Version of Record: https://www.sciencedirect.

com/science/article/pii/S0001868617303196
Manuscript_ef1992586bdf478b8366017ed2603367

Improving Emulsion Formation, Stability and Performance using Mixed

Emulsifiers: A Review

David Julian McClements1* and Seid Mahdi Jafari2

1
Department of Food Science, University of Massachusetts, Chenoweth Laboratory, Amherst, MA, USA
2
Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural

Sciences and Natural Resources, Gorgan, Iran

*Corresponding Author

Address: Department of Food Science, University of Massachusetts, Chenoweth Laboratory, Amherst,

MA, 01003 USA

Abstract

The formation, stability, and performance of oil-in-water emulsions may be improved by using

combinations of two or more different emulsifiers, rather than an individual type. This article provides

a review of the physicochemical basis for the ability of mixed emulsifiers to enhance emulsion

properties. Initially, an overview of the most important physicochemical properties of emulsifiers is

given, and then the nature of emulsifier interactions in solution and at interfaces is discussed. The

impact of using mixed emulsifiers on the formation and stability of emulsions is then reviewed.

Finally, the impact of using mixed emulsifiers on the functional performance of emulsifiers is given,

including gastrointestinal fate, oxidative stability, antimicrobial activity, and release characteristics.

This information should facilitate the selection of combinations of emulsifiers that will have improved

performance in emulsion-based products.

Keywords: emulsifiers; mixed; emulsions; nanoemulsions; competitive adsorption; performance.

1
© 2017 published by Elsevier. This manuscript is made available under the Elsevier user license
https://www.elsevier.com/open-access/userlicense/1.0/
1. Introduction

Oil-in-water emulsions are important constituents of many commercial products, including foods,

supplements, pharmaceuticals, cosmetics, personal care products, and agrochemicals [1-3]. This type

of colloidal dispersion consists of small oil droplets dispersed within an aqueous continuous phase [4].

Emulsions are thermodynamically unstable, and therefore tend to breakdown over time due to various

physicochemical mechanisms, including gravitational separation, flocculation, coalescence, particle

coalescence, Ostwald ripening, and phase separation [1, 5, 6]. For this reason, stabilizers are included

in emulsion formulations to improve their long-term stability, such as emulsifiers, texture modifiers,

ripening inhibitors, and weighting agents [4]. Emulsifiers are one of the most important stabilizers

used in any emulsion formulation. In addition to their ability to stabilize emulsions, the nature of the

emulsifier used also determines the ease of emulsion formation and the functional attributes of the final

product. Consequently, selection of an appropriate emulsifier is one of the most important decisions

when formulating emulsion-based products [7]. Emulsifiers are typically amphiphilic molecules that

have both hydrophilic and hydrophobic groups on the same molecule, such as small molecule

surfactants, phospholipids, proteins, polysaccharides, and other surface-active polymers [8-10].

In some applications, an emulsion can be formulated using a single type of emulsifier. However,

in many applications, the formation, stability, and functional attributes of emulsions can be improved

by using combinations of emulsifiers, rather than individual ones [11-13]. Nevertheless, there is

currently a relatively poor understanding of the impact of mixed emulsifier systems on emulsion

formation and properties. This knowledge is holding back the rational design of emulsion-based

products with improved physicochemical and functional properties. The aim of this article is therefore

to review the current knowledge of the impact of mixed emulsifier systems on emulsion formation and

stability. Initially, an overview of emulsifier properties is given, and then the impact of mixed

emulsifiers on emulsion formation and stability is discussed. This article focuses on the utilization of

2
molecular-based emulsifiers, because these are currently the most commonly used in industry.

However, it should be noted that there has been considerable interest in the development and utilization

of particle-based emulsifiers recently, which are capable of stabilizing emulsions through a Pickering

stabilization mechanism [14-17]. These particles may also be used in mixtures with molecular

emulsifiers or with other particle-based emulsifiers to improve emulsion stability or create improved

functional attributes, however, this area subject was beyond the scope of the current article.

2. Emulsifier Properties

Emulsifiers vary considerably in their molecular and physicochemical properties depending on

their origin [8, 10, 18, 19]. An understanding of how combinations of emulsifiers behave depends on

understanding how individual emulsifiers behave, and so we begin by giving a brief overview of the

most important properties of individual emulsfiers. Nevertheless, a discussion of the impact of using

combined emulsifiers on these properties is also given where appropriate. More detailed information

about the properties of specific emulsifiers can be found elsewhere [7, 8, 19, 20].

2.1. Emulsifier Types

The main focus of this article is on emulsifiers that are suitable for utilization in food products [10,

21], such as small molecule surfactants, phospholipids, proteins, and polysaccharides (Figure 1).

Consequently, a short overview of the key characteristics of this type of emulsifier is given in this

section.

Fig. 1

Small molecule surfactants: Most small molecule surfactants used in the food industry are

synthetic molecules (such as Tweens, Spans, CITREM, and DATEM) [7], but recently some natural

versions have been identified and approved for use in commercial products (such as quillaja saponins)

[10, 22, 23]. Small molecule surfactants typically have a non-polar tail group and a polar head group,

although this is not always the case (Figure 1). For example, some natural surfactants have ellipsoid

3
structures with a hydrophobic side and a hydrophilic side (such as bile salts and saponins) [24-27]. For

conventional surfactants, the non-polar tail may vary in the number of chains, the chain length, and the

degree of unsaturation, whereas the polar head group may vary in its charge charactersitics (positive,

negative, zwitterionic, or neutral) and dimensions [28, 29].

Phospholipids: Phospholipids are a special kind of natural small molecule surfactants that are

usually derived from the cell membranes of plant, animal, or microbial tissues [30-32]. Naturally,

phospholipids are comprised of a glycerol moiety with a phosphate group usually located at the sn-3

position, and two fatty acid chains located at the sn-1 and sn-2 positions [33]. The fatty acid chains may

vary in their relative positions, chain lengths and degree of unsaturation depending on the biological

origin of the phospholipids. The nature of functional groups attached to the phosphate group may also

vary considerably depending on the origin of the phospholipids. Some of the most common

phospholipids with different head groups are phosphatidylcholine (PC), phosphatidylethanolamine

(PE), phosphatidylglycerol (PG), phosphastidylinositol (PI), phosphatidylserine (PS) and phosphatidic

acid (PA). The functional performance of a phospholipid as an emulsifier depends strongly on the

nature of the head group and tail groups attached to the glycerol backbone. The phospholipids used in

food and beverage applications are usually referred to as lecithins, which consist of a complex mixture

of acetone-insoluble phospholipid and non-phospholipid components [33-37]. Natural lecithin

ingredients usually have two fatty acids attached to the glycerol backbone, but one of the chains can be

chemically or enzymatically removed to form ingredients (lyso-lecithins) with different functional

attributes [38]. For example, lyso-lecithins tend to be more hydrophilic than conventional lecithins,

and therefore are often more suitable for stabilizing oil-in-water emulsions. The electrical charge on

phospholipids may vary from negative to positive depending on the nature of the head group and

solution pH. The chemical stability of phospholipids depends on the degree of unsaturation of the fatty

acid chains, since this impacts their susceptibility to oxidation [33].

4
 Proteins: Many food-grade proteins are amphiphilic molecules that have both polar and non-polar

groups on their surfaces, such as whey proteins, caseins, egg proteins, soy proteins, gelatin, meat

proteins, and pea proteins [22, 23]. To be a good emulsifier a protein should have an appropriate

balance of polar and non-polar groups on its surface, so as to produce good water-solubility and good

surface activity. If the surface of a protein is too non-polar then it will not dissolve in water, but if it is

too polar then it will not be surface active. Proteins vary considerably in their molecular weights,

conformations, surface hydrophobicities, and electrical charge characteristics [39, 40]. Typically,

protein-based emulsifiers have either globular or flexible random coil structures, which impacts the

properties of the interfaces formed after they adsorb to droplet surfaces. The electrical properties of

proteins play a major role in determining the physical and chemical stability of emulsions, and typically

move from positive at low pH to negative at high pH, with a point of zero charge at the isoelectric point

(pI) [41]. The pI of many food proteins is around pH 5, but some proteins have considerably higher or

lower pI values depending on their amino acid compositions [39, 42].

Polysaccharides: Many polysaccharides used as food ingredients are highly hydrophilic molecules

with poor surface activity, and are therefore unsuitable as emulsifiers [10, 22, 43]. However, a number

of natural or modified polysaccharides have hydrophobic groups attached to a hydrophilic chain, which

makes them amphiphilic, e.g., gum arabic, modified starch, modified cellulose, and beet pectin [10,

22]. These hydrophobic groups may be covalently or physically attached phenolic groups,

hydrocarbons, or proteins depending on the nature of the polysaccharide. For example, beet pectin has

phenolic groups (ferulic acid) and protein moieties attached to the polysaccharide backbone [30],

whereas modified starch has octenyl succinate groups covalently attached to starch molecules [44].

Most commonly used surface-active polysaccharides have a negative charge across a wide pH range

because of the presence of anionic functional groups, such as carboxyl, phosphate, or sulfate groups.

5
2.2. Solubility and Partitioning

The solubility and partitioning of emulsifiers have a pronounced impact on their functional

performance in emulsions [7, 45]. The formation of oil-in-water emulsions usually requires that an

emulsifier has good water solubility, and preferentially partitions into the water phase (rather than the

oil phase). However, this is not always the case, as it is better to disperse certain types of

phospholipids (lecithins) in the oil phase prior to the formation of oil-in-water emulsions [46]. For

small molecule surfactants and phospholipids, the hydrophile-lipophile balance (HLB) number is often

used to describe their solubility and partitioning characteristics [45]. Surfactants with low HLB

numbers (2-6) partition into the oil phase and stabilize water-in-oil emulsions, whereas surfactants with

high HLB numbers (8-18) partition into the water phase and stabilize oil-in-water emulsions. For

mixed surfactants, an effective HLB can be calculated from knowledge of the concentrations and HLB

numbers of the individual emulsifiers [45]. The HLB number does not always give a good prediction of

the performance of individual or mixed surfactants in emulsions, e.g, two systems with the same HLB

number may perform quite differently because other factors are also important (such as molecular

geometry or specific interactions). For amphiphilic biopolymers, there is no equivalent to the HLB

system, but solubulity and partitioning characteristic are still important. The vast majority of surface-

active biopolymers are predominately hydrophilic, and should be dissolved in the aqueous phase prior

to forming an oil-in-water emulsion [22].

Differences in the solubility charactersitics of emulsifiers can be utilized when forming emulsions

from combinations of emulsifiers. For example, a water-soluble emulsifier can be dissolved in the

aqueous phase (such as a biopolymer) and an oil-soluble emulsifier can be dispersed in the oil phase

(such as a lecithin) prior to homogenization [47]. Alternativley, two water-soluble emulsifiers could be

dissolved in the aqueous phase prior to forming an emulsion, e.g., an ionic and a non-ionic surfactant

6
[48]. The combination of two different emulsifiers may lead to the formation of emulsions with

different droplet sizes and stabilities than either of the individual emulsifiers used separately [13, 49].

2.3. Surface Pressure, Load, and Activity

There is often a relatively high interfacial tension at an oil-water interface because of the

thermodynamically unfavorable contact between the oil and water molecules associated with the

hydrophobic effect [50]. Emulsifiers reduce the interfacial tension by adsorbing onto the interface and

screening the unfavorable interactions between the oil and water molecules [45, 51]. The ability of an

emulsifier to reduce the interfacial tension is therefore highly dependent on its molecular

characteristics, such as its molecular weight and the number and location of hydrophilic and

hydrophobic groups. One of the main effects opposing the adsorption of emulsifiers is the reduction in

their translational entropy, and therefore the tendency to adsorb to an interface is also highly dependent

on the molecular weight of an emulsifier [52]. Typically, the lower the molecular weight, the greater

the entropy of mixing effect opposing adsorption.

The interfacial charcteristics of an individual emulsifier can be conveniently characterized by

measuring the change in interfacial tension with emulsifier concentration at an oil-water interface [10,

53] (Figure 2).

Fig. 2

When the concentration of an emulsifier in the aqueous phase is increased, some of it adsorbs onto the

interface, which causes a reduction in the interfacial tension. Typically, the interfacial tension does not

change appreciably at very low emulisifier concentrations, then it decreases steeply with increasing

emulsifier concentration at adsorbed occurs, and then it reaches a relatively constant level when the

interface becomes saturated with emulsifier. At saturation, the interfacial tension depends on how

effective the emulsifier molecules are at screening the unfavorable interactions between the oil and

water phases. There are often appreciable changes in the conformation of biopolymer emulifiers at

7
interfaces after they adsorb and try to minimize their free energy in their new surroundings, e.g., by

maximizing the number of hydrophobic interactions with the oil phase [54].

To a first approximation, the tendency for an individual emulsifier to adsorb to an oil-water

interface can be described using the Gibbs adsorption isotherm [45]. This equation relates the amount

of emulsifier adsorbed at the interface () to the interfacial tension () and the amount of emulsifier in

the bulk solution (c):

1 𝑑
Γ = − 𝑝𝑅𝑇 (𝑑ln(𝑐)) (1)

Here,  is the emulsifier concentration at the interface, c is the emulsifier concentration in the

continuous phase, R is the gas constant, T is the absolute temperature, and p is a parameter that depends

on the ionic properties of the emulsifier. For neutral molecules, p = 1, but for charged molecules it may

be >1 depending on the number of counter-ions released when the emulsifier adsorbs to the interface

[55]. The Gibbs adsorption isotherm equation is useful for determining the surface excess concentration

or surface load () of an emulsifier from experimental measurements of the interfacial tension versus

emulsifier concentration, since  is related to the slope of a plot of  versus ln(c) (Figure 2). Plots of

the surface pressure, which is the difference between the interfacial tension in the presence and absence

of emulsifier ( =  – 0), versus emulsifier concentration also provide valuable information about

emulsifier properties (Figure 2). Typically, the surface pressure increases with increasing emulsifier

level until a constant value is reached. The maximum surface pressure achieved at saturation ()

provides information about how effective an emulsifier is at reducing the interfacial tension: the

smaller  the more effective is the emulsifier at screeing unfavorable interactions between oil and

water phases. A measure of the surface activity (SA) can also be obtained from a plot of surface

pressure versus emulsifier concentration. The recipricol of the emulsifier concentration where the

8
surface pressure reaches half of its saturation value (SA = 1/c½) can be taken to be the surface activity of

the emulsifier: the higher the value of SA, the stronger the tendency for emulsifier adsorption to occur.

Again, to a first approximation, the fraction of an oil-water interface that is covered by emulsifier

can be described using the Langmuir adsorption isotherm:

 c / c1 / 2
  (2)
 1  c / c1 / 2

Here,  is the fraction of adsorption sites at the interface occupied by emulsifier molecules,  is the

surface excess concentration when the interface is saturated with emulsifier, and c½ is the emulsifier

concentration in the aqueous phase where  = ½. As mentioned earlier, recipricol of c½ provides a

measure of the surface activity of the emulsifier. It should be noted that the Gibbs and Langmuir

adsorption isotherms only give first approximations of the behavior of real systems, and ignore many

important factors, such as the orientation, interactions and conformational changes of emulsifiers at an

interface. Neverthless, more sophisticated equations are available that take into account many of these

factors, which have been reviewed in detail elsewhere [52, 54, 56].

The surface activities of individual emulsifiers are particularly important when determining their

tendency to adsorb onto oil-water interfaces in mixed systems [57, 58]. At the same concentration, one

would expect an emulsifier with a higher surface activity (smaller c½) to preferentially adsorb to an oil-

water interface. Emulsifiers vary greatly in their surface activities, and therefore knowledge of these

values are important when designing mixed interfaces with specific compositions and functional

attributes. A discussion of the impact of emulsifier concentration and surface activity on the

composition of the interfaces formed by mixed emulsifiers is given in a later section (Section 3.2).

In summary, interfacial tension versus concentration measurements can therefore provide a lot of

useful information about the interfacial properties of individual emulsifiers: surface pressure at

saturation (); surface acivity (SA); and, surface load at saturation (). The utilization of mixed

9
emulsifiers to formulate an emulsion may alter the values of  and  depending on the

concentrations and surface activities of the emulsifiers used, as well as order of addition effects (see

later) [56, 59].

2.4. Self-association

Many types of emulsifier have a tendency to self-associate when they are dispersed in aqueous

solutions due to the hydrophobic effect, i.e., the tendency for the system to reduce the unfavorable

contact area between the non-polar groups on the emulsifier molecules and the surrounding water

molecules [28, 29, 45]. Small molecule surfactants have a tendency to form micelles, whereas some

biopolymers may form molecular clusters (such as caseins). At relatively low surfactant

concentrations, surfactant molecules tend to exist as monomers in solution because entropy of mixing

effects dominate [45]. However, when surfactant level exceeds a certain value, known as the critical

micelle concentration (CMC), the surfactant molecules spontaneously self-associate and form small

colloidal particles known as micelles [28, 29]. Knowledge of the CMC of surfactants, as well as the

size, shape, and charge characteristics of the micelles formed, is important for understanding the

functional performance of emulsifiers.

The utilization of combined emulsifiers may change the CMC and micelle properties depending on

the type and concentration of the emulsifiers involved [28, 29, 59]. For example, surfactants or

phospholipids may bind to biopolymers through electrostatic or hydrophobic interactions and either

increase or decrease their surface activity. Moreover, the formation of complexes in the bulk aqueous

phase may alter the kinetics of adsorption of the emulsifier molecules to oil-water interfaces, which

could impact emulsion formation.

2.5. Interfacial properties: thickness, packing and rheology

The physicochemical properties of the interfacial layer formed when emulsifier molecules adsorb

to an oil-water interface largely determine the stability and functionality of emulsions [1]. The

10
interface may vary in its thickness, density, and rheology depending on the molecular dimensions,

packing and interactions of the adsorbed emulsifier molecules [54, 56, 57, 59, 60]. These differences in

interfacial properties have a pronounced influence on the physical stability of emulsions. For instance,

the thickness of the interface has a strong impact on the strength and range of the steric interactions

between emulsion droplets, while the electrical characteristics have a strong influence on the strength

of the electrostatic interactions [1]. Emulsifiers that form thick interfaces (such as polysaccharides) are

often able to stabilize emulsions entirely through steric repulsion, whereas those that form thin

interfaces (such as globular proteins) require a combination of both electrostatic and steric repulsion to

stabilize droplets [1, 40].

The thickness, packing, and rheology of interfacial layers can often be manipulated by using

combinations of emulsifiers [54, 59]. Small emulsifiers (such as small molecule surfactants) can pack

between large emulsifiers (such as biopolymers) at oil-water interfaces, thereby leading to a higher

surface load and lower interfacial tension. Mixtures of small molecule surfactants with different head

group sizes can alter the optimum curvature of the interface, thereby influencing emulsion formation

and stability [61]. For example, Tweens (large hydrophilic heads) and Spans (small hydrophilic head

groups) can be used in combination to produce emulsions with smaller droplets than are possible by

using either of the individual emulsifiers due to their impact on interfacial curvature and tension [62,

63]. Some combinations of emulsifiers interact with each other at oil-water interfaces and change

interfacial properties (such as charge, thickness, and rheology), e.g., proteins and ionic surfactants can

interact strongly through electrostatic and hydrophobic attraction [54, 59, 64].

2.6. Interfacial charge

The interfacial layers formed by emulsifiers may have charges that range from strongly negative,

to neutral, to strongly positive depending on emulsifier type and solution conditions such as pH and

ionic composition [1]. Most food-grade surfactants tend to be either non-ionic (such as Tweens) or

11
anionic (such as DATEM), but there are a limited number of cationic ones (such as lauric arginate) [7,

40]. The charge on amphiphilic proteins usually goes from positive to negative as the pH is raised

from below to above the isoelectric point [40, 65]. Most food-grade amphiphilic polysaccharides have

a negative charge (such as gum arabic, modified starch, and beet pectin) due to the presence of anionic

groups (such as carboxyl, sulfate, or phosphate groups) [1]. The electrical properties of interfacial

layers have a pronounced impact on the physical and chemical properties of emulsions. The droplets in

many emulsions are stabilized against aggregation due to the presence of a charged interface that

generates a strong electrostatic repulsion [1]. The chemical stability of lipids inside the droplets may

also be influenced by interfacial charge, e.g., anionic interfaces can adsorb cationic transition metals

(such as iron or copper) to their surfaces, which promotes lipid oxidation [66].

The electrical characteristics of emulsion droplets can be modulated by using combinations of

emulsifiers with different charge charactersitics [67]. The emulisifers can either be mixed together

before homogenization, or one emulsifier can be added before and another after homogenization [12].

For example, oil droplets with a wide range of charges, ranging from highly negative to highly positive

(Figure 3), have been prepared by mixing emulsions stabilized by non-ionic surfactants with different

levels of anionic or cationic surfactants after homogenization [67]. The electrical charge on protein-

stabilized emulsions has also been altered by mixing them with ionic surfactants, e.g., addition of an

anionic surfactant (SDS) after homogenization increased the negative charge on droplets initially

coated by globular proteins [68, 69].

Fig. 3

2.7. Adsorption kinetics

The rate at which emulsifier molecules adsorb to oil-water interfaces is particularly important

during emulsion formation [70, 71]. As will be discussed later, the rate at which emulsifier molecules

adsorb to droplet surfaces during homogenization impacts the tendency for droplet coalescence to

12
occur [72]. If emulsifier adsorption occurs more rapidly than droplet collisions, then the droplets will

be stabilized against coalescence. Conversely, if the emulsifiers adsorb too slowly the droplets will not

be coated by emulsifier molecules when they collide, which leads to coalescence and reduces the

efficiency of homogenization. The rate at which emulsifiers adsorb to oil-water interfaces during

homogenization depends on their molecular characteristics and their tendency so self-associate in

solution. Typically, a small monomer will diffuse more quickly to an interface than a large polymer or

molecular cluster. Moreover, the collision efficiency, that is the number of emulsifier-droplet

encounters that actually leads to attachment, depends on the distribution of non-polar groups on the

surface of the emulsifier, which could also be altered by complexation of emulsifiers in the aqueous

phase. However, it should be noted that the adsorption kinetics of emulsifiers to droplet surfaces may

be very different inside the highly turbulent conditions occurring during homogenization than in the

static conditions occurring in a surface tension meter [71]. At present, there are few analytical

techniques that can be used to accurately monitor the adsorption kinetics of emulsifiers under realistic

homogenization conditions.

Some emulsifiers are highly effective at stabilizing emulsions once they adsorb to oil droplet

surfaces, but they only adsorb to the droplet surfaces relatively slowly during homogenization, e.g.,

amphilic polysaccharides such as gum Arabic [73]. As a result, it is difficult to form small droplets in

the initial emulsions produced by homogenization [73, 74]. This problem may be overcome by using

combinations of emulsifiers. A small emulsifier that rapidly adsorbs onto the droplet surfaces but does

not provide good emulsion stability could be used in combination with a large emulsifier that does not

adsorb rapidly but that does provide good stability.

2.8. Interfacial conformation

Unlike small molecule surfactants, many types of biopolymers undergo appreciably changes in

their conformation after adsorption onto an oil-water interface so that their three-dimension structure

13
and flexibility may be quite different at an interface than in the bulk solution [22, 55, 75]. These

conformational changes may have a pronounced impact on the ability of a biopolymer to form and

stabilize emulsions. For example, many globular proteins unfold after they adsorb to oil-water

interfaces and expose non-polar and sulfur-containing groups, which leads to hydrophobic interactions

and disulfide bond formation between neighboring molecules [76, 77]. As a result, a two-dimensional

network of aggregated proteins may form with some elastic-like characteristics. The formation of this

covalently cross-linked interfacial layer can make it more difficult to displace the proteins from the

interface after they have been adsorbed, e.g., by adding surfactants or phospholipids to the aqueous

phase [78].

In mixed emulsifier systems, the conformation of biopolymers at an interface may also be

influenced by the presence of another emulsifier [54, 56]. Small molecule surfactants may directly

interact with biopolymers to alter their three-dimensional conformation either before or after

adsorption. For example, ionic surfactants may promote the denaturation of globular proteins due to

their ability to interact with charged and non-polar regions on the protein surface [79, 80]. These

interactions will alter interfacial properties, such as charge, thickness, and rheology, which will in turn

impact emulsion properties.

2.9. Chemical reactivity

Many emulsifiers have functional groups with the capability to participate in chemical reactions

that may occur within emulsions, e.g., oxidation or reduction reactions. In some cases, careful

selection of emulsifier type can be utilized to improve the chemical stability of emulsions, e.g.,

antioxidant proteins or phospholipids can be used to inhibit the oxidation of lipids in the oil phase [33,

66]. Lipid droplets coated with lactoferrin have been reported to have good oxidative stability, which

was mainly attributed to the strong antioxidant properties of this protein [81]. It may therefore be

possible to modulate the chemical reactivity of the oil-water interface by using blends of emulsifiers

14
with different characteristics. For instance, one emulsifier may be used to facilitate the formation and

physical stability of an emulsion, whereas another emulsifier is used to improve its chemical stability.

3. Emulsifier Interactions in Solution and at Interfaces

When two or more emulsifiers are mixed together in solution or in an emulsion, they may interact

with each other through various types of molecular interactions, such as hydrophobic, electrostatic, or

hydrogen bonding [6, 53, 55, 56, 59]. As a result, these interactions may alter the structural

organization, physicochemical properties, and functional attributes of the emulsifier molecules [59]. In

this section, we consider surfactant-surfactant, surfactant-biopolymer and biopolymer-biopolymer

systems dispersed in bulk aqueous solutions and at interfaces. Here, small molecule surfactants and

phospholipids are considered to be surfactants, whereas proteins and polysaccharides are considered to

be biopolymers.

3.1. In solution

When two or more hydrophilic emulsifiers are dispersed into an aqueous solution, a number of

different physicochemical phenomena may occur (Figure 4) [54, 59].

Fig. 4

3.1.1. Non-interacting systems

Surfactant-surfactant: In principle, when two surfactant solutions are mixed together they could

exist as two different types of surfactant monomers or micelles that do not interact with each other

(Figure 4a). Below the CMC of both surfactants, the surfactant monomers are likely to exist

independently of each other. However, above the CMC of at least one of the surfactants it is likely that

mixed micelles are formed that contain a blend of the two surfactants due to entropy of mixing effects

[25, 82]. Consequently, it is unlikely that two surfactants would not interact with each other at the

levels used in most commercial products.

15
 Surfactant-biopolymer: In principle, surfactants and biopolymers may not interact with each other

when they are mixed together in aqueous solutions [54, 59]. Instead, the surfactant may exist as non-

adsorbed monomers or micelles, while the biopolymer exists as individual molecules or clusters,

similar to how they would behave if they had simply been dispersed individually in solution. This type

of situation may occur when there are no strong attractive interactions between the different kinds of

emulsifier, e.g., hydrophobic or electrostatic attraction. Conversely, it may also occur when there are

strong repulsive interactions between the different kinds of emusifier, e.g., electrostatic repulsion. In

practice, one would expect surfactants and biopolymers to interact with each other, because the

biopolymers used as emulsifiers are amphiphilic molecules that have non-polar regions on their

surfaces, and so they may interact with surfactants through hydrophobic attraction [59]. Moreover,

most biopolymer-based emulsifiers have an electrical charge and could therefore interact with ionic

surfactants through electrostatic interactions.

Biopolymer-biopolymer: Combinations of certain types of amphiphilic biopolymer may exist

independently of each other when they are mixed together in an aqueous solution [83, 84]. This

situation may occur when there is a strong steric or electrostatic repulsion between the different kinds

of biopolymers, e.g., if they are both neutral or both have a strong negative charge. However, they may

associate with each other and form soluble or insoluble complexes if there is a sufficiently strong

attraction between them [85, 86]. In particular, biopolymers are relatively large molecules and so the

entropy of mixing effects opposing complexation may be relatively small.

In summary, non-interacting systems are probably fairly unfrequent in practice because emulsifiers

are amphiphilic molecules that often have a charge, and so they may interact with each other through

hydrophobic or electrostatic interactions.

16
3.1.2. Interacting systems

Surfactant-surfactant: As mentioned earlier, when two types of surfactant are mixed together

above the CMC of at least one of them, they may form mixed micelles [25, 82]. The size, shape, charge

and CMC of the mixed micelles are usually different from those of the simple micelles formed by the

individual surfactants [87]. As a result, one would expect that the mixed surfactant system would

behave differently than either of the single surfactant systems in terms of emulsion formation and

stability. For example, the movement of the surfactants to the oil-water interface and their attachment

their may be effected by the size of the mixed micelles formed.

Surfactant-biopolymer: Surfactant-polymer complexes may be formed in mixed systems

containing small molecule surfactants and amphiphilic polymers, provided there is a sufficiently strong

attraction between them [59, 80]. This may be a hydrophobic attraction between non-polar surfactant

tails and non-polar patches on biopolymer surfaces, or it may be electrostatic attraction between

charged surfactant headgroups and oppositely chared groups on biopolymer surfaces. The formation

and properties of surfactant complexes have been comprehensively reviewed elsewhere [54, 56, 59].

Recently, a study was carried out to characterize the properties of aqueous solutions containing

mixtures of a natural surfactant (quillaja saponins) and various amphiphilic biopolymers (proteins)

[88]. This study showed that the two emulsifiers may be miscible, flocculate, or precipitate depending

on emulsifier type and solution pH. In particular, electrostatic and hydrophobic interactions were

proposed to have a major impact on the tendency for the emulsifiers to associate with each other.

Biopolymer-biopolymer: Biopolymers may interact with each other through either attractive or

repulsive interactions in aqueous solutions leading to phase separation through either complexation or

thermodynamic incompatibility, respectively [89, 90]. Mixed biopolymer complexes are formed when

there is a sufficiently strong attraction between the two different kinds of biopolymer molecules [86].

Electrostatic attraction between oppositely charged groups is one of the most common forms of

17
attractive interaction encountered between amphiphilic biopolymers in aqueous solutions [91]. In

particular, there may be a relatively strong electrostatic attraction between anionic polysaccharides and

cationic proteins when the pH is below the pI of the protein, or between cationic polysaccacharides and

anionic proteins when the pH is above the pI of the protein [84]. Moreover, their may be a strong

electrostatic attraction between two different types of proteins when the solution pH is between their pI

values [92, 93]. These interactions may lead to the formation of soluble complexes, coacervates, or

precipitates depending on the charge characteristics, concentrations, and molar ratio of the different

types of biopolymers [91]. Amphiphilic biopolymers may also associate with each other due to

hydrophobic interactions between the non-polar patches on their surfaces.

When certain types of biopolymer are mixed together at sufficiently high concentrations, the

resulting solution separates into two different phases [94-96]. This type of behavior tends to occur in

systems where there is not a strong attraction between the biopolymers, and there are appreciable steric

exclusion effects. For example, when relatively high levels of two biopolymers (A and B) are

dissolved in an aqueous solution, the mixture may separate into two phases: one of the phases is

enriched with biopolymer A and depleted in biopolymer B, whereas the other phase is enriched in

biopolymer B and depleted in biopolymer A. This type of behavior has been reported when one of the

biopolymers is an amphiphilic protein (such as a casein, whey protein, or gelatin), and therefore

thermodynamic incompatibility may impact their performance in emulsions. This effect may be less

important when both biopolymers are amphiphilic, since then there would be a hydrophobic attraction

between them.

The structural organization and interactions of the emulsifiers in mixed systems may lead to

different functional properties than are obtained with the individual emulsifiers. For example, there

may be changes in emulsion formation, stabilization, and functionality, which could be either beneficial

18
or detrimental to emulsion performance. An improved understanding of these kinds of interactions

may therefore lead to emulsifier blends with improved functionality.

3.2. At interfaces

The behavior of mixed emulsifiers at droplet surfaces impacts their functional performance in

emulsions, and consequently it is important to understand the relationship between interfacial and

emulsion properties [11, 56, 59]. This section highlights the most important phenomena that may occur

at interfaces comprised of mixtures of emulsifiers, and provides examples of research work with

special emphasis on natural emulsifiers. Typically, there is a large specific surface area (AS / m2 kg-1)

of the oil phase in an emulsion due to the small diameter of the droplets: AS = 6/d (here  is the

density of the oil phase). Consequently, there is often sufficient oil-water interface present for different

types of emulsifier to adsorb onto the droplet surfaces. The interfacial layer may therefore consist of a

mixture of both types of emulsifiers, with the interfacial composition and structural organization of the

interface depending on the concentration and type of emulsifiers used (Figure 5). The composition and

structure of mixed interfaces impacts the packing, thickness, charge, and rheology of the interfacial

layer at droplet surfaces [54, 59, 97], and may therefore impact emulsion performance [11].

Fig. 5

3.2.1. Miscible systems

The two types of emulsifier may adsorb to the oil-water interface and form a two-dimensional

miscible system consisting of a random distribution of the emulsifiers [58]. This type of behavior is

analogous to a bulk solution containing two emulsifiers that do not strongly interact with each other,

and therefore form an intimate mixture. Interfacial properties, such as thickness, charge, and rheology,

may be modulated by varying the ratio of the two emulsifiers used to form the interfacial layer.

The concentrations of the two types of emulsifier at the interface depends on their surface activities

and concentrations. To a first approximation, the interfacial composition can be related to the

19
concentrations and surface activities of the different kinds of emulsifiers using the extended Langmuir

adsorption isotherm [98]:

1 c1 / c1,1 / 2
 (3a)
TOT c1 / c1,1 / 2  c2 / c2,1 / 2

2 c2 / c2,1 / 2
 (3b)
TOT c1 / c1,1 / 2  c2 / c2,1 / 2

Here c1 and c2 are the emulsifier concentrations in the bulk solution, 1 and 2 are the excess emulsifier

concentrations at the interface, and 1/c1,½ and 1/c2,½ are the surface activities of the emulsifiers. As

mentioned earlier, the surface activities can be estimated from plots of the surface pressure against

emulsifier concentration (Figure 2), where c½ is the emulsifier level where the surface pressure reaches

half its saturation value (Sat). This equation assumes that the two emulsifiers form an ideal mixture

and that they have similar dimensions, which is not the case in many situations. For instance, there may

be strong interactions between the emulsifier molecules in the bulk solution or at the surface, the

emulsifier molecules may be able to adopt different configurations at the interface, or the adsorption-

desorption process may not be fully reversible because one or both of the emulsifiers undergoes surface

denaturation [52, 56]. Nevertheless, the above equation does provide some valuable insights into the

expected change in interfacial composition with emulsifier concentration and surface activity (Figure

6). More sophisticated models have been developed to take into account emulsifiers with different

dimensions, post-adsorption conformational changes, and emulsifier interactions [52, 53, 56, 59].

These models require greater knowledge about the molecular and physicochemical properties of the

system, but they can provide more accurate descriptions of the factors affecting emulsifier composition.

Fig. 6

If the system is at equilibrium, then the composition of the interfacial layer should be

independent of the order of addition of the emulsifier molecules. In this case, the emulsfiiers could be

20
added either before or after homogenization, as long as the overall types and levels of emulsifiers used

are kept constant (and the particle size remains the same). In addition, this phenomenon makes it

possible to manipulate the interfacial properties after an emulsion has been formed by adding another

emulsifier to fully or partially displace the original emulsifier [99]. Thus, one emulsifier could be used

to form an emulsion, and another one to improve its stability or functional performance. This method

has been used to create emulsion droplets with different electrical charge characteristics [67], as shown

in Figure 3. If the system is not at equilibrium, then the interfacial composition depends on order of

addition effects [54, 56]. For example, it has been shown that when globular proteins are allowed to

age at an oil-water interface for several hours or when they are heated above their thermal denaturation

temperature, they are more difficult to displace with small molecule surfactants than from a fresh

interface [100, 101]. This effect is attributed to the fact that interfacial cross-linking occurs between

neighboring proteins after adsorption because they unfold and expose reactive non-polar and sulfhydryl

groups that lead to strong hydrophobic and disulfide bonds between neighboring protein molecules [77,

102]. Consequently, it is important to take into account these interfacial aging effects when using

mixed emulsifiers in certain systems [103, 104].

3.2.2. Immiscible systems

Experimental and computational studies have shown that two-dimensional phase separation

may occur at an oil-water interface for certain types of emulsifier mixtures, such as proteins and

surfactants [58, 105]. For example, adding increasing amounts of a small molecule surfactant to an

aqueous solution in contact with an oil-water interface saturated with protein molecules results in this

type of interfacial phase separation (Figure 7). At low surfactant concentrations, the surfactant

molecules penetrate between the protein molecules, but at higher levels the system phase separates into

domains rich in proteins and other domains rich in surfactant. If the proteins are strongly associated

with each other (e.g., whey proteins held together by strong hydrophobic or disulfide bonds), then the

21
protein-rich region may buckle and form irregular shaped domains that protrude from the interface

[106, 107]. Conversely, if the proteins do not strongly associate with each other (e.g., casein

molecules), then the protein-rich region may form circular domains [108]. Selected studies on the

interactions of biopolymers and surfactants at an oil-water interface are given in Table 1.

Fig. 7

Table 1

Table 1- Some selected studies on competitive adsorption of mixed emulsifiers within oil-in-water emulsions.

Type of emulsifying ingredients Analyzed Technique Reference

Protein Polysaccharide Surfactant
β-lactoglobulin; Methylcellulose; - Surface tension; Surface [109]
β-Casein Hydroxypropyl methyl cellulose rheology
- Modified starch Tween 20 Emulsion droplet size; [110]
Emulsion stability
β-lactoglobulin - Tween 20 Confocal microscopy [111]
β-lactoglobulin - Tween 20 Atomic force microscopy [106]
β-Casein Surface tension
- Sugar beet pectin; - Extraction of adsorbed [112]
Hydroxypropyl methylcellulose fractions; Emulsion
droplet size
β-Casein and - - Protein surface coverage [11]
αs1-Casein
Soy protein - Tween 40 Adsorption of protein by [113]
isolate SDS-PAGE
Sodium - Diglycerol Interfacial tension; [114]
caseinate esters of Protein surface coverage
fatty acids
Sodium Fucoidan; Gum Arabic - Isothermal titration [115]
caseinate calorimetry; Changes in
zeta-potential

Interestingly, studies have shown that systems that are believed to be prone to this phenomenon

are highly susceptible to droplet coalescence in the surfactant range where emulsifier phase separation

at the interface would be expected to occur [116]. Thus, both the surfactant-coated and protein-coated

droplets may be stable to coalescence, but the emulsions containing both surfactant and protein are

22
unstable. This phenomenon highlights that fact that emulsifier interactions may lead to undesirable as

well as desirable effects in terms of emulsion stability.

3.2.3. Interfacial complexation

Certain types of mixed emulsifier systems exist as complexes at the oil-water interface due to

strong attractive interactions between the different types of emulsifiers present [11, 54, 59, 117].

Complexation may occur in the aqueous phase before emulsifier adsorption to the droplet surfaces, or it

may occur at the droplet surfaces after adsorption has occurred. The conformation and surface

chemistry of biopolymers may be changed appreciably when they interact with surfactants, which will

impact their adsorption behavior and the properties of the interface formed. Various types of physical

and chemical interactions may be responsible for interfacial complexation, including electrostatic,

hydrophobic, hydrogen bonding, and disulfide interactions, as well as entropy effects associated with

counter-ion release [59]. The nature of the interactions involved, and their impact on interfacial

properties, depends on the type of emulsifiers involved. Both surfactants and amphiphilic biopolymers

may be anionic, cationic, zwiterionic, or non-ionic, depending on their functional groups. Emulsifiers

with oppositely charged groups may interact with each other through electrostatic attraction, while

those with similarly charged groups may interact via electrostatic repulsion. Electrostatic complexation

between charged emulsifiers and oppositely charged polysaccharides is one of the most commonly

used methods of forming interfacial complexes. This approach has been used to form interfacial

complexes between ionic polysaccharides and oppositely charged small molecule surfactants [118],

phospholipids [119], and proteins [120, 121]. In principle, two neutral emulsifiers could interact with

each other through hydrophobic interactions or hydrogen bonding at an interface, but to the authors

knowledge there has been little work in this area. The formation of emulsifier complexes at droplet

surfaces alters interfacial properties, such as charge, thickness, and rheology, which may impact

emulsion stability and other characteristics [11, 12, 122]. For instance, addition of small molecule

23
surfactants to an interface originally containing protein molecules leads to an increase in interfacial

thickness [107]. The complexation of emulsifiers at an interface may also lead to pronounced changes

in the mechanical properties of interfacial layers, such as the shear or dilational rheology [54, 117]. A

thorough review of the complexation of surfactants and polymers at interfaces has recently been

published, and the reader is referred there for more information [59].

3.2.4. Multilayer formation

Multiple layers of emulsifiers can be formed at an interface when there is a strong attraction

between the different types of emulsifiers using sequential deposition methods [11, 123-125]. Initially,

one type of emulsifier is adsorbed onto the droplet surfaces, and then another type is adsorbed on top of

it. This process can be repeated multiple times to form nanolaminated interfacial layers whose

thickness, composition, charge, and environmental responsiveness can be fine tuned for particular

applications [126]. The most commonly used means of forming multilayer interfaces is based on

sequential electrostatic deposition of oppositely charged molecules [12, 127]. This method has been

widely used to improve the physicochemical stability of oil-in-water emulsions containing protein-

coated oil droplets. In particular, it has been utilized to improve their resistance to flocculation at pH

values near the isoelectric point, at high salt levels, at elevated temperatures, during freezing-thawing,

and during dehydration [128-133]. In addition, this approach has been used to improve the chemical

stability of polyunsaturated lipids by locating antioxidant biopolymers at the oil-water interface or by

inhibiting the ability of cationic transition metals to come into contact with the lipids [134-137].

Finally, it has also been used to control the gastrointestinal fate of lipid droplets by designing interfaces

that inhibit the adsorption of bile salts or digestive enzymes to the droplet surfaces [119, 138, 139].

There is therefore considerable flexibility in being able to use mixed emulsifiers to engineer

interfacial properties so as to modify emulsion stability and functionality.

24
3.2.5. Equilibrium and Kinetic Aspects of Mixed Emulsifier Systems

One of the main challenges associated with working on any mixed systems, whether in the bulk or

at an interface, is that they are often non-equilibrium systems whose properties change over time. The

nature and rate of the changes in the structural organization of the molecules in these systems may then

impact the physicochemical properties and stability of a material. One of the main reasons for these

non-equilibrium effects is that many materials contain domains that are molecularly crowded, have

high local viscosities, or contain large folded and/or entangled polymer molecules. Consequently,

alterations in the structural organization and location of the molecules in the system may change

relatively slowly in response to environmental changes. For instance, emulsified food products may be

subjected to mechanical stresses, temperature changes, or compositional changes during their lifetime,

which may promote a change in the structural organization of particular domains within the system

(such as the interface) over time. In other words, a new equilibrium state is favored under the new

environmental conditions, and so there is a thermodynamic driving force favoring the movement of the

system into this new state. The rate and extent of the changes in the system depend on the presence of

any kinetic energy barriers. If these kinetic energy barriers are sufficiently large, then the system may

be trapped in a metastable state, rather than in a thermodynamically stable one. In future, it will

therefore be important to examine the rate at which materials respond to changes in environmental

conditions, and how this depends on system composition and structure. In the case of mixed

emulsifiers, this will involve measuring changes in the interfacial and bulk emulsifier composition and

structure over time after some well-defined environmental change has been imposed on the system. At

present, one of the main challenges in this area is the lack of analytical tools to provide detailed

information about the composition and structural organization of emulsifiers present in very thin

interfacial layers in emulsified systems in real time. Some progress can be made using existing

analytical techniques, such as microscopy, spectroscopy, scattering, rheometry, -potential, and

25
chemical methods that can measure changes in interfacial composition or properties in emulsions and at

planar interfaces. However, the authors encourage further research in this area because it will improve

our understanding of how mixed emulsifier interfaces behave in commercial products during long-term

storage and in response to changes in their environmental conditions.

4. Impact of Mixed Emulsifiers on Emulsion Formation

In general, emulsions can be formed using either high- or low-energy methods depending on the

ingredients and approaches used [4]. High-energy methods, such as high shear mixers, high-pressure

homogenizers, colloid mills and sonicators, generate intense disruptive forces that mechanically

breakup the oil and water phases [140-142]. Low-energy methods, such as phase inversion temperature

(PIT), phase inversion composition (PIC), and spontaneous emulsification (SE), rely on the

spontaneous generation of fine droplets in certain surfactant-oil-water mixtures when system conditions

(such as temperature or composition) are changed in a specific manner [143, 144]. The utilization of

mixed emulsifiers may impact the particle size distribution and stability of the droplets produced using

both of these methods.

4.1. High-energy methods

The potential impact of mixed emulsifiers on the formation of emulsions using high-energy

methods depends on their influence on the physicochemical processes occurring inside a homogenizer

[140, 145, 146]. In this article, only a high-pressure homogenizer is considered for the sake of brevity

because this type of device is currently the most frequently used equipment in industry to form

emulsions. However, similar physicochemical events will occur inside most other mechanical devices

used for homogenization. As shown in Fig. 8, the production of emulsions using this type of

homogenizer usually involves a two-step process [141]. First, a coarse emulsion containing relatively

large droplets (d > 1 m) is formed by blending oil and aqueous phases together in the presence of one

or more emulsifiers using a high-shear mixer. After this process, all of the large droplets formed

26
should be fully coated by a layer of emulsifier molecules, but there should also be a substantial amount

of free emulsifier remaining in the aqueous phase. Second, an emulsion containing relatively small

droplets (d < 1 m) is formed by forcing the coarse emulsion through a small valve inside the

homogenizer at high pressure, which generates powerful disruptive forces such as turbulence, shear,

and cavitation [142, 145, 146]. The size of the droplets emerging from the device depends on the

magnitude of the disruptive forces generated by the homogenizer compared to the magnitude of the

interfacial restoring forces, as well as the stability of the droplets to coalescence when they collide with

each other [145].

Fig. 8

4.1.1. Impact of emulsifier on droplet disruption

The droplets in an emulsion tend to adopt a spherical shape because of the imbalance of molecular

forces operating at the oil-water interface, i.e., the hydrophobic effect [147]. The positive free energy

associated with the unfavorable interactions at the oil-water interface means the system adopts a

structure that minimizes the contact area between the oil and water phases. The magnitude of this

effect depends on the interfacial tension, which can be moduluated by the types and amounts of

emulsifiers used. To deform and disrupt the droplets inside a homogenizer, the device must generate

disruptive forces that are sufficiently large to overcome the interfacial restoring forces:

4
PL  (4)
d

Here, PL is the Laplace pressure,  is the oil-water interfacial tension, and d is the diameter of the

droplets. This equation shows that the intensity of the disruptive forces needed to promote droplet

disruption increases as increases or d decreases. Under fixed homogenization conditions (such as

operating pressure and number of passes), this expression indicates that it should be possible to

generate smaller droplets by reducing the interfacial tension. Some emulsifiers are more effective at

27
reducing the interfacial tension than others, and would therefore be expected to produce smaller

droplets during homogenization. However, the effectiveness of an emulsifier also depends on how

quickly it adsorbs onto the droplet surfaces (Figure 9). All of the droplets are coated by a layer of

emulsifier when a coarse emulsion first enters the homogenizer, and therefore the interfacial tension of

all the droplets should be relatively low. After the larger droplets are fragmented, there is an increase

in total surface area, and so the droplets may not be completely covered by emulsifier, leading to an

increase in interfacial tension. If emulsifier molecules in the aqueous phase are able to adsorb to the

droplet surfaces faster than a subsequent fragmentation event, then the interfacial tension will be

relatively low and droplet breakdown favored [140]. However, if the emulsifier molecules adsorb too

slowly, then droplet breakdown will be less efficient. Consquently, the ability of the droplets to be

disrupted within a homogenizer depends on the adsorption kinetics of the emulsifiers, as well as their

ability to suppress the interfacial tension.

Fig. 9

Individual food-grade emulsifiers vary considerably in their adsorption kinetics and their ability to

reduce interfacial tension [10]. Small molecule surfactants (such as Tweens and saponins) adsorb

rapidly to oil droplet surfaces during homogenization and decrease the interfacial tension by an

appreciable amount. Consequently, they are highly efficient at forming emulsions containing small

droplets [148, 149]. Conversely, large amphiphilic biopolymers (such as polysaccharides) adsorb

relatively slowly and do not cause as large a reduction in interfacial tension, and they are therefore

much less effective at producing small droplets during homogenization [21, 99].

Utilizing a mixture of emulsifiers during the homogenization process may be able to reduce the size

of the droplets produced. For example, one type of emulsifier may be able to adsorb rapidly and reduce

the interfacial tension effectively, but may be less effective at stabilizing the droplets against

coalescence. Conversely, another type of emulsifier may not be as effective at reducing the interfacial

28
tension, but it may be highly effective at inhibiting droplet coalescence. By utilizing these two

emulsifiers in combination it may be possible to produce small stable droplets.

The impact of using mixtures of a small molecule surfactant (Tween 20) and a biopolymer (whey

protein isolate) on the formation and stability of oil-in-water emulsions has been reported [150]. At a

constant total emulsifier concentration, increasing the ratio of Tween 20-to-WPI led to a decrease in

mean droplet diameter. In a related study, the effect of using different ratios of Tween 20 and -

lactoglobulin (a globular protein) on the droplet size produced by high-pressure homogenization was

investigated [151]. This study showed that smaller droplets could be produced when a mixture of

emulsifiers was used than when either of the individual emulsifiers was used under the same

homogenization conditions. The impact of using combinations of Tween 20 and -casein (a flexible

protein) on the formation of emulsions has also been examined [152]. The effect of using mixtures of

lecithin and gum arabic (two natural emulsifiers) on the formation and properties of eugenol

nanoemulsions by high shear mixing has been carried out [153]. This study reported that the smallest

droplets were produced at a specific ratio of lecithin-to-gum arabic. Mixtures of a protein (sodium

caseinate) and soy lecithin have been shown to form thyme oil-in-water nanoemulsions with smaller

droplets than could be achieved using either emulsifier individually when produced using a high-shear

mixer [154]. The same group reported that thyme oil nanoemulsions with smaller droplet sizes could

be produced using a mixture of a cationic surfactant (lauric arginate) and soy lecithin than using the

individual emulsifiers [155]

Hydrophobic surfactants (sorbitan monolaurate) present within the oil phase, have also been shown

to impact the size of the droplets produced by homogenization when amphiphilic proteins (-

lactoglobulin or -casein) are present in the water phase [116]. The presence of the hydrophobic

surfactant decreased the particle size when used at intermediate levels (by adsorbing rapidly and

reducing the interfacial tension), but increased it when used at high levels (by increasing the oil phase

29
viscosity). The addition of lecithin to the oil phase used to produce oil-in-water emulsions from -

casein has also been shown to influence droplet size and interfacial composition [156]. In the studies

reported, the level of amphiphilic biopolymer at the oil-water interface after homogenization usually

decreases as the concentration of small molecule surfactants in the system increases. In conclusion, a

number of studies have shown that it may be possible to reduce the size of the droplets produced during

homogenization by using mixtures of emulsifiers, rather than individual emulsifiers. However, the

types and levels of the two emulsifiers utilized must be carefully controlled, since certain combinatons

of emulsifiers can actually interfere with the formation of small droplets during homogenization.

In future studies, it would be interesting to investigate the relative point of addition of mixed

emulsifiers during homogenization. For example, for two hydrophilic emulsifiers, both emulsifiers

could be added to the aqueous phase prior to homogenization, or one of them could be added before

homogenization and one after homogenization. Similarly, for a hydrophobic and hydrophilic

emulsifier, both emulsifiers could be added before homogenization, or the hydrophobic one could be

added to the oil phase before homogenization, and the hydrophilic one added to the aqueous phase after

homogenization.

4.1.2. Impact of emulsifier on droplet coalescence

After converting large droplets into smaller droplets inside a homogenizer, it is necessary to prevent

them from coalescing with each other when they collide, otherwise the droplet size will increase and

some of the energy used will be wasted (Figure 9). When large droplets are fragmented into smaller

ones, there is an increase in surface area, and so not all of the droplet surfaces are saturated with

emulsifier [145, 157]. The tendency for droplets to coalesce with each other after a collision within a

homogenizer depends on the extent of surface coverage, with the coalescence frequency increasing

with decreasing surface coverage [158]. For this reason, it is important that the droplet surfaces are

fully covered by a layer of emulsifier molecules before they collide with each other [140, 157, 159].

30
The rate of surface coverage depends on the amount of emulsifier present, as well as the emulsifier

adsorption rate. Droplet coalescence can therefore be reduced by ensuring there is sufficient emulsifier

present to cover all of the newly formed droplet surfaces, that the emulsifier molecules adsorb rapidly

relative to droplet-droplet collisions, and that the interfacial layers formed generate repulsive

interactions between the droplets [72, 160]. Small molecule surfactants are particularly effective at

inhibiting coalescence, and therefore producing small droplets, because they can rapidly adsorb onto

the droplet surfaces [161-163]. Conversely, many amphiphilic polymers (such as large proteins and

polysaccharides) are less effective because they adsorb relatively slowly and so the droplets collide

before they are fully covered with emulsifier [74, 164]. In a mixed emulsifier system, the small

molecules may adsorb to the oil droplet surfaces first, and then the larger biopolymer molecules may

attach to the droplet surfaces later, thereby changing the interfacial composition.

One of the most important factors determining the minimum size of the droplets that can be

produced during homogenization is the surface load of the emulsifier [158]. The smallest mean droplet

diameter that can be created in an oil-in-water emulsion containing a fixed amount of oil phase is given

by the following expression [1]:

6  sat  
d min  (5)
cS

Here, dmin is the surface-weighted mean droplet diameter (d32), sat is the surface load of the

emulsifier at saturation (in kg m-2),  is the disperse phase volume fraction (unitless), and cS is the total

emulsifier concentration in the emulsion (in kg m-3). This equation assumes that droplets are only

stable to coalescence when their surfaces are saturated with emulsifier, that the droplet size is not

limited by the disruptive forces generated by the homogenizer, and that all the emulsifier molecules

adsorb onto the droplet surfaces. The change in d32 with emulsifier concentration is shown in Figure 10

for a series of emulsifiers with differing surface loads. The droplet diameter decreases as the amount of

31
emulsifier increases, and the minimum droplet size that can be stabilized at a particular emulsifier

concentration rises with increasing surface load. The surface load of some commonly used food

emulsifiers increases in the following order: small molecule surfactants < phospholipids < globular

proteins < flexible proteins < polysaccharides [10]. Thus, under fixed homogenization conditions

(pressure and number of passes), small molecule surfactants (such as Tweens or saponins) should

produce smaller droplets than polysaccharides (such as gum Arabic) when utilized at similar levels.

These predictions are supported by empirical measurements of droplet diameter versus emulsifier

profiles [73, 165]. In reality, the predicted minimum droplet diameter is often not achieved because

only a fraction of the added emulsifier adsorbs, some coalescence occurs within the homogenzation,

and/or the disruptive forces generated by the homogenization are not high enough to reach this droplet

size.

Fig. 10

The utilization of mixed emulsifiers may alter the tendency for droplet coalescence to occur during

and/or after homogenization [116]. It may be possible to include one emulsifier that adsorbs rapidly to

the droplet surfaces and lowers the interfacial tension (thereby facilitating droplet disruption), and then

include another emulsifier that adsorbs more slowly but that inhibits droplet coalescence even at levels

below that required to cause saturation.

A number of studies have shown that the coalescence stability of emulsions formed from mixed

emulsifiers is worse than that of emulsions formed from either individual emulsifier. This is

particularly the case for emulsions formed using mixtures of proteins and small molecule surfactants.

The origin of this effect has been attributed to differences in the physicochemical mechanisms that

these two types of emulsifiers use to inhibit coalescence [116]. Proteins form interfacial layers that are

resitant to coalescence due to the fact that there are strong interactions between neighboring protein

molecules that produce an interfacial layer that has sufficient mechanical strength to resist deformation.

32
Conversely, surfactants form interfacial layers that can inhibit coalescence due to the fact that the

surfactant molecules are highly mobile and free to move and therefore they can rapidly move to regions

of high surface tension (low surfactant concentration), i.e., the Marangoni effect. These mechanisms

are effective at inhibiting droplet coalescence in emulsions containing individual emulsifiers, but they

interfere with each other in emulsions containing mixed emulsifiers. For example, the surfactants

interfere with the interactions between neighboring proteins, whereas the proteins interfere with the

mobility of the surfactants. As a result, emulsions containing combinations of surfactants and proteins

may have worse coalescence stability than either one alone [166]. This factor has to be taken into

account when formulating emulsion-based products that are expected to have high coalescence

stability.

Studies have shown that the inclusion of a hydrophobic surfactant (Span) in the oil phase and

hydrophilic proteins (-lactoglobulin or -casein) in the aqueous phase leads to smaller oil droplets

being produced, than just using the proteins alone [116]. However, the emulsions containing the

hydrophobic surfactant were more prone to droplet coalescence, which can be attributed to the fact that

they partially displace the protein molecules from the droplet surfaces, and they only had small polar

head groups that did not prevent the droplets from coming into close proximity when present on their

own.

Another important factor to consider during emulsion formation is the dependence of the droplet

size on homogenization pressure [163, 167]. Typically, the mean droplet diameter decreases with

increasing pressure, but the dependence of this relationship depends on emulsifier type and

concentration [1]. A number of possible situations are highlighted in Figure 11:

Fig. 11

(i) Excess Emulsifier: If there is an excess of emulsifier present, then the droplet diameter will

continue to decrease with increasing homogenization pressure. Eventually, the upper limit for

33
 droplet disruption by the homogenizer is reached, and the droplet size will not decrease any

further. In this case, droplet size is determined by homogenization pressure and there is typically

a log-log relationship between them [147]. Droplet size also depends on the ease of droplet

disruption. In oil-in-water emulsions the ease of droplet disruption tends to increase with

diminishing interfacial tension and dispersed-to-continuous phase viscosity ratio [167, 168].

Thus, natural emulsifiers that are better at decreasing the interfacial tension tend to lead to

smaller droplets [73, 165].

(ii) Limited Emulsifier: If there is only a limited amount of emulsifier present, then the droplet size

decreases with increasing homogenization pressure until a certain droplet size is reached [158].

At this point, all of the emulsifier initially added to the system is adsorbed onto the droplet

surfaces, and so the droplet size cannot be reduced any further since there is not enough

emulsifier to cover newly formed droplets. As a result, any smaller droplets formed within the

homogenizer will not be fully covered with emulsifier, and so they will tend to coalesce with

each other. In this case, the minimum droplet size that can be produced is mainly determined by

the initial emulsifier concentration added.

(iii) Over-processing: In some situations, the droplet size may initially decrease with increasing

homogenization pressure, but then increases, which is often referred to as “over-processing” [72].

There is often a considerable increase in the temperature of a sample during homogenization at

high pressures due to frictional losses. High pressures and temperatures sometimes cause an

increase in droplet diameter due to a reduction in emulsifier functionality, e.g., due to

depolymerization or unfolding of biopolymer chains or due to dehydration of surfactant head-

groups. These effects are likely to be highly system specific. As mentioned earlier, some proteins

and polysaccharides are susceptible to depolymerization or unfolding in certain types of

34
 homogenizers, and therefore this effect has to be taken into account when deciding the most

appropriate homogenization method for a specific natural emulsifier.

There have been few rigorous studies of the impact of mixed emulsifier systems on the formation

of emulsions using high-energy methods. Clearly, further research is needed to determine changes in

droplet size and interfacial composition when emulsions are prepared using different homogenization

conditions, such as operating pressure and number of passes. The emulsifiers may adsorb

independently of each other, or they may form complexes that adsorb to the droplet surfaces. The

complexes formed in the aqueous phase under the highly turbuldent conditions inside a homogenizer

may be quite different from those formed in quiescent solutions under static conditions.

4.2. Low-energy methods

Emulsions can be formed from certain combinations of surfactant, oil, and water using low-energy

methods, such as the spontaneous emulsification (SE), phase inversion temperature (PIT), and phase

inversion composition (PIC) approaches [143, 144]. At present, these methods are mainly used to form

emulsions from small molecule surfactants, rather than amphiphilic biopolymers, although some recent

work suggests that certain types of proteins may also be used [169]. Studies have shown that the

formation and properties of emulsions fabricated using a PIT approach can be modulated by using

mixtures of non-ionic and cationic surfactants [170]. The phase inversion temperature increased with

increasing cationic surfactant concentration, as did the positive charge on the oil droplets after

emulsion formation. Studies have also shown that the size and stability of the oil droplets in emulsions

produced using various low methods could be tuned by varying the ratio of a hydrophilic (Tween 80)

and hydrophobic (Span 80) non-ionic surfactant, which was attributed to changes in the optimum

curvature of the surfactant monolayer [171-174]. There is an optimum hydrophilic/hydrophobic

surfactant ratio to produce stable emulsions containing small droplets. Recent studies have shown that

lemon oil emulsions can be produced using the PIT method by using combinations of sodium casinate

35
and Tween 20 [169]. The droplet size produced using the mixed emulsifier system was smaller than

that which could be produced using either emulsifier alone. This study therefore demonstrated that

some of the synthetic surfactants required to form emulsions using the PIT method could be replaced

with natural emulsifiers.

5. Impact of Mixed Emulsifiers on Emulsion Stability

As well as their ability to facilitate emulsion formation, emulsifiers also play an important role in

stabilizing emulsions by forming a resistant interfacial layer, generating strong repulsive forces, and

preventing droplet aggregation (Fig. 12).

Fig. 12

The major instability mechanisms that operate in emulsions are shown in Fig. 13 and will be discussed

in the following subsections for mixed emulsifier systems.

Fig. 13

5.1. Gravitational separation

To a first approximation the rate at which an isolated spherical droplet moves through an ideal

liquid is described by Stokes’ Law [5]:

2 gr 2 ( 2  1 )
v Stokes   (6)
9 1

Here, r is the droplet radius, g is the acceleration due to gravity,  is the density,  is the shear

viscosity, and the subscripts 1 and 2 refer to the continuous and dispersed phases, respectively. This

equation indicates that the creaming velocity increases with increasing droplet size, increasing density

contrast, and decreasing continuous phase viscosity. The utilization of mixed emulsifiers may alter the

creaming stability in a number of ways:

Initial droplet size: The size of the droplets produced during homogenization depends on the

nature of the emulsifiers used to stabilize the emulsion [1, 72, 141]. If a blend of emulsifiers produces

36
a smaller droplet size than an individual emulsifier, then the droplets will cream more slowly and the

emulsion will be more stable to gravitational separation.

Density Contrast: The overall density of an emulsion droplet depends on the densities and volume

fractions of both the lipid core and the emulsifier shell, which can be described by the following

equation [4]:

𝑟𝜌𝑐𝑜𝑟𝑒 +3𝛿𝜌𝑠ℎ𝑒𝑙𝑙
𝜌2 = (7)
𝑟+3𝛿

Here, core and shell are the densities of the material core inside the droplets (pure oil) and within the

interfacial layer (emulsifier), and  is the thickness of the interfacial layer. Typically, emulsifiers have

a higher density than either oil or water and therefore the presence of the interfacial layer will tend to

increase the overall density of the droplets. Emulsifiers that pack more tightly at the oil-water interface

will tend to produce denser interfaces, and will therefore have a bigger impact. The above equation

indicates that the overall density of an emulsion droplet may be appreciably higher than that of the oil

phase when the interfacial layer is relatively thick and dense. Consequently, it is possible to use

mixtures of emulsifiers to form this kind of thick dense interfacial layer so as to reduce the creaming

velocity of small oil droplets [4, 175].

Inhibition of aggregation: When the droplets in an emulsion aggregate with each other, they form

particles that are larger than the original droplets, which leads to more rapid creaming [1].

Consequently, if a blend of emulsifiers can inhibit droplet aggregation (flocculation or coalescence),

then it should improve the creaming stability of an emulsion [11]. The utilization of mixed emulsifiers

has been shown to improve the aggregation stability of emulsion droplets by forming interfacial

complexes that increase the electrostatic and/or steric repulsion between the oil droplets, as shown in

Fig. 14 [129, 130]. Consequently, they may be used to improve the creaming stability of emulsions by

this mechanism.
37
 Fig. 14

5.2. Flocculation

Droplets tend to flocculate when the attractive interactions (such as van der Waals, hydrophobic,

and depletion) acting between the droplets outweigh the repulsive interactions (such as steric and

electrostatic) [1]. The utilization of mixed emulsifiers may increase the repulsive interactions between

the droplets and therefore improve their flocculation stability. For example, the electrostatic deposition

method has been used to create multilayer interfaces around oil droplets to improve their stability by

increasing the steric and/or electrostatic repulsion between them [118, 129, 130]. In these cases, a

charged biopolymer is deposited onto an oppositely charged emulsifier-coated droplet. It has been

shown that the adsorption of gum Arabic onto the surfaces of protein-coated droplets improves their

stability to pH or salt-induced flocculation [176]. The adsorption of ionic surfactants to the surfaces of

protein-coated oil droplets has also been shown to improve their flocculation stability by increasing the

electrostatic repulsion between them [68, 177]. Similarly, the adsorption of non-ionic surfactants to the

surfaces of protein-coated droplets has been shown to improve their flocculation stability by increasing

the steric repulsion between them [178]. A recent study showed that more stable emulsions could be

formed by using a mixture of sodium caseinate and soy protein isolate than either emulisifer in

isolation [179]. On the other hand, the presence of high levels of non-adsorbed emulsifiers in mixed

systems can actually lead to emulsion instability due to a depletion flocculation mechanism. For

example, when high levels of a non-ionic surfactant (Tween 20) were mixed with a protein emulsifier

(caseinate) the emulsion formed became unstable to creaming, which was attributed to the formation of

casein micelles in the aqueous phase that promoted depletion flocculation [180].

5.3. Coalescence

Coalescence occurs when two or more droplets come into close proximity and fuse together to

form a larger droplet [158, 181]. This process tends to occur when the attractive forces acting between

38
the droplets outweigh the repulsive forces (similar to flocculation), and the interfacial layers around the

oil droplets rupture when the droplets come into contact. Coalescence may therefore be inhibited by

using mixed emulsifiers to form interfacial layers that are resistant to rupture. A number of studies

have shown that mixed emulsifiers can be used to design interfacial coatings around oil droplets that

improve their coalescence stability. For example, oil droplets coated by multiple layers of emulsifiers

and biopolymers have been shown to enhance coalescence stability when exposed to freezing/thawing

or dehydration [132, 133, 182, 183]. In addition, the coalescence stability of oil bodies stabilized by

oleosins and phospholipids was improved by addition of a non-ionic surfactant (Tween 80) [184].

Conversely, other studies have shown that the presence of a mixture different types of emulsifiers in

emulsions can actually promote coalescence. For example, the incorporation of a lipophilic non-ionic

surfactant (Spans) into an emulsion stabilized by milk proteins (-lactoglobulin) was shown to promote

coalescence by displacing some of the proteins from the oil droplet surfaces [185]. In this case, the

small hydrophilic head groups of the Span molecules would not be sufficient to protect the droplets

from coalescence. The introduction of hydrophilic non-ionic surfactants (Tweens) has also be shown

to promote coalescence in emulsions stabilized by milk proteins during shearing, which was attributed

to the ability of low levels of surfactant to increase the mobility of the interfacial layer [186]. The

addition of both hydrophilic (Tweens) and lipophilic (Spans) surfactants was shown to promote the

coalescence of oil droplets in the emulsions formed during peanut oil extraction [187]. In this case,

droplet coalescene was desirable since it facilitated isolation of the oil from the other components in the

system. Again, increased droplet coalescence in the systems containing mixed protein-surfactant

interfaces may have been because they are highly sensitive to disruption due to their increased mobility

[188].

39
5.4. Ostwald ripening

Ostwald ripening (OR) manifests itself as the growth of larger droplets and the shrinkage of

smaller droplets over time due to the diffusion of oil molecules through the intervening continuous

phase [189]. The driving force for this process is the higher solubility of the oil molecules in the

vicinity of the smaller droplets compared to the larger droplets due to curvature effects [190]. Once

steady state conditions have been realized, the rate of Ostwald ripening can be described by the

following equation [190]:

𝑑<𝑟>3 8𝛾𝑉𝑚 𝑆∞ 𝐷
= (8)
𝑑𝑡 9𝑅𝑇

Here S is the equilibrium solubility of the oil phase in the bulk aqueous phase, r is the droplet radius, 

is the interfacial tension, Vm is the molar volume of the oil phase, R is the gas constant, T is the absolute

temperature, and D is the translational diffusion coefficient of the oil molecules through the aqueous

phase. There are a number of ways that the utilization of mixed emulsifiers may impact the rate of OR.

Interfacial tension reduction: Equation 8 shows that the OR rate should decrease as the interfacial

tension decreases [191]. Consequently, OR may be inhibited by using a combination of emulsifiers that

reduces the interfacial tension [192].

Interfacial diffusion barrier: The movement of oil molecules from one droplet to another has also

been shown to depend on the rate that they diffuse through the interfacial layer coating the droplets

[190]. Ostwald ripening may therefore be reduced by decreasing the diffusion coefficient of the oil

molecules through this layer, or by increasing its thickness. To the author’s knowledge, there has been

little experimental work on this topic.

Interfacial mechanical strength: The OR rate may be reduced by increasing the mechanical

strength of the interfacial layer around the droplets so that it retards the growth/shrinkage of the oil

droplets [191, 192]. Cross-linking the proteins in interfacial layers has been shown to reduce the rate

of OR in oil-in-water emulsions [193]. Creating interfacial layers comprised of multiple layers of

40
biopolymers has also been shown to reduce the OR rate, presumably by providing mechanical

resistance to droplet growth/shrinkage [194, 195]. Similarly, the inhibition of OR in emulsions

containing a mixture of non-ionic surfactant and amphiphilic polyelectrolyte has been attributed to their

ability to form a rigid interfacial layer [196].

Interfacial compositional ripening: The OR rate can be retarded by using a mixture of emulsifiers

that have different water-solubilities or surface activities. One of the emulsifiers (E1) is selected so that

it remains strongly attacted to the original droplet surfaces (due to a very low water-solublity or high

surface activity) formed during homogenization, whereas the other emulsifier (E2) is selected so that it

is able to transfer from one droplet to another. If the droplets swell or shrink, then there will be a

change in the interfacial composition due to the fact that E1 cannot move between droplets, but E2 can.

For instance, there will be a higher concentration of E1 than E2 in the interfacial layer for a droplet that

shrinks, and vice versa. A change in interfacial composition between different droplets is

thermodynamically unfavorable due to an entropy of mixing effect, i.e., the system wants to maximize

its entropy by having the emulsifier molecules randomly distributed amongst all of the droplet surfaces.

Consequently, an increase in droplet size due to Ostwald ripening is opposed by this interfacial

compositional ripening effect. This phenomenon may account for the increased stability of thyme oil

emulsions to droplet growth when they were stabilized by a mixture of sodium caseinate and lecithin,

rather than either emulsifier alone [154]. The retardation of OR in emulsions containing mixtures of

non-ionic surfactants and amphiphilic polymers may also be partly due to this effect [197].

To the authors knowledge, there have been few studies that have attempted to systematically

investigate the relative impact of these different physicochemical mechanisms on the rate of Ostwald

ripening in emulsions containing mixtures of emulsifiers. This would therefore be a fruitful area for

research in the future.

41
6. Competitive adsorption of mixed emulsifiers at droplet surfaces

The competition between different surface-active ingredients is very important for the functionality

and stability of emulsions [11, 58, 116, 198]. As discussed previously, the utilization of mixed

emulsifiers in food emulsions may have beneficial or detrimental effects on both emulsion formation

and stability. Consequently, it is important to understand the factors that impact interfacial

composition. The competition between different types of emulsifiers is important in determining the

composition and structure of interfacial layers in emulsions. If the mixed emulsifiers are added prior to

homogenization, then they will compete for the interface inside the homogenizer. Typically, the

emulsifier that adsorbs more rapidly to the droplet surfaces will initially be present, but this may be

displaced during storage due to competitive adsorption with other emulsifiers present in the system.

Alternatively, one emulsifier may be added before homogenization, while another one is added after

homogenization. In this case, the original emulsifier may be fully or partially displaced from the

droplet surfaces by the added emulsifier depending on their relatively concentrations and surface

activities (Section 3.2.1).

A number of studies have shown that addition of small molecule surfactants to oil-in-water

emulsions containing protein-coated droplets can fully or partially displace the adsorbed proteins [58,

111, 199]. For flexible proteins, such as caseins, that do not form highly cross-linked interfacial layers,

the adsorbed proteins can be fully displaced by surfactants when they are present at sufficient quantity

and are given enough time [106, 109]. However, displacement by surfactants may be more difficult for

globular proteins, such as -lactoglobulin, that partially unfold after adsorption and then interact with

their neigbours to form cross-linked networks [58]. Globular proteins may be particularly difficult to

displace when the interface has been aged or heated, since this leads to a greater amount of interfacial

protein cross-linking due to surface or thermal denaturation [108]. Researchers have applied various

techniques including confocal microscopy, interfacial rheology, Atomic Force Microscopy (AFM) and

42
computer simulations to evaluate and visualize the structural changes involved at the oil-water interface

in this type of mixed system [58, 106, 109].

Fig. 15

The displacement of proteins from interfaces by small molecule surfactants may occur due to a

number of phenomena [52, 58, 60]: (a) Solubilization (mainly for ionic surfactants) - surfactants bind to

proteins and form soluble protein-surfactant complexes that have relatively low surface-activity; (b)

Replacement (for all surfactants) - surfactants displace proteins from the interface due to an entropy of

mixing effect, i.e., they are both surface active molecules that compete for the same interface. Proteins

typically have a much higher adsorption energy per molecule than surfactants, and therefore tend to

saturate fluid interfaces at much lower concentrations than surfactants [110]. However, at higher

concentrations, because of their more effective packing, the interfacial tension is lowered more by

surfactants than by proteins. Hence, proteins are competitively displaced by surfactants from the oil–

water interface, as has been reported in many studies for mixtures of surfactants and amphiphilic

biopolymers (Table 1).

The adsorption of surfactants to an interface originally coated by proteins can cause the formation

of defects in the interfacial layer [58], which has been described as "orogenic displacement", which is

illustrated schematically in Figure 7 [188]. Three stages of orogenic displacement of proteins by small

molecule surfactants have been identified [106, 200]. In the first stage (compression phase), the

surfactant adsorbs into small gaps in the protein film, resulting in the formation of small surfactant

domains, which increase the surface pressure of the film. As surfactant adsorption continues, the

surfactant domains grow, but the protein layer thickness is largely unaffected. In the second stage

(collapse phase), the surfactant domains continue to grow, but the decrease in protein film area is

compensated for by a corresponding increase in protein film thickness, and thus, the volume of

adsorbed protein remains relatively constant. In the third stage (separation phase), the aggregated

43
protein molecules break away from the interface, leading to the formation of a continuous surfactant

phase.

7. Impact of Mixed Emulsifiers on Emulsion Functionality

7.1. Antioxidant activity

It is well established that the nature of the interfacial layer between the oil and water phases can

have a pronounced impact on the rate and extent of lipid oxidation in emulsions [66, 201]. Various

studies have shown that the oxidative stability of emulsions can be modulated by using combinations of

emulsifiers to alter the properties of the interfacial layer around the lipid droplets. Electrostatic

deposition of beet pectin onto the surfaces of silk fibroin-coated lipid droplets was shown to reduce

lipid oxidation, i.e., it increased the lag phase before oxidation was observed [136]. This effect may

have been due to the fact that beet pectin contains antioxidant phenolic groups (ferulic acid), or because

it can physically hinder the ability of transition metal ions reaching the lipid phase. Multilayer coatings

consisting of an anionic protein (BSA) and successive layers of anionic (dextran sulfate) and cationic

(poly-L-arginine) layers have been used to trap an antioxidant (tannic acid) within the interfacial layer,

which was shown to protect the lipid droplets (linseed oil) from oxidation [202]. A similar approach

has been used to improve the chemical stability of emulsified polyunsaturated lipids by locating

antioxidant biopolymers at the oil-water interface or by inhibiting the ability of cationic transition

metals to come into contact with the lipids [134-137].

7.2. Antimicrobial activity

There is increasing interest in the utilization of emulsion-based delivery systems to enhance the

handling and efficacy of antimicrobials [203-205]. Emulsions are particularly suitable for this purpose

because they can be used to encapsulate hydrophobic, hydrophilic, and amphiphilic antimicrobial

agents in a single system. A number of studies have shown that using mixed emulsifiers to form

44
emulsion-based antimicrobial delivery systems can improve their physical stability and antimicrobial

activity.

Thyme oil nanoemulsions produced using a mixture of a cationic surfactant (lauric arginate) and

anionic phospholipids (soy lecithin) contained smaller droplets than those produced using either

emulsifier individually, however, the antimicrobial activity of the system was not improved using

mixed emulsifiers [155]. Cinnamon oil nanoemulsions have been formed using a mixture of lauric

arginate and a non-ionic surfactant (Tween 80) as emulsifiers [206]. This study showed that physically

stable antimicrobial nanoemulsions could be formed from these mixed emulsifiers that were efficacious

against Salmonella enteritidis, Escherichia coli, and Listeria monocytogenes. Gum Arabic and lecithin

have been used in combination to form antimicrobial eugenol nanoemulsions that were shown to be

effective against Listeria monocytogenes and Salmonella enteritides [153]. The smallest initial droplet

sizes produced by high shear mixing were obtained at a particular ratio of gum Arabic to lecithin.

Sodium caseinate and soybean lecithin are two natural emulsifiers that have been used in combination

to form antimicrobial thyme oil nanoemulsions that are effective against a variety of bacteria, i.e.,

Escherichia coli, Listeria monocytogenes, and Salmonella enteritidis [207]. Thyme oil emulsions

stabilized by ovalbumin/gum Arabic electrostatic complexes were shown to have good storage stability

and antimicrobial efficacy against E. coli [208]. Interestingly, this study showed that emulsions

fabricated by forming the electrostatic complexes before or after homogenization behaved fairly

similarly.

7.3. Flavor encapsulation

A number of studies have shown that mixed emulsifiers can be used to formulate emulsion-based

delivery systems for non-polar flavor oils. Combinations of sucrose monopalmitate and lysolecithin

have been used to form orange oil emulsions [209, 210]. Sucrose monopalmitate could be used to form

small droplets at neutral pH when used in isolation, but the emulsions were highly unstable to

45
aggregation at acidic pH because of the reduction in electrostatic repulsion between the oil droplets.

The addition of lecithin to the oil phase of the emulsions prior to homogenization did not impact the

initial size of the droplets formed, but it did improve their acid-stability by generating a strong negative

charge that increased the electrostatic repulsion between the droplets. Mixtures of sodium caseinate

and Tween 20 have been used to form lemon oil nanoemulsions using a low-energy (PIT) method

[169]. The size of the droplets produced was smaller for certain caseinate/Tween combinations than

for either of the individual emulsifiers, and had good storage stability. The electrostatic deposition of

beet pectin onto the surfaces of the protein-coated oil droplets in citral emulsions was shown to

improve their stability to pH and ionic strength, and to protect the citral from chemical degradation

[211]. Similarly, the electrostatic deposition of either soybean soluble polysaccharides or beet pectin

onto lactoferrin-coated oil droplets in orange oil emulsions improved their physical and chemical

stability [212].

7.4. Nutraceutical encapsulation

Nutraceuticals are bioactive agents found in foods that have beneficial effects on human health and

wellbeing [213]. Many nutraceuticals are highly hydrophobic molecules that cannot simply be

incorporated into aqueous-based food products, and consequently they need to be encapsulated in a

suitable delivery system [214]. Emulsion-based delivery systems, such as oil-in-water emulsions and

nanoemulsions, are particularly suitable for this purpose because they can be fabricated on a

commercial scale using food-grade ingredients and existing technologies [215-217]. The utilization of

combinations of emulsifiers have been shown to be able to improve the formation, stability, and

performance of emulsion-based delivery systems.

The physical and chemical stability of -carotene delivery systems has been improved by covering

the carotenoid-loaded lipid droplets with a bilayer of milk proteins and chitosan-polypenol complexes

[218]. In particular, the rate of -carotene degradation when exposed to UV-light or heat was reduced

46
for the mixed emulsifier systems compared to the single emulsifier ones. Proteins with different charge

characteristics (anionic -lactoglobulin and cationic lactoferrin) have been used to create mixed

emulsifier interfacial layers utilizing the electrostatic deposition method, which were shown to be

effective at inhibiting -carotene degradation [219]. This effect was mainly attributed to the fact that

the interface contained lactoferrin, which is known to have strong antioxidant properties. The

formation of multilayers consisting of chitosan, whey protein, and flaxseed gum around lutein-loaded

oil droplets has been shown to reduce the chemical degradation of the encapsulated lutein [220].

7.5. Formation of multiple emulsions

Mixed emulsifiers can also be used in the formation of multiple emulsions, which have been

receiving increased interest for the encapsulation and delivery of nutraceuticals, and for the production

of low-fat foods [221, 222]. Multiple emulsions with different structures can be formed, such as oil-in-

water-in-oil (O1/W/O2) and water-in-oil-in-water (W1/O/W2) emulsions. In the latter case, small water

droplets (W1) are dispersed in larger oil droplets (O), which are themselves dispersed within a

continuous aqueous phase (W2). W1/O/W2 emulsions are typically formed using a two-step process.

First, a W1/O emulsion is formed by homogenizing a water phase (W1) with an oil phase (O) containing

a lipophilic emulsifier. Second, the W1/O/W2 emulsion is formed by homogenizing the W1/O emulsion

with an aqueous phase (W2) containing a hydrophilic emulsifier. These types of multiple emulsion

therefore require the utilization of two different types of emulsifier [223, 224] (Fig. 15). Lipophilic

surfactants (such as Spans or PGPR) are often used to stabilize the W1-O interfaces formed in the

water-in-oil emulsions, whereas hydrophilic emulsifiers (such as Tweens, proteins and

polysaccharides) are utilized to stabilize the O-W2 interface in the multiple emulsions [225-227]. Since

there are two separate interfaces, there may be no competition between the mixed emulsifiers for the

interfaces. However, if small molecule surfactants are used, then they may be able to diffuse through

the oil phase and therefore move from one interface to another.

47
 Fig. 15

Multiple emulsions typically have a lower thermodynamic and kinetic stability than single

emulsions because of their increased complexity [228]. In addition, to destabilization by coalescence,

flocculation and gravitational seperation, they may also breakdown due to migration of water

molecules between the inner and the outer aqueous phases [229]. Neverthless, multiple emulsions do

have some advantages over single emulsions for certain applications. The inner water (W1) phase can

be used as a carrier of hydrophilic flavors, aromas, colors, preservatives, antimicrobials, vitamins,

minerals, polyphenols, amino acids, bioactive proteins, enzymes, or peptides (Table 2). Entrapment of

these ingredients within the internal water phase can protect them from environmental degradation, can

control their release profile, or can mask undesirable sensory attributes [216].

Table 2

Table 2- Some selected studies on application of mixed emulsifiers in W1/O/W2 double emulsions for encapsulation

purposes

Emulsifier type at the W1/O Emulsifier type at the O/W2 Encapsulated Reference
interface interface ingredient within W1
droplets
Span 80 WPCa+ pectin Saffron extract [222, 228]
Span 80 WPC+ pectin Olive leaf phenolics [230, 231]
b
PGPR WPC + Arabic Gum Crocin [224, 227]
WPC + Angum gum
Span 80; PGPR WPC+ pectin Folic acid [221, 223, 226, 229]
Span 80 Tween 80 + β-Cyclodextrin Lactobacillus [232]
dellbrueckii

Tween 80; PGPR Tween 80 + PGPR Apigenin [233]

Sodium Caseinate + PGPR Sodium Caseinate + PGPR Mg2+ [234]
Gelatin + PGPR Kafirin nanoparticles Anthocyanin [235]
c
Beetroot juice powder + WPI Beetroot juice [236]
PGPR
a
Whey protein concentrate
b
Polyglycerol polyricinoleate
c
Whey protein isolate

48
 The stability of W1/O/W2 emulsions can be improved by careful selection of individual or mixed

biopolymer emulsifiers to stabilize the oil droplets [230, 231]. Recently, a number of studies have been

carrioud out on using mixed emulsifiers for the encapsulation of hydrophilic bioactive agents in

multiple emulsions (Table 2), including olive leaf phenolics, saffron extracts, folic acid, and crocin.

These studies have shown that multiple emulsions can often protect encapsulated bioactive ingredients

better than single emulsions, and can also be designed to control the release behavior of the bioactives.

For instance, Mohammadi et al (2016) showed that multiple emulsions stabilized with with a mixture

of hydrophilic emulsifiers (pectin-whey protein complexes) and lipophilic emulsifiers (Span 80) could

retard the oxidation of soybean oil much better than single emulsions. Assadpour et al (2016) reported

that the same type of multiple emulsion could be used to deliver folic acid to the large intestine, while

Faridi et al (2015) showed that they could be used to improve the stability of encapsulated saffron

extracts during spray drying.

7.6. Gastrointestinal fate

The nature of the interfacial layer in oil-in-water emulsions impacts their gastrointestinal (GIT)

fate after oral ingestion, which can be utilized to design functional foods, supplements, and

pharmaceutical products with desirable attributes [237-239]. The bioavailability of hydrophobic

nutraceuticals and drugs can be increased by designing emulsions that are rapidly digested in the small

intestine and form mixed micelles that can solubilize and transport the bioactive agents [240].

Conversely, slowing down the rate of lipid digestion may be utilized to design foods that induce satiety

or satiation, which can help tackle overeating and obesity [237]. Finally, it is possible to design

emulsion-based delivery systems that protect bioactive agents within the upper GIT tract, but that

release them in the colon [241].

A number of studies have shown that interfacial coatings formed from mixed emulsifiers can be

used to retard the rate of lipid digestion in emulsions by inhibiting the adsorption of bile salts or

49
digestive enzymes to the lipid droplet surfaces [119, 138]. Mixing a non-ionic surfactant (Tween 20)

with emulsions containing β-lactoglobulin/alginate coated oil droplets was shown to alter their fate in a

simulated GIT [242]. The presence of the non-ionic surfactant was found to increase the rate of lipid

digestion, which was attributed to its ability to inhibit extensive droplet flocculation in the small

intestine.

The rate of lipid digestion can also be modulated by using mixtures of surfactants to coat the oil

droplets in the original emulsions. In one study, different ratios of phospholipids (soybean lecithin)

and non-ionic surfactants (Pluronic F68) were used to form the initial emulsions [243]. Pluronic F68

binds strongly to the oil droplet surfaces and thereby inhibits lipase adsorption and lipid digestion,

wherease the lecithin only binds weakly and is therefore not as effective at inhibiting lipid digestion.

Consequently, the rate of lipid digestion could be controlled by changing the ratio of these two

surfactants. However, another study using oil droplets coated by lecithin and Pluronic F68 indicated

that the non-ionic surfactant was not effective at inhibiting lipid digestion, but Pluronic F127 was

[244]. This observation was supported by earlier work that also showed that Pluronic F127 was more

effective at inhibiting lipid digestion than Pluronic F68, due to its ability to strongly bind to the

interface and sterically hinder lipase adsorption [245, 246]. In the same study, the authors showed that

the lipid digestion profile could be controlled by varying the ratio of Pluronic F127 to F68, with

increasing inhibition of lipid digestion occurring as the fraction of Pluronic F127 used to form the

emulsions was increased.

Another study examined the impact of three types of surfactants, CTAB (cationic), anionic (SDS),

and non-ionic (Tween 20), on the simulated GIT fate of gum Arabic (GA)-coated droplets [247]. The

interaction of the surfactants with the polysaccharide-coated droplets depended on their charge: the

cationic surfactant formed interfacial electrostatic complxes with anionic GA; the anionic surfactant

displaced the GA from the droplet surfaces; and the non-ionic surfactant co-adsorbed with the GA.

50
The lipid digestion rate could be controlled to be either faster or slower than the control (GA-coated

droplets) by altering the type and amounts of surfactants added. This effect was attributed to the

influence of the surfactants on droplet flocculation and their ability to interfere with the adsorption of

bile salts and lipase onto the lipid droplet surfaces.

7.7. Rheological properties

The rheological properties of emulsions, such as viscosity, viscoelasticity, yield stress, or elastic

modulus, play an important role in determining their functional performance in many commercial

applications [1]. The rheological properties of emulsions are strongly influenced by the nature of the

interactions between the droplets they contain, which can be modulated by altering interfacial

properties, such as thickness, charge, and hydrophobicity. Consequently, the rheological properties of

emulsions can be modulated by using mixed emulsifiers that alter their interfacial characteristics.

Studies have shown that there may be a steep increase in the viscosity of oil-in-water emulsions

containing a mixture of surfactants and polymers that interact with each other due to their ability of the

surfactant-polymer complexes to adsorb to the surfaces of two or more droplets and induce bridging

flocculation [248]. The addition of non-ionic surfactants (such as Tween 20) to oil-in-water emulsions

containing protein-coated droplets near their isoelectric point has been shown to reduce their viscosity

by disrupting the flocs formed [178]. The non-ionic surfactant adsorbs onto the droplet surfaces and

displaces some of the adsorbed proteins, which increases the steric repulsion between the droplets.

Mixing an emulsion containing oil droplets coated by cationic emulsifiers with another emulsion

containing oil droplets coated by anionic emulsifiers has been shown to lead to a large increase in

viscosity or to gelation due to extensive droplet aggregation promoted by electrostatic attraction

between the oppositely charged droplets [249-251].

51
7.8. Powder formation

It is well established that there is a strong relationship between the surface composition of powder

particles, their drying performance and their physiochemical properties e.g., cohesiveness,

dispersibility, and shelf-life [252]. In the production of fat-rich powders, a high surface fat content can

lead to a number of problems, including powder stickiness, low powder recovery, production down-

time, reduced shelf life, and undesirable functional attributes. The surface composition of emulsion-

based powders is mainly governed by the emulsifier system used [253]. Air/liquid interfaces are

generated when an emulsion is atomized within a spray dryer. Any surface-active components present

in the emulsion will tend to migrate towards this interface, and will therefore be located at the exterior

of any powder particles formed.

There have been a number of studies showing that the properties of the particles in powders

formed by spray drying emulsions depend on whether single or mixed emulsifiers are used. For

instance, Drapala et al (2017) reported that differences in the wettability, surface topography, and

particle size distribution of the dried and reconstituted powder were linked to the emulsifier system

used. Also, they showed that the build-up of powder on the walls of the spray dryer during the

dehydration process depended on emulsifier type: utilization of a mixture of whey protein hydrolysate

and Citrem led to a greater build-up than a mixture of whey protein hydrolysate and maltodextrin.

8. Conclusions

Mixtures of different kinds of emulsifiers can be used for the formulation of emulsion-based

products, such as those found in the food, cosmetics, agrochemical, and pharmaceutical industries. In

the food industry, these emulsifiers are usually selected from either high molecular weight biopolymers

(such as proteins or polysaccharides) or low molecular weight surfactants (such as lecithins, saponins,

Tweens, or Spans). Each type of emulsifier has its own unique molecular and physicochemical

properties that can be used to modulate the interfacial properties of emulsion droplets. The utilization

52
of mixed emulsifiers may be either beneficial or detrimental to emulsion formation and stability, and

therefore the type and level of emulsifiers employed must be carefully selected. Often this involves

systematically characterizing the interfacial properties of mixed emulsifier systems, such as adsorption

kinetics, and interfacial tension, rheology, thickness, composition, and charge. Novel or improved

functional attributes can often be obtained by using emulsifier mixtures rather than single emulsifiers,

e.g., enhancements in antioxidant activity, flavor encapsulation, nutraceutical delivery, or textural

attributes. At present, there is still a relatively poor understanding of the impact of different

combinations of emulsifiers on emulsion formation and stability, and this will be important are of

future studies. In particular, emphasis should be placed on the utilization of mixtures of natural

emulsifiers, such as proteins, polysaccharides, phospholipids and saponins, since there is increasing

demand from consumers for clean-label products.

9. Acknowledgements

This material was partly based upon work supported by the National Institute of Food and

Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491) and USDA, AFRI

Grants (2013-03795, 2014-67021 and 2016-25147).

10. References

[1] McClements DJ. Food Emulsions: Principles, Practices, and Techniques. Third Edition ed. Boca
Raton, FL: CRC Press; 2015.
[2] Puri A, Loomis K, Smith B, Lee JH, Yavlovich A, Heldman E, et al. Lipid-Based Nanoparticles as
Pharmaceutical Drug Carriers: From Concepts to Clinic. Critical Reviews in Therapeutic Drug Carrier
Systems. 2009;26:523-80.
[3] Yukuyama MN, Ghisleni DDM, Pinto TJA, Bou-Chacra NA. Nanoemulsion: process selection and
application in cosmetics - a review. International Journal of Cosmetic Science. 2016;38:13-24.

53
[4] McClements DJ. Edible nanoemulsions: fabrication, properties, and functional performance. Soft
Matter. 2011;7:2297-316.
[5] Hiemenz PC, Rajagopalan R. Principles of Colloid and Surface Chemistry. Third Edition ed. New
York: Marcel Dekker; 1997.
[6] Israelachvili J. Intermolecular and Surface Forces, Third Edition. Third Edition ed. London, UK:
Academic Press; 2011.
[7] Kralova I, Sjoblom J. Surfactants Used in Food Industry: A Review. Journal of Dispersion Science
and Technology. 2009;30:1363-83.
[8] Fitzgerald A. Emulsifiers: Properties, Functions and Applications. Hauppauge, NY Nova Science
Publishers; 2015.
[9] Hasenhuettl GL. Overview of food emulsifiers2008.
[10] McClements DJ, Gumus CE. Natural emulsifiers - Biosurfactants, phospholipids, biopolymers,
and colloidal particles: Molecular and physicochemical basis of functional performance. Advances in
Colloid and Interface Science. 2016;234:3-26.
[11] Dickinson E. Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures.
Food Hydrocolloids. 2011;25:1966-83.
[12] Guzey D, McClements DJ. Formation, stability and properties of multilayer emulsions for
application in the food industry. Advances in Colloid and Interface Science. 2006;128:227-48.
[13] Forgiarini A, Esquena J, Gonzalez C, Solans C. Formation and stability of nano-emulsions in
mixed nonionic surfactant systems. In: Koutsoukos PG, (editor). Trends in Colloid and Interface
Science Xv. Vol. 1182001. p. 184-9.
[14] Xiao J, Li YQ, Huang QR. Recent advances on food-grade particles stabilized Pickering
emulsions: Fabrication, characterization and research trends. Trends in Food Science & Technology.
2016;55:48-60.
[15] Berton-Carabin CC, Schroen K. Pickering Emulsions for Food Applications: Background, Trends,
and Challenges. In: Doyle MP, Klaenhammer TR, (editors). Annual Review of Food Science and
Technology, Vol 6. Vol. 62015. p. 263-97.
[16] Lam S, Velikov KP, Velev OD. Pickering stabilization of foams and emulsions with particles of
biological origin. Current Opinion in Colloid & Interface Science. 2014;19:490-500.
[17] Tavernier I, Wijaya W, Van der Meeren P, Dewettinck K, Patel AR. Food-grade particles for
emulsion stabilization. Trends in Food Science & Technology. 2016;50:159-74.

54
[18] Tcholakova S, Denkov ND, Lips A. Comparison of solid particles, globular proteins and
surfactants as emulsifiers. Physical Chemistry Chemical Physics. 2008;10:1608-27.
[19] Stauffer C. Emulsifiers. St. Paul, MN: Eagan Press 1999.
[20] Krog NJ, Sparso FV. Food emulsifiers: Their chemical and physical properties. Food emulsions.
2004;12.
[21] Ozturk B, McClements DJ. Progress in natural emulsifiers for utilization in food emulsions.
Current Opinion in Food Science. 2016;7:1-6.
[22] Dickinson E. Hydrocolloids at interfaces and the influence on the properties of dispersed systems.
Food hydrocolloids. 2003;17:25-39.
[23] Lam RSH, Nickerson MT. Food proteins: A review on their emulsifying properties using a
structure-function approach. Food Chemistry. 2013;141:975-84.
[24] Wu TH, Wang ZN. Micellization and Phase Behavior of Biosurfactant Bile Salts. Progress in
Chemistry. 2011;23:80-9.
[25] Malik NA. Solubilization and Interaction Studies of Bile Salts with Surfactants and Drugs: a
Review. Applied Biochemistry and Biotechnology. 2016;179:179-201.
[26] Stanimirova R, Marinova K, Tcholakova S, Denkov ND, Stoyanov S, Pelan E. Surface Rheology
of Saponin Adsorption Layers. Langmuir. 2011;27:12486-98.
[27] Pagureva N, Tcholakova S, Golemanov K, Denkov N, Pelan E, Stoyanov SD. Surface properties
of adsorption layers formed from triterpenoid and steroid saponins. Colloids and Surfaces a-
Physicochemical and Engineering Aspects. 2016;491:18-28.
[28] Holmberg K, Jonsson B, Kronberg B, Lindman B. Surfactants and Polymers in Aqueous Solution.
Second Edition ed. New York, N.Y.: Wiley; 2002.
[29] Krongberg B, Holmberg K, Lindman B. Surface Chemistry of Surfactants and Polymers.
Chichester, West Sussex, UK: John Wiley and Sons; 2014.
[30] Ngouemazong ED, Christiaens S, Shpigelman A, Van Loey A, Hendrickx M. The Emulsifying
and Emulsion-Stabilizing Properties of Pectin: A Review. Comprehensive Reviews in Food Science
and Food Safety. 2015;14:705-18.
[31] Klang V, Valenta C. Lecithin-based nanoemulsions. Journal of Drug Delivery Science and
Technology. 2011;21:55-76.
[32] van Nieuwenhuyzen W, Tomas MC. Update on vegetable lecithin and phospholipid technologies.
European Journal of Lipid Science and Technology. 2008;110:472-86.

55
[33] Cui L, Decker EA. Phospholipids in foods: prooxidants or antioxidants? Journal of the Science of
Food and Agriculture. 2016;96:18-31.
[34] Joshi A, Paratkar SG, Thorat BN. Modification of lecithin by physical, chemical and enzymatic
methods. European journal of lipid science and technology. 2006;108:363-73.
[35] Pichot R, Watson RL, Norton IT. Phospholipids at the interface: current trends and challenges.
International journal of molecular sciences. 2013;14:11767-94.
[36] Zhu D, Damodaran S. Dairy Lecithin from Cheese Whey Fat Globule Membrane: Its Extraction,
Composition, Oxidative Stability, and Emulsifying Properties. Journal of the American Oil Chemists'
Society. 2013;90:217-24.
[37] Cabezas D, Diehl BW, Tomás MC. Emulsifying properties of hydrolysed and low HLB sunflower
lecithin mixtures. European Journal of Lipid Science and Technology. 2016;118:975 - 83.
[38] Gunstone FD. Phospholipid Technology and Applications. Cambridge, UK: Woodhead
Publishing; 2008.
[39] Damodaran S, Parkin KL, Fennema OR. Fennema's Food Chemistry. Fourth ed. Boca Raton, FL.:
CRC Press; 2007.
[40] Dickinson E. Protein-stabilized emulsions. Journal of Food Engineering. 1994;22:59-74.
[41] McClements DJ. Protein-stabilized emulsions. Current Opinion in Colloid & Interface Science.
2004;9:305-13.
[42] Righetti PG, Tudor G, Ek K. ISOELECTRIC POINTS AND MOLECULAR-WEIGHTS OF
PROTEINS - A NEW TABLE. Journal of Chromatography. 1981;220:115-94.
[43] Garti N, Leser ME. Emulsification properties of hydrocolloids. Polymers for Advanced
Technologies. 2001;12:123-35.
[44] Sweedman MC, Tizzotti MJ, Schaefer C, Gilbert RG. Structure and physicochemical properties of
octenyl succinic anhydride modified starches: A review. Carbohydrate Polymers. 2013;92:905-20.
[45] Meyers D. Surfactant Science and Technology. Hoboken, NJ: John Wiley & Sons; 2006.
[46] Magnusson E, Nilsson L, Bergenstahl B. Effect of the dispersed state of phospholipids on
emulsification-Part 1. Phosphatidylcholine. Colloids and Surfaces a-Physicochemical and Engineering
Aspects. 2016;506:794-803.
[47] Drapala KP, Auty MAE, Mulvihill DM, O'Mahony JA. Influence of lecithin on the processing
stability of model whey protein hydrolysate-based infant formula emulsions. International Journal of
Dairy Technology. 2015;68:322-33.

56
[48] Xin X, Zhang HX, Xu GY, Tan YB, Zhang J, Lv X. Influence of CTAB and SDS on the properties
of oil-in-water nano-emulsion with paraffin and span 20/Tween 20. Colloids and Surfaces a-
Physicochemical and Engineering Aspects. 2013;418:60-7.
[49] Liu Y, Wei FL, Wang YY, Zhu GN. Studies on the formation of bifenthrin oil-in-water nano-
emulsions prepared with mixed surfactants. Colloids and Surfaces a-Physicochemical and Engineering
Aspects. 2011;389:90-6.
[50] van Oss CJ. Development and applications of the interfacial tension between water and organic or
biological surfaces. Colloids and Surfaces B-Biointerfaces. 2007;54:2-9.
[51] Somasundaran P, Huang L. Adsorption/aggregation of surfactants and their mixtures at solid-
liquid interfaces. Advances in Colloid and Interface Science. 2000;88:179-208.
[52] Fainerman VB, Lucassen-Reynders E, Miller R. Adsorption of surfactants and proteins at fluid
interfaces. Colloids and Surfaces a-Physicochemical and Engineering Aspects. 1998;143:141-65.
[53] Fainerman VB, Lucassen-Reynders EH. Adsorption of single and mixed ionic surfactants at fluid
interfaces. Advances in Colloid and Interface Science. 2002;96:295-323.
[54] Fainerman VB, Aksenenko EV, Kragel J, Miller R. Thermodynamics, interfacial pressure
isotherms and dilational rheology of mixed protein-surfactant adsorption layers. Advances in Colloid
and Interface Science. 2016;233:200-22.
[55] Norde W. Colloids and Interfaces in Life Sciences and Bionanotechnology. Second Edition ed.
Boca Raton, FL: CRC Press; 2011.
[56] Kotsmar C, Pradines V, Alahverdjieva VS, Aksenenko EV, Fainerman VB, Kovalchuk VI, et al.
Thermodynamics, adsorption kinetics and rheology of mixed protein-surfactant interfacial layers.
Advances in Colloid and Interface Science. 2009;150:41-54.
[57] Dickinson E. Colloid science of mixed ingredients. Soft Matter. 2006;2:642-52.
[58] Pugnaloni LA, Dickinson E, Ettelaie R, Mackie AR, Wilde PJ. Competitive adsorption of proteins
and low-molecular-weight surfactants: computer simulation and microscopic imaging. Advances in
Colloid and Interface Science. 2004;107:27-49.
[59] Guzman E, Llamas S, Maestro A, Fernandez-Pena L, Akanno A, Miller R, et al. Polymer-
surfactant systems in bulk and at fluid interfaces. Advances in Colloid and Interface Science.
2016;233:38-64.
[60] Bos MA, van Vliet T. Interfacial rheological properties of adsorbed protein layers and surfactants:
a review. Advances in Colloid and Interface Science. 2001;91:437-71.

57
[61] Acosta EJ, Bhakta AS. The HLD-NAC Model for Mixtures of Ionic and Nonionic Surfactants.
Journal of Surfactants and Detergents. 2009;12:7-19.
[62] Liu WR, Sun DJ, Li CF, Liu Q, Xu H. Formation and stability of paraffin oil-in-water nano-
emulsions prepared by the emulsion inversion point method. Journal of Colloid and Interface Science.
2006;303:557-63.
[63] Li CF, Mei Z, Liu Q, Wang J, Xu J, Sun DJ. Formation and properties of paraffin wax submicron
emulsions prepared by the emulsion inversion point method. Colloids and Surfaces a-Physicochemical
and Engineering Aspects. 2010;356:71-7.
[64] Derkach SR. Interfacial layers of complex-forming ionic surfactants with gelatin. Advances in
Colloid and Interface Science. 2015;222:172-98.
[65] Dalgleish DG. Adsorption of protein and the stability of emulsions. Trends in Food Science &
Technology. 1997;8:1-6.
[66] McClements DJ, Decker EA. Lipid oxidation in oil-in-water emulsions: Impact of molecular
environment on chemical reactions in heterogeneous food systems. Journal of Food Science.
2000;65:1270-82.
[67] Ziani K, Chang Y, McLandsborough L, McClements DJ. Influence of Surfactant Charge on
Antimicrobial Efficacy of Surfactant-Stabilized Thyme Oil Nanoemulsions. Journal of Agricultural and
Food Chemistry. 2011;59:6247-55.
[68] Demetriades K, McClements DJ. Influence of sodium dodecyl sulfate on the physicochemical
properties of whey protein-stabilized emulsions. Colloids and Surfaces a-Physicochemical and
Engineering Aspects. 2000;161:391-400.
[69] Kong LG, Beattie JK, Hunter RJ. Electroacoustic study of BSA or lecithin stabilised soybean
oilin-water emulsions and SDS effect. Colloids and Surfaces B-Biointerfaces. 2003;27:11-21.
[70] Karbstein H, Schubert H. Developments in the continuous mechanical production of oil-in-water
macro-emulsions. Chemical Engineering and Processing. 1995;34:205-11.
[71] Stang M, Karbstein H, Schubert H. Adsorption-kinetics of emulsifiers at oil-water interfaces and
their effect on mechanical emulsification. Chemical Engineering and Processing. 1994;33:307-11.
[72] Jafari SM, Assadpoor E, He YH, Bhandari B. Re-coalescence of emulsion droplets during high-
energy emulsification. Food Hydrocolloids. 2008;22:1191-202.
[73] Ozturk B, Argin S, Ozilgen M, McClements DJ. Formation and stabilization of nanoemulsion-
based vitamin E delivery systems using natural biopolymers: Whey protein isolate and gum arabic.
Food Chemistry. 2015;188:256-63.

58
[74] Chanamai R, McClements DJ. Comparison of gum arabic, modified starch, and whey protein
isolate as emulsifiers: Influence of pH, CaCl(2) and temperature. Journal of Food Science.
2002;67:120-5.
[75] Beverung CJ, Radke CJ, Blanch HW. Protein adsorption at the oil/water interface: characterization
of adsorption kinetics by dynamic interfacial tension measurements. Biophysical Chemistry.
1999;81:59-80.
[76] McClements DJ, Monahan FJ, Kinsella JE. Disulfide bond formation affects stability of whey-
protein isolate emulsions. Journal of Food Science. 1993;58:1036-9.
[77] Monahan FJ, McClements DJ, German JB. Disulfide-mediated polymerization reactions and
physical properties of heated WPI-stabilized emulsions. Journal of Food Science. 1996;61:504-9.
[78] Courthaudon JL, Dickinson E, Matsumura Y, Clark DC. Competitive adsorption of beta-
lactoglobulin + tween 20 at the oil-water interface. Colloids and Surfaces. 1991;56:293-300.
[79] Kelley D, McClements DJ. Interactions of bovine serum albumin with ionic surfactants in aqueous
solutions. Food Hydrocolloids. 2003;17:73-85.
[80] Otzen D. Protein-surfactant interactions: A tale of many states. Biochimica Et Biophysica Acta-
Proteins and Proteomics. 2011;1814:562-91.
[81] Lesmes U, Sandra S, Decker EA, McClements DJ. Impact of surface deposition of lactoferrin on
physical and chemical stability of omega-3 rich lipid droplets stabilised by caseinate. Food Chemistry.
2010;123:99-106.
[82] Kralchevsky PA, Danov KD, Anachkov SE. Micellar solutions of ionic surfactants and their
mixtures with nonionic surfactants: Theoretical modeling vs. Experiment. Colloid Journal.
2014;76:255-70.
[83] Benichou A, Aserin A, Garti N. Protein-polysaccharide interactions for stabilization of food
emulsions. Journal of Dispersion Science and Technology. 2002;23:93-123.
[84] Ye AQ. Complexation between milk proteins and polysaccharides via electrostatic interaction:
principles and applications - a review. International Journal of Food Science and Technology.
2008;43:406-15.
[85] Fuenzalida JP, Goycoolea FM. Polysaccharide-Protein Nanoassemblies: Novel Soft Materials for
Biomedical and Biotechnological Applications. Current Protein & Peptide Science. 2015;16:89-99.
[86] Semenova MG. Thermodynamic analysis of the impact of molecular interactions on the
functionality of food biopolymers in solution and in colloidal systems. Food Hydrocolloids.
2007;21:23-45.

59
[87] Khan A, Marques EF. Synergism and polymorphism in mixed surfactant systems. Current Opinion
in Colloid & Interface Science. 1999;4:402-10.
[88] Reichert CL, Salminen H, Leuenberger BH, Hinrichs J, Weiss J. Miscibility of Quillaja Saponins
with other Co-surfactants under Different pH Values. Journal of Food Science. 2015;80:E2495-E503.
[89] Schmitt C, Sanchez C, Desobry-Banon S, Hardy J. Structure and technofunctional properties of
protein-polysaccharide complexes: A review. Critical Reviews in Food Science and Nutrition.
1998;38:689-753.
[90] van de Velde F, de Hoog EHA, Oosterveld A, Tromp RH. Protein-Polysaccharide Interactions to
Alter Texture. In: Doyle MP, Klaenhammer TR, (editors). Annual Review of Food Science and
Technology, Vol 6. Vol. 62015. p. 371-88.
[91] Kayitmazer AB, Seeman D, Minsky BB, Dubin PL, Xu YS. Protein-polyelectrolyte interactions.
Soft Matter. 2013;9:2553-83.
[92] Flanagan SE, Malanowski AJ, Kizilay E, Seeman D, Dubin PL, Donato-Capel L, et al. Complex
Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and beta-Lactoglobulin.
Langmuir. 2015;31:1776-83.
[93] Kizilay E, Seeman D, Yan YF, Du XS, Dubin PL, Donato-Capel L, et al. Structure of bovine beta-
lactoglobulin-lactoferrin coacervates. Soft Matter. 2014;10:7262-8.
[94] Tolstoguzov VB. Functional properties of food proteins and role of protein-polysaccharide
interaction. Food Hydrocolloids. 1991;4:429-68.
[95] Schmitt C, Turgeon SL. Protein/polysaccharide complexes and coacervates in food systems.
Advances in Colloid and Interface Science. 2011;167:63-70.
[96] Turgeon SL, Schmitt C, Sanchez C. Protein-polysaccharide complexes and coacervates. Current
Opinion in Colloid & Interface Science. 2007;12:166-78.
[97] Fischer P. Rheology of interfacial protein-polysaccharide composites. European Physical Journal-
Special Topics. 2013;222:73-81.
[98] Razumovsky L, Damodaran S. Incompatibility of mixing of proteins in adsorbed binary protein
films at the air-water interface. Journal of Agricultural and Food Chemistry. 2001;49:3080-6.
[99] Bai L, McClements DJ. Extending Emulsion Functionality: Post-Homogenization Modification of
Droplet Properties. Processes. 2016;4.
[100] Chen JS, Dickinson E. Time-dependent competitive adsorption of milk-proteins and surfactants
in oil-in-water emulsions. Journal of the Science of Food and Agriculture. 1993;62:283-9.

60
[101] Dickinson E, Hong ST. Surface coverage of beta-lactoglobulin at the oil-water interface -
influence of protein heat-treatment and various emulsifiers. Journal of Agricultural and Food
Chemistry. 1994;42:1602-6.
[102] Monahan FJ, German JB, Kinsella JE. Effect of ph and temperature on protein unfolding and
thiol-disulfide interchange reactions during heat-induced gelation of whey proteins. Journal of
Agricultural and Food Chemistry. 1995;43:46-52.
[103] Courthaudon JL, Dickinson E, Matsumura Y, Williams A. Influence of emulsifier on the
competitive adsorption of whey proteins in emulsions. Food Structure. 1991;10:109-15.
[104] Dickinson E, Matsumura Y. Time-dependent polymerization of beta-lactoglobulin through
disulfide bonds at the oil-water interface in emulsions. International Journal of Biological
Macromolecules. 1991;13:26-30.
[105] Mackie A, Wilde P. The role of interactions in defining the structure of mixed protein-surfactant
interfaces. Advances in Colloid and Interface Science. 2005;117:3-13.
[106] Mackie AR, Gunning AP, Wilde PJ, Morris VJ. Orogenic Displacement of Protein from the
Oil/Water Interface. Langmuir. 2000;16:2242-7.
[107] Horne DS, Atkinson PJ, Dickinson E, Pinfield VJ, Richardson RM. Neutron reflectivity study of
competitive adsorption of beta-lactoglobulin and nonionic surfactant at the air-water interface.
International Dairy Journal. 1998;8:73-7.
[108] Wilde P, Mackie A, Husband F, Gunning P, Morris V. Proteins and emulsifiers at liquid
interfaces. Advances in Colloid and Interface Science. 2004;108:63-71.
[109] Arboleya J-C, Wilde PJ. Competitive adsorption of proteins with methylcellulose and
hydroxypropyl methylcellulose. Food Hydrocolloids. 2005;19:485-91.
[110] Jafari SM, He Y, Bhandari B. Effectiveness of encapsulating biopolymers to produce sub-micron
emulsions by high energy emulsification techniques. Food Research International. 2007;40:862-73.
[111] Kerstens S, Murray BS, Dickinson E. Microstructure of β-lactoglobulin-stabilized emulsions
containing non-ionic surfactant and excess free protein: Influence of heating. Journal of Colloid and
Interface Science. 2006;296:332-41.
[112] Li X, Al-Assaf S, Fang Y, Phillips GO. Competitive adsorption between sugar beet pectin (SBP)
and hydroxypropyl methylcellulose (HPMC) at the oil/water interface. Carbohydrate Polymers.
2013;91:573-80.
[113] Diftis N, Kiosseoglou V. Competitive adsorption between a dry-heated soy protein–dextran
mixture and surface-active materials in oil-in-water emulsions. Food Hydrocolloids. 2004;18:639-46.

61
[114] Matsumiya K, Takahashi Y, Nakanishi K, Dotsu N, Matsumura Y. Diglycerol esters of fatty
acids promote severe coalescence between protein-stabilized oil droplets by emulsifier–protein
competitive interactions. Food Hydrocolloids. 2014;42:397-402.
[115] Chang Y, Hu Y, McClements DJ. Competitive adsorption and displacement of anionic
polysaccharides (fucoidan and gum arabic) on the surface of protein-coated lipid droplets. Food
Hydrocolloids. 2016;52:820-6.
[116] Cornec M, Wilde PJ, Gunning PA, Mackie AR, Husband FA, Parker ML, et al. Emulsion
stability as affected by competitive adsorption between an oil-soluble emulsifier and milk proteins at
the interface. Journal of Food Science. 1998;63:39-43.
[117] Patino JMR, Pilosof AMR. Protein-polysaccharide interactions at fluid interfaces. Food
Hydrocolloids. 2011;25:1925-37.
[118] Mun S, Decker EA, McClements DJ. Effect of molecular weight and degree of deacetylation of
chitosan on the formation of oil-in-water emulsions stabilized by surfactant-chitosan membranes.
Journal of Colloid and Interface Science. 2006;296:581-90.
[119] Klinkesorn U, McClements DJ. Impact of Lipase, Bile Salts, and Polysaccharides on Properties
and Digestibility of Tuna Oil Multilayer Emulsions Stabilized by Lecithin-Chitosan. Food Biophysics.
2010;5:73-81.
[120] Jourdain LS, Schmitt C, Leser ME, Murray BS, Dickinson E. Mixed Layers of Sodium Caseinate
plus Dextran Sulfate: Influence of Order of Addition to Oil-Water Interface. Langmuir. 2009;25:10026-
37.
[121] Jourdain L, Leser ME, Schmitt C, Michel M, Dickinson E. Stability of emulsions containing
sodium caseinate and dextran sulfate: Relationship to complexation in solution. Food Hydrocolloids.
2008;22:647-59.
[122] Dickinson E. Properties of emulsions stabilized with milk proteins: Overview of some recent
developments. Journal of Dairy Science. 1997;80:2607-19.
[123] Bortnowska G. Multi layer Oil-in-Water Emulsions: Formation, Characteristics and Application
as the Carriers for Lipophilic Bioactive Food Components - a Review. Polish Journal of Food and
Nutrition Sciences. 2015;65:157-66.
[124] Grigoriev DO, Miller R. Mono- and multilayer covered drops as carriers. Current Opinion in
Colloid & Interface Science. 2009;14:48-59.
[125] Shchukina EM, Shchukin DG. Layer-by-layer coated emulsion microparticles as storage and
delivery tool. Current Opinion in Colloid & Interface Science. 2012;17:281-9.

62
[126] McClements DJ. Design of Nano-Laminated Coatings to Control Bioavailability of Lipophilic
Food Components. Journal of Food Science. 2010;75:R30-R42.
[127] Hammond PT. Form and function in multilayer assembly: New applications at the nanoscale.
Advanced Materials. 2004;16:1271-93.
[128] Chang YG, McClements DJ. Interfacial deposition of an anionic polysaccharide (fucoidan) on
protein-coated lipid droplets: Impact on the stability of fish oil-in-water emulsions. Food
Hydrocolloids. 2015;51:252-60.
[129] Guzey D, McClements DJ. Impact of electrostatic interactions on formation and stability of
emulsions containing oil droplets coated by beta-lactoglobulin-pectin complexes. Journal of
Agricultural and Food Chemistry. 2007;55:475-85.
[130] Surh J, Decker EA, McClements DJ. Influence of pH and pectin type on properties and stability
of sodium-caseinate stabilized oil-in-water emulsions. Food Hydrocolloids. 2006;20:607-18.
[131] Zeeb B, Lopez-Pena CL, Weiss J, McClements DJ. Controlling lipid digestion using enzyme-
induced crosslinking of biopolymer interfacial layers in multilayer emulsions. Food Hydrocolloids.
2015;46:125-33.
[132] Gu YS, Decker EA, McClements DJ. Application of multi-component biopolymer layers to
improve the freeze-thaw stability of oil-in-water emulsions: beta-lactoglobulin-iota-carrageenan-
gelatin. Journal of Food Engineering. 2007;80:1246-54.
[133] Shaw LA, McClements DJ, Decker EA. Spray-dried multilayered emulsions as a delivery method
for omega-3 fatty acids into food systems. Journal of Agricultural and Food Chemistry. 2007;55:3112-
9.
[134] Fustier P, Achouri A, Taherian AR, Britten M, Pelletier M, Sabik H, et al. Protein-Protein
Multilayer Oil-in-Water Emulsions for the Microencapsulation of Flaxseed Oil: Effect of Whey and
Fish Gelatin Concentration. Journal of Agricultural and Food Chemistry. 2015;63:9239-50.
[135] Kartal C, Unal MK, Otles S. Flaxseed Oil-In-Water Emulsions Stabilized by Multilayer
Membranes: Oxidative Stability and the Effects of pH. Journal of Dispersion Science and Technology.
2016;37:1683-91.
[136] Chen BC, Li HJ, Ding YP, Rao JJ. Improvement of physicochemical stabilities of emulsions
containing oil droplets coated by non-globular protein-beet pectin complex membranes. Food Research
International. 2011;44:1468-75.

63
[137] Gudipati V, Sandra S, McClements DJ, Decker EA. Oxidative Stability and in Vitro Digestibility
of Fish Oil-in-Water Emulsions Containing Multilayered Membranes. Journal of Agricultural and Food
Chemistry. 2010;58:8093-9.
[138] Hu M, Li Y, Decker EA, Xiao H, McClements DJ. Impact of Layer Structure on Physical
Stability and Lipase Digestibility of Lipid Droplets Coated by Biopolymer Nanolaminated Coatings.
Food Biophysics. 2011;6:37-48.
[139] Zeeb B, Weiss J, McClements DJ. Electrostatic modulation and enzymatic cross-linking of
interfacial layers impacts gastrointestinal fate of multilayer emulsions. Food Chemistry. 2015;180:257-
64.
[140] Hakansson A, Tragardh C, Bergenstahl B. Studying the effects of adsorption, recoalescence and
fragmentation in a high pressure homogenizer using a dynamic simulation model. Food Hydrocolloids.
2009;23:1177-83.
[141] Santana RC, Perrechil FA, Cunha RL. High- and Low-Energy Emulsifications for Food
Applications: A Focus on Process Parameters. Food Engineering Reviews. 2013;5:107-22.
[142] Hakansson A, Fuchs L, Innings F, Revstedt J, Tragardh C, Bergenstahl B. On flow-fields in a
high pressure homogenizer and its implication on drop fragmentation. In: Saravacos G, Taoukis P,
Krokida M, Karathanos V, Lazarides H, Stoforos N, et al., (editors). 11th International Congress on
Engineering and Food. Vol. 12011. p. 1353-8.
[143] Komaiko JS, McClements DJ. Formation of Food-Grade Nanoemulsions Using Low-Energy
Preparation Methods: A Review of Available Methods. Comprehensive Reviews in Food Science and
Food Safety. 2016;15:331-52.
[144] Solans C, Sole I. Nano-emulsions: Formation by low-energy methods. Current Opinion in
Colloid & Interface Science. 2012;17:246-54.
[145] Hakansson A, Innings F, Tragardh C, Bergenstahl B. A high-pressure homogenization
emulsification model-Improved emulsifier transport and hydrodynamic coupling. Chemical
Engineering Science. 2013;91:44-53.
[146] Hakansson A, Tragardh C, Bergenstahl B. Dynamic simulation of emulsion formation in a high
pressure homogenizer. Chemical Engineering Science. 2009;64:2915-25.
[147] Walstra P. Principles of emulsion formation. Chemical Engineering Science. 1993;48:333-49.
[148] Bai L, Huan SQ, Gu JY, McClements DJ. Fabrication of oil-in-water nanoemulsions by dual-
channel microfluidization using natural emulsifiers: Saponins, phospholipids, proteins, and
polysaccharides. Food Hydrocolloids. 2016;61:703-11.

64
[149] Yang Y, Leser ME, Sher AA, McClements DJ. Formation and stability of emulsions using a
natural small molecule surfactant: Quillaja saponin (Q-Naturale (R)). Food Hydrocolloids.
2013;30:589-96.
[150] Mao LK, Xu DX, Yang J, Yuan F, Gao YX, Zhao J. Effects of Small and Large Molecule
Emulsifiers on the Characteristics of beta-Carotene Nanoemulsions Prepared by High Pressure
Homogenization. Food Technology and Biotechnology. 2009;47:336-42.
[151] Mackie AR, Wilde PJ, Wilson DR, Clark DC. Competitive effects in the adsorbed layer of oil-in-
water emulsions stabilized by beta-lactoglobulin-tween 20 mixtures. Journal of the Chemical Society-
Faraday Transactions. 1993;89:2755-9.
[152] Mackie AR, Nativel S, Wilson DR, Ladha S, Clark DC. Process-induced changes in molecular
structure that alter adsorbed layer properties in oil-in-water emulsions stabilised by beta-
casein/Tween20 mixtures. Journal of the Science of Food and Agriculture. 1996;70:413-21.
[153] Hu QB, Gerhard H, Upadhyaya I, Venkitanarayanan K, Luo YC. Antimicrobial eugenol
nanoemulsion prepared by gum arabic and lecithin and evaluation of drying technologies. International
Journal of Biological Macromolecules. 2016;87:130-40.
[154] Xue J, Zhong QX. Thyme Oil Nanoemulsions Coemulsified by Sodium Caseinate and Lecithin.
Journal of Agricultural and Food Chemistry. 2014;62:9900-7.
[155] Ma QM, Davidson PM, Zhong QX. Nanoemulsions of thymol and eugenol co-emulsified by
lauric arginate and lecithin. Food Chemistry. 2016;206:167-73.
[156] Courthaudon JL, Dickinson E, Christie WW. Competitive adsorption of lecithin and beta-casein
in oil in water emulsions. Journal of Agricultural and Food Chemistry. 1991;39:1365-8.
[157] Maindarkar SN, Hoogland H, Henson MA. Predicting the combined effects of oil and surfactant
concentrations on the drop size distributions of homogenized emulsions. Colloids and Surfaces a-
Physicochemical and Engineering Aspects. 2015;467:18-30.
[158] Tcholakova S, Denkov ND, Ivanov IB, Campbell B. Coalescence stability of emulsions
containing globular milk proteins. Advances in Colloid and Interface Science. 2006;123:259-93.
[159] Maindarkar SN, Bongers P, Henson MA. Predicting the effects of surfactant coverage on drop
size distributions of homogenized emulsions. Chemical Engineering Science. 2013;89:102-14.
[160] Hakansson A, Hounslow MJ. Simultaneous determination of fragmentation and coalescence rates
during pilot-scale high-pressure homogenization. Journal of Food Engineering. 2013;116:7-13.

65
[161] Brosel S, Schubert H. Investigations on the role of surfactants in mechanical emulsification using
a high-pressure homogenizer with an orifice valve. Chemical Engineering and Processing.
1999;38:533-40.
[162] Donsi F, Sessa M, Ferrari G. Effect of Emulsifier Type and Disruption Chamber Geometry on
the Fabrication of Food Nanoemulsions by High Pressure Homogenization. Industrial & Engineering
Chemistry Research. 2012;51:7606-18.
[163] Lee LL, Niknafs N, Hancocks RD, Norton IT. Emulsification: Mechanistic understanding.
Trends in Food Science & Technology. 2013;31:72-8.
[164] Charoen R, Jangchud A, Jangchud K, Harnsilawat T, Naivikul O, McClements DJ. Influence of
Biopolymer Emulsifier Type on Formation and Stability of Rice Bran Oil-in-Water Emulsions: Whey
Protein, Gum Arabic, and Modified Starch. Journal of Food Science. 2011;76:E165-E72.
[165] Ozturk B, Argin S, Ozilgen M, McClements DJ. Formation and stabilization of nanoemulsion-
based vitamin E delivery systems using natural surfactants: Quillaja saponin and lecithin. Journal of
Food Engineering. 2014;142:57-63.
[166] Dickinson E, Ritzoulis C. Creaming and rheology of oil-in-water emulsions containing sodium
dodecyl sulfate and sodium caseinate. Journal of Colloid and Interface Science. 2000;224:148-54.
[167] Qian C, McClements DJ. Formation of nanoemulsions stabilized by model food-grade
emulsifiers using high-pressure homogenization: Factors affecting particle size. Food Hydrocolloids.
2011;25:1000-8.
[168] Wooster TJ, Golding M, Sanguansri P. Impact of Oil Type on Nanoemulsion Formation and
Ostwald Ripening Stability. Langmuir. 2008;24:12758-65.
[169] Su D, Zhong QX. Lemon oil nanoemulsions fabricated with sodium caseinate and Tween 20
using phase inversion temperature method. Journal of Food Engineering. 2016;171:214-21.
[170] Mei Z, Liu SY, Wang L, Jiang JJ, Xu J, Sun DJ. Preparation of positively charged oil/water
nano-emulsions with a sub-PIT method. Journal of Colloid and Interface Science. 2011;361:565-72.
[171] Hessien M, Singh N, Kim C, Prouzet E. Stability and Tunability of O/W Nanoemulsions
Prepared by Phase Inversion Composition. Langmuir. 2011;27:2299-307.
[172] Yu LJ, Li C, Xu J, Hao JC, Sun DJ. Highly Stable Concentrated Nanoemulsions by the Phase
Inversion Composition Method at Elevated Temperature. Langmuir. 2012;28:14547-52.
[173] Lv GJ, Wang FM, Cai WF, Zhang XB. Characterization of the emulsions formed by catastrophic
phase inversion. Colloids and Surfaces a-Physicochemical and Engineering Aspects. 2014;450:141-7.

66
[174] Bullon J, Molina J, Marquez R, Vejar F, Scorzza C, Forgiarini A. Nano-emulsification of
triglyceride oils for parenteral use by mean of a low-energy emulsification method. Revista Tecnica De
La Facultad De Ingenieria Universidad Del Zulia. 2007;30:428-36.
[175] McClements DJ, Rao J. Food-Grade Nanoemulsions: Formulation, Fabrication, Properties,
Performance, Biological Fate, and Potential Toxicity. Critical Reviews in Food Science and Nutrition.
2011;51:285-330.
[176] Wang B, Wang LJ, Li D, Adhikari B, Shi J. Effect of gum Arabic on stability of oil-in-water
emulsion stabilized by flaxseed and soybean protein. Carbohydrate Polymers. 2011;86:343-51.
[177] Kelley D, McClements DJ. Influence of sodium dodecyl sulfate on the thermal stability of bovine
serum albumin stabilized oil-in-water emulsions. Food Hydrocolloids. 2003;17:87-93.
[178] Demetriades K, McClements DJ. Influence of pH and heating on physicochemical properties of
whey protein-stabilized emulsions containing a nonionic surfactant. Journal of Agricultural and Food
Chemistry. 1998;46:3936-42.
[179] Ji J, Zhang JP, Chen JS, Wang YL, Dong N, Hu CL, et al. Preparation and stabilization of
emulsions stabilized by mixed sodium caseinate and soy protein isolate. Food Hydrocolloids.
2015;51:156-65.
[180] Dickinson E, Ritzoulis C, Povey MJW. Stability of emulsions containing both sodium caseinate
and Tween 20. Journal of Colloid and Interface Science. 1999;212:466-73.
[181] Kabalnov AS. Coalescence in emulsions. In: Binks BP, (editor). Modern Aspects of Emulsion
Science. Cambridge, UK: The Royal Society of Chemistry 1998. Chapter Chapter 7.
[182] Thanasukarn P, Pongsawatmanit R, McClements DJ. Utilization of layer-by-layer interfacial
deposition technique to improve freeze-thaw stability of oil-in-water emulsions. Food Research
International. 2006;39:721-9.
[183] Noshad M, Mohebbi M, Shahidi F, Koocheki A. Freeze-thaw stability of emulsions with soy
protein isolate through interfacial engineering. International Journal of Refrigeration-Revue
Internationale Du Froid. 2015;58:253-60.
[184] Nikiforidis CV, Kiosseoglou V. Competitive displacement of oil body surface proteins by Tween
80-Effect on physical stability. Food Hydrocolloids. 2011;25:1063-8.
[185] van Aken GA. Competitive adsorption of protein and surfactants in highly concentrated
emulsions: effect on coalescence mechanisms. Colloids and Surfaces a-Physicochemical and
Engineering Aspects. 2003;213:209-19.

67
[186] Dickinson E, Owusu RK, Williams A. Orthokinetic destabilization of a protein-stabilized
emulsion by a water-soluble surfactant. Journal of the Chemical Society-Faraday Transactions.
1993;89:865-6.
[187] Zhang SB, Wang T. Destabilization of Emulsion Formed During Aqueous Extraction of Peanut
Oil: Synergistic Effect of Tween 20 and pH. Journal of the American Oil Chemists Society.
2016;93:1551-61.
[188] Mackie AR. Structure of adsorbed layers of mixtures of proteins and surfactants. Current Opinion
in Colloid & Interface Science. 2004;9:357-61.
[189] Taylor P. Ostwald ripening in emulsions. Advances in Colloid and Interface Science.
1998;75:107-63.
[190] Kabalnov AS, Shchukin ED. Ostwald ripening theory - applications to fluorocarbon emulsion
stability. Advances in Colloid and Interface Science. 1992;38:69-97.
[191] Capek I. Degradation of kinetically-stable o/w emulsions. Advances in Colloid and Interface
Science. 2004;107:125-55.
[192] Weers JG. Ostwald ripening in emulsions. In: Binks BP, (editor). Modern Aspects of Emulsion
Science. Cambridge, UK: Royal Society of Chemistry; 1998. p. 292-327.
[193] Dickinson E, Ritzoulis C, Yamamoto Y, Logan H. Ostwald ripening of protein-stabilized
emulsions: effect of transglutaminase crosslinking. Colloids and Surfaces B-Biointerfaces.
1999;12:139-46.
[194] Mun SH, McClements DJ. Influence of interfacial characteristics on Ostwald ripening in
hydrocarbon oil-in-water emulsions. Langmuir. 2006;22:1551-4.
[195] Zeeb B, Gibis M, Fischer L, Weiss J. Influence of interfacial properties on Ostwald ripening in
crosslinked multilayered oil-in-water emulsions. Journal of Colloid and Interface Science.
2012;387:65-73.
[196] Galindo-Alvarez J, Le KA, Sadtler V, Marchal P, Perrin P, Tribet C, et al. Enhanced stability of
nanoemulsions using mixtures of non-ionic surfactant and amphiphilic polyelectrolyte. Colloids and
Surfaces a-Physicochemical and Engineering Aspects. 2011;389:237-45.
[197] Chebil A, Desbrieres J, Nouvel C, Six JL, Durand A. Ostwald ripening of nanoemulsions stopped
by combined interfacial adsorptions of molecular and macromolecular nonionic stabilizers. Colloids
and Surfaces a-Physicochemical and Engineering Aspects. 2013;425:24-30.
[198] Maldonado-Valderrama J, Patino JMR. Interfacial rheology of protein-surfactant mixtures.
Current Opinion in Colloid & Interface Science. 2010;15:271-82.

68
[199] Wilde P, Mackie A, Husband F, Gunning P, Morris V. Proteins and emulsifiers at liquid
interfaces. Advances in Colloid and Interface Science. 2004;108–109:63-71.
[200] Mackie AR, Gunning AP, Wilde PJ, Morris VJ. Orogenic Displacement of Protein from the
Air/Water Interface by Competitive Adsorption. Journal of Colloid and Interface Science.
1999;210:157-66.
[201] Berton-Carabin CC, Ropers MH, Genot C. Lipid Oxidation in Oil-in-Water Emulsions:
Involvement of the Interfacial Layer. Comprehensive Reviews in Food Science and Food Safety.
2014;13:945-77.
[202] Lomova MV, Sukhorukov GB, Antipina MN. Antioxidant Coating of Micronsize Droplets for
Prevention of Lipid Peroxidation in Oil-in-Water Emulsion. Acs Applied Materials & Interfaces.
2010;2:3669-76.
[203] Donsi F, Ferrari G. Essential oil nanoemulsions as antimicrobial agents in food. Journal of
Biotechnology. 2016;233:106-20.
[204] Franklyne J, Mukherjee A, Chandrasekaran N. Essential oil micro- and nanoemulsions:
promising roles in antimicrobial therapy targeting human pathogens. Letters in Applied Microbiology.
2016;63:322-34.
[205] Salvia-Trujillo L, Soliva-Fortuny R, Rojas-Grau MA, McClements DJ, Martin-Belloso O. Edible
Nanoemulsions as Carriers of Active Ingredients: A Review. In: Doyle MP, Klaenhammer TR,
(editors). Annual Review of Food Science and Technology, Vol 8. Vol. 82017. p. 439-66.
[206] Hilbig J, Ma QM, Davidson PM, Weiss J, Zhong QX. Physical and antimicrobial properties of
cinnamon bark oil co-nanoemulsified by lauric arginate and Tween 80. International Journal of Food
Microbiology. 2016;233:52-9.
[207] Xue J, Davidson PM, Zhong QX. Antimicrobial activity of thyme oil co-nanoemulsified with
sodium caseinate and lecithin. International Journal of Food Microbiology. 2015;210:1-8.
[208] Niu FG, Pan WC, Su YJ, Yang YJ. Physical and antimicrobial properties of thyme oil emulsions
stabilized by ovalbumin and gum arabic. Food Chemistry. 2016;212:138-45.
[209] Choi SJ, Decker EA, Henson L, Popplewell LM, Xiao H, McClements DJ. Formulation and
properties of model beverage emulsions stabilized by sucrose monopalmitate: Influence of pH and
lyso-lecithin addition. Food Research International. 2011;44:3006-12.
[210] McClements DJ, Decker EA, Choi SJ. Impact of Environmental Stresses on Orange Oil-in-Water
Emulsions Stabilized by Sucrose Monopalmitate and Lysolecithin. Journal of Agricultural and Food
Chemistry. 2014;62:3257-61.

69
[211] Xiang J, Liu FG, Fan R, Gao YX. Physicochemical stability of citral emulsions stabilized by milk
proteins (lactoferrin, alpha-lactalbumin, beta-lactoglobulin) and beet pectin. Colloids and Surfaces a-
Physicochemical and Engineering Aspects. 2015;487:104-12.
[212] Zhao JJ, Wei T, Wei ZH, Yuan F, Gao YX. Influence of soybean soluble polysaccharides and
beet pectin on the physicochemical properties of lactoferrin-coated orange oil emulsion. Food
Hydrocolloids. 2015;44:443-52.
[213] McClements DJ, Li F, Xiao H. The Nutraceutical Bioavailability Classification Scheme:
Classifying Nutraceuticals According to Factors Limiting their Oral Bioavailability. In: Doyle MP,
Klaenhammer TR, (editors). Annual Review of Food Science and Technology, Vol 6. Vol. 62015. p.
299-327.
[214] McClements DJ. Enhancing nutraceutical bioavailability through food matrix design. Current
Opinion in Food Science. 2015;4:1-6.
[215] Huang QR, Yu HL, Ru QM. Bioavailability and Delivery of Nutraceuticals Using
Nanotechnology. Journal of Food Science. 2010;75:R50-R7.
[216] McClements DJ. Emulsion Design to Improve the Delivery of Functional Lipophilic
Components. In: Doyle MP, Klaenhammer TR, (editors). Annual Review of Food Science and
Technology, Vol 1. Vol. 12010. p. 241-69.
[217] Velikov KP, Pelan E. Colloidal delivery systems for micronutrients and nutraceuticals. Soft
Matter. 2008;4:1964-80.
[218] Wei ZH, Gao YX. Physicochemical properties of beta-carotene bilayer emulsions coated by milk
proteins and chitosan-EGCG conjugates. Food Hydrocolloids. 2016;52:590-9.
[219] Mao YY, Dubot M, Xiao H, McClements DJ. Interfacial Engineering Using Mixed Protein
Systems: Emulsion-Based Delivery Systems for Encapsulation and Stabilization of beta-Carotene.
Journal of Agricultural and Food Chemistry. 2013;61:5163-9.
[220] Xu DX, Aihemaiti Z, Cao YP, Teng C, Li XT. Physicochemical stability, microrheological
properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of
whey protein isolate, flaxseed gum and chitosan. Food Chemistry. 2016;202:156-64.
[221] Assadpour E, Maghsoudlou Y, Jafari S-M, Ghorbani M, Aalami M. Evaluation of Folic Acid
Nano-encapsulation by Double Emulsions. Food and Bioprocess Technology. 2016;9:2024-32.
[222] Faridi Esfanjani A, Jafari SM, Assadpour E. Preparation of a multiple emulsion based on pectin-
whey protein complex for encapsulation of saffron extract nanodroplets. Food Chemistry.
2017;221:1962-9.

70
[223] Assadpour E, Jafari S-M, Maghsoudlou Y. Evaluation of folic acid release from spray dried
powder particles of pectin-whey protein nano-capsules. International Journal of Biological
Macromolecules. 2017;95:238-47.
[224] Mehrnia M-A, Jafari S-M, Makhmal-Zadeh BS, Maghsoudlou Y. Rheological and release
properties of double nano-emulsions containing crocin prepared with Angum gum, Arabic gum and
whey protein. Food Hydrocolloids. 2017;66:259-67.
[225] Jafari SM, Paximada P, Mandala I, Assadpour E, Mehrnia M-A. Encapsulation by nano-
emulsions. In: Jafari SM, (editor). Nano-encapsulation Technologies for the Food and Nutraceutical
Industries: Elsevier; 2017. Chapter 2. p. doi: B978-0-12-809436-5.00002-1.
[226] Assadpour E, Jafari S-M. Spray Drying of Folic Acid within Nano-Emulsions; Optimization by
Taguchi Approach. Drying Technology. 2017:null-null.
[227] Mehrnia MA, Jafari SM, Makhmal-Zadeh BS, Maghsoudlou Y. Crocin loaded nano-emulsions:
Factors affecting emulsion properties in spontaneous emulsification. International journal of biological
macromolecules. 2016;84:261-7.
[228] Esfanjani AF, Jafari SM, Assadpoor E, Mohammadi A. Nano-encapsulation of saffron extract
through double-layered multiple emulsions of pectin and whey protein concentrate. Journal of Food
Engineering. 2015;165:149-55.
[229] Assadpour E, Maghsoudlou Y, Jafari S-M, Ghorbani M, Aalami M. Optimization of folic acid
nano-emulsification and encapsulation by maltodextrin-whey protein double emulsions. International
Journal of Biological Macromolecules. 2016;86:197-207.
[230] Mohammadi A, Jafari SM, Assadpour E, Faridi Esfanjani A. Nano-encapsulation of olive leaf
phenolic compounds through WPC-pectin complexes and evaluating their release rate. International
journal of biological macromolecules. 2016;82:816-22.
[231] Mohammadi A, Jafari SM, Esfanjani AF, Akhavan S. Application of nano-encapsulated olive
leaf extract in controlling the oxidative stability of soybean oil. Food chemistry. 2016;190:513-9.
[232] Eslami P, Davarpanah L, Vahabzadeh F. Encapsulating role of β-cyclodextrin in formation of
pickering water-in-oil-in-water (W1/O/W2) double emulsions containing Lactobacillus dellbrueckii.
Food Hydrocolloids. 2017;64:133-48.
[233] Kim B-K, Cho A-R, Park D-J. Enhancing oral bioavailability using preparations of apigenin-
loaded W/O/W emulsions: In vitro and in vivo evaluations. Food Chemistry. 2016;206:85-91.
[234] Andrade J, Corredig M. Vitamin D3 and phytosterols affect the properties of polyglycerol
polyricinoleate (PGPR) and protein interfaces. Food Hydrocolloids. 2016;54:278-83.

71
[235] Xiao J, Lu X, Huang Q. Double emulsion derived from kafirin nanoparticles stabilized Pickering
emulsion: Fabrication, microstructure, stability and in vitro digestion profile. Food Hydrocolloids.
2017;62:230-8.
[236] Eisinaite V, Juraite D, Schroën K, Leskauskaite D. Preparation of stable food-grade double
emulsions with a hybrid premix membrane emulsification system. Food Chemistry. 2016;206:59-66.
[237] Golding M, Wooster TJ, Day L, Xu M, Lundin L, Keogh J, et al. Impact of gastric structuring on
the lipolysis of emulsified lipids. Soft Matter. 2011;7:3513-23.
[238] Mackie A, Macierzanka A. Colloidal aspects of protein digestion. Current Opinion in Colloid &
Interface Science. 2010;15:102-8.
[239] Singh H, Sarkar A. Behaviour of protein-stabilised emulsions under various physiological
conditions. Advances in Colloid and Interface Science. 2011;165:47-57.
[240] McClements DJ, Xiao H. Potential biological fate of ingested nanoemulsions: influence of
particle characteristics. Food & Function. 2012;3:202-20.
[241] Zhang ZP, Zhang RJ, Chen L, Tong QY, McClements DJ. Designing hydrogel particles for
controlled or targeted release of lipophilic bioactive agents in the gastrointestinal tract. European
Polymer Journal. 2015;72:698-716.
[242] Li Y, McClements DJ. Modulating lipid droplet intestinal lipolysis by electrostatic complexation
with anionic polysaccharides: Influence of cosurfactants. Food Hydrocolloids. 2014;35:367-74.
[243] Wulff-Perez M, Galvez-Ruiz MJ, de Vicente J, Martin-Rodriguez A. Delaying lipid digestion
through steric surfactant Pluronic F68: A novel in vitro approach. Food Research International.
2010;43:1629-33.
[244] Plaza-Oliver M, de Baranda JFS, Robledo VR, Castro-Vazquez L, Gonzalez-Fuentes J, Marcos
P, et al. Design of the interface of edible nanoemulsions to modulate the bioaccessibility of
neuroprotective antioxidants. International Journal of Pharmaceutics. 2015;490:209-18.
[245] Wulff-Perez M, de Vicente J, Martin-Rodriguez A, Galvez-Ruiz MJ. Controlling lipolysis
through steric surfactants: New insights on the controlled degradation of submicron emulsions after
oral and intravenous administration. International Journal of Pharmaceutics. 2012;423:161-6.
[246] Torcello-Gomez A, Maldonado-Valderrama J, Jodar-Reyes AB, Foster TJ. Interactions between
Pluronics (F127 and F68) and Bile Salts (NaTDC) in the Aqueous Phase and the Interface of Oil-in-
Water Emulsions. Langmuir. 2013;29:2520-9.
[247] Yao XL, Wang N, Fang YP, Phillips GO, Jiang FT, Hu JZ, et al. Impact of surfactants on the
lipase digestibility of gum arabic-stabilized O/W emulsions. Food Hydrocolloids. 2013;33:393-401.

72
[248] Nambam JS, Philip J. Competitive adsorption of polymer and surfactant at a liquid droplet
interface and its effect on flocculation of emulsion. Journal of Colloid and Interface Science.
2012;366:88-95.
[249] Mao YY, McClements DJ. Modulation of bulk physicochemical properties of emulsions by
hetero-aggregation of oppositely charged protein-coated lipid droplets. Food Hydrocolloids.
2011;25:1201-9.
[250] Mao YY, McClements DJ. Modulation of emulsion rheology through electrostatic
heteroaggregation of oppositely charged lipid droplets: Influence of particle size and emulsifier
content. Journal of Colloid and Interface Science. 2012;380:60-6.
[251] Mao YY, McClements DJ. Fabrication of viscous and paste-like materials by controlled
heteroaggregation of oppositely charged lipid droplets. Food Chemistry. 2012;134:872-9.
[252] Jafari SM, Assadpoor E, Bhandari B, He Y. Nano-particle encapsulation of fish oil by spray
drying. Food Research International. 2008;41:172-83.
[253] Drapala KP, Auty MAE, Mulvihill DM, O’Mahony JA. Influence of emulsifier type on the
spray-drying properties of model infant formula emulsions. Food Hydrocolloids. 2017;69:56-66.

73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
Graphical abstract

Emulsion Emulsion
Formation Stabilization

92
Highlights

 Mixed emulsifiers may give better or worse performance than single ones
 Mixtures may involve proteins, polysaccharides, phospholipids, surfactants etc.
 Emulsifier type and levels impacts emulsion formation and stability
 Mixed emulsifiers can be used to provide novel functional attributes

93