J. Agric. Food Chem. 2007, 55, 475−485
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Impact of Electrostatic Interactions on Formation and Stability of Emulsions Containing Oil Droplets Coated by β-Lactoglobulin−Pectin Complexes DEMET GUZEY
AND
DAVID JULIAN MCCLEMENTS*
Biopolymers and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003
Interfacial protein-polysaccharide complexes can be used to improve the physical stability of oil-inwater emulsions. The purpose of this study was to examine the impact of ionic strength on the formation and stability of oil-in-water emulsions containing polysaccharide-protein-coated droplets. Emulsions were prepared that contained 0.1 wt % corn oil, 0.05 wt % β-lactoglobulin, and 0.02 wt % pectin at pH 7. The emulsions were then adjusted to pH 4 to promote electrostatic deposition of the pectin molecules onto the surfaces of the protein-coated droplets. The salt concentration of the aqueous phase (0 or 50 mM NaCl) was adjusted either before or after deposition of the pectin molecules onto the droplet surfaces. We found that stable emulsions containing polysaccharideprotein-coated droplets could be formed when the salt was added after pectin adsorption but not when it was added before pectin adsorption. This phenomenon was attributed to the ability of NaCl to promote droplet flocculation in the protein-coated droplets so that the pectin molecules adsorbed onto the surfaces of flocs rather than individual droplets when salt was added before pectin adsorption. We also found that polysaccharide-protein-coated droplets had a much improved stability to saltinduced flocculation than protein-coated droplets with the same droplet charge (ζ-potential). Theoretical predictions indicated that this was due to the ability of the adsorbed polysaccharide layer to strongly diminish the van der Waals attraction between the droplets. KEYWORDS: Emulsion; β-lactoglobulin; pectin; multilayer; stability; electrostatic
INTRODUCTION
The stability of oil-in-water emulsions containing proteincoated droplets can often be improved by adding a polysaccharide that adsorbs onto the droplet surfaces and forms a protective layer (1-5). This process is normally achieved by mixing a protein-stabilized emulsion and a polysaccharide solution under conditions where there is an attractive force between the polysaccharide molecules and the protein-coated droplet surfaces. The most commonly utilized attractive force is the electrostatic interaction between the charged groups on polysaccharide molecules and the oppositely charged groups on adsorbed protein molecules. The charge on the protein-coated droplets can be varied from positive to negative by adjusting the pH from below to above the isoelectric point (pI) of the adsorbed proteins (6). On the other hand, the charge characteristics of the polysaccharide molecules can be controlled by selecting polysaccharides with different numbers, distributions, and types of ionizable functional groups (7). Thus, anionic polysaccharides can be adsorbed onto the surfaces of proteincoated droplets at pH values where the droplets either have a net positive charge or have a significant amount of positively * To whom correspondence should be addressed. Tel: 413-545-1019. Fax: 413-545-1262. E-mail:
[email protected].
charged patches on their surfaces (8-11). Conversely, cationic polysaccharides can be adsorbed onto the surfaces of proteincoated droplets at pH values where the droplets have either a net negative charge or an appreciable amount of negatively charged patches on their surfaces (12, 13). The resulting “multilayer emulsions” contain oil droplets coated by proteinpolysaccharide interfacial complexes. Previously, it has been shown that oil droplets coated by protein-polysaccharide complexes often have superior stability to environmental stresses than those coated by proteins alone because of the alteration in interfacial charge, structure, and thickness (1, 2). For example, the stability of emulsions to pH, ionic strength, thermal processing, freezing, and dehydration can be improved using the multilayer approach (1, 9, 11, 1418). Although the multilayer technology provides scientists with a powerful method to improve the resistance of emulsions to environmental stresses, it is still essential to select the most appropriate system composition and preparation conditions to create stable multilayer emulsions with the desired physicochemical characteristics. A significant problem reported in studies that have used this approach to create multilayer-coated droplets is the tendency for extensive aggregation of the droplets to occur (19). Different kinds of droplet aggregation may occur depending on the system conditions: (i) Bridging flocculation
10.1021/jf062342f CCC: $37.00 © 2007 American Chemical Society Published on Web 12/19/2006
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occurs if there is insufficient polyelectrolyte to completely coat all of the droplet surfaces when they collide, which may occur either because there is insufficient polyelectrolyte present or because it does not adsorb fast enough relative to dropletdroplet collisions; (ii) depletion flocculation occurs if there is too much excess polyelectrolyte present in the continuous phase; and (iii) flocculation may also occur if the repulsive interactions between the coated droplets are not strong enough to overcome the attractive interactions (19). Droplet aggregation therefore has to be retarded by carefully controlling preparation conditions, such as droplet characteristics (e.g., concentration, size, and charge), polyelectrolyte characteristics (e.g., concentration, molar mass, and charge density), solvent properties (e.g., ionic strength, pH, and dielectric constant), and/or the mixing method (e.g., order of addition, mixing speed, and flow profile). The main driving force for polyelectrolyte adsorption to the droplet surfaces is electrostatic; hence, multilayer formation depends strongly on solution pH and ionic strength because these parameters influence the magnitude of the electrical charge on the droplets and polyelectrolyte molecules, as well as the range of the electrostatic interactions in the system (20, 21). Previous studies have shown that the amount of polyelectrolyte adsorbed onto colloidal particles and the properties of the interfaces formed are strongly influenced by the ionic strength of the aqueous phase, as well as on whether the ionic strength was adjusted before or after polyelectrolyte adsorption (21-26). The purpose of this study was therefore to examine the influence of electrostatic interactions on the formation and properties of emulsions containing protein-polysaccharide-coated droplets. In this study, we prepared oil-in-water emulsions containing droplets coated by an interfacial layer comprised of two natural polymers: a globular protein [β-lactoglobulin (β-Lg)] and an anionic polysaccharide (pectin). β-Lg was used because its molecular properties are well-known and it is the major component in whey protein, which is commonly used as an emulsifier in the food industry (6). The isoelectric point of β-Lg has been reported to be around 4.7-5.2 (27-29). Pectin was used because it is an anionic polysaccharide (pKa ≈ 3.5) that is already widely used as a functional ingredient by the food industry (30). The major objective of this study was to examine the influence of electrostatic interactions, modulated by changing the pH and ionic strength, on the formation and stability of oilin-water emulsions containing lipid droplets coated by β-Lgpectin layers. Electrostatic interactions would be expected to influence the adsorption of charged polysaccharide molecules onto the surfaces of oppositely charged lipid droplets, as well as influencing the strength of the electrostatic repulsion between oil droplets. MATERIALS AND METHODS Materials. Powdered β-Lg was obtained from Davisco Foods International (Lot #JE 001-1-922, Le Sueur, MN). The protein content was reported to be 98.3% (dry basis) by the supplier, with β-Lg making up 95.5% of the total protein. The moisture content of the protein powder was reported to be 4.9%. The fat, ash, and lactose contents of this product were reported to be 0.3 ( 0.1, 2.5 ( 0.2, and 5 wt %) there may be problems with preparing stable multilayer emulsions (45). In conclusion, in this manuscript, the influence of electrostatic interactions on the formation of stable multilayer emulsions based on the electrostatic deposition of anionic polysaccharide molecules onto the surfaces of protein-coated droplets was examined. At relatively high pH values, the protein-coated droplets and polysaccharide molecules were both strongly negatively charged and so no adsorption occurred. When the pH was decreased, positive patches developed on the surface of the protein-coated droplets, which promoted adsorption of the negatively charged pectin molecules. In the absence of salt, the protein-coated droplets had a strong electrostatic repulsion between them that prevented flocculation. Consequently, the pectin molecules adsorbed onto the surfaces of individual droplets leading to the formation of stable multilayer emulsions. On the other hand, in the presence of salt, the electrostatic repulsion between the protein-coated droplets was reduced, which promoted flocculation. In this case, the pectin molecules adsorbed onto the surface of flocs, rather than individual droplets. This study indicates that one must therefore be careful to ensure that the primary emulsion is stable prior to adsorption of the next polyelectrolyte layer. In future studies, it would be useful to systematically identify and quantify the key molecular characteristics of pectin required to provide a good protective layer around protein-coated droplets, e.g., molecular weight, charge density (DE), and structure.
We greatly thank Danisco for donating the β-Lg used in this study and Profs. Paul Dubin and Eric Decker for useful discussions. APPENDIX I
The Hamaker functions appropriate for a system consisting of droplets (material 1) coated by interfacial membranes (material 2) that are suspended in a continuous phase (material 3) can be calculated using the equations given below (Figure 1) (46). The Hamaker function can be divided into a “zerofrequency” and a “frequency-dependent” contribution due to differences in the origin of the various van der Waal interactions (46):
Aijk ) Aijk,V)0 + Aijk,V>0
(I.1)
where
( )( )
i - j k - j 3 Aijk,V)0 ) kT 4 i + j k + j
(I.2)
Aijk,V>0 ) 3hVe
dielectric constant, n is the refractive index, and Ve is the major electronic absorption frequency in the ultraviolet region of the electromagnetic spectrum. The influence of electrostatic screening and retardation can be taken into account using the following expression (41, 46):
[
(
Aijk ) [e -2κh]Aijk,V)0 + 1 - 5.32 × 107 h ln 1 +
(ni2 - nj2)(nk2 - nj2)
2 2 1/2 2 2 1/2 2 2 1/2 2 2 1/2 8x2 (ni + nj ) (nk + nj ) {(ni + nj ) + (nk + nj ) } (I.3)
The Aijk,V)0 term represents the nonscreened “zero-frequency” contribution, and the Aijk,V>0 term represents the nonretarded “frequency-dependent” contribution (46). Here, k is Boltzmann’s constant (1.381 × 10-23 J K-1), T is the absolute temperature, h is Planck’s constant (6.626 × 10-34 J s), is the static relative
)]
1 Aijk,V>0 (I.4) 5.32 × 107h
The prefactors in the square brackets take into account the influence of electrostatic screening on Aν)0 and retardation on Aν>0, respectively. Here, κ-1 is the Debye screening length (see text). APPENDIX II
When the electrostatic interaction between a charged surface and the counterions is relatively weak and there is no surface charge regulation, the surface charge density is simply related to the surface potential (39):
σ ) R0κΨδ
(II.1)
For aqueous solutions at room temperatures, κ ≈ 3.29 × 109xI nm-1 and R ≈ 80 where I is the ionic strength expressed in moles per liter (31). Experimentally, one can usually replace the surface potential with the ζ-potential, i.e., Ψδ ≈ ζ . Hence, the above equation can be approximated by:
σ ≈ 0.233xI ζ
ACKNOWLEDGMENT
483
(II.2)
If one assumes that the surface charge density is unaffected by salt, which is usually the case for relatively low concentrations of indifferent ions (39), then a simple relationship can be developed between the ζ-potential values measured in the absence (ζ[0]) and presence (ζ[m]) of a known amount of added salt (m):
ζ(0)xIb ) ζ(m)xIb + m
(II.3)
This equation can be rearranged to give an expression for the background ionic strength:
Ib ) m
[( ) ] ζ(0) -1 ζ(m)
-1
(II.4)
This equation was used to predict the background ionic strength of primary and secondary emulsions containing 0 or 50 mM as a function of pH. Over the pH range 3-7, we found that Ib was equal to 23 ( 5 mM for the primary emulsion (ignoring pH 4.0 and 4.5) and 23 ( 4 mM for the secondary emulsion (ignoring pH 3.0 and 3.5). Some values were not included in the calculations because the magnitude of the ζ-potential was relatively small (
