Science 128–130 (2006) 227–248www.elsevier.com/locate/cis

Advances in Colloid and Interface

Formation, stability and properties of multilayer emulsionsfor application in the food industry

Demet Guzey, D. Julian McClements ⁎

Biopolymer and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States

Available online 16 January 2007

Abstract

The formation of multilayered interfaces around oil droplets in oil-in-water emulsions provides food technologists with a novel means ofimproving the quality and stability of many food products, as well as the ability to develop novel encapsulation and delivery systems. This articlereviews the basic principles of multilayer emulsion formation, discusses the factors that influence the characteristics of the interfaces formed, andhighlights the relationship between interfacial properties and emulsion functionality. Finally, it highlights some potential applications of themultilayer emulsion technology in the food industry for improving the stability of emulsions to environmental stresses or for developing controlledor triggered release systems.© 2006 Elsevier B.V. All rights reserved.

Keywords: Layer-by-layer deposition; Emulsions; Electrostatic attraction; Colloidal dispersions; Multilayer formation; Bridging flocculation; Depletion flocculation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2281.1. Importance of emulsions in the food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2281.2. Limitations of current emulsifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

2. Potential of LBL technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2283. Principles of LBL technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2284. Factors effecting properties of layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

4.1. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2314.2. Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2314.3. Solvent quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2324.4. Emulsifier characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2324.5. Polyelectrolyte characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

5. Factors effecting stability of multilayer emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2335.1. Theoretical stability maps for optimizing multilayer formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2345.2. Numerical calculation of factors affecting formation of multilayer systems . . . . . . . . . . . . . . . . . . . . . . . . . . 235

5.2.1. Influence of droplet concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2355.2.2. Influence of droplet size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2355.2.3. Influence of polyelectrolyte characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2365.2.4. Comparison of theory and experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

6. Characterization methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2377. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

7.1. Improved stability against environmental stresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

⁎ Corresponding author. Tel.: +1 413 545 1019; fax: +1 413 545 1262.E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).

0001-8686/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.cis.2006.11.021

228 D. Guzey, D.J. McClements / Advances in Colloid and Interface Science 128–130 (2006) 227–248

7.1.1. pH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2397.1.2. Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2407.1.3. Thermal processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2427.1.4. Chilling and freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2437.1.5. Lipid oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2447.1.6. Dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

7.2. Controlled release/triggered release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2457.3. Hollow (nano)capsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

1. Introduction

1.1. Importance of emulsions in the food industry

The food industry is one of many industries that heavily relyon the use of emulsions and emulsifiers. Products such as softdrinks, milk, cream, salad dressings, mayonnaise, soups,sauces, dips, butter and margarine are all examples ofemulsions. Traditionally, oil-in-water (O/W) emulsions areproduced by homogenizing oil and aqueous phases together inthe presence of one or more emulsifiers [1,2]. Duringhomogenization, emulsifiers adsorb to the surfaces of freshlyformed droplets and reduce the interfacial tension, whichfacilitates further droplet disruption. In addition, they form aprotective layer around the droplets that may help protect themfrom aggregation by generating repulsive interactions. The mostcommon emulsifiers used in the food industry are amphiphilicproteins, polysaccharides, phospholipids and small moleculesurfactants [2,3]. Emulsifiers vary greatly in their effectivenessat producing small oil droplets during homogenization, and intheir ability to prevent droplet aggregation under differentenvironmental stresses, such as pH, ionic strength, heating, andfreezing [2,4]. They also differ in cost, availability, ease of use,compatibility with other ingredients and “label friendliness” [5].For these reasons, there is no single emulsifier that is ideal foruse in every type of food product. The most appropriateemulsifier or combination of emulsifiers for a particular foodproduct depends on the type and concentration of otheringredients that it contains, the way that it was produced, andthe environmental conditions that it experiences during itsmanufacture, storage and utilization.

1.2. Limitations of current emulsifiers

There are limitations to the functional properties that can beachieved using existing food emulsifiers and the conventionalmethod of creating emulsions, for example, limited stability topH, salt, heating, dehydration, freezing and chilling. Theselimitations have led to research being carried out to findalternative methods of improving emulsion stability bydeveloping novel emulsifier-based strategies. One strategy hasbeen to create covalent protein–polysaccharide complexes thathave good surface activity and provide improved protection

against environmental stresses [6,7]. The amphiphilic proteinfragment anchors the complexes to the interface, while thehydrophilic polysaccharide fragment protrudes into the aqueousphase and provides stability against droplet aggregation bygenerating a long-range steric repulsion.

An alternative strategy is to create an interfacial layer aroundoil droplets that consists of multiple layers of emulsifiers and/orpolyelectrolytes using a layer-by-layer (LBL) electrostaticdeposition technique [8–16].

2. Potential of LBL technique

In the LBL approach, an ionic emulsifier that rapidly adsorbsto the surface of lipid droplets during homogenization is used toproduce a “primary” emulsion containing small droplets, thenan oppositely charged polyelectrolyte is added to the systemthat adsorbs to the droplet surfaces and produces a “secondary”emulsion containing droplets coated with a two-layer interface.This procedure can be repeated to form oil droplets coated byinterfaces containing three or more layers. Under certaincircumstances, emulsions containing oil droplets surroundedby multilayer interfaces have been found to have better stabilityto environmental stresses than conventional oil-in-water emul-sions with single-layer interfaces [15,17–22]. Knowledge of thephysicochemical principles governing the formation andproperties of these multilayer interfaces is important to establishthe optimum conditions required for producing multilayeremulsions with desirable properties.

3. Principles of LBL technique

In the 1960s, Iler reported the adsorption of charged colloidalparticles onto oppositely charged solid surfaces when hedeposited alternating layers of cationic molecules (surfactants,proteins and alumina) and anionic silica onto glass surfaces [23].Nevertheless, it was only in the early 1990s that LBLelectrostatic assembly research was revolutionized when Decheret al. demonstrated electrostatic multilayer adsorption of chargedpolymers onto solid surfaces. In this technique surface chargereversal was the basis of the assembling technique which did notrequire special equipment or procedures. Molecular films on flator curved surfaces could be prepared from aqueous solutions atroom temperature [24–26].

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Since then the LBL method has been used in a broad range ofapplications to form multilayered films on planar surfaces,biological cells and colloidal particles. For example, thinmultilayer films have been produced on planar supports such asglass, gold and graphite and on inorganic particles such as latex[27–29], gold [28,30–33], polystyrene [34], and organicparticles such as proteins [35], blood cells [36,37], and DNA[38]. In the late 1990s Caruso et al. extensively investigated theadsorption of polyelectrolytes onto colloidal particles using theLBL technique, which opened the way to many applications ofthis technique including encapsulation of enzymes [39,40] andproduction of hollow nanoparticles [31,33].

In the LBL electrostatic deposition technique a polyelectro-lyte layer is formed on a charged surface due to strongelectrostatic attraction between the surface and oppositelycharged polyelectrolyte molecules in solution. The fact that thetotal number of charges on the adsorbed polyelectrolytemolecules is greater than that required to neutralize the oppositecharges on the surface, means that charge reversal occurs. Thisovercompensation of the surface charge has two importantconsequences. First, it means that the adsorbing polyelectrolytestend to form mono-layers because once the surface has beensaturated with polyelectrolytes there is an electrostatic repulsionbetween it and the non-adsorbed polyelectrolytes that preventsfurther adsorption. Second, it means that further layers can beformed by adsorbing oppositely charged polyelectrolytes on topof the first layer, e.g., S–P1–P2, where S is the substrate, and P1

and P2 are two oppositely charged polyelectrolytes. Repetitionof both adsorption steps leads to the formation of multilayerstructures: S–[P1–P2]n–P1–P2 or S–[P1–P2]n–P1 [41]. Thepolyelectrolyte that forms the outer layer usually determinesthe net charge on the overall interface.

For planar surfaces, the method of preparing multilayerstructures simply consists of consecutive immersion of thesubstrate into two or more coating solutions containingoppositely charged species (Fig. 1). The basic method ofproducing multilayer colloidal suspensions is highlighted inFig. 2. First, a suspension of charged colloidal particles is

Fig. 1. Layer-by-layer deposition of oppositely charged polyelectrolytes on aplanar surface. Steps 1 and 3 represent the adsorption of a polyanion andpolycation, respectively. Steps 2 and 4 are washing steps where the excesspolyelectrolytes are washed. Repetition of Steps 1–4 leads to the adsorption ofone polyanion/cation bi-layer per cycle [41].

prepared, which could be solid particles or liquid droplets.Then, an aqueous solution containing oppositely chargedpolyelectrolytes is mixed with the colloidal suspension. Thepolyelectrolytes adsorb to the particle surfaces due toelectrostatic attraction, and if there is sufficient polyelectrolytepresent they will become saturated and charge reversal willoccur. Before another layer is formed around the particles itis often necessary to ensure that there is little or no freepolyelectrolyte present in the aqueous solution. Otherwise, itwill interact with the oppositely charged polyelectrolytes in thenext solution, which may interfere with the formation of themultilayers around the particles. If the polyelectrolyte solutionused is going to form the outer coating, then it is often un-necessary to remove any excess polyelectrolyte since it will notinterfere with subsequent film formation.

A number of preparation strategies that have been developedto produce stable multilayer systems without promoting dropletaggregation due to the presence of excess free polyelectrolyte[42]:

(1) Saturation method. It is possible to add just enoughpolyelectrolyte to completely coat all of the particlespresent in the system, so that there is little freepolyelectrolyte remaining in the aqueous phase. Thesaturation concentration for a particular system has to bedetermined empirically (for example using ζ potentialmeasurements). One has to be careful to add enoughpolyelectrolyte to prevent bridging flocculation, but not toomuch as to cause depletion flocculation (see later). Bridgingflocculation occurs when a polyelectrolyte adsorbs to thesurface of more than one droplet and links them together.Depletion flocculation occurs when the free polyelectrolyteconcentration in the continuous phase generates anattractive osmotic force that is strong enough to overcomethe various repulsive forces. The origin of this osmotic forceis the exclusion of polyelectrolyte molecules from a narrowregion surrounding the droplet surfaces.

(2) Centrifugation method. In this method a solution thatcontains more than enough polyelectrolyte than required tocompletely saturate the particles present is added to acolloidal suspension. Any excess non-adsorbed polyelec-trolyte molecules are then removed by centrifuging thecolloidal suspension, collecting the particles, and re-suspending them in an appropriate buffer solution. Thisprocedure can be repeated a number of times to ensure thatall of the free polyelectrolyte has been removed, before thenext polyelectrolyte solution is added. The main problemwith this method is that it can promote particle aggregationduring the centrifugation step because the particles areforced into close proximity.

(3) Filtration method. In this method a solution that containsmore than enough polyelectrolyte than required to com-pletely saturate the particles present is also added to thecolloidal suspension. However, in this case the excess non-adsorbed polyelectrolyte molecules are removed by mem-brane filtration of the colloidal suspension. A filter is usedthat allows the polyelectrolyte molecules to pass through,

Fig. 2. Utilization of LBL technique for the production of O/W emulsions [16].

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but not the colloidal particles. The colloidal suspension isput under pressure, which forces the aqueous phasecontaining the excess polyelectrolyte through the filter. Atthe same time, a buffer solution can be added to thecolloidal suspension to keep the overall volume of thesystem constant. In this way, the colloidal particles arenever forced into close proximity, which reduces theamount of particle aggregation that occurs in the system.Another advantage of this method is that it is not necessaryto have a density difference between the particles and thesurrounding liquid.

We should mention that in some systems it is possible to mixthe colloidal particles and polyelectrolyte molecules together ata pH where they do not have opposite charges, and then adjustthe pH to a value where they do have opposite charges so as topromote polyelectrolyte adsorption. This procedure can becarried out when the charge on the polyelectrolyte or colloidalparticle can be varied by altering the pH, e.g., the charge on aprotein goes from positive to negative when the pH is adjustedfrom below to above its isoelectric point. The advantage of thismethod is that the polyelectrolytes are evenly distributedthroughout the continuous phase before adsorption occurs,which should ensure uniform and rapid polyelectrolyte adsorp-tion and help prevent droplet aggregation.

For all of the methods mentioned above one must carefullycontrol the system composition and preparation conditions inorder to form stable multilayer colloidal particles. For example,it is important to ensure: that there is sufficient polyelectrolytepresent to cover all of the surfaces present; that there is not toomuch free polyelectrolyte present to promote depletion floccu-lation; and, that the polyelectrolyte adsorbs more rapidlythan when particle–particle collisions occur (see later). Theseprocesses depend on the size and concentration of thepolyelectrolyte molecules and colloidal particles present, aswell as on the solution conditions (e.g., pH, ionic strength,dielectric constant, temperature, and stirring— see later). In thefollowing sections we review progress that has been made in

understanding the influence of the properties of polyelectrolytesand colloidal particles on the formation of stable multilayeremulsions.

The LBL technology can simply be adapted to producemultilayer food emulsions using a step-by-step method thatshould be fairly easy, fast and cheap (Fig. 2). In this method aprimary emulsion is prepared by homogenizing oil and waterphases in the presence of a positive or negatively chargedemulsifier. The resulting primary emulsion is then mixed intoan oppositely charged polyelectrolyte solution to create a sec-ondary emulsion. The secondary emulsion is then mixed intoanother solution containing polyelectrolytes that have anopposite charge to the previous one to create a tertiary emul-sion, and so on. As mentioned earlier, it may be necessary toremove any excess polyelectrolyte between each adsorptionstep, although this can often be avoided by selection of anappropriate initial polyelectrolyte concentration. In a series ofinvestigations, O/W emulsions have been prepared with smallmolecule surfactants, proteins, polysaccharides and phospholi-pids using the LBL technique [11,13–19,43–46]. There areadditional production costs associated with utilizing the LBLtechnique due to the additional ingredients (usually proteinsand/or polysaccharides) and processing operations (mixing,homogenization, centrifugation and/or filtration) used, and so itis only likely to be used on food products where the financialbenefits overcome these additional costs. For example, it maybe possible to reduce the amount of product normally lost dueto poor or unreliable performance of existing emulsifiers, or itmay be possible to sell a product at a higher cost due toimproved functionality or extended shelf life.

It should be noted that the stabilization of foods by theadsorption of polyelectrolytes onto the surfaces of oppositelycharged colloidal particles is already utilized in some foodproducts, e.g., in the stabilization of acid beverages [47].Nevertheless, the LBL approach extends this concept bysystematically utilizing the principles of directed self-assemblyof polyelectrolytes to form multiple layers at an interface, ratherthan relying on a more empirical mix-and-see approach.

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4. Factors effecting properties of layers

4.1. pH

The electrical charge on emulsion droplets primarily comesfrom adsorption of ionized species from the surroundingsolution onto the surface, e.g., ionic emulsifiers, polyelectro-lytes, or mineral ions. The magnitude and sign of the dropletcharge therefore depend on the type and concentration ofmolecules adsorbed to the surface, as well as the prevailingsolution conditions. One of the most important factorsdetermining the formation and properties of multilayer colloidsis the solution pH, both during and after formation of theinterface. The pH of the solution determines the ionization ofsurface groups and therefore the final surface charge density[48]. Normally for the LBL deposition technique, the pH of thesolution should be selected so that the signs of the electricalcharges on the particle surface and adsorbing polyelectrolyte areopposite and the magnitude of the charges on both species issufficiently high [35]. Many of the polyelectrolytes used in thefood industry have ionizable groups that are relatively weakacids or bases. The negative charges on most anionic polyelec-trolytes come from sulfate, phosphate, or carbonate groups withpKa values around 1–2 and 4–5, respectively, whereas thepositive charges on cationic polyelectrolytes come from aminoor imino groups with pKa values around 7–11 [35]. Bymanipulating the solution pH it is possible to control the degreeof polyelectrolyte adsorption to the particle surfaces. Forexample, anionic pectin will not adsorb to the surface of anionicβ-lactoglobulin-coated droplets at pH 7 because of the largeelectrostatic repulsion between the polysaccharide and droplets,however, it will adsorb at pH 3 because the anionicpolysaccharide and cationic droplets then have opposite charges[11]. There is often a critical pH where adsorption is firstobserved, which depends on the relative magnitude and sign ofthe charges on the polyelectrolyte molecules and the colloidalparticles. It should be noted that a polyelectrolyte can adsorb to aparticle with the same net charge under certain circumstances,e.g., when the particle surface has some oppositely chargedpatches. An example of this phenomenon is the adsorption ofanionic carrageenan onto the surface of anionic β-lactoglobulin-coated droplets at pH 6, which has been attributed to thepresence of some positive patches on the surface of the adsorbedprotein molecules [44].

The properties of the multilayered interfaces surroundingcolloidal particles can be adjusted by small variations in pHafter their formation [49]. Alterations in solution pH can changethe electrostatic interactions between the particle surface and theadsorbed polyelectrolyte, between two or more adsorbedpolyelectrolytes in the layer, or between adsorbed and non-adsorbed polyelectrolytes. These changes can alter the thick-ness, packing and integrity of the multilayered interface. Forexample, changing external pH conditions can be used to causeone or more of the polyelectrolytes in the interface to fall off,thereby providing a means of selective triggering of release ofcharged molecules [50]. Alternatively, pH changes during orafter multilayer formation can result in reversible changes in

interfacial porosity [51], which can provide a valuable means ofselectively controlling the transport of different sized molecularspecies across the interface. For example, it has been shown thatproteins and carbohydrates can be trapped within a multilayersystem at high pH, but released when the pH is reduced becauseof an increase in shell permeability [51]. Finally, pH inducedalterations in the thickness of the interfacial layer may providebetter or worse stability to droplet aggregation by altering themagnitude and range of the steric repulsion and van der Waalsattraction between droplets.

4.2. Salt

The ionic strength of the solution determines the strength andrange of intra- and inter-molecular electrostatic interactions andhence multilayer film formation, structure and thickness[26,52]. The magnitude and range of electrostatic interactionsbetween a polyelectrolyte and a droplet decrease as the ionicstrength of the solution increases because of the accumulation ofcounter-ions around the surfaces, which is usually referred to aselectrostatic screening [2]. Electrostatic screening becomesstronger as the concentration and valency of the counter-ions inthe solution increases. The range of this effect is characterizedby the Debye screening length (κ−1), which varies with theinverse square root of the ionic strength:

j−1 ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffie0eRkT

e2P

n0iz2i

s

For aqueous solutions at room temperature j−1 ¼0:304=

ffiffiI

pnm, where I is the ionic strength of 1:1 electrolytes

(e.g., NaCl) in moles per liter [48]. Multivalent counter-ions(e.g., Ca2+, Fe2+, Fe3+) are much more effective at screeningelectrostatic interactions than monovalent counter-ions (e.g.,Na+, Cl−, K+). Thus, smaller concentrations of multivalentcounter-ions would screen more charges than monovalentcounter-ions. Multivalent counter-ions can also bind to thesurface of polyelectrolytes and change the surface chargedensity [2].

The presence of salt during multilayer buildup can affect thecomposition, structure and thickness of the adsorbed polymerlayers [53]. In the absence of salt, polyelectrolytes tend to formthin layers with the chains being flat against the surface (Fig. 3),which has been attributed to the highly extended conformation ofthe polyelectrolyte molecules in solution (due to strong intra-molecular electrostatic repulsion) and the restriction of post-adsorption molecular rearrangements due to the extremely strongelectrostatic attraction between charged polyelectrolyte andsurface groups. In the presence of salt, polyelectrolytes oftenform thicker layers because they have a more compact chainconformation in solution (due to weaker intra-molecular repul-sion) and because the weaker electrostatic attraction betweencharged polyelectrolyte and surface groups allows post-adsorp-tion molecular rearrangements [54]. For the same reasons, thetotal amount of polyelectrolyte adsorbed to the surface tends to begreater in the presence of salt than in its absence.

Fig. 3. Effect of ionic strength on layer thickness and polymer orientation at the substrate surface; (A) at low ionic strength, and (B) at high ionic strength.

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The characteristics of a polyelectrolyte multilayer may alsobe altered by adjustments in the ionic strength of an aqueoussolution after the polyelectrolyte layer has been formed. Forexample the permeability of a polyelectrolyte multilayer tosmall molecules can often be increased by increasing the ionicstrength, since this weakens the attractive interactions betweenpolyelectrolyte molecules in different layers thereby causing anincrease in interfacial porosity [54]. At sufficiently high ionicstrength one or more polyelectrolyte layers may becomedetached from the droplet surfaces because of the weakeningin attractive electrostatic interactions.

Consequently, the properties of multilayer emulsions can bemanipulated by carefully controlling the ionic composition ofthe aqueous solution both during and after multilayer formation.A great deal of research in this area has been carried out usingsynthetic polymers, but more systematic research is requiredusing food-grade polyelectrolytes with well-defined properties.

4.3. Solvent quality

Solvent quality is another important parameter in multilayerformation. Changing the dielectric constant of the solvent willchange the strength of the electrostatic interactions, as well asthe influence of any hydrophobic interactions [55]. Poptoshev etal. studied the effect of solvent quality on the formation ofmultilayer polyelectrolyte films by adding ethanol to thesolution. They found that increasing the amount of ethanolincreased the interfacial layer thickness and the amount ofmaterial adsorbed, which was attributed to a reduction in theinter-chain electrostatic repulsion. Exposure to a solvent such asethanol after multilayer formation however was reported tocause the film to collapse instead of swelling, which wasexplained by unfavorable segment–solvent interactions [52]. Itis clear that further work is needed to examine the influence ofsolvent quality on the formation and properties of multilayerfood emulsions.

4.4. Emulsifier characteristics

The electrical properties of the first layer of a multilayeremulsion are determined by the emulsifier, and so can becontrolled by selecting different types of emulsifier. In the foodindustry, a variety of different emulsifiers can be used, includingsurfactants, phospholipids, proteins and polysaccharides. Eachof these emulsifiers has different electrical characteristics,which can influence the formation and properties of multilayerinterfaces. In principle, non-ionic surfactants should formuncharged droplets, but in practice the droplets often do havean electrical charge (negative at high pH and positive at low

pH). This has been attributed to the presence of chargedimpurities in the oils used to prepare the emulsions (e.g.,phospholipids or free fatty acids) or due to preferentialadsorption of small ions from the aqueous phase (e.g., OH−

or H3O+ ions) [2]. A number of different food-grade anionic

surfactants and phospholipids are available to prepare primaryemulsions with negatively charged droplets, including lecithin,fatty acid salts, Diacetyl Tartaric Acid Esters of Monoglycerides(DATEM), and stearoyl-lactylates [2]. On the other hand, thereare few examples of food-grade cationic surfactants, and so it isdifficult to prepare primary emulsions containing positivelycharged droplets using surfactants. This severely limits thepolyelectrolytes that can be used to form multilayer emulsionswhen surfactants are used to form the primary emulsion, sincemost food-grade polyelectrolytes are anionic. It should also benoted that the magnitude of the surface charge on surfactant-coated droplets can be controlled by using mixtures ofsurfactants with different electrical characteristics.

Protein emulsifiers are particularly useful for controlling thedroplet charge in the primary emulsion because the sign andmagnitude of their charge can be altered simply by varyingsolution pH. Proteins are positively charged below theirisoelectric point (pI ) and negatively charged above theirisoelectric point. By varying the solution pH it is thereforepossible to “tune” the electrostatic interactions between apolyelectrolyte and a protein-coated droplet surface. In addition,different proteins have different isoelectric points, so it is oftenpossible to alter the electrical characteristics of the droplets inprimary emulsions by selecting proteins that have the requiredelectrical properties at the desired solution pH.

The two most widely used polysaccharide emulsifiers in thefood industry are gum Arabic and modified starch, which tendto form relatively thick anionic interfaces. These emulsifiers arenot strongly surface active and therefore have to be used at fairlyhigh emulsifier-to-oil ratios, but they do form oil-in-wateremulsions with good stability to environmental stresses. To theauthors' knowledge these polysaccharide emulsifiers have notpreviously been used to form multilayer emulsions.

A minimum charge density of the first layer has beenreported as necessary for multilayer formation [56,57]. Such athreshold charge density is different for different systems, yetthe ratio between the charge densities of the emulsifier andadsorbing polyelectrolyte is important for the optimization of amultilayer system [58].

Finally, it should be noted that the selection of an appropriateemulsifier to form the first layer does not only depend on itselectrical characteristics. The thickness, structure and environ-mental sensitivity of the layers formed by emulsifiers may alsoaffect the formation and stability of multilayer emulsions. For

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example, caseins tend to form relatively thick open interfaciallayers that are fairly resistant to heating, whereas whey proteinstend to form relatively thin dense interfacial layers that becomemore hydrophobic upon heating due to protein denaturation.

4.5. Polyelectrolyte characteristics

The formation, stability and properties of multilayercolloidal systems also depend on the nature of the polyelec-trolytes used to adsorb to the surfaces of the charged particles.The chain length, charge density, charge distribution, rigidity,and degree of branching of polyelectrolytes influence theirtendency to adsorb to surfaces and the characteristics of theadsorbed layers formed, such as thickness, structure, porosityand environmental sensitivity [59].

The thickness of an interfacial layer is often governed by thecharge density of the polyelectrolyte chain. At sufficiently lowsalt concentrations, highly charged polyelectrolyte chains tendto form relatively thin layers when they adsorb to oppositelycharge surfaces, which can be attributed to the presence ofstrong intra-molecular electrostatic interactions [60]. In solu-tion, there is a strong electrostatic repulsion between differentsegments of the same polyelectrolyte chain, which causes themolecule to become highly extended. Consequently, when itfirst adsorbs to an oppositely charged surface it tends to lie flatand be spread out. In addition, after adsorption there is a strongattraction between segments of the polyelectrolyte chains andoppositely charged groups on the surface, which causes them tocome into close proximity, thus forming a thin interface. Thiseffect is less prevalent at high salt concentrations becauseelectrostatic screening effects will cause the polyelectrolyte toadsorb in a more compact structure, and the interactionsbetween segments of the polyelectrolyte chains and oppositelycharged groups on the surface will be weakened [54].

The dependence of multilayer interfacial thickness onpolyelectrolyte chain length depends on the conformation ofthe polyelectrolyte in solution. If the polyelectrolyte has ahighly extended conformation in solution (e.g., due to low salt,high charge density or high molecular rigidity) it tends to adsorbflat against the surface, and so the chain length has little impacton interfacial thickness. In this case, the thickness is determinedby the width of the polyelectrolyte chain, rather than its length(Fig. 3A). On the other hand, if a polyelectrolyte has a randomcoil conformation in solution (e.g., due to high salt, low chargedensity, or high molecular flexibility) it will tend to adsorb tothe surface in a more ball-like conformation, and so the inter-facial thickness increases with increasing chain length (Fig. 3B)[60]. It should be noted that the packing and flexibility of anadsorbed polyelectrolyte also depend on its relative positionwithin the multilayer. Polyelectrolyte molecules within the innerlayers tend to be more densely packed and have more restrictedmotion than those in the outermost layer [60].

The amount of polyelectrolyte adsorbed to the surface andthe thickness, structure and porosity of the interfaces formed arestrongly determined by the polyelectrolytes sensitivity to itsenvironment, e.g., pH, ionic strength and temperature. Differentpolyelectrolytes have different environmental sensitivities, i.e.,

changes in electrical charge, conformation, hydrophobicity,self-association and binding with temperature, pH or salt.Consequently, it is possible to create multilayer interfaces withdifferent environmental responsiveness by selecting differentkinds and combinations of polyelectrolytes to form theinterfaces.

It should be noted that the electrical characteristics of apolyelectrolyte molecule trapped within a multilayer interfacemay be appreciably different from those of the same individualmolecule suspended in an aqueous solution. The pKa of theionizable groups on a polyelectrolyte can be shifted from theirvalues in solution due to their local electrostatic environment[60]. This shift in pKa can alter the pH where one would expecta polyelectrolyte layer to become desorbed from an oil–waterinterface [22,45].

Finally, it is important to use a polyelectrolyte concentrationthat is high enough to completely saturate the surface of thecharged particles, and that will reverse the surface charge of theprevious layer by an amount that is sufficiently high to create astrong electrostatic repulsion between the final droplets [61].The amount of polyelectrolyte needed to saturate the surfaceand the final charge on the particle–polyelectrolyte complexdepends on the molecular characteristics of the polyelectrolyteused, e.g., chain length, conformation, charge density, andflexibility.

5. Factors effecting stability of multilayer emulsions

The main problem with using the LBL technique to preparemultilayer emulsions is the tendency for droplet aggregation tooccur during preparation. This loss of droplets to aggregationdepends on the method used to prepare and wash them, with thefiltration method being better than the saturation method, whichin turn is better than the centrifugation method [42]. Tominimize or avoid droplet aggregation using any of thesemethods it is important to carefully control solution composi-tion and preparation conditions: there should be sufficientpolyelectrolyte present to saturate all of the droplet surfaces; thepolyelectrolyte molecules should adsorb to the droplet surfacesmore rapidly than when droplet–droplet collisions occur; thereshould not be too much polyelectrolyte present to promotedepletion flocculation; the repulsive interactions between thecoated droplets should be strong enough to prevent dropletaggregation. These physicochemical phenomena depend on thesize and concentration of both the polyelectrolyte molecules andthe emulsion droplets present, as well as on the solutionconditions (e.g., pH, ionic strength, dielectric constant,temperature, and stirring). If droplet aggregation does occurduring the preparation of multilayer systems it is sometimespossible to disrupt the flocs formed by including an additionalmechanical agitation step after addition of each layer, e.g.,homogenization, blending or high intensity ultrasound[11,13,14,19].

The influence of interfacial properties on the stability ofmultilayer colloidal dispersions was recently assessed theoret-ically by calculating the colloidal interactions between themultilayer-coated droplets, i.e., van der Waals, electrostatic,

Fig. 4. Schematic diagram of the different events that occur when an electricallycharged polyelectrolyte is added to a colloidal dispersion containing oppositely

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steric and depletion [59]. These calculations will be reviewed inthe following section.

5.1. Theoretical stability maps for optimizing multilayerformation

Recently, a simple mathematical model has been developedto predict the stability of multilayer systems formed byadsorbing charged polyelectrolytes onto oppositely chargedparticles [59]. This model is based on a consideration of asystem consisting of a suspension of charged monodispersespherical particles to which oppositely charged polyelectrolytemolecules are added (Fig. 4). The model also assumes that theadsorption of polyelectrolyte molecules to the particle surfacesis diffusion limited and that the dominant particle–particlecollision mechanism is Brownian motion. There will be anelectrostatic attraction between the polyelectrolyte moleculesand the bare surfaces of the particles, which will causepolyelectrolyte molecules to adsorb to the particle surfaces.Polyelectrolyte adsorption could lead to emulsion stability dueto multilayer formation or emulsion instability due toflocculation. The model assumes that the system is designedso that the particles are completely stable to aggregation whenthere are no polyelectrolyte molecules present (bare particles)and when the particles are completely saturated with polyelec-trolyte molecules (provided the droplet repulsion is sufficientlyhigh, see below). Practically, this could be achieved by ensuringthat the magnitude of the electrical charge density on both thebare and saturated particles was high enough to generate astrong electrostatic repulsion between the particles. The stabilityof the particles to aggregation can then be divided into a numberof different regimes depending on the polyelectrolyte concen-tration, C:

(I). C=0. The particles are stable to aggregation because ofthe strong electrostatic repulsion between them.

(II). 0bCb (CSat or CAds). Bridging flocculation occurs whenthe polyelectrolyte concentration is insufficient to com-pletely saturate the particle surfaces (CSat) or when thepolyelectrolyte concentration is too low to ensure that thedroplets are completely saturated with polyelectrolytebefore a droplet collision occurs (CAds). This is becausethere are both positive and negative patches on the dropletsurfaces, which promote droplet aggregation.

(III). (CSat and CAds)bCbCDep. The particles should be stableto droplet aggregation when their surfaces are completelysaturated with polyelectrolyte, and there is not enoughfree polyelectrolyte present in the continuous phase topromote depletion flocculation. Under these circum-stances it should be possible to prepare stable multilayerparticles consisting of particles completely surrounded bya polyelectrolyte layer.

(IV). CNCDep. When the concentration of free polyelectrolyteexceeds some critical value (CDep) depletion flocculationoccurs because the attractive depletion forces are strongenough to overcome the various repulsive forces (e.g.,electrostatic and steric).

The following expressions were derived for the criticalpolyelectrolyte concentrations outlined above [62]:

CSat ¼ 3/CSat

rð1Þ

CAds ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi60C2

SatrPE/r3

sð2Þ

CDep ¼ MNA

−1þ ffiffiffiffiffiffiffiffiffiffiffiffiffiffi1−8vX

p

4v

� �ð3Þ

where:

X ¼ wDep

kBT

� �Crit

1

2pr2PE r þ 23 rPE

� �where ϕ is the volume fraction of the particles, ΓSat is thesurface load of the polyelectrolyte at saturation (in kg m−2), r isthe radius of the spherical particles (in m), assuming amonodisperse solution, rPE is the effective radius of thepolyelectrolyte molecules in solution (in m),M is the molecularweight of the polyelectrolyte (in kg mol−1), v (=4πrPE

3 /3) is theeffective molar volume of the polyelectrolyte in solution (in m3),NA is Avogadro's number, wDep is the strength of depletionattraction at droplet contact, which depends on droplet size andnon-adsorbing concentration of the polyelectrolyte, kB is theBoltzmann's constant, and T is the absolute temperature.

These equations can be used to develop “stability maps”,which indicate the region where stable multilayer emulsions canpotentially be created [59]. To produce a colloidal dispersionthat is stable to flocculation it is necessary to ensure that (CSat

and CAds)bCbCDep, i.e., that there is enough polyelectrolyte tocompletely saturate the surfaces of the particles, but not toomuch free polyelectrolyte to promote depletion flocculation.Using the various equations derived above for CSat, CDep andCAds it is possible to generate stability maps for formation of

charged spherical particles [59].

Fig. 7. Stability map showing the influence of the effective radius of thepolyelectrolyte molecules on the critical polyelectrolyte concentrations forsaturation, depletion and adsorption. It was assumed that the droplets had aradius of 0.5 μm and a volume fraction of 0.05 (5 vol.%), and that the non-adsorbed polyelectrolyte had a molecular weight of 100 kDa [59]. The shadedarea highlights the range of conditions where it should be possible to producenon-flocculated droplets.

Fig. 6. Stability map showing the influence of droplet size on the criticalpolyelectrolyte concentrations for saturation, depletion and adsorption. It wasassumed that the droplets had a volume fraction of 0.03 (3 vol.%), and the non-adsorbed polyelectrolyte had a molecular weight of 100 kDa and effective radiusof 30 nm [59]. The shaded area highlights the range of conditions where itshould be possible to produce non-flocculated droplets.

Fig. 5. Stability map showing the influence of droplet concentration on thecritical polyelectrolyte concentrations for saturation, depletion and adsorption. Itwas assumed that the droplets had a radius of 0.3 μm, and the non-adsorbedpolyelectrolyte had a molecular weight of 100 kDa and effective radius of 30 nm[59]. The shaded area highlights the range of conditions where it should bepossible to produce non-flocculated droplets.

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multilayer particles without promoting bridging flocculationand depletion flocculation. Further details about the construc-tion of stability maps are given in an earlier reference [59].

5.2. Numerical calculation of factors affecting formation ofmultilayer systems

In this section, we examine some of the major factors thatwould be expected to influence the formation of stablemultilayer colloidal dispersions, including polyelectrolyteconcentration and size (C and rPE), and particle concentrationand size (ϕ and r).

5.2.1. Influence of droplet concentrationA stability map showing the dependence of the critical

saturation, depletion and adsorption concentrations on dropletand polyelectrolyte concentration is shown in Fig. 5. In thesecalculations it was assumed that the droplets had a radius of0.3 μm, and that the polyelectrolyte had a molecular weight of100 kDa and a radius of gyration of 30 nm (which arerepresentative values for some natural polyelectrolytes). Thestability map indicates that at low polyelectrolyte concentrationsthe particles should be susceptible to bridging flocculationbecause there is insufficient polyelectrolyte present to com-pletely saturate the surfaces of all the particles (CbCSat).Nevertheless, even when the polyelectrolyte concentrationexceeds CSat, the particles may still be susceptible to bridgingflocculation because the polyelectrolyte does not adsorb rapidlyenough to saturate the particle surfaces before a particle–particlecollision occurs (CbCAds). At relatively low particle concentra-tions (ϕb0.11), it should be possible to make stable multilayer-coated particles without flocculation occurring over a range ofintermediate polyelectrolyte concentrations (CAdsbCbCDep).However, if the polyelectrolyte concentration is increasedfurther so that CDep is exceeded then the particles will be

susceptible to depletion flocculation, and it will not be possibleto make a stable multilayer system. In addition, at relatively highparticle concentrations (ϕN0.11), there is no range of polyelec-trolyte concentrations where stable multilayer-coated particlescan be formed, since depletion flocculation tends to occur atthe polyelectrolyte concentration required to ensure rapidadsorption.

5.2.2. Influence of droplet sizeA stability map showing the dependence of the critical

saturation, depletion and adsorption concentrations on dropletsize is shown in Fig. 6. In these calculations it was assumed that

Fig. 8. Stability map showing the influence of the molecular weight of thepolyelectrolyte molecules on the critical polyelectrolyte concentrations forsaturation, depletion and adsorption. It was assumed that the droplets had aradius of 0.3 μm and a volume fraction of 0.05 (5 vol.%), and that the non-adsorbed polyelectrolyte had an effective radius of 30 nm [59]. The shaded areahighlights the range of conditions where it should be possible to produce non-flocculated droplets.

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the droplet concentration was ϕ=0.03 (3 vol.%), and that thepolyelectrolyte had a molecular weight of 100 kDa and a radiusof gyration of 30 nm. The stability map predicts that it shouldnot be possible to make stable multilayer colloidal dispersions at

Table 1Stability map of the SDS–chitosan emulsions (3 wt.% corn oil, 100 mM acetic acidstorage at room temperature (C: clumps, S: stable, F: flocculated) [63]

relatively low particle radii (rb0.15 μm), even above CSat,because the particles collide with each other before theirsurfaces are completely saturated with polyelectrolyte. Howev-er, at sufficiently high particle sizes there should be a range ofintermediate polyelectrolyte concentrations (CAdsbCbCDep)where non-flocculated multilayer-coated particles can beproduced.

5.2.3. Influence of polyelectrolyte characteristicsStability maps showing the dependence of the critical

saturation, depletion and adsorption concentrations on polyelec-trolyte radius of gyration and molecular weight are shown inFigs. 7 and 8. In these calculations it was assumed that thedroplets had a radius of 0.5 μm and a volume fraction of 0.05.The stability map in Fig. 7 assumes that the molecular weight ofthe polyelectrolyte stays constant (100 kDa) while the effectiveradius varies, which could occur if the temperature or solventconditions (e.g., pH or ionic strength) were varied. For allpolyelectrolyte effective radii there is an intermediate range ofpolyelectrolyte concentrations where stable multilayer-coatedparticles could be formed (CAdsbCbCDep). The stability map inFig. 8 assumes that the molecular weight of the polyelectrolytevaries while the effective radius remains constant (30 nm), whichcould occur if different kinds of polyelectrolytes were used. Thecritical saturation, depletion and adsorption concentrations all

, pH 3.0) containing different chitosan concentrations (0 to 1 wt.%) after 24 h

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increased linearly with increasing polyelectrolyte molecularweight so that the range of polyelectrolyte concentrations wherestable multilayer-coated particles could be formed becomeswider as the conformation of the polyelectrolytes becomes morecompact, i.e., same effective polyelectrolyte radius for increas-ing molecular weight.

5.2.4. Comparison of theory and experimentExperiments with oil-in-water emulsions stabilized by an

anionic surfactant (SDS) to which a cationic polyelectrolyte(chitosan) was added qualitatively support the theoreticalpredictions made above [63]. This study showed that extensivedroplet flocculation occurred for CbCSat at all dropletconcentrations, and that it was not possible to make non-aggregated emulsions when the droplet concentration exceededa particular value (5 vol.%) at any polyelectrolyte concentration(Table 1). The experimental data from this study thereforequalitatively supported the predictions of the stability mapshown in Fig. 5. Nevertheless, further systematic studies arerequired to establish the influence of polyelectrolyte character-istics, such as molecular weight, stiffness and charge density onthe formation of stable multilayer emulsions.

6. Characterization methods

A wide variety of different analytical techniques andexperimental protocols have been used to characterize theproperties of multilayer interfaces and emulsions. The mostimportant characteristics of multilayer emulsions that usuallyneed to be established are the net electrical charge on thedroplets, the thickness and the composition of the interface, theextent of flocculation in the system during preparation, and thelong-term stability of the system after preparation, e.g., todroplet aggregation, creaming and Ostwald ripening. Inaddition, it is often important to establish how thesephysicochemical properties of interfaces and emulsions respondto changes in environmental conditions, such as pH, ionicstrength, solvent quality or temperature. In this section, weprovide a brief overview of the kinds of analytical techniquesthat can be used to provide this information.

Multilayer buildup and properties in planar surfaces or incolloidal systems can be characterized using a variety oftechniques including, spectroscopy, scattering, microscopy,gravimetric and reflectivity techniques. The thickness of thinfilms coated on planar supports has been characterized by re-flectivity techniques, such as ellipsometry [64–66], X-rayreflectometry [56,67,68] and neutron reflectometry [69]. Spec-troscopic techniques such as UV–Visible [67], surface plasmonresonance spectroscopy (SPRS) [52,70], and Fourier TransformInfrared (FTIR) spectroscopy [71] and gravimetric techniquessuch as Quartz Crystal Microbalance (QCM) [52,72] have alsobeen used to monitor multilayer buildup of thin films at planarsurfaces. In this section, we will primarily focus on thosetechniques that are used to characterize the multilayer filmsformed around colloidal particles, rather than at planar surfaces,since these are most applicable to understanding multilayeremulsions.

The multilayer buildup on colloidal particles has been mostlycharacterized using techniques based on measurements ofelectrophoretic mobility [27,29,73,74], scattering (such assingle particle light scattering, SPLS [29,60,73,74], dynamiclight scattering, DLS [75–77], and small-angle neutronscattering, SANS [78]), microscopy (such as scanning electronmicroscopy (SEM) [27,79], transmission electron microscopy(TEM) [27,29,49], confocal laser scanning microscopy (CLSM)[80,81] and atomic force microscopy (AFM) [27,74,82]) andspectroscopy (FTIR [71,79] and fluorescence spectroscopy[82–85]).

The adsorbed amount of polyelectrolytes on colloidalparticles can be determined using FTIR (Fourier TransformInfrared) spectroscopy. FTIR spectroscopy provides the inte-grated band area from the band positions assigned to infraredvibrations of the polyelectrolyte, which can be used as a linearscale of the surface concentration of the adsorbed polymer[71,79].

Particle electrophoretic instruments are used to measure thedirection and velocity of droplet movement in a well-definedelectric field. The velocity and direction are measures of thesign and magnitude of the electrical charge on the particlesurface and can be used to calculate their zeta (ζ) potential. ζpotential can be defined as the electrical potential at the shearplane, which is the distance from the charged surface belowwhich the counter-ions remain strongly attached and isapproximately equal to the diameter of the hydrated ions [2].ζ potential is calculated from Henry's equation using measuredelectrophoretic mobility, viscosity of the surrounding liquid,dielectric constant of the material and the dielectric constant ofvacuum and using either the Huckel or Smoluchowski ap-proximation for Henry's Function [86]. Emulsions need to bediluted to a droplet concentration of approximately 0.005 wt.%oil using buffer solution to avoid multiple scattering effects [2].Measurements of the change in ζ potential along with thechange in the mean particle diameter when a polyelectrolyte isadded to an emulsion containing oppositely charged dropletscan be used to monitor the adsorption of the polyelectrolyte tothe droplet surfaces (Fig. 9A and B).

DLS, also referred to as Photon Correlation Spectroscopy,can be used to study the increase in particle diameter as polymerlayers are adsorbed. DLS measures the Brownian motion andrelates it to particle size— the larger the particle the smaller theBrownian motion, which is measured as the translationaldiffusion coefficient. Hydrodynamic radius of colloidal parti-cles can be determined from Stoke's equation using themeasured translational diffusion coefficient, absolute tempera-ture and viscosity of the surrounding liquid. The disadvantageof DLS is that it cannot distinguish between single particles andaggregates [76].

Multilayer growth on colloidal particles can also bemonitored using SPLS. SPLS records the light scattered froma single particle at a given moment in time at a 1–2 nmthickness resolution [65]. With this technique light scatteredfrom single particles and aggregates (doublets, triplets, andhigher order) can be distinguished provided the single particlediameter is larger than about 200 nm [76]. Layer thicknesses

Fig. 9. Change in (A) electrical charge (ζ-potential) and (B) mean particlediameter (d32) of emulsion droplets as cationic chitosan (0–0.25 wt.%) is addedto the Blg/pectin anionic secondary emulsion (5 wt.% corn oil, 0.225 wt.% Blg,0.2% pectin, 100 mMNa-acetate buffer) at pH 4. A photograph of the emulsionsafter 24 h of storage at room temperature is shown [22].

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can be determined using Rayleigh–Debye–Gans theory andrefractive index values of core particle and adjacent layers[65,76].

The thickness of the polyelectrolyte multilayers on colloidscan also be measured directly in aqueous solution using SANS[78]. The volume fraction profile of adsorbed polymers at

interfaces may be derived from SANS. The scattering lengthdensity of the cores of the polymer-coated colloids can bematched to that of the solution, so that the core becomes“invisible” and only the scattering from the polymer layer ispresent.

Microscopy (SEM, TEM, AFM) can be used to providevisual evidence for the formation of multilayer interfaces aroundcolloidal particles. SEM and TEM provide visual information onthe wall thickness, size, shape and morphology of multilayeredcolloids [27,29,34,35,38]. The resolution of TEM is about anorder of magnitude better than the SEM, so the latter technique ismore effective at determining the thickness of individual layers[30]. Both methods require fixation of samples by drying orfreezing which makes these methods difficult to use tocharacterize emulsion multilayer systems. The AFM consistsof a cantilever with a sharp tip at its end, which is brought intoclose proximity of a sample surface. The interaction forcebetween the tip and the sample leads to a deflection of thecantilever, which can be detected using a laser beam. The tip isscanned over the sample in the horizontal direction and itsdeflection in the vertical direction is measured. AFM thereforegives 3D information on the topography of the sample surface;adsorbed layer thickness, aggregation, homogeneity, roughnessand porosity and coverage of the surface [87]. AFM can be usedto visually characterize multilayer systems as affected byassembly parameters such as the number of layers, pH, andsalt concentration during assembly or after multilayer buildup[27,74,87]. An important advantage of AFM over SEM andTEM is that it is non-destructive to the sample which makes it asuitable tool to study liquid systems such as emulsions.

In addition, the release properties of multilayer colloids(microcapsules) are important to determine their potentialapplication as delivery systems. The permeability of multilayermicrocapsules can be studied using fluorescence spectroscopy[88] and CLSM [81,83,89]. Fluorophores, such as fluoresceinand rhodamin 6G, are weak electrolytes that could be used asprobes to bind to a component in the capsule. In fluorescencespectroscopy the fluorescence intensity is recorded as a functionof time as the dye is dissolved and released into the bulk solutionwhich gives information on the amount of the fluorescent dyereleased, thereby the permeability of the capsules under certainconditions [88]. CLSM provides information on the position ofthe probe under fluorescence, which can be used to obtaininformation on the permeability of the capsules at variousconditions e.g., pH and ionic strength [81].

Several techniques can be used to determine the extent offlocculation that occurs during and after multilayer formation.The droplet size distribution can be measured using lightscattering, electrical pulse counting, and ultrasound. Creamingstability can be measured using techniques by placing thesample in a transparent test tube and observing the creamseparation over the period of storage, either visually orinstrumentally. Rheology measurements on emulsions can beused to provide information about the tendency for flocculationto occur— the higher the apparent viscosity and degree of shearthinning the more extensive and stronger the flocs formed in thesystem [2].

Fig. 10. (A) Dependence of particle electrical charge (ζ-potential) on pH forprimary, secondary and tertiary emulsions at pH 3. (B) Dependence of meanparticle diameter (d32) on pH for primary, secondary and tertiary emulsions atpH 3 (primary emulsion, 1°: 5 wt.% corn oil, 5 mM SDS, secondary emulsion,2°: 1 wt.% corn oil, 1 mM SDS, 0.006 wt.% chitosan, and tertiary emulsion, 3°:0.2 wt.% corn oil, 0.2 mM SDS, 0.0012 wt.% chitosan, 0.04 wt.% pectin,100 mM acetate buffer, pH 3) [19].

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7. Applications

Multilayer emulsions may have a number of potentialapplications in the food industry. For example, thick highlycharged multilayer interfaces may be useful in protectingdroplets against aggregation or in preventing lipid oxidation. Onthe other hand, multilayer interfaces that change their propertiesin a controlled fashion in response to some environmentalcondition could be used for controlled or triggered release ofactive ingredients. In this section, we provide a brief overviewof some of the potential applications of multilayer technology inthe food industry.

7.1. Improved stability against environmental stresses

Food emulsions experience a variety of different environ-mental stresses during their manufacture, storage, transport andutilization, including pH extremes, high ionic strengths, thermalprocessing, freeze–thaw cycling, drying and mechanicalagitation. Many of the emulsifiers currently available forutilization within the food industry can only provide limitedprotection against these environmental stresses.

7.1.1. pHThe aqueous solution surrounding the oil droplets in food O/

Wemulsions may vary from acidic (e.g., pH 2.5–4 in some softdrinks) to slightly alkaline (e.g., pH 7–7.4 in some dairyproducts) depending on the nature of the product. In addition,the aqueous phase pH may vary during the production, storageor utilization of the product. For example, in coffee creamers thepH of the aqueous solution surrounding the protein-coated oildroplets changes from around pH 7 in the original product toaround pH 5 when the creamer is poured into hot coffee [90]. Itis often important to ensure that that the oil droplets do notaggregate when they are exposed to variations in pH. Theinfluence of pH on the tendency of oil droplets to aggregate isnormally determined by how the various repulsive forcesgenerated by the interfacial layer vary with pH, e.g.,electrostatic and steric repulsion. Multilayer interfaces arenormally produced using weak polyelectrolytes, and so theirthickness, structure and electrical characteristics are stronglydependent on solution pH. By manipulating the type ofpolyelectrolytes used to prepare multilayer emulsions it istherefore possible to control the influence of pH on dropletaggregation. In this section, we highlight some of the work thathas been carried out in our laboratory on the influence ofsolution pH on the adsorption of polyelectrolytes to charged oildroplets, and on its impact on the stability of the multilayeremulsions formed to droplet aggregation.

The influence of pH on the ζ potential of primary (SDS),secondary (SDS–chitosan) and tertiary (SDS–chitosan–pectin)emulsions containing 0.2 wt.% corn oil is shown in Fig. 10A.The ζ potential of the SDS-coated droplets in the primaryemulsions was negative at all pH values due to the presence ofthe adsorbed anionic surfactant. The ζ potential of the SDS–chitosan-coated droplets in the secondary emulsions waspositive at relatively low pH values (≤pH 6), but became

negative at higher pH. Charge reversal probably occurredbecause chitosan lost some of its positive charge at high pHvalues (pKa≈6.5), which may also have caused it to desorbfrom the droplet surfaces. The ζ potential of the SDS–chitosan–pectin-coated droplets in the tertiary emulsion was negative atall pHs, which suggested that the chitosan layer did not desorbfrom the surface of the emulsion droplets at higher pH values asit did in the secondary emulsions. This may have been becausethe pKa value of the positively charged groups on the chitosanmolecules was increased appreciably when the chitosan wassandwiched between two negatively charged polyelectrolytelayers, as has been reported for other polyelectrolytes [91,92].There was a substantial increase in the magnitude of thenegative charge on the droplets in the tertiary emulsion withincreasing pH, which probably occurred because of the increasein negative charge on the pectin molecules (pKa∼4.5) anddecrease in positive charge on the chitosan molecules

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(pKa∼6.5). Multilayer emulsions initially stabilized by lecithin,rather than SDS, exhibit similar characteristics [14,15,20].

The change in droplet charge with pH had a large impact onthe stability of the emulsions to droplet aggregation (Fig. 10B).The droplets in the primary and tertiary emulsions were stableacross the whole pH range studied, which can be attributed tothe strong electrostatic repulsion between them. On the otherhand, the secondary emulsions were stable to droplet aggrega-tion at pHs 3 to 6, but exhibited extensive droplet aggregationand rapid creaming at pH ≥7. This was probably because themagnitude of the droplet ζ potential in the secondary emulsionsbecame relatively low at the higher pH values due to loss ofpositive charge on the chitosan molecules (pKa∼6.5) (Fig. 10A).Consequently, there was a reduction in the strength of theelectrostatic repulsion between the droplets, which would havepromoted droplet flocculation. In addition, there may have beensome bridging flocculation if the chitosan molecules onlyadsorbed weakly to the droplet surfaces.

The pH dependence of polyelectrolyte adsorption to chargeddroplets and on the subsequent stability of the multilayeremulsions formed to droplet aggregation depends on theelectrical characteristics of the polyelectrolytes and dropletsused. Thus, the range of pH where stable emulsions can beformed is highly system dependent. In the remainder of thissection we provide a brief overview of other studies on theinfluence of pH on multilayer stability.

Fish gelatin has been shown to adsorb to the surfaces ofSDS-coated droplets from pH 3 to 7 [17]. Under theseconditions the SDS-coated droplets are negatively chargedand the gelatin is either positively charged or slightly negativelycharged (pI around 6). It was postulated that adsorptionoccurred due to electrostatic attraction between positive regionson the gelatin and the negatively charged headgroups of theadsorbed surfactants. The adsorption of gelatin greatlyimproved the stability of the emulsions to droplet aggregationand oiling off at all pH values.

A series of experiments have examined the influence ofsolution pH on the adsorption of carrageenan to the surfaces ofβ-lactoglobulin-coated droplets [44–46]. ι-Carrageenan has beenshown to adsorb to the surface of protein-coated droplets from pH3 to 6, but not at pH 7. β-Lactoglobulin-coated droplets arepositively charged below the pI of the adsorbed proteins (∼pH 5),but negatively charged above, whereas the ι-carrageenan isstrongly negatively charged across the whole pH range. Theadsorption of carrageenan at pHs 3 and 4 can therefore beattributed to a strong electrostatic attraction between the anioniccarrageenan and the cationic protein-coated droplets. On the otherhand, the adsorption of carrageenan at pH 5 and 6 can beattributed to binding of segments of carrageenan molecules topositive patches on the surfaces of the β-lactoglobulin molecules.The adsorption of carrageenan to the droplet surfaces improvedthe stability of the emulsions to droplet aggregation at pHs 5 and6, but promoted extensive droplet flocculation at pHs 3 and 4.This latter effect was attributed to charge neutralization andpolymer bridging effects [17,46]. The influence of carrageenan onthe pH stability of protein-coated droplets also depended on theconformation and electrical characteristics of the carrageenan

molecules used. Studies have shown that ι-carrageenan adsorbsmore strongly and provides better stability than κ- or λ-carrageenan, which was attributed to its high charge density andhelical structure [46].

Pectin has also been shown to adsorb strongly to β-lactoglobulin-coated droplets at pH values from 3 to 5[11,13], which was attributed to the electrostatic attractionbetween the anionic polysaccharides and the cationic droplets.The presence of the pectin improved the stability of theemulsions to droplet aggregation at pH 4 and 5, but not at pH 3.This was attributed to the fact that pectin starts to lose itsnegative charge around this pH (pKa∼3.5), so the magnitude ofthe charge on the emulsion droplets decreases. It is interesting tonote, and of considerable practical importance, that the stabilityof multilayer emulsions is strongly dependent on the pH atwhich the polysaccharide and protein-coated droplets are mixed[11]. If a multilayer emulsion is formed by mixing pectin and β-lactoglobulin-coated droplets together at pH 7 (where they areboth anionic) and then reducing the pH to 4 (where the protein iscationic and the polysaccharide is anionic), the overall stabilityto droplet aggregation is better than directly mixing themtogether at pH 4. Presumably, this is because the polysaccharideis evenly distributed around the droplets when the latter gaintheir positive charge, which facilitates adsorption.

In summary, to form stable multilayer emulsions at aparticular pH, it is necessary to use preparation conditions thatdo not promote droplet flocculation, and to select a polysac-charide/emulsifier combination that provides a strong enoughelectrostatic and/or steric repulsion between the droplets.

7.1.2. SaltThe type and concentration of ions in a food emulsion may

vary considerably depending on the nature of the food, thepurity of the functional ingredients, and the hardness of thewater used to prepare the emulsion. In many situations it isimportant to ensure that the presence of ions does not promoteemulsion instability, e.g., by screening electrostatic interactionsor by binding to oppositely charged groups. The presence ofsalts can alter interfacial and emulsion properties through avariety of physicochemical mechanisms, including changingthe amount of polyelectrolyte adsorbed, altering the structure ofthe interfacial layer, or modulating the strength and range of thevarious colloidal interactions between the droplets.

The influence of salts on the properties of multilayeremulsions depends on the electrical characteristics of theemulsifier-coated droplets and polyelectrolytes involved. Anumber of studies have shown that emulsions containingmultilayer-coated droplets are more stable to high saltconcentrations than those containing single-layer-coated dro-plets [11,13,15,19–21]. An example of the effectiveness ofmultilayer interfaces at increasing the stability of emulsions tohigh salt concentrations is shown in Fig. 11. Primary (β-lactoglobulin-coated) and secondary (β-lactoglobulin–carra-geenan-coated) emulsions were prepared at pH 6, and thendifferent amounts of NaCl were added to the aqueous phase[21]. The primary emulsions showed evidence of dropletaggregation and creaming at ≥100 mM NaCl, whereas the

Fig. 12. (A) Dependence of the particle electrical charge (ζ-potential) onisothermal heat treatment (30–90 °C, 20 min), salt concentration (0 or 150 mMNaCl) for primary and secondary emulsions at pH 6. (B) Dependence of themean particle diameter (d32) on isothermal heat treatment (30–90 °C, 20 min),salt concentration (0 or 150 mM NaCl) for primary and secondary emulsions atpH 6 (primary emulsions, 1°: 5 wt.% corn oil, 0.5 wt.% Blg and secondaryemulsion, 2°: 5 wt.% corn oil, 0.5 wt.% Blg+0.1 wt.% ι-carrageenan, 5 mMphosphate buffer, pH 6). Data of primary (○) and secondary (□) emulsions inthe absence of NaCl fall close to the x-axis [21].

Fig. 11. (A) Dependence of the mean particle diameter (d32) on NaClconcentration for primary and secondary emulsions at pH 6. (B) Dependence ofthe emulsion creaming stability on NaCl concentration for primary andsecondary emulsions at pH 6 (primary emulsion, 1°: 5 wt.% corn oil, 0.5 wt.%Blg and secondary emulsion, 2°: 5 wt.% corn oil, 0.5 wt.% Blg+0.1 wt.%ι-carrageenan). Creaming index=100×(height of serum/height of emulsion)in a test tube [21].

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secondary emulsions were stable at all salt levels studied(≤500 mM NaCl). The origin of this effect was attributed to thefact that the steric repulsion is a longer range due to the thickerinterfacial membranes in the secondary emulsions. Similarresults were also found with β-lactoglobulin–pectin-coateddroplets at pH 3 [13].

Other studies have shown that emulsions containing dropletscoated by interfacial layers formed from an anionic surfactantand cationic chitosan have better stability to high saltconcentrations than those coated by anionic surfactants alone[15,19]. For example, lecithin–chitosan-coated droplets (pH 3)were stable to droplet aggregation at ≤500 mM CaCl2, whereaslecithin-coated droplets were unstable at≥3 mM [19]. This waspartly because the lecithin–chitosan-coated droplets werecationic and so the counter-ions were monovalent (Cl−),whereas the lecithin-coated droplets were anionic and so thecounter-ions were multivalent (Ca2+). Multivalent ions areknown to be much more effective at binding and screening than

monovalent ions [2,48]. Nevertheless, studies with purelymonovalent salts (NaCl) have also shown that emulsion dropletscoated by interfacial layers formed from an anionic surfactantand cationic chitosan have better stability than those coated byanionic surfactants alone, which has been attributed to anincreased steric repulsion between the droplets [14].

There is evidence that high levels of salt can causedesorption of polyelectrolytes from droplet surfaces, whichhas been attributed to a weakening of the electrostatic attractionbetween the adsorbed polyelectrolyte and the droplet surface.For example, the desorption of cationic chitosan from thesurfaces of anionic SDS droplets has been reported at 1 M NaCland pH 3 [14]. Partial or complete desorption of polyelectrolytemolecules often promotes droplet flocculation due to charge

Fig. 13. (A) Dependence of mean particle diameter (d32) of primary, secondary and tertiary emulsions on number of freeze–thaw cycles. (B) Dependence of emulsioncreaming stability of primary, secondary and tertiary emulsions on number of freeze–thaw cycles (primary emulsion, 1°: 5 wt.% corn oil, 5 mM SDS, secondaryemulsion, 2°: 1 wt.% corn oil, 1 mM SDS, 0.006 wt.% chitosan, and tertiary emulsion, 3°: 0.2 wt.% corn oil, 0.2 mM SDS, 0.0012 wt.% chitosan, 0.04 wt.% pectin,100 mM acetate buffer, pH 3). Creaming instability is indicated by a low turbidity at 600 nm as measured at 30% of emulsion height after 24 h storage. A photograph ofthe primary, secondary and tertiary emulsion after 1 freeze–thaw cycle is shown [19].

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neutralization and bridging effects. On the other hand, it may beused as a triggered release mechanism, where one or morelayers are induced to desorb from the droplet surfaces when aparticular ionic environment is encountered.

7.1.3. Thermal processingMany food emulsions undergo some form of thermal

processing during their production, storage or utilization, e.g.,

pasteurization, sterilization or cooking [2]. It is usuallyimportant that an emulsion is capable of withstanding thesethermal treatments without breaking down due to dropletflocculation or coalescence. Many emulsifiers are unsuitable forcreating droplets that are resistant to thermal processing becausethey undergo changes in their ability to prevent dropletaggregation with temperature. For example, many smallmolecule surfactants have a phase inversion temperature

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(PIT) around which extensive droplet coalescence occurs,whereas many globular proteins have a thermal denaturationtemperature (Tm) around which extensive droplet flocculationoccurs [2]. Studies in our laboratory have shown that wrappinga natural polyelectrolyte around an emulsion droplet may beable to improve its stability to thermal processing.

Multilayer emulsions containing droplets coated by ananionic surfactant and a cationic polyelectrolyte have beenshown to be stable to thermal processing from 30 to 90 °C, e.g.,SDS–chitosan [19], lecithin–chitosan [15] and SDS–gelatin[17]. For a number of these systems, the stability of thesecondary emulsions to heating was better than that of theprimary emulsion, which was partly attributed to the increasedsteric repulsion between the droplets. Multilayer emulsionscontaining droplets coated by protein–polysaccharide com-plexes have also been shown to have better stability to thermalprocessing than those stabilized by proteins alone. For example,it has recently been shown that an adsorbed pectin layer canimprove the thermal stability of β-lactoglobulin-coated dropletsat pH 4 [22]. It should be noted that the tendency forpolyelectrolytes to adsorb to the surfaces of emulsifier-coateddroplets may be altered by changes in temperature. This isillustrated in recent studies that have shown the adsorption ofcarrageenan to the surfaces of β-lactoglobulin-coated dropletswas strongly influenced by thermal processing and saltconcentration [21]. Secondary emulsions containing β-lacto-globulin–carrageenan-coated droplets (pH 6) were prepared atroom temperature and then subjected to an isothermal heattreatment (30 to 90 °C for 20 min), before being cooled to roomtemperature and analyzed (Fig. 12A and B). In the absence ofsalt, carrageenan remained adsorbed to the β-lactoglobulin-coated droplets after all thermal treatments, which wasattributed to the strong electrostatic attraction between the twokinds of molecules at low ionic strengths. On the other hand, inthe presence of 150 mM NaCl, the carrageenan moleculesdesorbed from the β-lactoglobulin-coated droplet surfaces attemperatures of 60 °C and higher. This was attributed to theweakening of the electrostatic attraction by the presence of thesalt, in combination with an alteration in the conformation of theadsorbed β-lactoglobulin molecules upon heating (e.g., due to achange in the charge density of the positive patches on theproteins surface). Consequently, the carrageenan layer was ableto prevent droplet flocculation at relatively low temperatures(≤50 °C) where it remained adsorbed to the protein-coateddroplet surfaces, but not at higher temperatures because itbecame desorbed [21].

It is clear that different emulsifier–polyelectrolyte combina-tions have different thermal stabilities and that furthersystematic research is needed to identify the molecular andphysicochemical basis of these effects.

7.1.4. Chilling and freezingThere are many potential applications for oil-in-water

emulsions that can be chilled or frozen during storage andthen warmed prior to use in the food industry, e.g., dairyproducts, desserts, sauces, and ice cream [2]. Cold storage isoften used to protect product quality by retarding microbial

growth and undesirable chemical reactions, such as lipidoxidation. Nevertheless, many oil-in-water emulsions becomephysically unstable when they are chilled and/or frozen, andrapidly break down after reheating. It is therefore important tohave technologies improve the stability of food emulsions tochilling, freezing and thawing.

A variety of different physicochemical processes may occurwhen a food emulsion is cooled, including fat crystallization,ice formation, freeze-concentration, interfacial phase transi-tions, and biopolymer conformational changes. When oil-in-water emulsions are cooled to temperatures where the fat phasebecomes partially crystallized (but the aqueous phase remainsliquid) they become susceptible to a phenomenon known aspartial coalescence [2]. Partial coalescence is the processwhereby a fat crystal from one partially crystalline dropletpenetrates into a liquid region of another partially crystallinedroplet [93]. This process results in the formation of irregularlyshaped aggregates that usually decrease the creaming stabilityand increase the shear viscosity (i.e., “thickening”) of theemulsion. Partial coalescence is a critical step in the productionof some types of food emulsions, including ice cream, whippedcream, margarine and butter but is undesirable in other foodemulsions because it leads to deterioration of product quality,e.g., thickening of creams.

When oil-in-water emulsions are cooled to a temperaturewhere the water crystallizes, there are a number of additionalphysicochemical processes that occur that can also promoteemulsion instability. First, when ice crystals form in the aqueousphase the oil droplets are forced closer together. Second, theremay be insufficient free water present to fully hydrate theemulsifier molecules adsorbed to the droplet surfaces, whichcan promote droplet–droplet interactions. Third, there is anincrease in the ionic strength in the unfrozen aqueous phasesurrounding the emulsion droplets due to ice crystal formation,which screens any electrostatic repulsion between the droplets.Fourth, ice crystals may physically penetrate into oil dropletsand disrupt their interfacial membranes, thereby making themmore prone to coalescence once they are thawed. Fifth,emulsifiers may adsorb to ice crystal surfaces, and hencethere may be competition between emulsifier adsorption to oildroplet and ice crystal surfaces. Finally, emulsifiers may losetheir functionality when the temperature is decreased below acertain level, e.g., globular proteins may be denatured or theremay be a change in the optimal curvature of small moleculesurfactants. At present there is still a relatively poor under-standing of the relative importance of these various mechanismsof emulsion instability for particular systems. Nevertheless,preliminary studies in our laboratory have indicated thatmultilayer technology can improve the stability of oil-in-wateremulsions to chilling and freezing processes, which suggests itis able to retard or prevent some of these instability mechanisms[15,19].

A clear example of the ability of multilayer technology toimprove the stability of emulsions to cold storage is provided bythe SDS–chitosan–pectin system [19]. Primary (SDS-coated),secondary (SDS–chitosan-coated) and tertiary (SDS–chitosan–pectin-coated) emulsions were prepared at pH 3. The emulsion

Fig. 14. Formation of TBARS in primary (1°: 5 wt.% corn oil, 5 mM SDS),secondary (2°: 1 wt.% corn oil, 1 mM SDS, 0.006 wt.% chitosan), and tertiary(3°: 0.2 wt.% corn oil, 0.2 mM SDS, 0.0012 wt.% chitosan, 0.04 wt.% pectin)emulsions (100 mM acetic acid buffer, pH 3) during storage at 37 °C for 8 days(unpublished data).

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samples were then subjected to a freeze–thaw cycle thatinvolved incubation at −20 °C for 22 h, followed by thawing at30 °C for 2 h. This freeze–thaw cycle was repeated six timesand its influence on emulsion properties was measured aftereach cycle (Fig. 13A and B). The primary emulsion exhibitedextensive oiling-off after only one freeze–thaw cycle, suggest-ing that extensive droplet coalescence had occurred. Thesecondary emulsion exhibited extensive droplet flocculation(but no coalescence or oiling-off) after only one freeze–thawcycle, which led to rapid droplet creaming. On the other hand,the mean diameter of the particles in the tertiary emulsionsremained stable after 6 freeze–thaw cycles (∼0.3±0.05 μm),and there was no evidence of creaming. These results suggestthat it is possible to create emulsions that are highly resistant todroplet aggregation during freeze–thaw cycling using the LBLelectrostatic deposition technique. The high stability of thetertiary emulsions to freeze–thaw cycling may be because thethick interfacial membrane is resistant to rupture by oil or fatcrystals, or because the repulsive colloidal interactionsgenerated by the thick electrically charged interfacial mem-branes are sufficiently large to overcome any attractive colloidinteractions or mechanical forces that tend to push the dropletstogether during the freezing process.

7.1.5. Lipid oxidationThere is considerable interest in the incorporation of

polyunsaturated fats (such as ω-3 fatty acids) into food productsbecause of their potential health benefits, e.g., there is evidencethat they decrease the risk of coronary heart disease, immuneresponse disorders, and mental illnesses [94]. Nevertheless,incorporation of these oils into food products is problematicbecause they are highly susceptible to oxidative degradationresulting in rancid off-flavors, which has greatly limited theirmore widespread usage. The multilayer technology described inthis review provides the food industry with a powerful tool foraltering the electrical charge and thickness of the interfaciallayer surrounding the lipids, and thereby improving the stabilityof the encapsulated lipids to oxidation.

Experiments in our laboratory have shown that multilayeremulsions have better stability to lipid oxidation than single-layer emulsions [15,18]. The lipid oxidation rates in primary(SDS), secondary (SDS–chitosan) and tertiary (SDS–chit-osan–pectin) emulsions containing fish oil are compared inFig. 14 (unpublished data). The primary emulsions wereconsiderably more unstable to lipid oxidation than thesecondary or tertiary emulsions. The instability of the primaryemulsions to lipid oxidation has been attributed to the abilityof positively charged Fe2+ ions to adsorb to the surface of thenegatively charged SDS-coated droplets, where they comeinto close proximity to the unsaturated lipids within the oildroplets. The greater stability of the secondary emulsions canbe attributed to the fact that the SDS–chitosan-coated dropletsare positively charged, and therefore electrostatically repel theFe2+. In addition, the greater thickness of the interfacialmembrane may help to physically prevent the iron catalystcoming into close contact with the lipid substrate. It wasinteresting to note that the tertiary emulsions were still stable

to lipid oxidation even though they contained negativelycharged droplets, which again suggested that the thickness ofthe interfacial membrane plays an important role in retardingiron-catalyzed lipid oxidation in O/W emulsions.

7.1.6. DehydrationOil-in-water emulsions are often converted into a powdered

form in the food industry to increase their shelf life, reducetransport costs and/or facilitate their utilization. This micro-encapsulation process is normally carried out by evaporating themajority of water from the emulsion using a suitabledehydration method, such as spray drying or freeze drying[95]. After dehydration it is important that the resulting powderhas good functional characteristics, e.g., density, flow, handling,strength, stability, and re-dispersion. Dehydration often pro-motes emulsion instability by adversely affecting the propertiesof the interfacial layers surrounding the oil droplets, e.g., byphysically disrupting them or by promoting cross-linking ofemulsifiers adsorbed onto different droplets. It is important thatthe powder remains physically and chemically stable duringstorage, and that the oil droplets can be rapidly and completelydispersed into an aqueous solution at the time of utilization.Normally, a relatively high concentration of sugars, polyols,polysaccharides or proteins has to be added to an oil-in-wateremulsion prior to dehydration to improve the formation andstability of the powder. After dehydration these substances formpart of the “wall material” that surrounds the fat droplets in thepowder.

Experiments have been carried out in our laboratorycomparing the stability of oil-in-water emulsions containingsingle-layer and multiple-layer droplets to freeze drying andspray drying [18,96]. Secondary emulsions (lecithin–chitosan)had a much better stability to droplet aggregation than primaryemulsions (lecithin), especially when maltodextrin was incor-porated into the emulsions. For example, in the presence ofmaltodextrin there was no change in the mean particle diameter

Fig. 15. Production of hollow (nano)capsules [99].

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or microstructure of the emulsions after freeze drying, whereasextensive droplet aggregation was observed in its absence [18].We believe that it may be possible to create high qualitypowdered emulsions with good functional properties using themultilayer technique. In particular, we believe that it may bepossible to reduce the total amount of wall material required tocreate the powder, and thereby increase the load of the lipidphase, which may reduce production and transport costs.Nevertheless, further studies are required using differentcombinations of emulsifiers and polyelectrolytes to establishthe optimum formulation of these products.

7.2. Controlled release/triggered release

Delivery systems can be used to encapsulate, stabilize anddeliver a variety of functional food components e.g., flavors,bioactive lipids, enzymes, peptides, antimicrobials and anti-oxidants [97]. Oil-in-water emulsions, used in either their wet ordehydrated states, can be used as delivery systems, because theyare capable of containing oil-soluble, water-soluble andamphiphilic functional agents in a single system. Nevertheless,there are a number of limitations associated with the utilizationof conventional single-layer emulsions for delivery systems,such as the lack of control over the charge, thickness andenvironmental responsiveness of the interfaces surrounding theoil droplets [33]. Research on non-food systems has demon-strated the potential of the multilayer technology for thedevelopment of delivery systems with improved functionalcharacteristics.

The micro-encapsulation of various materials using poly-electrolyte multilayer self-assembly has been reported[32,39,65,73,98–108]. The multilayer technique has the majoradvantage that wall characteristics, such as thickness, chargeand permeability, can be finely tuned by careful selection ofpolyelectrolytes and preparation conditions. Caruso et al.demonstrated the use of the LBL approach for the encapsulationof hydrophobic, low molecular weight compounds, as well asproviding a novel pathway to the fabrication of polymermultilayered microcapsules with controlled release propertiesfor their delivery. The release time depends on the nature of thefirst adsorbed layer and the number of polyelectrolyte layerscomposing the shell wall [85].

In the food industry, the use of the LBL technique forencapsulation of sensitive food components, such as enzymesand bioactive compounds, holds great promise. Research in ourlaboratory has shown that the outer polyelectrolyte layer inmultilayer emulsions can be made to detach from the droplet

surfaces in response to alterations in pH, ionic strength ortemperature [15,21,22]. It may be possible to use this approachto encapsulate one or more charged functional componentsbetween the interfacial layers, so that they can be released inresponse to specific environmental triggers.

7.3. Hollow (nano)capsules

Hollow capsules can be produced by adsorbing layers ofpolymers onto a colloidal template, which can then be removedby chemical or physical methods [108] (Fig. 15). Caruso et al.[99] examined the release behavior of solubilized pyrene andfluorescein diacetate from the crystalline core by monitoringtheir fluorescence after dissolution with ethanol. Completeremoval of the core yielded hollow polymer capsules ofmicrometer dimensions. The capsule porosity was found to beinfluenced by the selection of polyelectrolytes used, the wallthickness and the ambient conditions. The pores of the shell wallcan be selectively opened and closed by chemically modifyingor adjusting the ambient conditions. A high salt concentration ofthe medium used for the deposition of the polyelectrolyte resultsin a low packing density and a high permeability of the shellwall. On the other hand, a low salt concentration of the mediumused for the deposition results in a high packing density and thuslow permeability of the particles. Therefore, by adjusting the saltconcentrations in the deposition medium the permeability of theshell wall can be controlled [109].

The hollow capsules prepared are attractive for theencapsulation and release of various substances; for example,the release of active compounds can occur when loadedcapsules are exposed to environmental conditions that decom-pose them, e.g., in the bloodstream [38].

8. Conclusions

Work so far has shown that stable emulsions containingmultilayered lipid droplets can be prepared using a simple cost-effective method and food-grade ingredients. These multilay-ered emulsions have better stability to environmental stressesthan conventional emulsions under certain conditions. Moreresearch is still needed to establish, at a fundamental level, thefactors that influence the preparation of stable multilayeredemulsions with specific functional attributes, including emul-sifier characteristics (e.g., sign and magnitude of dropletcharge), polyelectrolyte characteristics (e.g., molecular weight,charge density and flexibility), and mixing conditions (e.g.,ionic strength, pH, temperature, stirring, order of addition), and

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washing conditions (e.g., ionic strength, pH, temperature,stirring, filtration and centrifugation). In addition, researchneeds to be carried out to establish where these multilayeredemulsions can be practically used within the food industry.

One of the most important factors that will need to beaddressed before this technology can be successfully utilized bythe food industry is the cost–benefit analysis. The implemen-tation of the LBL method involves using one or more additionalingredients (e.g., proteins or polysaccharides) and one or moreadditional unit operations (e.g., mixing, homogenization,centrifugation and/or filtration). Consequently, it will benecessary to show that the financial benefits accruing fromutilizing this technology (e.g., bigger market share or higherproduct pricing) are more than the additional costs. We believethat this technology will be suitable for many food productsbecause the additional ingredients are only needed at relativelylow levels (b1 g per 10 g of oil) and the additional processingoperations are simple and widely used.

Acknowledgements

This material is based upon work supported by theCooperative State Research, Extension, Education Service,United State Department of Agriculture (USDA), Massachu-setts Agricultural Experiment Station (project no. 831), by anUSDA, CREES, NRI Grant (Award Number 2005-35503-16164) and an USDA Seafood Safety Grant.

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