A variety of different emulsifiers can be used to prepare O/W emulsions containing electrically charged droplets, such as proteins, polysaccharides, and ionic surfactants (Figure 4). Each type of emulsifier has its own advantages and disadvantages for particular applications. Selection of the most appropriate emulsifier is one of the most important factors influencing the formation of heteroaggregated emulsions. In this section, we provide an overview of a number of electrically charged food-grade emulsifiers that can be used.
Protein-based emulsifiers can be isolated from various natural sources, including various animal, plant, and marine products. However, the most commonly used protein-based emulsifiers in the food industry are those derived from bovine milk because of their relatively low cost and ease of isolation, that is, caseins and whey proteins. Caseins are relatively flexible and disordered proteins that make up about 80% of milk proteins, and include four main fractions: αS1-casein, αS2-casein, β-casein, and κ-casein. Whey proteins are compact globular proteins that make up about 20% of milk proteins, and also include a number of different fractions, such as β-lactoglobulin (β-Lg), α-lactalbumin, bovine serum albumin (BSA), lactoferrin, and various other minor components.
These proteins are amphiphilic molecules that have both nonpolar and polar groups on the same molecule, and can therefore adsorb to oil–water interfaces. They normally provide stabilization against droplet aggregation by a combination of electrostatic and steric repulsion. The electrical characteristics of different proteins are determined by their primary sequence, especially the type, number, and location of ionizable amino acid side groups and other charged groups (such as phosphates) along the polypeptide backbone. Each type of protein can be characterized by its isoelectric point (pI), which is the pH where the net charge on the protein is zero (that is, the number of positive and negative charges are balanced). Below the pI the electrical charge on the proteins is positive, but above this pH it is negative. The isoelectric points and acid dissociation constant point of some common biopolymers are summarized in Table 1. The variation of the electrical charge on protein-coated fat droplets stabilized by different proteins (lactoferrin and β-Lg) is shown in Figure 5.
Table 1. Summary of the Isoelectric Points and Acid Dissociation Constants (pKa) of Some Common Food-Grade Biopolymers that can Be Used to Form Electrically Charged Emulsion Droplets
| ||Types of emulsifiers|| |
|Proteins (isoelectric points)||Polysaccharides (acid-dissociation constants)|
|Casein||β-Lg||Lactoferrin||WPI||Modified starch||Gum arabic||Pectin||Chitosan|
Figure 5. Change in electrical charge (z-potential) of protein-coated fat droplets with pH: β-Lg, β-lactoglobulin; LF, lactoferrin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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When the adsorbed proteins form a relatively thin interfacial coating around the fat droplets, the primary stabilization mechanism is electrostatic repulsion.[40, 41] For this type of system, the stability of the emulsion is particularly sensitive to changes in pH and ionic strength. Droplet aggregation tends to occur when the pH is close to the isoelectric point (low net droplet charge) or at high salt concentrations (strong electrostatic screening). β-Lg is a commonly used globular protein that forms thin interfacial coatings around fat droplets, and is therefore highly sensitive to solution pH and ionic strength. However, when the adsorbed proteins form a relatively thick hydrophilic coating around the fat droplets, the stabilization mechanism is a combination of electrostatic and steric repulsion. Emulsions stabilized by this kind of protein are much more resistant to alterations in pH and ionic strength. Lactoferrin is an example of a globular protein that forms thick interfacial coatings around fat droplets because of its high molecular weight and the fact it contains hydrophilic carbohydrate side chains that protrude into the aqueous phase.[43, 44] Experimental studies have shown that lactoferrin-coated fat droplets are highly stable to changes in pH and salt concentration, provided there is sufficient protein present to fully coat the droplet surfaces. If there is insufficient surface coverage, the lactoferrin-coated droplets do aggregate.
Another factor that is important for determining the functional performance of globular protein-coated fat droplets is their response to temperature changes. Globular proteins (such as β-Lg, BSA, and lactoferrin) unfold when they are heated above their thermal denaturation temperature (Tm). These conformational changes expose reactive amino acid groups, such as those containing nonpolar or sulfhydryl groups, which promote protein–protein interactions. As a result, fat droplets coated by these proteins may aggregate at elevated temperatures because of increases in the hydrophobic attraction or disulfide bond formation between proteins on different droplets. On the other hand, there are no major changes in the conformation of caseins when they are heated, and therefore they are more stable to thermal processing.
Previous studies have shown that heteroaggregation can be induced by mixing together two protein-stabilized emulsions: one containing β-Lg-coated droplets and one containing lactoferrin-coated droplets. β-Lg has an isoelectric point around pH 4.5, whereas lactoferrin has a pI around 8.5 (Figure 5). Consequently, there is a range of intermediate pH values where the two types of droplets have opposite charges and will tend to associate with each other through electrostatic attraction.
A number of natural and modified polysaccharides are amphiphilic molecules that are capable of stabilizing O/W emulsions, such as gum arabic (GA) and modified starch (MS). MS is produced by chemically modifying natural starches so that they gain some nonpolar groups. This is normally achieved by covalently attaching nonpolar octenyl succinic anhydride (OSA) side groups to the polar starch backbone. This leads to an amphiphilic biopolymer molecule that can adsorb to oil–water interfaces and stabilize fat droplets against aggregation.[49, 50] The non-polar OSA groups tend to penetrate into the oil droplets, while the polar starch molecules protrude into the surrounding aqueous phase. Previous studies have shown that MS-coated fat droplets are negatively charged over a wide pH range (pH 2–9) because of the presence of anionic groups on the OSA side chains. MS molecules form a relatively thick hydrophilic layer at the droplet surfaces and therefore can prevent droplet aggregation through a combination of steric and electrostatic repulsion.
GA is isolated from the exudate of a shrub (acacia tree) and is surface active because of the presence of polysaccharide and protein moieties on the same molecule. The protein part is believed to be nonpolar and anchors the molecule to the fat droplet surface, whereas the polysaccharide part is polar and protrudes into the aqueous phase. GA is negatively charged form around pH 2–9 and can therefore be used to create anionic droplets suitable for fabricating heteroaggregates. The fact that GA forms a thick negatively charged interfacial coating around fat droplets means that it mainly provides stabilization against aggregation through a combination of steric and electrostatic repulsion. In general, polysaccharides-based emulsifiers tend to be more stable to pH, ionic strength, and thermal treatment than protein-based emulsifiers.
There are a number of food-grade surfactants that can also be used to form electrically charged fat droplets in O/W emulsions.[56, 57] These surfactants consist of a hydrophilic head group that protrudes into the aqueous phase, and a hydrophobic tail group that protrudes into the oil phase. Most of the ionic surfactants available for utilization in the food industry are negatively charged, such as DATEM, CITREM, and lysolecithin.[56, 57] Nevertheless, lauric arginate is a cationic surfactant that is capable of producing stable positively charged droplets at relatively low pH values (pH < 7). Ionic surfactants can be used in isolation, or they can be mixed with nonionic surfactants to improve emulsion stability.
A potential problem with using two different kinds of fat droplets stabilized with different emulsifiers is the exchange of emulsifiers between them. When one mixes droplets coated by different emulsifiers together then the emulsifier from one droplet may exchange with the emulsifier from a different droplet. This process is likely to occur via the bulk aqueous phase that separates the droplets, that is, an adsorbed emulsifier exchanges with a nonadsorbed emulsifier in the surrounding aqueous phase. As a result, the electrical charge on the two kinds of droplets will become more similar. If complete mixing of the emulsifiers occurs at the droplet interfaces, then all the droplets will eventually have the same charge, which may prevent heteroaggregation. Emulsifier exchange may limit the types of emulsifiers that can be used to form heteroaggregated systems. Polymeric emulsifiers tend to be more resistant to exchange than small molecule surfactants,[59, 60] particularly if they can be cross-linked at the interface, for example, by thermal, chemical, or enzymatic treatment.