- Email: [email protected]
Influence of pH and CaCl2 on the stability of dilute whey protein stabilized emulsions
Food Research International 33 (2000) 15±20
www.elsevier.com/locate/foodres
In¯uence of pH and CaCl2 on the stability of dilute whey protein stabilized emulsions Asylbek Kulmyrzaev, Ratjika Chanamai, D. Julian McClements * Biopolymer and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
Abstract The in¯uence of pH and CaCl2 on the physical stability of dilute oil-in-water emulsions stabilized by whey protein isolate has been studied. The particle size, zeta potential and creaming stability of 0.05 wt% soy bean oil-in-water emulsions (d 0.53 mm) were measured with varying pH (3 to 7) and CaCl2 concentration (0 to 20 mM). In the absence of CaCl2 extensive droplet aggregation occurred around the isoelectric point of the whey proteins (4 < pH < 6) because of their low electrical charge, which led to creaming instability. Droplet aggregation occurred at higher pH when CaCl2 was added to the emulsions. The minimum concentration of CaCl2 required to promote aggregation increased as the pH increased. Aggregation was induced in the presence of CaCl2 probably because of the reduction in electrostatic repulsion between droplets, caused by binding of counter ions to droplet surfaces and electrostatic screening eects. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Emulsion stability; Electrostatic interactions; Aggregation; Calcium; pH
1. Introduction Whey protein ingredients are frequently used in foods as emulsi®ers because of their ability to facilitate the formation and stabilization of oil-in-water emulsions (Dickinson, 1997; Human, 1996; Phillips, Whitehead & Kinsella, 1994). These ingredients are dissolved in an aqueous phase that is then homogenized with an oil phase. During homogenization the amphiphilic whey protein molecules rapidly adsorb to the surface of the freshly created oil droplets. Protein adsorption reduces the interfacial tension, which facilitates the further disruption of droplets and therefore increases homogenization eciency (Walstra, 1996). Proteins also form a protective membrane around droplets that prevents them from aggregating with their neighbors both during and after homogenization (Dalgleish, 1996; Dickinson, 1997). The aggregation of droplets has a pronounced in¯uence on the appearance, shelf life and texture of emulsions (Dickinson & McClements, 1995; McClements, 1999; Phillips et al., 1994). In relatively dilute emulsions droplet aggregation decreases creaming stability. In concentrated emulsions it causes a pronounced increase in emulsion viscosity and may actually increase cream* Corresponding author. Tel.: +1-413-545-1019; fax: +1-413-5451262. E-mail address: [email protected] (D.J. McClements).
ing stability because droplet movement is restricted by network formation (McClements, 1999). The production of high quality food emulsions therefore depends on a good understanding of the factors that determine the aggregation of protein-stabilized emulsion droplets. A number of important protein-stabilized food emulsions are supplemented with minerals, e.g. infant formulations, sports drinks and beverages. It is particularly important for manufacturers of these products to understand the in¯uence that minerals have on emulsion stability. Minerals may in¯uence emulsion stability in a variety of dierent ways depending on their valence, size and concentration (McClements, 1999). Minerals increase the ionic strength of the aqueous phase, which reduces the electrostatic repulsion between droplets through electrostatic screening (Evans & Wennerstrom, 1994; Hunter, 1986; Israelachvili, 1992). Some minerals bind to oppositely charged groups on the surface of emulsion droplets, decreasing the magnitude of their -potential and thereby reducing the electrostatic repulsion between droplets (Hunter, 1986). Ion binding can increase the short-range hydration repulsion between droplets because of the additional energy required to disrupt the sheath of water molecules associated with them (Israelachvili, 1992). At suciently high concentrations minerals cause alterations in the structural organization of the water molecules, which alters the strength of the hydrophobic interactions between
0963-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(00)00018-1
16
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
non-polar groups (Israelachvili, 1992). It is clear that the in¯uence of mineral ions on the stability of proteinstabilized emulsions is complex and depends on the precise nature of the ions, the electrical characteristics of the droplet surface and the prevailing environmental conditions (McClements, 1999). Recent studies have examined the eect of sodium ions (Agboola & Dalgleish, 1996; Demetriades, Coupland & McClements, 1997a,b; Demetriades & McClements, 1998), calcium ions (Hunt & Dalgleish, 1995) and copper ions (Silvestre, Decker & McClements, 1999) on the stability of oil-in-water emulsions stabilized by whey proteins. These studies were carried out over narrow ranges of pH (usually either 3 or 7), which limits their applicability to a wide variety of food products. In this study, we examine the in¯uence of both pH and calcium ion concentration on the stability of dilute whey protein stabilized emulsions. 2. Materials and methods 2.1. Materials Whey protein isolate (WPI) powder was obtained from New Zealand Milk Proteins (Santa Rosa, CA) and was used with no further puri®cation. The protein content, determined by Kjeldahl, was 93.4%. The total solid content, determined after heating the powder to 102 C for 5 h, was 960 g kgÿ1, 40 g kgÿ1 moisture. The ash content, determined by combustion at 550 C for 12 h, was 16 g kgÿ1. The cation content of the powder, determined by atomic absorption, was: Na, 4.5 g kgÿ1; Ca, 1.2 g kgÿ1; K, 1.0 g kgÿ1; and P, 0.5 g kgÿ1. Calcium chloride and sodium azide were purchased from Sigma Chemical Company (St. Louis, MO). Soy bean oil was obtained from a local supermarket. Double distilled water was used to prepare the aqueous phase of the emulsions. 2.2. Emulsion preparation An aqueous phase was prepared by dispersing 0.5 wt% whey protein isolate and 0.02 wt% sodium azide in distilled water and stirring for 1 hour to ensure complete dissolution of the protein. An oil-in-water emulsion was prepared by homogenizing 10 wt% soy bean oil with 90% aqueous phase in a high-speed blender (M133/ 1281-0, Biospec Products, Inc., ESGC, Switzerland) followed by three passes at 5000 psi through a highpressure valve homogenizer (APV-Gaulin, Model MiniLab 8.30H, Wilmington, MA). A series of 0.05 wt% emulsions with dierent CaCl2 concentrations were prepared by diluting the 10 wt% emulsion 250-fold using distilled water containing dierent calcium chloride concentrations.
2.3. Particle size measurements The particle size distribution of the non-aggregated emulsions was measured using a laser light scattering instrument (Horiba LA-900, Irvine, CA). A refractive index ratio of 1.08 was used in the calculations of the particle size. The emulsions were placed directly into the measurement cell of the instrument and stirred slowly during the measurement. The initial mean particle diameter of the emulsion droplets was measured as 0.53 mm. Droplet aggregation was monitored by measuring the turbidity spectra of undiluted emulsions using a UV± visible spectrophotometer (UV-2101PC, Shimadzu Scienti®c Instruments, Columbia, MD). The 0.05 wt% emulsions were placed directly in 1-cm quartz cuvettes and their turbidity was measured from 790 to 810 nm. The particle size was then determined using the following equation (Reddy & Fogler, 1981): r mÿ1
r m0 ÿ1 mÿm0 r l 0 0
where r is the mean radius, is the turbidity, l is the measurement wavelength, m is the slope of a plot of log( ) versus log(l) at the measurement wavelength, and the subscript 0 refers to the values for the initial nonaggregated emulsion. In this study turbidity measurements at a wavelength of 800 nm were used, and the initial radius was measured by the light scattering technique. 2.4. z-Potential measurements Oil-in-water emulsions (0.05 wt%) were injected directly into the measurement chamber of a particle electrophoresis instrument capable of measuring the potential of emulsion droplets (ZEM5003, Zetamaster, Malvern Instruments, Worcs., UK). The -potential measurements are reported as the average of two separate injections, with ®ve readings made per injection. 2.5. Creaming stability measurements Ten grams of emulsion were transferred into a test tube (internal diameter 16 mm, height 160 mm), and then stored for 24 h at 30 C. After storage a number of emulsions separated into a thin ``creamed'' 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.
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
3. Results and discussion 3.1. In¯uence of pH and CaCl2 on z-potential
n o1=2 X ÿsgn 2"0 "R kT n0i exp ÿzi e=kT ÿ 1
17
1
The dependence of droplet -potential on pH and CaCl2 concentration is shown in Fig. 1. At the lowest pH the droplets had a relatively high positive -potential because the pH was below the isoelectric point of the whey proteins. Under these conditions the amino groups are positively charged (±NH+ 3 ), whereas the carboxyl groups are neutral (±COOH). When the pH was increased the magnitude of the positive charge on the droplets decreased, partly because carboxyl groups became negatively charged (±COOÿ) and partly because some of the amino groups become neutral (±NH2). Eventually the -potential of the droplets becomes zero, which indicates that the number of positively charged groups balances the number of negatively charged groups. A further increase in pH causes the droplets to gain a net negative charge, which increases as the number of negatively charged groups increases and positively charged groups decreases. The addition of CaCl2 to the emulsions caused a pronounced alteration in the -potential vs. pH curves. At low pH, CaCl2 caused a decrease in positive charge on the droplets, whereas at high pH it caused a decrease in negative charge. It was interesting to note that there was a shift in the isoelectric point of the droplets to a higher pH when the CaCl2 concentration was increased from 0 to 1 mM, but that the isoelectric point remained constant at higher CaCl2 concentrations. The alteration in the potential and isoelectric point of the droplets could have occurred because of two dierent phenomena: electrostatic screening and/or ion binding (Hunter, 1986). The relative importance of these two phenomena can be assessed using the principles of colloid science. The potential of a charged surface is related to its surface charge density through the following relationship (Hunter, 1986):
where, sgn means ``sign of'', e0 is the dielectric constant of a vacuum, eR is the relative dielectric constant of the surrounding medium, k is Boltzman's constant, T is the absolute temperature, noi is the number of ions of type i per unit volume, zi is the valancy of the ions of type i, and e is the unit electrical charge. This equation was used to calculate the surface charge density of the emulsion droplets from the measured -potential and known ion concentration (Fig. 2). The ion concentrations were calculated from the amounts of CaCl2, HCl, and NaN3 present in the emulsions. It was not possible to reliably calculate the surface charge density in the emulsion containing no CaCl2 because the ionic strength was so low that a small uncertainty in the ion concentration caused a dramatic change in the surface charge density. Between pH 3.5 and 7 the surface charge density of the emulsion droplets was fairly insensitive to CaCl2 concentrations in excess of 1 mM (Fig. 2), which indicated that the measured decrease in magnitude of the -potential in these systems was due to electrostatic screening. At lower pH values there was a decrease in the positive charge on the emulsion droplets as the calcium chloride concentration increased above 10 mM, which suggests that there may have been some binding of Clÿ ions to the ±NH+ 3 groups on the proteins. The increase in isoelectric point of the droplets that occurred when the CaCl2 was increased from 0 to 1 mM (Fig. 1) suggests that there may have been some binding of Ca2+ to the droplets at low concentrations. If Ca2+ ions bound to some of the negatively charged groups on the droplet surfaces, then it would be necessary to reach a higher pH before the negatively charged groups cancelled out the positively charged ones. Our results suggest that the adsorbed proteins became saturated with
Fig. 1. Dependence of droplet -potential (determined using particle electrophoresis) of 0.05 wt% soy bean oil-in-water emulsions on pH and CaCl2 concentration.
Fig. 2. Dependence of droplet surface charge density of 0.05 wt% soy bean oil-in-water emulsions on pH and CaCl2 concentration.
18
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
bound calcium ions at low concentrations (< 1 mM), and that further addition of calcium just caused electrostatic screening. It is useful to compare the values of the surface charge densities determined from the -potential measurements with those calculated from knowledge of the surface coverage and electrical charge characteristics of whey protein molecules. Typically, whey proteins have a surface load of about 2 mg mÿ2 (Damodaran, 1996), which corresponds to an electrical charge per unit surface area of 0.0104n C mÿ2, where n is the net number of charged groups per protein molecule. The value of n depends on the number of ionizable groups per molecule, the sign of their charge, and the pH of the aqueous solution. blactoglobulin has 26 ionizable negative and 20 ionizable positive side-groups, whereas a-lactalbumin has 17 ionizable negative and 16 ionizable positive side groups (Swaisgood, 1996). The net surface charge density on the emulsion droplets should therefore be somewhere between +0.2 (at low pH) and ÿ0.2 C mÿ2 (at high pH). The measured surface charge density of the protein-stabilized droplets ranged from +0.05 to ÿ0.01 C mÿ2, which is about an order of magnitude lower than the calculated values. There are a number of possible reasons for this discrepancy. Firstly, not all of the ionizable groups would be ionized at the lowest and highest pH used in this study. Secondly, the net charge on the protein is the sum of the positively and negatively charged side-groups and therefore some of the positive charges would be cancelled out by negative charges and vice versa. Thirdly, the binding of counter-ions to the ionized surface groups will partly neutralize the charge on the droplet surfaces. 3.2. In¯uence of pH and CaCl2 on droplet aggregation The pH dependence of the mean particle size of protein stabilized emulsions determined by turbidity measurements is shown in Fig. 3. These measurements were carried out 24 h after the CaCl2 was added to the emulsions and they had been stored at 30 C. In the absence of calcium chloride there was a signi®cant increase in mean particle size around the isoelectric point (4 < pH < 6) of the whey proteins, which suggested that appreciable droplet aggregation occurred. The addition of CaCl2 to the emulsions signi®cantly altered the extent of droplet aggregation. When the CaCl2 concentration was increased from 0 to 1 mM the pH at which the droplets aggregated shifted to higher values (Fig. 3). This is because the isoelectric point of the emulsion droplets shifted to higher pH (Fig. 1), presumably because of binding of Ca2+ ions at very low calcium chloride concentrations. As the CaCl2 concentration was increased above 1mM the range of pH values above the isoelectric point over which the emulsions became unstable grew wider. At 20 mM CaCl2 the
Fig. 3. Dependence of mean droplet diameter (determined using turbidity measurements) of 0.05 wt% soy bean oil-in-water emulsions on pH and CaCl2 concentration.
emulsions were unstable to aggregation at all pH values above the isoelectric point. 3.3. In¯uence of pH and CaCl2 on droplet creaming Droplet ¯occulation caused a signi®cant increase in the creaming rate of the droplets in the emulsions (Fig. 4). In the absence of CaCl2 the emulsions were relatively stable to creaming at low and high pH, but were highly unstable around the isoelectric point of the proteins. The addition of CaCl2 had a signi®cant impact on the pH dependence of the creaming stability of the emulsions. At pH values signi®cantly below the isoelectric point of the proteins (pH < 4.5) all of the emulsions were relatively stable to creaming. At pH values above the isoelectric point the emulsions were relatively stable to creaming below a certain CaCl2 concentration, but became unstable when this concentration was exceeded. This critical CaCl2 concentration increased as the pH moved further away from the isoelectric point. Above about 5 mM CaCl2 all of the emulsions were unstable to creaming above the isoelectric point. 3.4. Prediction of emulsion stability to droplet aggregation To a ®rst approximation, the dependence of the interaction potential (V) between two protein-stabilized droplets on the surface-to-surface separation (h) can be represented by summing the contributions of the van der Waals attraction (VVDW) and electrostatic repulsion (VE) (Evans & Wennerstrom, 1994; Hiemenz & Rajagopalan, 1997; Hunter, 1986; Israelachvili, 1992): V h VVDW h VE h
2
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
19
Fig. 4. Dependence of creaming stability of 0.05 wt% soy bean oil-inwater emulsions on pH and CaCl2 concentration.
Expressions for the van der Waals and electrostatic interactions given in a recent publication (Silvestre et. al., 1999) were used to calculate the in¯uence of pH and CaCl2 on the interactions between the emulsion droplets used in this work (Fig. 5). A value of the Hamaker function of A=1.3 kT and a droplet radius of 0.26 mm were used in the calculations. There is a large maximum in the interaction potential when the droplets have a high -potential and the electrolyte concentration is low. This energy barrier is responsible for preventing droplets coming close enough together to aggregate. As the -potential decreases or the electrolyte concentration increases the height of the energy barrier decreases, until it is not suciently high to prevent the droplets from aggregating. Firstly, we assumed that the salt concentration remained constant (1 mM CaCl2), but the -potential varied due to changes in pH (Fig. 5a). The predictions show that the height of the energy barrier decreased as the -potential decreased, until eventually it became relatively small compared to the thermal energy for < 10 mV. These calculations account for the droplet aggregation that occurred near the isoelectric point of the proteins because they had a low -potential (Fig. 3). Secondly, we assumed that the pH remained constant (pH 7) as the electrolyte concentration was increased (Fig. 5b). The values of the measured -potential and the known electrolyte concentrations were used in these calculations. These calculations show that the height of the energy barrier became relatively small compared to the thermal energy when the CaCl2 concentration exceeded about 5 mM, which accounts for the observed aggregation of the emulsion droplets around this CaCl2 concentration that was observed at high pH values (Fig. 3).
Fig. 5. Calculations of the in¯uence of pH and CaCl2 concentration on the interaction potential of oil droplets suspended in water: (a) variable -potential (due to pH alterations), CaCl2=1 mM; (b) variable CaCl2 and -potential as determined experimentally.
4. Conclusion This study shows that the aggregation and creaming stability of whey-protein stabilized emulsions is strongly dependent on pH and calcium chloride concentration. Emulsion droplets were highly unstable to aggregation near the isolectric point of the whey proteins because of the relatively low electrostatic repulsion between the droplets. The emulsion stability was relatively insensitive to CaCl2 (< 20 mM) when the pH was below the isoelectric point. On the other hand, fairly low CaCl2 concentrations were capable of promoting droplet ¯occulation and creaming instability when the pH was above the isoelectric point. The pH dependence of the ¯occulation stability can be accounted for by the fact that the counter-ions are monovalent (Clÿ) below the isoelectric point and divalent (Ca2+) above it. Multivalent ions are much more eective at screening electrostatic interactions and at binding to oppositely charged surfaces than monovalent ions. Our results have important consequences for the application of whey proteins in food products. To produce an emulsion that is stable to ¯occulation, it is important to ensure that the pH is suciently far from the isoelectric point of the proteins and that the calcium ion concentration is less than that required to promote ¯occulation. Higher calcium concentrations could be used if some other compound could be added to the aqueous phase of the emulsions to complex the calcium ions or if
20
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
a surfactant was used to stabilize the emulsions rather than a protein. It is possible that the critical ¯occulation concentration would be increased at higher droplet concentrations because the calcium ions would be distributed over a larger number of droplets and therefore the reduction of -potential due to ion binding would be less for each individual droplet. Acknowledgements We thank the Massachusetts Agricultural Experiment Station (Project number MAS00745) and Dairy Management Inc., for support of this project. One of us (A.K.) thanks the Islamic Development Bank for a scholarship to support his work on this project. References Agboola, S. O., & Dalgleish, D. G. (1996). Kinetics of the calciuminduced instability of oil-in-water emulsions: studies under quiescent and shearing conditions. Lebensmittel-Wissenschaft und-Technologie, 29, 425±432. Dalgleish, D. G. (1996). Food emulsions. In J. Sjoblom, Emulsions and emulsion stability (pp. 287±325). New York: Marcel Dekker. Damodaran, S. (1996). Amino acids, peptides and proteins. In O. R. Fennema, Food chemistry (3rd ed.) (pp. 321±429). New York: Marcel Dekker. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997a). Physical properties of whey protein stabilized emulsions as related to pH and ionic strength. Journal of Food Science, 62, 342±347. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997b). Physicochemical properties of whey protein stabilized emulsions as aected by heating and ionic strength. Journal of Food Science, 62, 462±467.
Demetriades, K., & McClements, D. J. (1998). In¯uence of pH and heating on the physicochemical properties of whey protein stabilized emulsions containing a non-ionic surfactant. Journal of Agricricultural Food Chemistry, 46, 3936±3942. Dickinson, E. (1997). Properties of emulsions stabilized with milk proteins: overview of some recent developments. Journal of Dairy Science, 80, 2607±2619. Dickinson, E., & McClements, D. J. (1995). Advances in food colloids. Glasgow: Blackie Academic & Professional. Evans, D. F., & Wennerstrom, H. (1994). The colloidal domain: Where physics, chemistry, biology and technology meet. New York: VCH Publishers. Hiemenz, P. C., & Rajagopalan, R. (1997). Principles of colloid and surface chemistry (3rd ed.). New York: Marcel Dekker. Human, L. M. (1996). Processing whey protein for use as a food ingredient. Food Technology, 50, 49±52. Hunt, J. A., & Dalgleish, D. G. (1995). Heat stability of oil-in-water emulsions containing milk proteins: eect of ionic strength and pH. Journal of Food Science, 60, 1120±1123. Hunter, R. J. (1986). Foundations of colloid science Vol. 1. Oxford: Oxford University Press. Israelachvili, J. N. (1992). Intermolecular and surface forces. London: Academic Press. McClements, D. J. (1999). Food emulsions: Principles, practice and techniques. Boca Raton: CRC Press. Phillips, L. G., Whitehead, D. M., & Kinsella, J. E. (1994). Structure± function properties of food proteins. San Diego: Academic Press. Reddy, S. R., & Fogler. (1981). Emulsion stability: determination from turbidity. Journal of Colloid and Interface Science, 79, 101± 104. Silvestre, M. P. C., Decker, E. A., & McClements, D. J. (1999). In¯uence of copper on the stability of whey protein stabilized emulsions. Food Hydrocolloids, 13, 419±424. Swaisgood, H. E. (1996). Characteristics of milk. In O. R. Fennema, Food chemistry (3rd ed.). (pp. 841±878). New York: Marcel Dekker. Walstra, P. (1996). Dispersed systems: basic considerations. In O. R. Fennema, Food chemistry (pp. 95±155). New York: Marcel Dekker.
www.elsevier.com/locate/foodres
In¯uence of pH and CaCl2 on the stability of dilute whey protein stabilized emulsions Asylbek Kulmyrzaev, Ratjika Chanamai, D. Julian McClements * Biopolymer and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
Abstract The in¯uence of pH and CaCl2 on the physical stability of dilute oil-in-water emulsions stabilized by whey protein isolate has been studied. The particle size, zeta potential and creaming stability of 0.05 wt% soy bean oil-in-water emulsions (d 0.53 mm) were measured with varying pH (3 to 7) and CaCl2 concentration (0 to 20 mM). In the absence of CaCl2 extensive droplet aggregation occurred around the isoelectric point of the whey proteins (4 < pH < 6) because of their low electrical charge, which led to creaming instability. Droplet aggregation occurred at higher pH when CaCl2 was added to the emulsions. The minimum concentration of CaCl2 required to promote aggregation increased as the pH increased. Aggregation was induced in the presence of CaCl2 probably because of the reduction in electrostatic repulsion between droplets, caused by binding of counter ions to droplet surfaces and electrostatic screening eects. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Emulsion stability; Electrostatic interactions; Aggregation; Calcium; pH
1. Introduction Whey protein ingredients are frequently used in foods as emulsi®ers because of their ability to facilitate the formation and stabilization of oil-in-water emulsions (Dickinson, 1997; Human, 1996; Phillips, Whitehead & Kinsella, 1994). These ingredients are dissolved in an aqueous phase that is then homogenized with an oil phase. During homogenization the amphiphilic whey protein molecules rapidly adsorb to the surface of the freshly created oil droplets. Protein adsorption reduces the interfacial tension, which facilitates the further disruption of droplets and therefore increases homogenization eciency (Walstra, 1996). Proteins also form a protective membrane around droplets that prevents them from aggregating with their neighbors both during and after homogenization (Dalgleish, 1996; Dickinson, 1997). The aggregation of droplets has a pronounced in¯uence on the appearance, shelf life and texture of emulsions (Dickinson & McClements, 1995; McClements, 1999; Phillips et al., 1994). In relatively dilute emulsions droplet aggregation decreases creaming stability. In concentrated emulsions it causes a pronounced increase in emulsion viscosity and may actually increase cream* Corresponding author. Tel.: +1-413-545-1019; fax: +1-413-5451262. E-mail address: [email protected] (D.J. McClements).
ing stability because droplet movement is restricted by network formation (McClements, 1999). The production of high quality food emulsions therefore depends on a good understanding of the factors that determine the aggregation of protein-stabilized emulsion droplets. A number of important protein-stabilized food emulsions are supplemented with minerals, e.g. infant formulations, sports drinks and beverages. It is particularly important for manufacturers of these products to understand the in¯uence that minerals have on emulsion stability. Minerals may in¯uence emulsion stability in a variety of dierent ways depending on their valence, size and concentration (McClements, 1999). Minerals increase the ionic strength of the aqueous phase, which reduces the electrostatic repulsion between droplets through electrostatic screening (Evans & Wennerstrom, 1994; Hunter, 1986; Israelachvili, 1992). Some minerals bind to oppositely charged groups on the surface of emulsion droplets, decreasing the magnitude of their -potential and thereby reducing the electrostatic repulsion between droplets (Hunter, 1986). Ion binding can increase the short-range hydration repulsion between droplets because of the additional energy required to disrupt the sheath of water molecules associated with them (Israelachvili, 1992). At suciently high concentrations minerals cause alterations in the structural organization of the water molecules, which alters the strength of the hydrophobic interactions between
0963-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(00)00018-1
16
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
non-polar groups (Israelachvili, 1992). It is clear that the in¯uence of mineral ions on the stability of proteinstabilized emulsions is complex and depends on the precise nature of the ions, the electrical characteristics of the droplet surface and the prevailing environmental conditions (McClements, 1999). Recent studies have examined the eect of sodium ions (Agboola & Dalgleish, 1996; Demetriades, Coupland & McClements, 1997a,b; Demetriades & McClements, 1998), calcium ions (Hunt & Dalgleish, 1995) and copper ions (Silvestre, Decker & McClements, 1999) on the stability of oil-in-water emulsions stabilized by whey proteins. These studies were carried out over narrow ranges of pH (usually either 3 or 7), which limits their applicability to a wide variety of food products. In this study, we examine the in¯uence of both pH and calcium ion concentration on the stability of dilute whey protein stabilized emulsions. 2. Materials and methods 2.1. Materials Whey protein isolate (WPI) powder was obtained from New Zealand Milk Proteins (Santa Rosa, CA) and was used with no further puri®cation. The protein content, determined by Kjeldahl, was 93.4%. The total solid content, determined after heating the powder to 102 C for 5 h, was 960 g kgÿ1, 40 g kgÿ1 moisture. The ash content, determined by combustion at 550 C for 12 h, was 16 g kgÿ1. The cation content of the powder, determined by atomic absorption, was: Na, 4.5 g kgÿ1; Ca, 1.2 g kgÿ1; K, 1.0 g kgÿ1; and P, 0.5 g kgÿ1. Calcium chloride and sodium azide were purchased from Sigma Chemical Company (St. Louis, MO). Soy bean oil was obtained from a local supermarket. Double distilled water was used to prepare the aqueous phase of the emulsions. 2.2. Emulsion preparation An aqueous phase was prepared by dispersing 0.5 wt% whey protein isolate and 0.02 wt% sodium azide in distilled water and stirring for 1 hour to ensure complete dissolution of the protein. An oil-in-water emulsion was prepared by homogenizing 10 wt% soy bean oil with 90% aqueous phase in a high-speed blender (M133/ 1281-0, Biospec Products, Inc., ESGC, Switzerland) followed by three passes at 5000 psi through a highpressure valve homogenizer (APV-Gaulin, Model MiniLab 8.30H, Wilmington, MA). A series of 0.05 wt% emulsions with dierent CaCl2 concentrations were prepared by diluting the 10 wt% emulsion 250-fold using distilled water containing dierent calcium chloride concentrations.
2.3. Particle size measurements The particle size distribution of the non-aggregated emulsions was measured using a laser light scattering instrument (Horiba LA-900, Irvine, CA). A refractive index ratio of 1.08 was used in the calculations of the particle size. The emulsions were placed directly into the measurement cell of the instrument and stirred slowly during the measurement. The initial mean particle diameter of the emulsion droplets was measured as 0.53 mm. Droplet aggregation was monitored by measuring the turbidity spectra of undiluted emulsions using a UV± visible spectrophotometer (UV-2101PC, Shimadzu Scienti®c Instruments, Columbia, MD). The 0.05 wt% emulsions were placed directly in 1-cm quartz cuvettes and their turbidity was measured from 790 to 810 nm. The particle size was then determined using the following equation (Reddy & Fogler, 1981): r mÿ1
r m0 ÿ1 mÿm0 r l 0 0
where r is the mean radius, is the turbidity, l is the measurement wavelength, m is the slope of a plot of log( ) versus log(l) at the measurement wavelength, and the subscript 0 refers to the values for the initial nonaggregated emulsion. In this study turbidity measurements at a wavelength of 800 nm were used, and the initial radius was measured by the light scattering technique. 2.4. z-Potential measurements Oil-in-water emulsions (0.05 wt%) were injected directly into the measurement chamber of a particle electrophoresis instrument capable of measuring the potential of emulsion droplets (ZEM5003, Zetamaster, Malvern Instruments, Worcs., UK). The -potential measurements are reported as the average of two separate injections, with ®ve readings made per injection. 2.5. Creaming stability measurements Ten grams of emulsion were transferred into a test tube (internal diameter 16 mm, height 160 mm), and then stored for 24 h at 30 C. After storage a number of emulsions separated into a thin ``creamed'' 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.
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
3. Results and discussion 3.1. In¯uence of pH and CaCl2 on z-potential
n o1=2 X ÿsgn 2"0 "R kT n0i exp ÿzi e=kT ÿ 1
17
1
The dependence of droplet -potential on pH and CaCl2 concentration is shown in Fig. 1. At the lowest pH the droplets had a relatively high positive -potential because the pH was below the isoelectric point of the whey proteins. Under these conditions the amino groups are positively charged (±NH+ 3 ), whereas the carboxyl groups are neutral (±COOH). When the pH was increased the magnitude of the positive charge on the droplets decreased, partly because carboxyl groups became negatively charged (±COOÿ) and partly because some of the amino groups become neutral (±NH2). Eventually the -potential of the droplets becomes zero, which indicates that the number of positively charged groups balances the number of negatively charged groups. A further increase in pH causes the droplets to gain a net negative charge, which increases as the number of negatively charged groups increases and positively charged groups decreases. The addition of CaCl2 to the emulsions caused a pronounced alteration in the -potential vs. pH curves. At low pH, CaCl2 caused a decrease in positive charge on the droplets, whereas at high pH it caused a decrease in negative charge. It was interesting to note that there was a shift in the isoelectric point of the droplets to a higher pH when the CaCl2 concentration was increased from 0 to 1 mM, but that the isoelectric point remained constant at higher CaCl2 concentrations. The alteration in the potential and isoelectric point of the droplets could have occurred because of two dierent phenomena: electrostatic screening and/or ion binding (Hunter, 1986). The relative importance of these two phenomena can be assessed using the principles of colloid science. The potential of a charged surface is related to its surface charge density through the following relationship (Hunter, 1986):
where, sgn means ``sign of'', e0 is the dielectric constant of a vacuum, eR is the relative dielectric constant of the surrounding medium, k is Boltzman's constant, T is the absolute temperature, noi is the number of ions of type i per unit volume, zi is the valancy of the ions of type i, and e is the unit electrical charge. This equation was used to calculate the surface charge density of the emulsion droplets from the measured -potential and known ion concentration (Fig. 2). The ion concentrations were calculated from the amounts of CaCl2, HCl, and NaN3 present in the emulsions. It was not possible to reliably calculate the surface charge density in the emulsion containing no CaCl2 because the ionic strength was so low that a small uncertainty in the ion concentration caused a dramatic change in the surface charge density. Between pH 3.5 and 7 the surface charge density of the emulsion droplets was fairly insensitive to CaCl2 concentrations in excess of 1 mM (Fig. 2), which indicated that the measured decrease in magnitude of the -potential in these systems was due to electrostatic screening. At lower pH values there was a decrease in the positive charge on the emulsion droplets as the calcium chloride concentration increased above 10 mM, which suggests that there may have been some binding of Clÿ ions to the ±NH+ 3 groups on the proteins. The increase in isoelectric point of the droplets that occurred when the CaCl2 was increased from 0 to 1 mM (Fig. 1) suggests that there may have been some binding of Ca2+ to the droplets at low concentrations. If Ca2+ ions bound to some of the negatively charged groups on the droplet surfaces, then it would be necessary to reach a higher pH before the negatively charged groups cancelled out the positively charged ones. Our results suggest that the adsorbed proteins became saturated with
Fig. 1. Dependence of droplet -potential (determined using particle electrophoresis) of 0.05 wt% soy bean oil-in-water emulsions on pH and CaCl2 concentration.
Fig. 2. Dependence of droplet surface charge density of 0.05 wt% soy bean oil-in-water emulsions on pH and CaCl2 concentration.
18
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
bound calcium ions at low concentrations (< 1 mM), and that further addition of calcium just caused electrostatic screening. It is useful to compare the values of the surface charge densities determined from the -potential measurements with those calculated from knowledge of the surface coverage and electrical charge characteristics of whey protein molecules. Typically, whey proteins have a surface load of about 2 mg mÿ2 (Damodaran, 1996), which corresponds to an electrical charge per unit surface area of 0.0104n C mÿ2, where n is the net number of charged groups per protein molecule. The value of n depends on the number of ionizable groups per molecule, the sign of their charge, and the pH of the aqueous solution. blactoglobulin has 26 ionizable negative and 20 ionizable positive side-groups, whereas a-lactalbumin has 17 ionizable negative and 16 ionizable positive side groups (Swaisgood, 1996). The net surface charge density on the emulsion droplets should therefore be somewhere between +0.2 (at low pH) and ÿ0.2 C mÿ2 (at high pH). The measured surface charge density of the protein-stabilized droplets ranged from +0.05 to ÿ0.01 C mÿ2, which is about an order of magnitude lower than the calculated values. There are a number of possible reasons for this discrepancy. Firstly, not all of the ionizable groups would be ionized at the lowest and highest pH used in this study. Secondly, the net charge on the protein is the sum of the positively and negatively charged side-groups and therefore some of the positive charges would be cancelled out by negative charges and vice versa. Thirdly, the binding of counter-ions to the ionized surface groups will partly neutralize the charge on the droplet surfaces. 3.2. In¯uence of pH and CaCl2 on droplet aggregation The pH dependence of the mean particle size of protein stabilized emulsions determined by turbidity measurements is shown in Fig. 3. These measurements were carried out 24 h after the CaCl2 was added to the emulsions and they had been stored at 30 C. In the absence of calcium chloride there was a signi®cant increase in mean particle size around the isoelectric point (4 < pH < 6) of the whey proteins, which suggested that appreciable droplet aggregation occurred. The addition of CaCl2 to the emulsions signi®cantly altered the extent of droplet aggregation. When the CaCl2 concentration was increased from 0 to 1 mM the pH at which the droplets aggregated shifted to higher values (Fig. 3). This is because the isoelectric point of the emulsion droplets shifted to higher pH (Fig. 1), presumably because of binding of Ca2+ ions at very low calcium chloride concentrations. As the CaCl2 concentration was increased above 1mM the range of pH values above the isoelectric point over which the emulsions became unstable grew wider. At 20 mM CaCl2 the
Fig. 3. Dependence of mean droplet diameter (determined using turbidity measurements) of 0.05 wt% soy bean oil-in-water emulsions on pH and CaCl2 concentration.
emulsions were unstable to aggregation at all pH values above the isoelectric point. 3.3. In¯uence of pH and CaCl2 on droplet creaming Droplet ¯occulation caused a signi®cant increase in the creaming rate of the droplets in the emulsions (Fig. 4). In the absence of CaCl2 the emulsions were relatively stable to creaming at low and high pH, but were highly unstable around the isoelectric point of the proteins. The addition of CaCl2 had a signi®cant impact on the pH dependence of the creaming stability of the emulsions. At pH values signi®cantly below the isoelectric point of the proteins (pH < 4.5) all of the emulsions were relatively stable to creaming. At pH values above the isoelectric point the emulsions were relatively stable to creaming below a certain CaCl2 concentration, but became unstable when this concentration was exceeded. This critical CaCl2 concentration increased as the pH moved further away from the isoelectric point. Above about 5 mM CaCl2 all of the emulsions were unstable to creaming above the isoelectric point. 3.4. Prediction of emulsion stability to droplet aggregation To a ®rst approximation, the dependence of the interaction potential (V) between two protein-stabilized droplets on the surface-to-surface separation (h) can be represented by summing the contributions of the van der Waals attraction (VVDW) and electrostatic repulsion (VE) (Evans & Wennerstrom, 1994; Hiemenz & Rajagopalan, 1997; Hunter, 1986; Israelachvili, 1992): V h VVDW h VE h
2
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
19
Fig. 4. Dependence of creaming stability of 0.05 wt% soy bean oil-inwater emulsions on pH and CaCl2 concentration.
Expressions for the van der Waals and electrostatic interactions given in a recent publication (Silvestre et. al., 1999) were used to calculate the in¯uence of pH and CaCl2 on the interactions between the emulsion droplets used in this work (Fig. 5). A value of the Hamaker function of A=1.3 kT and a droplet radius of 0.26 mm were used in the calculations. There is a large maximum in the interaction potential when the droplets have a high -potential and the electrolyte concentration is low. This energy barrier is responsible for preventing droplets coming close enough together to aggregate. As the -potential decreases or the electrolyte concentration increases the height of the energy barrier decreases, until it is not suciently high to prevent the droplets from aggregating. Firstly, we assumed that the salt concentration remained constant (1 mM CaCl2), but the -potential varied due to changes in pH (Fig. 5a). The predictions show that the height of the energy barrier decreased as the -potential decreased, until eventually it became relatively small compared to the thermal energy for < 10 mV. These calculations account for the droplet aggregation that occurred near the isoelectric point of the proteins because they had a low -potential (Fig. 3). Secondly, we assumed that the pH remained constant (pH 7) as the electrolyte concentration was increased (Fig. 5b). The values of the measured -potential and the known electrolyte concentrations were used in these calculations. These calculations show that the height of the energy barrier became relatively small compared to the thermal energy when the CaCl2 concentration exceeded about 5 mM, which accounts for the observed aggregation of the emulsion droplets around this CaCl2 concentration that was observed at high pH values (Fig. 3).
Fig. 5. Calculations of the in¯uence of pH and CaCl2 concentration on the interaction potential of oil droplets suspended in water: (a) variable -potential (due to pH alterations), CaCl2=1 mM; (b) variable CaCl2 and -potential as determined experimentally.
4. Conclusion This study shows that the aggregation and creaming stability of whey-protein stabilized emulsions is strongly dependent on pH and calcium chloride concentration. Emulsion droplets were highly unstable to aggregation near the isolectric point of the whey proteins because of the relatively low electrostatic repulsion between the droplets. The emulsion stability was relatively insensitive to CaCl2 (< 20 mM) when the pH was below the isoelectric point. On the other hand, fairly low CaCl2 concentrations were capable of promoting droplet ¯occulation and creaming instability when the pH was above the isoelectric point. The pH dependence of the ¯occulation stability can be accounted for by the fact that the counter-ions are monovalent (Clÿ) below the isoelectric point and divalent (Ca2+) above it. Multivalent ions are much more eective at screening electrostatic interactions and at binding to oppositely charged surfaces than monovalent ions. Our results have important consequences for the application of whey proteins in food products. To produce an emulsion that is stable to ¯occulation, it is important to ensure that the pH is suciently far from the isoelectric point of the proteins and that the calcium ion concentration is less than that required to promote ¯occulation. Higher calcium concentrations could be used if some other compound could be added to the aqueous phase of the emulsions to complex the calcium ions or if
20
A. Kulmyrzaev et al. / Food Research International 33 (2000) 15±20
a surfactant was used to stabilize the emulsions rather than a protein. It is possible that the critical ¯occulation concentration would be increased at higher droplet concentrations because the calcium ions would be distributed over a larger number of droplets and therefore the reduction of -potential due to ion binding would be less for each individual droplet. Acknowledgements We thank the Massachusetts Agricultural Experiment Station (Project number MAS00745) and Dairy Management Inc., for support of this project. One of us (A.K.) thanks the Islamic Development Bank for a scholarship to support his work on this project. References Agboola, S. O., & Dalgleish, D. G. (1996). Kinetics of the calciuminduced instability of oil-in-water emulsions: studies under quiescent and shearing conditions. Lebensmittel-Wissenschaft und-Technologie, 29, 425±432. Dalgleish, D. G. (1996). Food emulsions. In J. Sjoblom, Emulsions and emulsion stability (pp. 287±325). New York: Marcel Dekker. Damodaran, S. (1996). Amino acids, peptides and proteins. In O. R. Fennema, Food chemistry (3rd ed.) (pp. 321±429). New York: Marcel Dekker. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997a). Physical properties of whey protein stabilized emulsions as related to pH and ionic strength. Journal of Food Science, 62, 342±347. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997b). Physicochemical properties of whey protein stabilized emulsions as aected by heating and ionic strength. Journal of Food Science, 62, 462±467.
Demetriades, K., & McClements, D. J. (1998). In¯uence of pH and heating on the physicochemical properties of whey protein stabilized emulsions containing a non-ionic surfactant. Journal of Agricricultural Food Chemistry, 46, 3936±3942. Dickinson, E. (1997). Properties of emulsions stabilized with milk proteins: overview of some recent developments. Journal of Dairy Science, 80, 2607±2619. Dickinson, E., & McClements, D. J. (1995). Advances in food colloids. Glasgow: Blackie Academic & Professional. Evans, D. F., & Wennerstrom, H. (1994). The colloidal domain: Where physics, chemistry, biology and technology meet. New York: VCH Publishers. Hiemenz, P. C., & Rajagopalan, R. (1997). Principles of colloid and surface chemistry (3rd ed.). New York: Marcel Dekker. Human, L. M. (1996). Processing whey protein for use as a food ingredient. Food Technology, 50, 49±52. Hunt, J. A., & Dalgleish, D. G. (1995). Heat stability of oil-in-water emulsions containing milk proteins: eect of ionic strength and pH. Journal of Food Science, 60, 1120±1123. Hunter, R. J. (1986). Foundations of colloid science Vol. 1. Oxford: Oxford University Press. Israelachvili, J. N. (1992). Intermolecular and surface forces. London: Academic Press. McClements, D. J. (1999). Food emulsions: Principles, practice and techniques. Boca Raton: CRC Press. Phillips, L. G., Whitehead, D. M., & Kinsella, J. E. (1994). Structure± function properties of food proteins. San Diego: Academic Press. Reddy, S. R., & Fogler. (1981). Emulsion stability: determination from turbidity. Journal of Colloid and Interface Science, 79, 101± 104. Silvestre, M. P. C., Decker, E. A., & McClements, D. J. (1999). In¯uence of copper on the stability of whey protein stabilized emulsions. Food Hydrocolloids, 13, 419±424. Swaisgood, H. E. (1996). Characteristics of milk. In O. R. Fennema, Food chemistry (3rd ed.). (pp. 841±878). New York: Marcel Dekker. Walstra, P. (1996). Dispersed systems: basic considerations. In O. R. Fennema, Food chemistry (pp. 95±155). New York: Marcel Dekker.