins causes an increase in the hydrophobic attraction and disulfide bond formation between droplets that are in close proximity, thereby promoting droplet flocculation. The aggregates in the pH 7 emulsions could be disrupted at relatively low temperatures (70 °C). When 1 wt % Tween 20 was mixed with the pH 3 emulsions prior to carrying out the laser diffraction experiments, we found that the mean particle diameter decreased appreciably and was relatively independent of temperature (0.61 ( 0.03 µm), which indicated that some droplet flocculation was present in the pH 3 emulsions. The addition of mercaptoethanol to the emulsions did not produce any further decrease in mean particle diameter, which suggests that the proteins at the droplet interface were not extensively cross-linked by disulfide bonds at any holding temperature.14,15 At first sight, these results suggest that the pH 3 emulsions are considerably more stable than pH 7 emulsions to droplet flocculation induced by thermal processing. Nevertheless, as we will see later, this is not necessarily the case and may be primarily due to limitations of the laser diffraction measurement technique. NaCl Added after Heating. The influence of pH on the mean particle diameters of β-Lg-stabilized oil-in-water emulsions to which 150 mM NaCl was added after holding the emulsions at different temperatures is shown in Figure
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2b. Under these conditions, there was a strong electrostatic repulsion between the droplets during thermal processing, which would have been expected to keep them apart while the emulsions were at elevated temperatures. In the pH 7 emulsions, appreciable droplet aggregation was observed when they were held at temperatures ranging from 30 to 65 °C, which can be attributed to surface denaturation of the adsorbed β-Lg.15 At temperatures above the thermal denaturation temperature of the adsorbed β-Lg (>70 °C), the extent of droplet aggregation in the pH 7 emulsions decreased appreciably, which was in stark contrast to the behavior observed when NaCl was added to this emulsion prior to heating (Figure 2a). This effect has been attributed to extensive hydrophobic and disulfide bond formation between protein molecules adsorbed to the same droplet during heating but little hydrophobic and disulfide bond formation between protein molecules adsorbed to different droplets because of the strong electrostatic repulsion between them.15 Extensive intradroplet protein-protein interactions reduce the hydrophobicity and chemical reactivity of the droplet surfaces. Hence, when the emulsions are cooled to room temperature and 150 mM NaCl is added, the attractive interactions between the droplets are no longer sufficient to promote droplet flocculation. In the pH 3 emulsions, the order of NaCl addition relative to thermal treatment had little impact on the temperature dependence of the mean particle diameter (Figure 2). The mean particle diameter was appreciably higher for emulsions held below the thermal denaturation temperature of the adsorbed β-Lg (d43 ) 1.04 ( 0.12 µm) than for those held above this temperature (d43 ) 0.67 ( 0.02 µm). This suggested that less droplet flocculation occurred in the pH 3 emulsions at higher temperatures, which is in qualitative agreement with data for the pH 7 emulsions. When 1 wt % Tween 20 was mixed with the pH 3 emulsions prior to carrying out the laser diffraction experiments, we found that the mean particle diameter decreased appreciably and was relatively independent of holding temperature (0.57 ( 0.02 µm), which indicated that there had been some flocculation in the pH 3 emulsions. The addition of mercaptoethanol to the emulsions did not produce any further decrease in mean particle diameter, which suggested that the proteins at the droplet interface were not extensively cross-linked by disulfide bonds at any holding temperature.14,15 These results suggest that emulsions to which NaCl is added after heating behave qualitatively the same, regardless of pH; that is, there is a decrease in droplet aggregation above the thermal denaturation temperature of the adsorbed proteins. Influence of Stirring Time on Droplet Aggregation Measured by Laser Diffraction. When we were performing the laser diffraction experiments on the pH 3 emulsions to determine their mean particle size, we noticed that it was difficult to obtain consistent results unless the emulsions were stirred for at least 5 min in the measurement chamber. We therefore postulated that there may have been some floc disruption occurring within the measurement chamber of the laser diffraction instrument due to dilution and shearing effects. For this reason, we examined the influence of holding time within the laser diffraction measurement chamber on the particle size determination. The particle size distribution (PSD) of emulsions containing either 0 or 150 mM NaCl stored at room temperature for 30 min after homogenization was measured. These measurements were made either immediately after placing the emulsions in the laser diffraction measurement chamber or after stirring the
Droplet Flocculation in Stabilized Emulsions
Figure 3. Particle size distributions of 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-Lg, pH 3.0, 150 mM NaCl) measured following storage at 30 °C for 30 min after homogenization. Measurements were made either immediately after introducing the emulsions into the measurement chamber of the laser diffraction instrument (0 min) or after 5 min of stirring (5 min). Measurements made on emulsions to which 1 wt % Tween 20 was added to disrupt any flocs formed are also shown.
emulsions for 5 min in the measurement chamber. To determine whether the emulsions were flocculated or coalesced, the PSDs of the same emulsions were measured after 1 wt % Tween 20 had been added to disrupt any flocs. The results for the emulsions containing 150 mM NaCl are shown in Figure 3, but similar results were also obtained in the absence of salt. Our measurements show that the emulsions were highly flocculated at the moment that they were first introduced into the laser diffraction instrument but that many of the larger flocs were disrupted during stirring within the instrument. Laser diffraction measurements carried out as a function of stirring time within the measurement chamber (0-20 min) indicated that there was little further change in the PSD at stirring times longer than 5 min (data not shown). The fact that there were a significant fraction of particles larger than those observed in the presence of Tween 20 suggests that some smaller flocs remained in the protein-stabilized emulsions even after 5 min of stirring. In summary, the PSD determined by laser diffraction was strongly dependent on the time that the emulsions were stirred in the measurement chamber prior to making the measurements (up to about 5 min), suggesting floc disruption occurred within the laser diffraction measurement chamber. In our previous studies at pH 7, we found that large flocs remained intact within the laser diffraction instrument.14,15,24 A possible explanation for the greater persistence of large flocs at pH 7 than at pH 3 is that extensive covalent bond formation (disulfide bonds) occurred between proteins adsorbed onto different droplets at pH 7 but not at pH 3. Previous studies have shown that disulfide bond formation occurs much more readily at neutral pH than at acid pH.28,29 In the presence of salt, emulsion droplets that have a relatively high surface hydrophobicity (e.g., due to surface or thermal denaturation of β-Lg) come into close proximity because the attractive van der Waals and hydrophobic interactions dominate the repulsive electrostatic interactions. At pH 7, disulfide bonds form between proteins adsorbed onto different droplets, which hold the droplets in the flocs tightly together. Thus, the (28) Croguennec, T.; Bouhallab, S.; Molle, D.; O’Kennedy, B. T.; Mehra, R. Biochem. Biophys. Res. Commun. 2003, 301, 465-471. (29) Magdassi, S.; Vinetsky, Y.; Relkin, P. Colloids Surf., B 1996, 6, 353-362.
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Figure 4. Influence of isothermal heat treatment (30-95 °C, 20 min) and order of salt addition on the creaming index of nondiluted 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-Lg, pH 3.0, 150 mM NaCl).
flocs formed in pH 7 emulsions are resistant to disruption during dilution and stirring in the laser diffraction measurement chamber. On the other hand, at pH 3, there are few disulfide bonds formed between emulsion droplets, so that the flocs formed are easily disrupted by dilution and stirring. Creaming Instability Studies. The major limitation of the laser diffraction technique for studying droplet aggregation is that emulsions must be highly diluted and stirred prior to analysis, which may alter the structure and size of any flocs present. We therefore utilized creaming measurements as an alternative means of studying the stability of nondiluted emulsions under quiescent conditions to droplet aggregation. After the thermal treatments had been carried out, the emulsions were placed in glass test tubes and stored for 24 h at room temperature prior to measuring the height of the serum layer. No evidence of creaming was observed in pH 3 emulsions containing no salt, suggesting that they were relatively stable to droplet aggregation, presumably because of the strong electrostatic repulsion between the droplets. On the other hand, appreciable droplet creaming was observed in the emulsions containing salt at certain temperatures, which suggested that they were susceptible to droplet aggregation. In the presence of 150 mM NaCl, the emulsions were highly unstable to creaming following heat treatments from 30 to 70 °C but relatively stable at temperatures from 75 to 95 °C (Figure 4). Whether the salt was added before or after heating seemed to make little difference to the temperature dependence of the creaming stability of the emulsions. These results suggest that the pH 3 emulsions are highly unstable to droplet aggregation at temperatures below the thermal denaturation temperature of β-Lg. A possible explanation of this phenomenon is that the globular proteins undergo a conformational transition after adsorption to the droplet surfaces, which increases the surface hydrophobicity of the droplets, thereby increasing the hydrophobic attraction between the droplets. Less droplet aggregation occurs at temperatures above the thermal denaturation temperature because the proteins are able to undergo a thermally induced conformational change that enables them to direct more hydrophobic groups toward the droplet surfaces (and away from the water), thereby reducing their surface hydrophobicity. Unlike at pH 7, strong flocs are not formed during heating in the presence of salt because disulfide bond formation cannot occur.
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Conclusions This study examined the influence of pH (3 and 7), salt (0 and 150 mM NaCl), storage temperature (30-90 °C), and order of salt addition relative to heating on droplet aggregation in β-Lg-stabilized emulsions. The major findings of this study are as follows: Disulfide bond formation plays an important role in holding emulsion droplets together in flocs at pH 7 but not at pH 3. At pH 3, the stability of emulsions to droplet aggregation improves when they are heated to temperatures above the thermal denaturation temperature of the adsorbed globular protein molecules. At pH 3, the order of addition of salt relative to thermal processing (before or after) has little influence on emulsion stability to droplet aggregation, whereas it has a profound influence at pH 7. Laser diffraction measurements made on weakly flocculated protein-stabilized emulsions should be treated with caution, since dilution and stirring may lead to
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considerable floc disruption. Consequently, it is important to back up laser diffraction measurements with a technique that does not rely on emulsion dilution, for example, creaming stability measurements. The data obtained in this study are important for understanding protein-based emulsion behavior and improving the functionality of globular proteins as emulsifiers in food, health care, and pharmaceutical products. Acknowledgment. This material is based upon work supported by the Cooperative State Research, Extension, Education Service, United States Department of Agriculture, Massachusetts Agricultural Experiment Station (Project No. 831), by a United States Department of Agriculture, CREES, IFAFS Grant (Award Number 20014526), and by a United States Department of Agriculture, CREES, NRI Grant (Award Number 2002-01655). We also thank Davisco Foods International for kindly donating the protein ingredients used in this study. LA048899B
