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Role of Postadsorption Conformation Changes of β-Lactoglobulin on Its Ability To Stabilize Oil Droplets against Flocculation during Heating at Neutral pH 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 April 24, 2002. In Final Form: July 10, 2002 The influence of heating on droplet aggregation in hydrocarbon oil-in-water emulsions stabilized by a globular protein was examined using laser diffraction. Different levels of salt (0 or 150 mM NaCl) were added to n-hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-Lg, pH 7.0) either before or after isothermal heat treatment (30-95 °C for 20 min). At 0 mM NaCl, no aggregation was observed in any of the emulsions because of strong electrostatic repulsion between the droplets. At 150 mM NaCl, droplet flocculation occurred between 30 and 65 °C due to surface denaturation of β-Lg after adsorption. Above 70 °C, droplet flocculation became less extensive when 150 mM NaCl was added to the emulsions after heating but more extensive when the salt was added before heating, which was attributed to thermal denaturation of adsorbed β-Lg. When the droplets are in close proximity during heating (150 mM NaCl), interactions between proteins adsorbed onto different droplets are favored, but when droplets are not in close proximity (0 mM NaCl), interactions between proteins adsorbed onto the same droplets are favored. Addition of N-ethylmaleimide, a sulfhydryl blocking agent, to the emulsions immediately after homogenization prevented droplet aggregation due to surface or thermal denaturation, highlighting the importance of disulfide bond formation on droplet flocculation stability. The mean droplet size decreased when small molecule surfactant (1 wt % Tween 20) and reducing agent (1 wt % 2-mercaptoethanol) were added to the emulsions, which indicated that the droplets were flocculated rather than coalesced. Our data show that the magnitude of droplet-droplet interactions during thermal denaturation of adsorbed globular proteins has a pronounced influence on the heat stability of protein-stabilized emulsions. This study has important implications for the formulation and production of protein stabilized oil-in-water emulsions.

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 surfaces of oil droplets formed when oilwater-protein mixtures are mechanically agitated 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 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.7-9 From a technological standpoint, many emulsion-based products must be capable of withstanding some form of thermal processing after they have been created, e.g., * Corresponding author: Tel +1-413-545-1019; Fax +1-413-5451262; 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 Publishers: New York, 1996. (5) Phillips, L. G.; Whitehead, D. M.; Kinsella, J. E. StructureFunction Properties of Food Proteins; Academic Press: San Diego, 1994. (6) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176-181. (7) Dickinson, E. Colloids Surf. B. 1999, 15, 161-176. (8) 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. (9) McClements, D. J. Food Emulsions: Principles, Practice and Techniques; CRC Press: Boca Raton, FL, 1999.

sterilization, pasteurization, cooking, etc.10,11 Previous studies have shown that emulsions stabilized by globular proteins are susceptible to droplet aggregation when they are heated above a particular temperature.12-17 The origin of this effect is believed to be the thermal denaturation of the adsorbed proteins, which leads to the exposure of amino acid residues originally located in the hydrophobic interior of the globular protein.18,19 Exposure of certain amino acids, such as those containing nonpolar or sulfhydryl groups, increases intermolecular protein interactions through hydrophobic attraction and thiol-disulfide interchange reactions.13,20-22 Intermolecular interactions (10) Arntfield, S. D.; Ismond, M. A. H.; Murray, E. D. In Thermal Analysis of Foods; Harwalkar, V. R., Ma, C. Y., Eds.; Elsevier: London, 1990; pp 51-91. (11) Harwalkar, V. R.; Ma, C. Y. In Food Proteins; Kinsella, J. E., Soucie, W. G., Eds.; American Oil Chemists Society: Champaign, IL, 1989; p 210. (12) Hunt, J. A.; Dalgleish, D. G. J. Food Sci. 1995, 60, 1120-1123, 1131. (13) Monahan, F. J.; McClements, D. J.; German, J. B. J. Food Sci. 1996, 61, 504-510. (14) Demetriades, K.; Coupland. J. N.; McClements, D. J. J. Food Sci. 1997, 62, 462-467. (15) Euston, S. R.; Finnigan, S. R.; Hirst, R. L. J. Agric. Food Chem. 2001, 49, 5576-5583. (16) Chen, J. S.; Dickinson, E. Int. J. Food Sci. Technol. 1999, 34, 493-501. (17) Chen, J. S.; Dickinson, E. Food Hydrocolloids 1999, 13, 363369. (18) Corredig, M.; Dalgleish, D. G. Colloids Surf. B 1995, 4, 411420. (19) Fang, Y.; Dalgleish, D. G. J. Colloid Interface Sci. 1997, 196, 292-298. (20) Sawyer, W. H. J. Dairy Sci. 1968, 51, 323-329. (21) De Wit, J. N. J. Dairy Sci. 1990, 73, 3602-3612. (22) Hoffmann, M. A. M.; van Mill, P. J. J. M. J. Agric. Food Chem. 1999, 47, 1898-1905.

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occur between protein molecules adsorbed onto different droplets when the droplets are in close proximity (e.g., when the repulsive interactions are relatively low or the attractive interactions are relatively high), which leads to extensive droplet aggregation.12-14 Droplet aggregation is usually undesirable because it leads to an increase in the creaming instability and viscosity of emulsion-based products.9 The objective of this study is to examine the influence of thermal processing conditions on the stability of oilin-water emulsions stabilized by a model globular protein at neutral pH. In particular, we intend to identify the physicochemical origin of droplet aggregation in heattreated emulsions stabilized by β-lactoglobulin (β-Lg) and to examine the role of droplet interactions during heating on the thermal stability of emulsions. β-Lactoglobulin was chosen as a model globular protein for this study because a great deal of information is available in the literature about its molecular and functional characteristics. The information obtained from this study has important implications for the formulation and production of oilin-water emulsions stabilized by globular proteins. Materials and Methods Materials. Analytical grade sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium azide (NaN3), N-ethylmaleimide (NEM), 2-mercaptoethanol, and nhexadecane were purchased from the Sigma Chemical Co. (St. Louis, MO). Powdered β-lactoglobulin was obtained from Davisco Foods International (Lot # 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 deionized and distilled water containing 0.04 wt % NaN3 (as an antimicrobial agent) and stirring for at least 2 h to ensure complete dispersion. Solutions containing different NaCl and NEM concentrations were prepared by dispersing weighed amounts of the powdered material into 5 mM phosphate buffer (pH 7.0). 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 distilled water) at room temperature. The oil and emulsifier solution were blended using a high-speed blender for 2 min (model 33BL79, Warring 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 emulsions were then diluted to 5 wt % oil with phosphate buffer (5 mM, pH 7.0) containing NaCl (0 or 150 mM) either before or after heat treatment and stored for 2.5 h before heating. Heat treatment involved placing emulsions in glass test tubes and holding them isothermally at selected temperatures (ranging from 30 to 95 °C) for 20 min and then placing them in water for 20 min to cool to room temperature. Emulsions containing added salt (150 mM NaCl) exhibited some creaming during heating at the higher temperatures, but all the other emulsions were stable to creaming during heating. Emulsions were then stored in a temperature-controlled water bath at 30 °C for 24 h, after which their particle size was measured. The final pH of the emulsions was measured to be 7.0 ( 0.2 (pH Meter 320, Corning Inc., Corning, NY). Evidence about the relative importance of covalent and noncovalent interactions on the flocculation stability of the emulsions was obtained by adding a nonionic surfactant (1 wt % Tween 20), a reducing agent (1 wt % 2-mercaptoethanol), or a mixture of the two (1 wt % Tween 20 + 1 wt % 2-mercaptoethanol) to the emulsions and shaking them for 5 h prior to making particle size measurements. Additional information about the role of thiol/disulfide interchange on droplet flocculation was

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Figure 1. Influence of isothermal heat treatment (30-95 °C, 20 min), salt concentration (0 or 150 mM NaCl), and order of salt addition (before or after heating) on the mean particle diameter (d43) of 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-lactoglobulin, pH 7.0). examined by adding NEM to one series of emulsions immediately after homogenization to block free thiol groups. These emulsions were incubated for 3 h in the presence of a freshly prepared NEM solution to give a final β-Lg to NEM molar ratio of 1:4. This concentration of NEM has previously been shown to be sufficient to block all free thiol groups on β-Lg molecules.23 These emulsions were then subjected to similar heat treatments as those received by the emulsions containing no NEM (see above). 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 and experimental measurements. A refractive index ratio of 1.08 was used in the particle size calculations. Emulsions were vortexed for 3 s, inverted, and then vortexed for another 3 s to ensure that they were homogeneous. They were then diluted (∼1:1000) with pH adjusted distilled water (pH 7) prior to making the particle size measurements to avoid multiple scattering effects. The emulsions were stirred continuously (at an instrument stirring speed of 50%) throughout the measurements to ensure the samples were homogeneous. Dilution and stirring may have partially disrupted weakly flocculated droplets, although it is unlikely that they will have disrupted any strongly flocculated droplets. 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 weight-average mean diameters, d43 ()∑nidi4/ ∑nidi3, where ni is the number of particles with diameter di). Particle diameters are reported as the average of measurements made on at least two freshly prepared samples, with standard deviations being less than 5%.

Results and Discussion Influence of Heating on Droplet Aggregation. In the absence of added salt (0 mM NaCl), no change in mean particle diameter was observed in the emulsions after heat treatment (30-95 °C) (Figure 1). Theoretical predictions of the droplet-droplet interaction potential for a similar system suggest that the electrostatic repulsion between the protein-stabilized emulsion droplets is sufficiently large to overcome the attractive interactions, e.g., van (23) Kitabatake, N.; Wada, R.; Fugita, Y. J. Agric. Food Chem. 2001, 49, 4011-4018.

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der Waals and hydrophobic attraction.24 On the other hand, extensive droplet aggregation was observed in emulsions containing 150 mM NaCl at certain temperatures, which was strongly dependent on the order of addition of the salt relative to the heat treatment (Figure 1). NaCl Added after Heating. The mean particle diameters of heat-treated β-Lg stabilized oil-in-water emulsions to which 150 mM NaCl was added after thermal treatment are shown in Figure 1. Under these conditions there is a relatively strong electrostatic repulsion between the emulsion droplets during thermal processing, and so no droplet aggregation occurs during heating. Any droplet aggregation observed in these emulsions is therefore the result of events that occur after the salt is added to the emulsions at room temperature. An appreciable amount of droplet aggregation was observed in emulsions that had been heated to temperatures ranging from 30 to 65 °C, which was presumably caused by surface denaturation of β-Lg after adsorption to the droplet surfaces.24,25 Surface denaturation causes increased exposure of nonpolar and sulfhydryl containing amino acids to the aqueous phase,26 which presumably promotes droplet aggregation through increased hydrophobic attraction and disulfide bond formation between proteins adsorbed to different droplets.24,27,28 Studies of changes in globular protein conformation after adsorption to droplet surfaces have shown that much of the secondary structure of the proteins is retained,18,19,29 which suggests that the adsorbed protein could be in a “molten globule state”.30 Surprisingly, we found that less droplet aggregation occurred in the emulsions after they had been heated to higher temperatures (70-95 °C). The most likely explanation of this phenomenon is that β-Lg adsorbed to oil droplets undergoes a conformational change around this temperature.18 Previous studies have found that the thermal denaturation temperature of β-lactaglobulin is around Tm ) 71-73 °C.18,31,32 Above their thermal denaturation temperature, the surface hydrophobicity and flexibility of the adsorbed β-Lg molecules increase, which increases their tendency to adopt conformations where the nonpolar amino acids are directed away from the aqueous phase. We postulate that when oil droplets are not in close proximity, which in these experiments is due to the strong electrostatic repulsion between the droplets during heating, the proteins are able to undergo a conformation change so that the exposed nonpolar amino acids either interact with other exposed nonpolar amino acids on neighboring protein molecules adsorbed to the same droplet or are redirected toward the oil phase. Consequently, the surface hydrophobicity of the droplets decreases (relative to droplets heated below the thermal denaturation temperature of the adsorbed proteins), which reduces the magnitude of the attractive forces between the droplets. Droplet aggregation does not occur when (24) Kim, H. J.; Decker, E. A.; McClements, D. J. J. Agric. Food Chem., submitted. (25) Lefebvre, J.; Relkin, P. In Surface Activity of Proteins; Magassi, S., Ed.; Marcel Dekker: New York, 1996; pp 181-236. (26) Dufour, E.; Dalgalarrondo, M.; Adam, L. J. Colloid Interface Sci. 1998, 207, 264-272. (27) McClements, D. J.; Monahan, F. J.; Kinsella, J. E. J. Food Sci. 1993, 58, 1036-1039. (28) Damodaran, S.; Anand, K. J. Agric. Food Chem. 1997, 45, 38133820. (29) Husband, F. A.; Garrood, M. J.; Mackie, A. R.; Burnett, G. R.; Wilde, P. J. J. Agric. Food Chem. 2001, 49, 859-866. (30) Nylander, T. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 27-54. (31) Ruegg, M.; Moor, U.; Blanc, B. J. Dairy Res. 1977, 44, 509-520. (32) Mulvihill, D. M.; Donovan, M. Irish J. Food Sci. Technol. 1987, 11, 43-75.

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salt is added to the emulsions after heating because the electrostatic repulsive forces are large enough to overcome the attractive van der Waals and (weakened) hydrophobic interactions.24 It is also possible that when the adsorbed proteins were heated above their thermal denaturation temperature, the number of thiol groups available for thiol-disulfide interchange reactions was reduced (either because they were redirected away from the aqueous phase or because they formed disulfide bonds with other thiol groups), which decreased the tendency for disulfide bonds to be formed between proteins adsorbed to different droplets. Nevertheless, it is not possible to identify the relative importance of changes in the location of nonpolar and sulfhydryl groups on droplet aggregation from the laser diffraction data alone. NaCl Added before Heating. The mean particle diameters of heat-treated β-Lg stabilized oil-in-water emulsions to which 150 mM NaCl was added before thermal treatment are shown in Figure 1. Under these conditions there is not a strong electrostatic repulsion between the droplets during thermal processing because these interactions are screened by salt.33 Consequently, any increase in the attractive interactions between the droplets as a result of heating would be expected to promote droplet aggregation.24 An appreciable amount of droplet aggregation was observed when the emulsions were heated at temperatures from 30 to 65 °C, due to the surface denaturation of β-Lg discussed above. At higher temperatures (70-95 °C), the extent of droplet aggregation increased appreciably, which is in complete contrast to the experiments where NaCl was added after heating. We postulate that when there is not a strong electrostatic repulsion between droplets during thermal treatment, the heat denaturation of β-Lg around 70 °C again leads to exposure of nonpolar and thiol containing amino acids. However, protein-protein interactions can now occur between β-Lg molecules adsorbed either to the same or to different droplets. In the latter case, these interactions led to extensive irreversible droplet aggregation. The fact that the particle diameter was relatively insensitive to temperature from 70 to 95 °C suggests that the attractive forces between the droplets were fairly temperature independent once the proteins had unfolded. In addition, the emulsions were diluted and stirred during the particle size measurements, which may have disrupted any larger flocs until a steady-state size was achieved. Influence of Reducing Agent and Surfactant on Aggregate Disruption. To gain insight into the origin of droplet aggregation (flocculation vs coalescence) and to establish the nature of the bonds holding the droplets in the aggregates together (hydrophobic vs disulfide), we examined the influence of a reducing agent and a surfactant on the disruption of aggregates formed during heat treatment (Figure 2). Nonionic surfactant (1 wt % Tween 20), reducing agent (1 wt % 2-mercaptoethanol), or nonionic surfactant + reducing agent (1 wt % Tween 20 + 1 wt % 2-mercaptoethanol) were stirred into heattreated emulsions for 1 h prior to making laser diffraction measurements. This concentration of Tween 20 has previously been shown to displace adsorbed protein molecules from droplet surfaces (provided that the proteins are not extensively cross-linked by covalent bonds) and form interfacial membranes that render the droplets stable to flocculation.1 This concentration of 2-mercaptoethanol has previously been shown to break any disulfide bonds (33) Israelachvili, J. N. Intermolecular and Surface Forces; Acadamic Press: London, 1992.

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Figure 2. Influence of reducing agent (1 wt % 2-mercaptoethanol) and surfactant (1 wt % Tween 20) on the disruption of aggregates formed in 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-lactoglobulin, pH 7.0) during isothermal heat treatment (30-95 °C, 20 min): (a) 150 mM NaCl added after heating; (b) 150 mM NaCl added before heating.

formed between protein molecules.34 The disruption of aggregates by reducing agent and surfactant depended on heating temperature and on whether the salt (150 mM NaCl) was added to the emulsions before or after heat treatment (Figure 2). NaCl Added after Heating. When the NaCl was added after heat treatment (Figure 2a), the incorporation of surfactant alone or surfactant + reducing agent together completely disrupted the aggregates formed at temperatures between 30 and 65 °C; i.e., the mean particle diameter (d43 ∼ 0.20 µm) was similar to that of the droplets in the original nonaggregated emulsion (d43 ∼ 0.20 µm). Incorporation of reducing agent alone caused a reduction in the amount of droplet aggregation, but the mean particle diameter (d43 ∼ 0.61 µm) was still appreciably higher than that of the original emulsion. These measurements clearly show that droplet aggregation was caused by flocculation, rather than by coalescence, since the aggregates could be completely disrupted, releasing droplets having the same size as those in the original emulsion. The measurements also suggest that disulfide bond formation played an important role in holding the droplets together in the aggregates but that they were not solely responsible, as evidenced by the ability of 2-mercaptoethanol alone to only partially disrupt the aggregates. We therefore believe that hydrophobic attraction between proteins adsorbed to different droplets also played an important role in holding the aggregates together. The fact that flocs could be disrupted equally well by surfactant alone as by surfactant + reducing agent in combination suggested that disulfide bond formation at the droplet surface was not so extensive at these temperatures that it prevented (34) Monahan, F. J.; McClements, D. J.; Kinsella, J. E. J. Agric. Food Chem. 1993, 41, 1826-1831.

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the proteins from being desorbed by surfactant, as is the case in emulsions heated above the thermal denaturation temperature of the adsorbed proteins.13 In the absence of surfactant or reducing agent, a small amount of droplet aggregation (d43 ∼ 0.25 µm) was observed in the emulsions heated to temperatures between 70 and 95 °C when 150 mM NaCl was added after heat treatment (Figure 2a). These aggregates could not be disrupted by adding Tween alone (d43 ∼ 0.25 µm at 95 °C) or 2-mercaptoethanol alone (d43 ∼ 0.24 µm at 95 °C) but could be completely disrupted by adding a combination of surfactant and reducing agent (d43 ∼ 0.20 µm at 95 °C). These results suggest that the protein molecules had become extensively cross-linked at the droplet surfaces through disulfide bond formation so that surfactant alone could not displace them, as shown previously for whey protein isolate.13 They also suggest that the aggregates are partly held together by hydrophobic interactions between proteins adsorbed onto different droplets as well as by disulfide bonds. (Otherwise, the droplets would have been totally disrupted by 2-mercaptoethanol alone.) The aggregates were completely disrupted when a combination of surfactant + reducing agent was added to the emulsions because the 2-mercaptoethanol broke any disulfide bonds; then the Tween 20 displaced the proteins from the droplet surfaces. NaCl Added before Heating. The disruption of aggregates by surfactant and reducing agent in emulsions heated between 30 and 65 °C was similar when salt (150 mM NaCl) was added before heating as when it was added after heating (see above). In the absence of surfactant or reducing agent, extensive droplet aggregation was observed in the emulsions heated to temperatures between 70 and 95 °C, e.g., d43 ∼ 48 µm at 95 °C (Figure 2b). The aggregate size increased when Tween was added alone (d43 ∼ 54 µm at 95 °C) but decreased appreciably when 2-mercaptoethanol was added alone (d43 ∼ 18 µm at 95 °C). The aggregates were almost completely disrupted by adding a combination of surfactant and reducing agent (d43 ∼ 0.31 µm at 95 °C). These results suggest that there was extensive cross-linking of proteins adsorbed to similar and to different droplets through disulfide bond formation so that surfactant alone could not displace the proteins or disrupt the flocs.13 An increase in aggregate size upon addition of Tween 20 alone has also been observed for heated emulsions stabilized by whey protein isolate, which was attributed to the ability of the surfactant molecules to displace some nonpolar segments of the protein from the droplet surfaces, thus increasing the attractive forces between the droplets.13 The ability of the reducing agent alone to partially disrupt the aggregates indicates that disulfide bonds play an important role in holding the droplets together in the flocs but that other forces must also be involved, such as increased hydrophobic interactions between the droplets, which is in agreement with studies of protein aggregation in aqueous solutions.22 The aggregates were almost completely disrupted when a combination of surfactant + reducing agent was added to the emulsions because the 2-mercaptoethanol broke any disulfide bonds; then the Tween 20 could displace the proteins from the droplet surfaces. Influence of Sulfhydryl Blocking Agent on Aggregate Formation. Further evidence of the role of disulfide bonds on droplet aggregation during thermal processing was obtained by examining the influence of a sulfhydryl blocking agent (NEM) on the size of the aggregates formed during heating (Figure 3). NEM was added to oil-in-water emulsions prepared at room temperature, at a molar ratio of 4 NEM molecules per β-Lg

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Figure 4. Influence of preheating (95 °C for 20 min in the absence of salt) on the formation of aggregates in 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-lactoglobulin, pH 7.0) containing 150 mM NaCl during isothermal heat treatment (30-95 °C, 20 min).

Figure 3. Influence of sulhydryl blocking agent (NEM) on the formation of aggregates in 5 wt % n-hexadecane oil-in-water emulsions (0.5 wt % β-lactoglobulin, pH 7.0) during isothermal heat treatment (30-95 °C, 20 min): (a) 150 mM NaCl added after heating; (b) 150 mM NaCl added before heating.

molecule, and the emulsions were then incubated for 3 h. This incubation period was chosen because previous studies have shown that over 90% of free sulfhydryl groups of β-Lg are blocked by NEM during this time.23 The emulsions were then held at temperatures ranging from 30 to 95 °C for 20 min either before or after 150 mM NaCl was added. Emulsions were then stored for 24 h, and their mean particle diameters were measured by laser diffraction (Figure 3). In the presence of NEM no droplet flocculation was observed in the emulsions at any temperature, regardless of whether the emulsions were heated before or after NaCl addition. These results indicated that sulfhydryl bonds played an important role in determining the flocculation stability of β-Lg-stabilized oil droplets. Studies of β-Lg-NEM complexes in aqueous solutions have shown that their thermal denaturation is reversible, in contrast to β-Lg molecules alone, because conformational changes are not restricted by the formation of intramolecular or intermolecular covalent bonds.23 We therefore postulate that blocking the sulfhydryl groups prevented disulfide bond formation between protein molecules adsorbed on the same or on different droplets, which prevented droplet flocculation. In addition, we postulate that the adsorbed β-Lg-NEM complexes were able to undergo conformational changes that enabled the nonpolar amino acid groups to interact with neighboring nonpolar amino acids or to be directed away from the droplet surface. Consequently, the surface hydrophobicity of the emulsion droplets was not appreciably increased, which prevented droplet flocculation through an increased hydrophobic attraction between the droplets. Influence of Previous Heat Treatment on Thermal Stability. The results reported above indicate that heating an emulsion in the absence of salt to a temperature above

the thermal denaturation temperature of the adsorbed protein (∼70 °C), and then adding salt afterward can produce emulsions that are stable to droplet aggregation (Figure 1). From a technological standpoint, however, it is often desirable to be able to thermally process oil-inwater emulsions in the presence of salt. Our results show that extensive droplet aggregation occurs when β-Lgstabilized emulsions are heated in the presence of salt (Figure 1). We therefore investigated the possibility that preheating a β-Lg-stabilized oil-in-water emulsion in the absence of salt may increase its subsequent stability to thermal processing in the presence of salt. We prepared an oil-in-water emulsion that was stable to droplet aggregation by heating it to 95 °C for 20 min. We then cooled the emulsion to room temperature, added either 0 or 150 mM NaCl, incubated it for 2.5 h and heated it at temperatures ranging from 30 to 95 °C for 20 min. The extent of droplet aggregation in the emulsions was then measured by laser diffraction after they had been cooled to room temperature and stored for 24 h (Figure 4). No droplet aggregation was observed when the preheated emulsions were heated again in the absence of salt (0 mM NaCl) at any temperature, indicating that electrostatic repulsion between the droplets was sufficient to prevent droplet aggregation (data not shown). The preheated emulsions containing 150 mM NaCl had considerably better thermal stability than the nonpreheated emulsions containing the same salt concentration (Figure 4). In the nonpreheated emulsions, droplet aggregation was observed when the emulsions were heated at temperatures from 30 to 65 °C due to surface denaturation of β-Lg (see above). At higher temperatures (70-95 °C), the extent of droplet aggregation increased appreciably, due to thermal denaturation of β-Lg leading to extensive hydrophobic attraction and disulfide bond formation between the droplets (see above). On the other hand, no aggregation was observed in the preheated emulsions when they were heated at temperatures from 30 to 65 °C. In addition, the temperature where extensive droplet aggregation occurred (>80 °C) in the preheated emulsions was much higher than the temperature where it occurred in the nonpreheated emulsions (>65 °C). These results show that preheating the emulsions in the absence of salt leads to irreversible changes of the interfacial membrane surrounding the droplets which improves the subsequent stability of the emulsion to thermal processing in the presence of salt.

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Figure 5. Schematic representation of the influence of surface and thermal denaturation of adsorbed proteins on droplet flocculation in β-Lg-stabilized emulsions in the presence and absence of strong droplet-droplet interactions.

Proposed Mechanism for Flocculation Stability of β-Lg-Stabilized Emulsions. We propose the following physicochemical mechanism to account for the heat stability of β-Lg-stabilized oil-in-water emulsions based on the results of this study and of previous studies (Figure 5): Step 1. Protein Adsorption. The increase in the surface area of the oil phase exposed to the aqueous phase when small droplets are created in a homogenizer results in the adsorption of protein to the oil-water interface.9 Immediately after adsorption, the β-Lg has a threedimensional structure that is similar to that of the native protein dispersed in the aqueous phase.19 Step 2. Surface Denaturation. When a globular protein, such as β-Lg, adsorbs to a droplet surface, its molecular environment changes from one in which it is surrounded completely by water molecules to one in which it is in contact with water molecules on one side and oil molecules on the other side.35 As a consequence of this change in molecular environment, the globular protein undergoes a slow conformational change, whose driving force is the tendency for the protein molecule to maximize the number of favorable interactions and minimize the number of unfavorable interactions with its environment.35,36 As a result of these conformational changes, the protein exposes nonpolar, thiol and disulfide amino acid groups that were originally located in the core of the native protein.26 Exposure of these reactive groups leads to increased protein-protein interactions due to attractive hydrophobic interactions, thiol-disulfide interchange reactions, and (35) Norde, W. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 27-54. (36) Stuart, M. A. C. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 1-25.

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disulfide bond formation through thiol-thiol reactions.27,28 The relatively short-range covalent interactions involving thiol and disulfide groups can only take place between protein molecules that are in close proximity. Consequently, β-Lg molecules can only interact with neighboring protein molecules adsorbed to the same droplet when the repulsion between emulsion droplets is sufficiently strong to prevent the droplets from coming into close proximity. These interactions led to polymerization of the β-Lg molecules through intermolecular disulfide bond formation37 and to an increase in interfacial elasticity due to network formation within the interfacial membrane.38 When the attraction between emulsion droplets is sufficiently high to allow the droplets to come into close proximity, then both interdroplet and intradroplet interactions occur between protein molecules. Interdroplet protein-protein interactions can lead to flocculation of the emulsion droplets, which alters the rheology and creaming stability of the emulsions.34 Our results indicate that interdroplet protein-protein interactions, probably involving a combination of hydrophobic and disulfide bond formation, occur regardless of whether salt is added before or after the heating process, provided the emulsions are heated below the thermal denaturation temperature of the adsorbed proteins. Step 3. Thermal Denaturation. When the emulsion is heated above the thermal denaturation temperature of the adsorbed β-Lg molecules, the proteins undergo a conformational change that is much more extensive than that associated with surface denaturation.18,19,30 Consequently, the exposure of reactive nonpolar, thiol and disulfide amino acid groups is much more substantial, which leads to more extensive protein-protein interactions. When droplet repulsion is strong enough to prevent the droplets from coming into close proximity during the heat treatment, the protein molecules can only interact with neighboring protein molecules adsorbed to the same droplet. Previous studies have shown that extensive polymerization of adsorbed whey protein molecules occurs during heating due to intermolecular disulfide bond formation.13 The formation of covalent cross-links between the adsorbed protein molecules within the interfacial membrane leads to a substantial increase in interfacial elasticity39 and prevents the proteins from being desorbed by nonionic surfactants.13 Our results show that β-Lgstabilized emulsions heated above the thermal denaturation temperature of the protein under conditions that did not allow droplets to be in close proximity (low salt) were more stable to droplet flocculation than emulsions heated below this temperature when 150 mM NaCl was added after heating (Figure 1). This result suggests that the conformational changes that take place at the interface as a result of thermal denaturation of the protein led to a decrease in the hydrophobic attraction between droplets and/or a decrease in the ability of disulfide bonds to form between droplets. One possible explanation of the decreased susceptibility of heat-treated droplets to flocculation in the presence of salt is that the polymerization of adsorbed proteins was so extensive that most of the free thiol groups had reacted with other free thiol groups to form disulfide bonds, thereby terminating any further intermolecular thiol-disulfide interchange reactions.40 In contrast, appreciable droplet flocculation did occur in (37) Dickinson, E.; Matsumura, Y. Int. J. Biol. Macromol. 1991, 13, 26-32. (38) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437-471. (39) Patino, J. M. R.; Nino, M. R. R.; Sanchez, C. C. J. Agric. Food Chem. 1999, 47, 2241-2248.

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emulsions heated below the thermal denaturation temperature of the proteins when salt was added either before or after heating (Figure 1), which suggests that there were still sufficient free thiol groups available to promote thioldisulfide bond interchange reactions between proteins adsorbed to different droplets. When droplet attraction is high enough to enable the droplets to be in close proximity during heating, then both interdroplet and intradroplet interactions occur between thermally denatured protein molecules. The interaction of proteins adsorbed to different droplets leads to extensive droplet flocculation through a combination of hydrophobic and disulfide bond formation, which substantially increases the viscosity of the emulsions and alters their creaming stability.13 In particular, our results highlight the important role of intermolecular disulfide bonds on the thermal stability of β-Lg-stabilized emulsions. In the presence of NEM, no flocculation was observed in the emulsions due to surface or thermal denaturation (Figure 3). Blocking the thiol groups with NEM prevented disulfide bonds from forming between proteins adsorbed onto different droplets and thus reduced the tendency for droplet flocculation to occur. In addition, we postulate that NEM prevented the formation of intermolecular disulfide bonds that would have trapped the adsorbed proteins into conformations that exposed nonpolar groups to the aqueous phase. In other words, the protein molecules were able to adopt conformations that enabled the nonpolar groups to be orientated either toward the droplet surface or to be in contact with nonpolar groups on neighboring protein molecules adsorbed to the same droplet, thus reducing the droplet hydrophobicity and the tendency for flocculation to occur. (40) Roefs, S. P. F. M.; de Kruif, K. G. Eur. J. Biochem. 1994, 226, 883-889.

Conclusions We have shown that surface or thermal denaturation of globular proteins after adsorption to oil droplet surfaces can lead to extensive flocculation in oil-in-water emulsions, provided that the repulsive interactions between the droplets are not so large that they prevent droplets from coming into close proximity. It is postulated that denaturation of adsorbed proteins increases the surface hydrophobicity of emulsion droplets, which increases the hydrophobic attraction between droplets. In addition, surface denaturation exposes protein sulfhydryl groups to the aqueous phase, which promotes disulfide bond formation between proteins adsorbed to the same or to different emulsion droplets. We have established a method of improving the heat stability of β-Lg-stabilized emulsions in the presence of salt by preheating the emulsion above the thermal denaturation temperature of the absorbed proteins in the absence of salt. The data obtained in this study are important for 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), and by a United States Department of Agriculture, CREES, IFAFS Grant (Award 2001-4526). This work was also supported by the Postdoctoral Fellowship Program of Korea Science & Engineering Foundation (KOSEF). We also thank Davisco Foods International for kindly donating the protein ingredients used in this study. LA020385U