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Droplet Surface Properties and Rheology of Concentrated Oil in Water Emulsions Stabilized by Heat-Modified β-Lactoglobulin B Jes C. Knudsen,*,† Lars H. Øgendal,‡ and Leif H. Skibsted† Department of Food Science, Faculty of Life Sciences, UniVersity of Copenhagen, RolighedsVej 30, DK-1958 Frederiksberg C, Denmark, and Department of Natural Sciences, Faculty of Life Sciences, UniVersity of Copenhagen, ThorValdsensVej 40, DK-1871 Frederiksberg C, Denmark ReceiVed June 8, 2007 Effects of substituting native β-lactoglobulin B (β-lactoglobulin) with heat-treated β-lactoglobulin as emulsifier in oil in water emulsions were investigated. The emulsions were prepared with a dispersed phase volume fraction of Φ ) 0.6, and accordingly, oil droplets rather closely packed. Native β-lactoglobulin and β-lactoglobulin heated at 69 °C for 30 and 45 min, respectively, in aqueous solution at pH 7.0 were compared. Molar mass determination of the species formed upon heating as well as measurements of surface hydrophobicity and adsorption to a planar air/water interface were made. The microstructure of the emulsions was characterized using confocal laser scanning microscopy, light scattering measurements of oil droplet sizes, and assessment of the amount of protein adsorbed to surfaces of oil droplets. Furthermore, oil droplet interactions in the emulsions were quantified rheologically by steady shear and small and large amplitude oscillatory shear measurements. Adsorption of heated and native β-lactoglobulin to oil droplet surfaces was found to be rather similar while the rheological properties of the emulsions stabilized by heated β-lactoglobulin and the emulsions stabilized by native β-lactoglobulin were remarkably different. A 200-fold increase in the zero-shear viscosity and elastic modulus and a 10-fold increase in yield stress were observed when emulsions were stabilized by heat-modified β-lactoglobulin instead of native β-lactoglobulin. Aggregates with a radius of gyration in the range from 25 to 40 nm, formed by heating of β-lactoglobulin, seem to increase oil droplet interactions. Small quantities of emulsifier substituted with aggregates have a major impact on the rheology of oil in water emulsions that consist of rather closely packed oil droplets.
Introduction Foods are complex materials composed of particles with different sizes, and their interactions determine the physical properties of food systems.1 A wide range of foods are emulsions where oil droplets and/or air cells of various volume fractions are dispersed in a continuous water phase. Modification and control of particle interactions can be used to obtain desired properties of food emulsions with respect to stability and rheology. By mixing of bulk phases of oil, water, and a water-soluble emulsifier, oil in water emulsions can be made using mechanical energy input, for example, high speed stirring or high-pressure homogenization.2,3 Such procedures create an emulsion that consists of oil droplets covered by a thin film of emulsifier that enables the oil droplets to be dispersed in water. Oil droplets in water are unstable and through several steps, which may include flocculation, creaming, and coalescence, the emulsion will ultimately phase separate.2,3 Hydrophobic interactions favor formation of larger oil droplets owing to their smaller surface area relative to their volume. Stability of emulsion droplets depends on the properties of the thin film of emulsifier, and the use of various emulsifiers and stabilizers can provide emulsions where droplet interactions are reduced and long time stability of emulsions can be obtained.2,3 Milk proteins are widely used as food ingredients, and oil in water emulsions stabilized by whey * To whom correspondence should be addressed. E-mail: jes@life. ku.dk. Phone: +45-35333144. Fax: +45-35333190. † Department of Food Science. ‡ Department of Natural Sciences. (1) Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Nat. Mater. 2005, 4, 729-740. (2) Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain where Physics, Chemistry, Biology and Technology Meet; Wiley-VCH: New York, 1999. (3) McClements, D. J. Food Emulsions. Principles, Practices, and Techniques; CRC Press: London, 2005.
protein and particularly by β-lactoglobulin, the most abundant protein in whey, have been investigated in detail.4-6 Investigations have mainly focused on flocculation and coalescence of oil droplets in emulsions consisting of a dispersed phase volume fraction (Φ) below 0.5. Less information exists about rheology of concentrated emulsions stabilized by β-lactoglobulin.7,8 β-Lactoglobulin forms aggregates upon heating in aqueous solution, and the aggregates coexist with oligomers and the fraction of nonaggregated species.9 Native dimeric β-lactoglobulin, which is the dominating species in aqueous solution with pH 7-9,9,10 has a diameter of about 5 nm,11 whereas the size of the aggregates formed upon heating of β-lactoglobulin is from 40 to 60 nm in diameter.9,12 The surface hydrophobicity of heat induced oligomers of β-lactoglobulin is higher than that of native β-lactoglobulin as has been shown by fluorescence spectroscopy using 1-anilino-naphthalene-8-sulfonate (ANS) as a probe.12 Heating is commonly used during processing of foods, and the effects of different molecular structures and surface properties of β-lactoglobulin on subsequent flow and viscoelastic properties of β-lactoglobulin stabilized emulsions are of interest to the food industry due to the wide applications of whey proteins as ingredient in various foods and food dispersions. Heat treatment (4) McClements, D. J. Curr. Opin. Colloid Interface Sci. 2004, 9, 305-313. (5) Dickinson, E. Soft Matter 2006, 2, 642-652. (6) Tcholakova, S.; Denkov, N. D.; Ivanov, I. B.; Campbell, B. AdV. Colloid Interface Sci. 2006, 123-126, 259-293. (7) Dimitrova, T. D.; Leal-Calderon, F. Langmuir 2001, 17, 3235-3244. (8) Dimitrova, T. D.; Leal-Calderon, F. AdV. Colloid Interface Sci. 2004, 108109, 49-61. (9) Bauer, R.; Carrotta, R.; Rischel, C.; Øgendal, L. Biophys. J. 2000, 79, 1030-1038. (10) Sawyer, L.; Kontopidis, G. Biochim. Biophys. Acta 2000, 1482, 136148. (11) Carrotta, R.; Arleth, L.; Pedersen, J. S.; Bauer, R. Biopolymers 2003, 70, 377-390. (12) Carrotta, R.; Bauer, R.; Waninge, R.; Rischel, C. Protein Sci. 2001, 10, 1312-1318.
10.1021/la703810g CCC: $40.75 © 2008 American Chemical Society Published on Web 02/21/2008
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of emulsions stabilized by β-lactoglobulin has been studied with respect to flocculation and coalescence of oil droplets;13-17 however, it is largely unexplored how rheology of concentrated emulsions can be modified by substituting native β-lactoglobulin with heat treated β-lactoglobulin as emulsifier. We have accordingly studied physical and chemical properties of oil in water emulsions with an oil volume fraction of Φ ) 0.6 and solely stabilized by β-lactoglobulin. The random close packing limit for equal sized spheres is a volume fraction of Φ ) 0.64,18 and the maximum close packing limit for equal-sized spheres is a volume fraction of Φ ) 0.74.2,18 Oil droplets in concentrated emulsions, with oil volume fractions about Φ ≈ 0.64, may adopt nonspherical shapes due to the dense packing. The possibility of modifying the oil droplet interactions and the emulsion rheology, through modification of β-lactoglobulin by heat treatment, prior to emulsification, was investigated. A molar mass determination of the species formed upon heating was made, and the surface hydrophobicity of heated β-lactoglobulin was measured as well. Heated and native β-lactoglobulin were also studied with respect to adsorption to a planar air/water interface. The emulsions were studied by confocal laser scanning microscopy, and sizes of oil droplets were measured by light scattering. Furthermore, the amount of protein adsorbed to surfaces of oil droplets was assessed, and finally oil droplet interactions in the emulsions were quantified by rheology, which included steady shear and small and large amplitude oscillatory shear measurements. Materials and Methods Materials. β-Lactoglobulin B (β-lactoglobulin) was purified according to Kristiansen et al.19 Protein content in dry matter was 96%, fat content in dry matter was 0.3%, calcium content was 0.6%, and sodium content was 0.2%. The fat fraction was removed by filtering, which is described below. Final concentrations of calcium and sodium are calculated to be below 1 mM in the 5 mg/mL protein samples. 1-Anilino-naphthalene-8-sulfonate (ANS) and Bodipy were from Molecular Probes, Eugene, OR. Bovine serum albumin (BSA), dimethyl sulfoxide, Rhodamine B, sodium azide, and n-tetradecane (99%) were from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. Imidazole, HCl, K2HPO4, KH2PO4, NaCl, and NaOH were from Merck, Darmstadt, Germany. All aqueous solutions were made from purified water (Milli-Q Plus, Millipore Corp., Bedford, MA). All chemicals employed were of analytical grade. Sample Preparation. Solutions of β-lactoglobulin were prepared in 20 mM imidazole buffer adjusted to pH 7.0 with HCl and added 0.5 mg/mL NaN3. The buffer was filtered through a 0.45 µm pore filter. Protein solutions were centrifuged at 11 000g for 5 min to remove large aggregates and further filtered through a 0.1 µm pore filter and subsequently through a 100 000 Da cutoff filter (Millipore Corporation, Bedford, MA) using an Amicon Ultrafiltration Cell (Amicon Corp., Scientific Systems Division, Danvers, MA). The filtered solution was finally diluted with buffer to 5.0 mg/mL. The concentration of β-lactoglobulin was determined from the absorbance at 280 nm employing an absorption coefficient of 0.96 mL mg-1 cm-1.20,21 Solutions were kept at 5 °C and used for experiments within 4 days. (13) Dickinson, E.; James, J. D. J. Agric. Food Chem. 1998, 46, 2565-2571. (14) Kim, H.-J.; Decker, E. A.; McClements, D. J. Langmuir 2002, 18, 75777583. (15) Sliwinski, E. L.; Roubos, P. J.; Zoet, F. D.; van Boekel, M. A. J. S.; Wouters, J. T. M. Colloids Surf., B 2003, 31, 231-242. (16) Kim, H.-J.; Decker, E. A.; McClements, D. J. Langmuir 2005, 21, 134139. (17) Tcholakova, S.; Denkov, N. D.; Sidzhakova, D.; Campbell, B. Langmuir 2006, 22, 6042-6052. (18) Larson, R. G. The Structure and Rheology of Complex Fluids; Oxford University Press: New York, 1999. (19) Kristiansen, K. R.; Otte, J.; Ipsen, R.; Qvist, K. B. Int. Dairy J. 1998, 8, 113-118. (20) Eigel, W. N.; Butler, J. E.; Ernstrom, C. A.; Farrell, H. M.; Harwalkar, V. R.; Jenness, R.; Whitney, R. M. J. Dairy Sci. 1984, 67, 1599-1631.
Knudsen et al. Heat Treatment. Samples of β-lactoglobulin, 5.0 mg/mL in 20 mM imidazole buffer pH 7.0 (containing 0.5 mg/mL NaN3), were transferred to glass tubes, which were tightly sealed to avoid evaporation. The glass tubes containing 2 mL sample were immersed in a water bath at 69 °C for 30 or 45 min. An ice bath was used to cool the samples to room temperature immediately after heating. The β-lactoglobulin samples were used for further analysis on the day of heat treatment. Emulsification. A preemulsion was made by mixing 60 vol % n-tetradecane and 40 vol % 5 mg/mL β-lactoglobulin in buffer (20 mM imidazole, pH 7.0, 0.5 mg/mL NaN3) using a high speed stirrer (Ultra-turrax T 25 from IKA Works, Inc., Wilmington, NC) operating at 24 000 rpm for 1 min. Either untreated or heated β-lactoglobulin was used. Immediately after preparation the preemulsion was subjected to one pass through a high-pressure homogenizer (EmulsiFlex-C5 from Avestin, Inc., Ottawa, ON, Canada) at ambient pressure and subsequently homogenized at 50 000 kPa. The emulsions were stored at room temperature until analysis, which was on the day of preparation. Size Exclusion Chromatography Followed by Simultaneous Static Light Scattering and Refractive Index Measurements. The β-lactoglobulin samples were injected on the size exclusion HPLC system less than 2 h after heating. The dilution of samples to 0.1 mg/mL was made with 0.020 M imidazole buffer, pH 7.0 (containing 0.5 mg/mL NaN3). A Superdex 200 column (300 mm × 7.8 mm i.d.) from Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden, was used. A 500 µL loop was used, and samples were injected at a concentration of either 5.0 or 0.1 mg/mL. The flow rate was 0.5 mL/min. The eluent was 0.001 M K2HPO4, 0.009 M KH2PO4, and 0.015 M NaCl adjusted with NaOH to pH 7.0. After separation on the column, static light scattering from the eluted components was measured by a Dawn EOS with a K2 cell, (Wyatt Technology, Santa Barbara, CA), and subsequently, a refractive index detector (RID-10A, Shimadzu, Japan) was used to determine the concentration of the eluted species. A Debye plot, i.e., light scattering signal versus q2 where q ) 4πn/λ0 sin θ/2, n being the refractive index of the buffer, λ0 the wavelength of the laser, and θ the scattering angle, was made to estimate radius of gyration of the aggregates. The molar mass of the aggregates was estimated using the intercept of the y-axis in the Debye plot divided by the refractive index. Bovine serum albumin was used for calibration of the light scattering signal. Further description of the method is given by Wen et al.,22 Folta-Stogniew, and Williams.23 Measurement of Surface Hydrophobicity. The 1-anilinonaphthalene-8-sulfonate (ANS) assay was performed with a LS 55 Luminescence Spectrometer from Perkin-Elmer Instruments, Beaconsfield, U.K. Excitation wavelength was 400 nm, and the emission spectra were recorded at 410-650 nm, with a scan speed of 50 nm/min and exitation and emission slit width of 5.0 nm. For all samples the β-lactoglobulin and ANS concentration were 2 and 100 µM, respectively, both in 20 mM imidazole pH 7.0, containing 0.5 mg/mL NaN3, and the scan was started 5 min after mixing of the two reagents. All measurements were made at 22 °C. Surface Tension. Surface tension of β-lactoglobulin solutions was measured using a Sigma 703 tensiometer (KSV instruments Ltd., Helsinki, Finland) equipped with a Wilhelmy plate. The surface layer of a solution of 0.01 mg/mL β-lactoglobulin in 20 mM imidazole buffer pH 7.0 containing 0.5 mg/mL NaN3 was removed by suction, and the Wilhelmy plate was immediately lowered in the air/water surface. Changes in surface tension were measured in a thermostated room at 21 °C for 100 min. All surface tension measurements of solutions of heated β-lactoglobulin were made on the same day as heating. Light Scattering Measurements of Oil Droplet Sizes. Distributions of oil droplet sizes in emulsions were obtained using a Malvern Mastersizer Microplus, Malvern Instruments Ltd., Malvern, U.K., (21) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci. 1995, 4, 2411-2423. (22) Wen, J.; Arakawa, T.; Philo, J. S. Anal. Biochem. 1996, 240, 155-166. (23) Folta-Stogniew, E.; Williams, K. R. J. Biomol. Technol. 1999, 10, 5163.
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connected to a 50 mL sample stirring unit. Measurements were made on emulsions diluted in buffer, 20 mM imidazole, pH 7.0, with 0.5 mg/mL NaN3. A model based on homogeneous oil spheres in water, with refractive indices of 1.46 and 1.33, respectively, was used for the calculation of oil droplet size distributions. The mean diameter, d32 ) Σnidi3/Σnidi2, where ni is the number of droplets within the size class of droplets with diameter di, was calculated from the size distribution. Measurements were conducted the day the emulsions were made. Protein Load Measurements. The surface load of an emulsion, i.e., the amount of emulsifier adsorbed/unit surface area of oil droplets, was calculated from3,24 Γ)
(Cinitial - Cserum)d32 6Φ
where d32 is the mean volume to surface area diameter of oil droplets, Cinitial is the initial protein concentration of β-lactoglobulin in the aqueous phase before emulsification, Cserum is the concentration of β-lactoglobulin in the emulsion serum phase, and Φ is the dispersed phase volume fraction in the emulsions. Emulsions were centrifuged at 3000g for 10 min followed by removal of subnatant, which was further centrifuged at 6500g for 10 min to separate the oil from the serum phase. Samples were carefully removed from the serum phase with a syringe, and the protein concentration was determined by size exclusion chromatography followed by refractive index measurements. Protein concentration was estimated as the peak areas in elution profiles for samples relative to the peak area for 5.0 mg/mL native β-lactoglobulin. All measurements were made at room temperature, and emulsions were analyzed the same day they were made. Confocal Laser Scanning Microscopy. The oil droplets in the β-lactoglobulin stabilized emulsions were visualized using a confocal laser scanning microscope, Leica DM IRBE, Leica Microsystems Wetzlar GmbH, Wetzlar, Germany, with an argon/krypton and a helium/neon laser. A Leica TCS SP2 confocal laser scanning microscope was used to investigate some samples (emulsions stabilized by β-lactoglobulin heated at 69 °C for 45 min). A water immersion objective enabling a magnification of 63× was used. Fluorescent dyes were dissolved to a concentration of 1 mg/mL Rhodamine B in 20 mM imidazole pH 7.0, containing 0.5 mg/mL NaN3 and Bodipy in dimethyl sulfoxide. Emulsion samples were diluted about 1% upon addition of fluorescent dyes. Excitation wavelength was 488 nm and 543/568 nm for Bodipy and Rhodamine B, respectively, and the emission was recorded at 494-550 and at 591-690 nm. Bodipy and Rhodamine B were used to stain the oil and the protein, respectively. Samples were placed on glass, and six images were recorded and averaged for each micrograph. Rheology. A Bohlin C-VOR rheometer, Bohlin Instruments Ltd., Malvern, U.K., with a measuring system consisting of a sandblasted vane (V25, radius 12.5 mm) and a sandblasted cup (radius 13.75 mm), was used in all experiments. Samples were gently poured into the cup, and an equilibration time of 5 min at 25 °C was applied before the measurements. All measurements were made at 25 °C. Shear flow experiments encompassed 30 steady shear rates (0.0001 s-1e γ˘ e 1000 s-1) each held for 1 min and with recording of the shear stress (σ) during the last 10 s from which the apparent viscosity η(γ˘ ) was calculated. Small and large amplitude oscillatory shear rheology was made at a constant shear frequency of 1 Hz. The amplitude was varied in 50 steps, where the stress was controlled and stepwise increased from 0.01 to 100 Pa. The stress was applied for 20 s at each step, and the strain amplitude was recorded and elastic (G′) and viscous (G′′) moduli were calculated.
Results and Discussion The light scattering and refractive index signal obtained after size exclusion HPLC separation of β-lactoglobulin are shown in Figure 1. (24) Nilsson, L.; Bergenståhl, B. Langmuir 2006, 22, 8770-8776.
Figure 1. Refractive index signal for 5.0 mg/mL native β-lactoglobulin and β-lactoglobulin heated at 69 °C for 30 and 45 min. The vertical lines indicate the elution volume of native β-lactoglobulin (N), dimer (D), and aggregates (A). The separations were conducted by SE-HPLC. Table 1. Molar Mass of β-Lactoglobulin Aggregates and Size of Aggregates after Heat Treatmenta conditions applied to protein soln (°C, min)
aggregates (kg/mol)
radius of gyration for aggregates (nm)
no heating 69, 30 69, 45
2500-104 3000-104
25-40 30-40
a Samples were 5.0 mg/mL β-lactoglobulin solutions. The separations were conducted by SE-HPLC.
An equilibrium distribution between dimers and monomers exists for native β-lactoglobulin at pH 7-9 with the dimer as the dominating species.9,10 The light scattering and refractive index signals in the figure are scaled to the same height at the peak for dimeric β-lactoglobulin. The refractive index signal is proportional to the particle weight concentration, and no aggregates were present in solutions with native β-lactoglobulin as seen from the absence of a refractive index signal at 7-8 mL retention volume (Figure 1). The tiny light scattering signal obtained at 7-8 mL retention volume for native β-lactoglobulin may arise from an extremely low concentration of large aggregates since the light scattering signal is proportional to the molecular weight of the particles and the particle concentration. The refractive index in Figure 1 shows that heating of β-lactoglobulin at 69 °C for 30 min formed a small fraction, ∼7%, of aggregates that coexisted with the nonaggregated species. Furthermore, heating for 45 min slightly increased the fraction of aggregates, ∼9%, compared to the fraction formed during heating for 30 min. The aggregates have a rather broad distribution of molecular masses, ranging from 2500 to 104 kg/mol (Table 1).
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Figure 3. ANS fluorescence emission spectra for native β-lactoglobulin (full line), β-lactoglobulin heated for 30 min at 69 °C (dash dot line), and β-lactoglobulin heated at 69 °C for 45 min (dashed line). The β-lactoglobulin and ANS concentrations were 2 and 100 µM, respectively.
Figure 2. Refractive index signal for 0.1 mg/mL native and heated β-lactoglobulin. Heating of 5.0 mg/mL β-lactoglobulin at 69 °C for 30 and 45 min was used. The vertical lines indicate the elution volume of native β-lactoglobulin (N), dimer (D), and aggregates (A). The separations were conducted by SE-HPLC.
Radii of gyration for the aggregates were derived from a Debye plot, i.e., the angular dependency of the light scattering, and were found to be in the range from 25 to 40 nm (Table 1). The elution volume for native β-lactoglobulin was increased when samples diluted to 0.1 mg/mL were applied to the column (Figure 2) compared to the elution volume observed when 5.0 mg/mL was applied to the column (Figure 1). This behavior has been observed previously for native β-lactoglobulin and explained by a shift in the equilibrium between dimers and monomers.9,25 When 0.1 mg/mL of the heated sample was injected on the column, a small fraction with a molar mass corresponding to a dimer appeared (Figure 2). This small fraction is most probably covalently linked dimers since the elution volume for this species has been shown to be stable upon dilution.9,25 Furthermore, the fraction of aggregates did not change elution volume upon dilution (Figures 1 and 2). Aggregates were the main species formed using the present mild conditions with respect to heating of β-lactoglobulin. Other studies of heat induced aggregation of β-lactoglobulin revealed formation of oligomers, mainly trimers, coexisting with aggregates and the fraction of native β-lactoglobulin.9,12 Oligomers were not observed in this study at neutral pH, most probably oligomers immediately reacted into aggregates. The heat-induced aggregation of β-lactoglobulin is faster at neutral pH, near the isoelectric point where aggregation is promoted, compared to higher pH where oligomers have been observed.9,12 The SE-HPLC analysis documented that a small, but significant, fraction of aggregates was present after heating of β-lactoglobulin, and effects of such a small quantity of aggregates were studied further with respect to surface and emulsifying properties of β-lactoglobulin. (25) Knudsen, J. C.; Lund, M.; Bauer, R.; Qvist, K. B. Langmuir 2004, 20, 2409-2415.
The change in surface hydrophobicity of β-lactoglobulin induced by heating was assessed by using the dye ANS, which fluoresces upon binding to hydrophobic domains.26,27 Heating of β-lactoglobulin at 69 °C for 30 and 45 min gradually increased the surface hydrophobicity compared to that of native β-lactoglobulin (Figure 3). However, the increase in surface hydrophobicity was much less pronounced in comparison to the large difference in the surface hydrophobicity that was observed between equimolar amounts of heat-induced oligomers alone, separated from aggregated β-lactoglobulin, and native β-lactoglobulin.12 The contribution to the increased ANS fluorescence intensity may predominantly arise from the fraction of aggregates since an increased fraction of aggregates, formed by longer heating time, was paralleled by an increase in ANS fluorescence intensity (Figures 1 and 3). The effects of the heat induced changes in the molecular properties of β-lactoglobulin, especially with regard to surface hydrophobicity and aggregation, were explored in a number of ways: by measurements of adsorption to planar air/water interfaces and adsorption to oil droplet surfaces in emulsions as well as by investigation of microstructure and rheology of emulsions stabilized by β-lactoglobulin, native or heated prior to emulsification. Development of the dynamic surface pressure at the air/water surface is shown for native and heated β-lactoglobulin in Figure 4. Previous results obtained with native β-lactoglobulin at neutral pH showed an upper level of approximately 20 mN/m for the surface pressure after long time adsorption,25,28 which agrees with the level found in the present study. A similar level was found for heat treated whey protein isolate at neutral pH.29 The growth of the dynamic surface pressure was similar for samples with heated β-lactoglobulin compared to that observed for the untreated samples indicating similar adsorption to the air/water surface for heated and native β-lactoglobulin. To measure the surface load (Γ), in this case the amount of protein adsorbed to surfaces of oil droplets in an emulsion relative to the surface area of the droplets, the protein concentration in the serum phase of the emulsions was determined as well as the sizes of the oil droplets. The β-lactoglobulin concentration in emulsion serum was slightly higher in emulsions stabilized by heated β-lactoglobulin compared to the serum concentration in (26) Engelhard, M.; Evans, P. A. Protein Sci. 1995, 4, 1553-1562. (27) Mataulis, D.; Lovrien, R. Biophys. J. 1998, 74, 422-429. (28) Bos, M. A.; Nylander, T. Langmuir 1996, 12, 2791-2797. (29) Schmitt, C.; Bovay, C.; Rouvet, M.; Shojaei-Rami, S.; Kolodziejczyk, E. Langmuir 2007, 23, 4155-4166.
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Table 2. Characteristics of Oil in Water Emulsions Made with an Oil Volume Fraction of Φ ) 0.6 and Stabilized by Native or Heat-Treated β-Lactoglobulina condition s applied to protein soln (°C, min)
initial β-lactoglobulin concn in aq phase before emulsification (mg/mL)
β-lactoglobulin concn in emulsion serum phase (mg/mL)
av oil droplet diameter (d32) (µm)
surface load (mg/m2)
no heating 69, 30 69, 45
5.0 5.0 5.0
0.34 ( 0.02 0.44 ( 0.02 0.46 ( 0.03
3.2 ( 0.05 3.7 ( 0.07 4.3 ( 0.06
4.2 ( 0.08 4.7 ( 0.08 5.4 ( 0.04
a Heating was applied to 5.0 mg/mL β-lactoglobulin solutions before emulsification. The surface load expresses the amount of protein adsorbed to the oil droplet surfaces in the emulsions relative to the surface area of the oil droplets. The average oil droplet size (d32) in emulsions was measured by light scattering. Results are means of triplicate determinations.
Figure 4. Time-dependent air/water surface pressure measured at 21 °C of 0.01 mg/mL β-lactoglobulin solutions at pH 7. The surface layer was removed at t ) 0. Native β-lactoglobulin (O); β-lactoglobulin heated at 69 °C for 30 min (0) and 45 min (4). Error bars show sample standard deviation.
Figure 5. Size distributions obtained from light scattering measurements of oil droplets in β-lactoglobulin stabilized oil in water emulsions. The oil droplets were stabilized by native β-lactoglobulin (full line) and β-lactoglobulin heated at 69 °C for 30 min (dashed line) or 45 min (dotted line) before emulsification.
emulsions stabilized by native β-lactoglobulin (Table 2). Sizes of oil droplets were determined by light scattering and small differences were observed between size distributions of oil droplets in emulsions made from native or heated β-lactoglobulin, as seen in Figure 5. The oil droplet sizes in emulsions stabilized by heat modified β-lactoglobulin were slightly larger compared to the sizes of oil droplets in emulsions made from native β-lactoglobulin as also seen from the derived mean diameters (d32), which are listed in Table 2. The emulsions were further investigated by confocal laser scanning microscopy. Each micrograph is an overlay of two images; the red color in the micrographs originates from staining of protein, and the green color originates from staining of oil (Figure 6). Oil droplet diameters of about 1-10 µm can be observed, and these sizes are within the distribution of oil droplet sizes obtained by light
scattering (Figure 5). The data in Table 2 were further used to calculate the surface load (Γ). A smaller amount of protein was adsorbed to surfaces of oil droplets in emulsions stabilized by heated β-lactoglobulin compared to emulsions stabilized by native β-lactoglobulin; however, the emulsion prepared with heated β-lactoglobulin contained larger oil droplet sizes and, thus, had a smaller total surface area. The calculations showed that a somewhat higher surface load was present, indicative of a slightly denser/thicker protein layer on oil droplets surfaces in the emulsions stabilized by heated β-lactoglobulin compared to the surface load in emulsions stabilized by native β-lactoglobulin (Table 2). In conclusion, subtle changes in the adsorption of β-lactoglobulin to oil/water surfaces are induced by heating of β-lactoglobulin. In contrast, remarkable differences between the properties of emulsions made with native β-lactoglobulin and emulsions made with heated β-lactoglobulin were observed by quantification of oil droplet interactions by rheological measurements, including viscosity measurements and determination of moduli of the emulsions. Figure 7 shows the apparent viscosity of the emulsions at a range of steady shear rates. The apparent viscosity in the low shear rate regime was increased about 50- and 200-fold for the emulsions stabilized by β-lactoglobulin heated at 30 and 45 min, respectively, compared to the emulsion stabilized by native β-lactoglobulin. Upon shearing of the samples at high rates, the viscosity became comparable, as expected, since the emulsions contained equal volume fraction Φ ) 0.6 of oil and the viscosity of a dispersion of particles at high shear rates is governed by the volume fraction of particles.30 The molecular events that may explain the shear thinning behavior, i.e., decreasing viscosity upon increasing shear rate, of the emulsions could be disentanglements of aggregates between oil droplets or weakening of interactions between surface molecules on different oil droplets.3 The apparent viscosity of the emulsions versus the applied shear stress, at a range of steady shear rates, is shown in Figure 8. The apparent viscosity decreases abruptly at a shear stress around 5 and 10 Pa, which are the yield stresses for the emulsions stabilized by β-lactoglobulin heated at 69 °C for 30 and 45 min, respectively. A more gradual decrease in the apparent viscosity appears for the emulsion stabilized by native β-lactoglobulin at a shear stress between 0.5 and 1 Pa. Small and large amplitude oscillatory shear rheology, at a constant frequency, was applied to determine the moduli of the two emulsions (Figure 9). The magnitude of the elastic modulus (G′), within the linear viscoelastic region, was about 1 Pa for the emulsion stabilized by native β-lactoglobulin and was increased 50- and 200-fold for the emulsions stabilized by β-lactoglobulin heated at 69 °C for 30 and 45 min, respectively. The viscous modulus (G′′) was increased about 6- and 30-fold when the (30) Moschakis, T.; Murray, B. S.; Dickinson, E. Langmuir 2006, 22, 47104719.
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Figure 7. Apparent viscosity at a range of steady shear rates (0.0001 s-1 e γ˘ e 1000 s-1) measured on oil in water emulsions stabilized by β-lactoglobulin. The emulsions contained an oil volume fraction of Φ ) 0.6 and were stabilized by native β-lactoglobulin (O) or β-lactoglobulin heated at 69 °C for 30 min (0) or 45 min (4) before emulsification.
Figure 8. Apparent viscosity versus shear stress at a range of steady shear rates (0.0001 s-1 e γ˘ e 1000 s-1) measured on oil in water emulsions stabilized by β-lactoglobulin. The emulsions contained an oil volume fraction of Φ ) 0.6 and were stabilized by native β-lactoglobulin (O) or β-lactoglobulin heated at 69 °C for 30 min (0) or 45 min (4) before emulsification. The same samples as used in Figure 7 are shown.
Figure 6. Overlay images obtained by confocal laser scanning microscopy of oil droplets in β-lactoglobulin stabilized oil in water emulsions. The emulsions contained an oil volume fraction of Φ ) 0.6 and were stabilized by native β-lactoglobulin (A) or β-lactoglobulin heated before emulsification for at 69 °C for 30 min (B) or 45 min (C). Fluorescent dyes were used to stain oil and protein, which appear green and red, respectively.
emulsions were stabilized by β-lactoglobulin heated for 30 and 45 min, respectively, instead of native. Thus, the emulsions
stabilized by heated β-lactoglobulin were much more solid like compared to the emulsions stabilized by native β-lactoglobulin. Furthermore, the crossover of the elastic (G′) and viscous modulus (G′′) occurred at a shear stress about 1 Pa, during small and large amplitude deformation of the emulsion stabilized by native β-lactoglobulin, while for the emulsions stabilized by β-lactoglobulin heated at 30 and 45 min the crossover occurred at a shear stress about 5 and 15 Pa, respectively. This study demonstrates that zero-shear viscosity and elastic modulus of β-lactoglobulin stabilized oil in water emulsions can be increased about 200-fold by a moderate heat modification of β-lactoglobulin prior to emulsification (Figures 7-9). Furthermore, the yield stress was increased about 10-fold in emulsions where heat modified β-lactoglobulin was used as emulsifier instead of native β-lactoglobulin (Figure 8). The emulsions were prepared with a dispersed phase volume fraction of Φ ) 0.6 leaving the oil droplets rather closely packed (Figure 6). The emulsions made with either native or heated β-lactoglobulin appeared similar with respect to their microstructure (Figure 6) and distribution of emulsifier between serum phase and oil droplet
β-Lactoglobulin-Stabilized Oil in Water Emulsions
Figure 9. Small amplitude oscillatory shear measurements on oil in water emulsions stabilized by β-lactoglobulin. The emulsions contained an oil volume fraction of Φ ) 0.6 and were stabilized by β-lactoglobulin heated at 69 °C for 45 min (A) or for 30 min (B) before emulsification or stabilized by native β-lactoglobulin (C).
surfaces (Table 2), while their rheology and accordingly oil droplet interactions differed considerably (Figures 7-9). In an attempt to clarify the underlying mechanism for the increase in oil droplet interactions, we have compared molecular properties of native and heat-modified β-lactoglobulin and resulting surface properties of the oil droplets as well as adsorption properties of native and heat-modified β-lactoglobulin. The applied heating at 69 °C does not facilitate adsorption of β-lactoglobulin to oil droplets; instead somewhat larger droplets, with a resulting smaller total surface area are formed, which in turn result in a higher surface load (Γ) in emulsions stabilized by heat-modified β-lactoglobulin compared to the surface load in emulsions stabilized by native β-lactoglobulin (Table 2). The equilibrium between the amount of β-lactoglobulin present in the emulsion serum and the amount of β-lactoglobulin adsorbed to surfaces of oil droplets seems mainly to be governed by the surface area of oil droplets generated upon homogenization. Heat modification of β-lactoglobulin seemed to affect the distribution between adsorbed and nonadsorbed β-lactoglobulin to a rather limited extent (Table 2). Similarly, the adsorption of β-lactoglobulin to a planar air/water interface was found to be almost unaffected by the heat modification (Figure 4). One would expect that the presence of aggregates decreases the diffusion and adsorption to surfaces. However, this effect may be more or less counteracted by the increased surface hydrophobicity of heatmodified β-lactoglobulin. Since adsorption properties of heated β-lactoglobulin were rather similar compared to that of native β-lactoglobulin, other factors may play a role in connection with the marked increase
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in viscosity, yield stress, and moduli, which occurred when emulsions were stabilized by heated instead of native β-lactoglobulin (Figures 7-9). The oil droplet surfaces are covered by protein molecules, and for the heat-modified β-lactoglobulin, the protein molecules possess a higher surface hydrophobicity (Figure 3). Furthermore, a fraction of the heat-modified molecules are aggregates with a radius of gyration of about 25-40 nm (Figures 1 and 2; Table 1). The oil droplet interactions can be increased as a result of interactions between the surface molecules that cover the oil droplets, and these interactions may be enhanced by the presence of aggregates as well as molecules with increased surface hydrophobicity as in the case with heat-modified β-lactoglobulin. The large dispersed phase volume fraction Φ ) 0.6, which is near the random close packing limit for equal sized spheres at 0.64,18 could also be of importance for oil droplet interactions and the rheology of the emulsions. An exact close packing limit for oil droplets in polydisperse emulsions, such as protein-stabilized emulsions, does not exist since oil droplets in polydisperse emulsions may pack more efficiently compared to oil droplets in monodisperse emulsions with equivalent oil volume fractions. The micrographs show indeed that the oil droplets in the emulsions are rather tightly packed (Figure 6). Accordingly, it seems reasonable to expect increased oil droplet interactions when aggregates of heated β-lactoglobulin with radii of 25-40 nm are adsorbed on oil droplet surfaces. The fraction of aggregates which coexisted with the nonaggregated species was in the range 7-9%. This small quantity of larger sized particles together with smaller sized molecules has large impact on the rheology of emulsions that consist of closely packed oil droplets. It has been shown, by interfacial rheology, that the shear viscosity of a planar oil/water surface was increased about 3-fold when the thin film at the interface was heated β-lactoglobulin instead of native β-lactoglobulin.31 However, when multiple oil droplet surfaces interact, as quantified with bulk rheology, the surface molecules on different droplets and their interactions may explain the oil droplet interactions rather than the rigidity of the single surface layer. The adsorption behavior of native and heated β-lactoglobulin to surfaces was rather similar; however, the results suggest that surface molecules consisting of aggregates, instead of smaller sized molecules, have a large contribution to the oil droplet interactions and rheology of emulsions. The magnitude of the droplet interactions and thus effects on rheology may vary depending on the volume fraction in the emulsions and the concentration of aggregates. The current investigation focused on two sets of aggregates and an oil volume fraction of Φ ) 0.6. Aggregates in larger concentrations may be used to increase viscosity and viscoelastic properties of emulsions with a lower oil volume fraction. The perspective, regarding food grade emulsions, is the possibility to enhance viscosity and viscoelasticity of low fat products. On the other hand, the concentration of aggregates needed to influence flow and viscoelastic properties of emulsions may be expected to decrease for emulsions with an oil droplet and/or air cell volume fraction in the vicinity of or above the close packing limit.
Conclusions Heating of β-lactoglobulin in aqueous solution at 69 °C for 30 and 45 min was used to modify β-lactoglobulin as emulsifier. Oil in water emulsions were made with an oil volume fraction of Φ ) 0.6. The two heat treatments of 30 and 45 min, respectively, induced a remarkably 50- and 200-fold increase in zero-shear viscosity and elastic modulus of oil in water emulsions (31) Roth, S.; Murray, B. S.; Dickinson, E. J. Agric. Food Chem. 2000, 48, 1491-1497.
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when the emulsions were stabilized by heated β-lactoglobulin instead of native. Furthermore, the yield stress was increased up to 10-fold in emulsions where heat-modified β-lactoglobulin was used as emulsifier instead of native β-lactoglobulin. Modification of β-lactoglobulin as emulsifier and subsequent effects on rheology of emulsions that consist of closely packed oil droplets seems mainly to be a result of heat-induced aggregates formed in a small quantity. The presence of aggregates at oil droplet surfaces increases oil droplet interactions. The adsorption of heated and native β-lactoglobulin to surfaces was found to be
Knudsen et al.
fairly similar. Controlled heat treatment of β-lactoglobulin may be relevant for industrial applications to tailor concentrated oil in water emulsions with respect to rheology. Acknowledgment. This study was supported by the Danish Dairy Research Foundation and The Ministry of Food, Agriculture, and Fisheries. The technical assistance from Pia S. Pedersen and Marianne L. Jensen is gratefully acknowledged. We also gratefully thank Ditte Arltoft for advice during the work. LA703810G
