Chapter 3

Emulsifying Properties of Proteins

3.1 Introduction

Emulsification is the most important process in the manufacturing of many

formulated foods. Food emulsions are classified as macroemulsions with droplet
size of 0.2 to 50 f..lm. Emulsion represents a heterogeneous mixture of fat globules.
Food emulsions can be of the oil in water (OfW) or water in oil (W /0) type. The
difference between OfW and W/0 emulsions is that an OfW emulsion commonly
exhibits a creamy texture, while a W/0 system has greasy textural properties.
Protein emulsifying activity is the ability of the protein to participate in
emulsion formation and to stabilize the newly created emulsion. The emulsifying
capacity is the ability of the protein solution or suspension to emulsify oil.
Emulsifying properties are useful functional characteristics which play an
important role in the development of new sources of plant protein products for
uses as foods. Proteins are the components that dominate in most food emulsions.
A significant number of foods are emulsions, dispersions, and foams, and in
these systems, proteins, in combination with lipids and carbohydrates, are
important stabilizers. The review of Halling [1] on the use of food proteins as
stabilizers of emulsions and foams, provides extensive references to earlier
reviews and reports. Walstra [2] presented the physical and colloidal aspects of
emulsification and whipping for milk and milk products.
Required functional properties of proteins are affected by product utilization,
for example in dairy foods, emulsifying properties are important, and milk
substitutes should possess proper emulsifying capacity (EC), color, mouthfeel,
flavor and solubility characteristics. In comminuted and other meats, limiting
functional properties are: water holding capacity, emulsifying capacity, and
emulsion stability (ES), fat binding and resistance of the functional properties to
heat treatment. Comparative studies of the emulsifying properties of different
proteins are difficult until all factors influencing protein emulsifying properties and
methods of testing are standardized.
The characteristics used to describe emulsifying properties of proteins are EC,
ES, and emulsifying activity (EA). They are used to describe the emulsifying
properties of proteins in food emulsion systems. EC is presented as the amount of
J. F. Zayas, Functionality of Proteins in Food
© Springer-Verlag Berlin Heidelberg 1997
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oil (ml) that is emulsified under specific conditions by 1 g protein. The

emulsifying capacity of an emulsifier depends on its ability I) to form the
adsorption films around the globules, and 2) to lower the interfacial tension at the
oil-water interface. Emulsion stability is the capacity of emulsion droplets to
remain dispersed without separation by creaming, coalescing, and flocculation.
Emulsifying activity is presented as the maximal interfacial area (cm2) per 1 g of
protein of a stabilized emulsion.
Many chemical and physical interrelated factors are involved in the formation,
stability, and textural properties of protein-fat-water emulsions. EC and ES depend
on the properties of proteins and conditions of emulsification and vary with the
source of protein, its concentration, pH, ionic strength (salt type and
concentration), and viscosity of the system. Nakai et al. [3] reported that solubility,
surface hydrophobicity, and molecular flexibility influence the emulsification
behavior of globular proteins. Emulsifying properties of proteins are influenced by
equipment design, temperature of oil, and protein solution.
In certain foods, a natural protein ingredient is an effective stabilizer. Proteins
are effective surface-active agents because they possess the capacity to lower the
interfacial tension between hydrophobic and hydrophilic components in foods.
Proteins participate in the formation of oil-in-water and water-in-oil emulsions and
stabilize the emulsions that are formed. A stabilizing effect of proteins in the
emulsion system results from the formation of a protective barrier around fat
droplets, preventing emulsion coalescence.
The emulsifying capacity of proteins depends on the shape, charge, and
hydrophobicity of the protein molecules, neutrality of dipoles, hydration of polar
groups. Emulsion stability depends on the magnitude of these interactions [4]. To
produce stable emulsions protein material should be selected that is soluble, has
the ability to adsorb rapidly at the interface, has well-distributed charged groups,
and has the ability to form a strong cohesive film. Stable emulsions are prepared
with milk proteins because components of micellar casein and whey proteins
possess the properties listed above.

3.2 Hydrophobic and Hydrophilic Properties of Proteins

The understanding of the emulsifying properties of plant and animal food proteins
was enhanced by establishing a correlation between surface hydrophobicity and
interfacial tension. Proteins with a large number of apolar amino acids, i.e. high
hydrophobicity are surface active. The attempt was made to explain protein
emulsifying properties by their surface hydrophobicity. The relationship between
protein structure and functional properties (emulsifying and foaming capacity) was
demonstrated by Kato and Nakai [5] as a result of determining protein surface
hydrophobicity. They reported strong correlation between EC and hydrophobicity
of proteins. Kato et al. [6] showed correlation between surface hydrophobicity and
interfacial tension of the protein solutions and emulsifying properties of proteins.
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Total hydrophobicity x surface hydrophobicity (SxS o) represents more than 71 %

of the variability in the emulsifying activity index [7]. A hypothesis was strongly
supported that hydrophobicity plays a crucial role in determining other functional
properties of proteins [8]. An effective hydrophobicity (So) measurement with cis-
parinaric acid has been extended to the insoluble proteins, and the value of So was
recommended as a predictor of some functional properties. Values of So have been
used to predict emulsifying capacity of proteins. According to the hydrophobicity
mechanism of protein emulsifying capacity, amphiphilic proteins having high
surface hydrophobicity are adsorbed at the oiVwater interface. Adsorbed proteins
reduce the interfacial or surface tension facilitating the formation of emulsions.
Proteins are surface active agents, and protein aqueous solution showed a surface
tension about 25 mN/m, lower than that of pure water. At the oiVwater interfaces,
the surface pressures of protein vary in the range 15-25 mN/m, depending on the
protein concentration, pH, ionic strength, temperature, etc. [9]. The concentration
of protein at the interface and decrease of the interfacial tension is affected by
protein hydrophobicity. The surface hydrophobicity of proteins correlated
significantly with an increasing index of emulsifying activity and decreasing
interfacial tension [5].
Globular proteins with a great surface hydrophobicity, such as lysozyme,
ovalbumin and whey proteins improve their emulsifying capacity by moderate
heating and partial unfolding. Proteins must possess a well-balanced distribution of
hydrophilic and hydrophobic domains in the molecules. The higher EC of
caseinates is related to their high solubility and to their dissociated and naturally
unfolded structure. Additionally, they possess relatively high hydrophobicity and a
separation of hydrophobic and hydrophilic regions of the polypeptide chains.
Molecular hydrophobicity and surface active properties are correlated. Effective
surface hydrophobicity (So) and interfacial tension influenced emulsifying activity.
A number of methods have been used to attach hydrophobic groups or change
the hydrophobicity of proteins to improve emulsifying and foaming properties:
attaching fatty acids, binding of hydrophobic amino acids by enzymatic or
chemical reactions, and deamidation of gluten [10]. An effective method of surface
hydrophobicity determination is the use of fluorescent probes which bind to the
hydrophobic regions on the surface of the protein molecule [5].
The surface activity, and emulsifying properties have been improved by
incorporation of the hydrophobic constituents to a hydrophilic protein. Alkyl esters
have been incorporated by using papain as a catalyst under mild alkaline
conditions (pH 9.0) [11]. In this treatment hydrolysis is carried out at a limited
amount of susceptible peptide bonds, and ester groups are linked by peptide bonds.
The EC increases gradually with alkyl chain length.
The side of the protein stabilizer, which is exposed to the discontinuous oil
phase should be hydrophobic. Protein adsorption and orientation at the oiVwater
interface is affected mostly by its hydrophobicity [12]. The side of the protein
stabilizer which is exposed to the continuous aqueous phase should be hydrophilic
and should have most of the polar and charged groups exposed. Hydrophobic
interactions stabilize the conformation of proteins in their native state in solution.
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The hydrophobicity of proteins is related to their content of apolar amino acids.

Bigelow [13] calculated the average hydrophobicity of several proteins by dividing
the total hydrophobicity by the total number of residues. The average
hydrophobicity of proteins ranged from 1000 to 121000 caUresidue.
The amino acid composition influences the folding and reactivity of food
proteins. Kato and Nakai [5] found that proteins with a high content of nonpolar
amino acid residues (more than 30% of the total amino acids) exhibit good surface
activity (emulsifying and foaming properties) and poor gelling ability. However,
prediction of the EC of proteins from amino acid composition showed low
correlation because it measured the total potential hydrophobicity of the protein
rather than hydrophobic groups oriented at the surface after unfolding. Only a
portion of nonpolar amino acid residues located in the interior of protein
molecules in solutions participate in the emulsification of oil into an aqueous
phase. Hydrophobicity of proteins influences protein solubility in water and
indirectly emulsifying properties of proteins. Proteins interact with the oil surface
more significantly if a large number of hydrophobic amino acids are at the surface.
Surface hydrophobicity is not the only one factor determining emulsifying
properties of proteins. p-lactoglobulin exhibited better emulsifying properties at
pH 7 than at pH 3, although surface hydrophobicity of p-lacto-globulin is higher at
pH < 3 than at pH> 3 [14].
The suitable hydrophilic-lipophilic balance (HLB) determines the emul-sifying
properties of proteins. Aoki et al. [15] suggested that emulsifying properties of
proteins are affected by a balance between the hydrophilic and lipophilic groups
and did not necessarily increase as the protein became more lipophilic. A proper
balance between hydrophilic and hydrophobic groups is necessary to lower the
surface and interfacial tension. The hydrophilic-lipophilic properties of proteins
enable them to orient at the oil-water interface with the lipophilic groups oriented
towards the oil droplets and hydrophilic groups oriented toward the water phase.
Despite the considerable lowering of the interfacial tension the emulsions are still
thermodynamically unstable.
Emulsions of high stability can be prepared with a combination of different
emulsifying agents exhibiting various HLB values. Maximum lowering of surface
and interfacial tension was achieved when the water-oil absorption index,
determined by spontaneous water and oil uptake, was nearly 2.0, Le. protein
absorbed twice as much water as oil [16]. If this ratio was higher than 2.0, proteins
showed higher hydrophilic properties and lower EC. Elizalde et al. [16] supported
observations of Kato and Nakai [5] that the greater the decrease in interfacial
tensions, the higher the emulsifying activity of proteins. At the same time, Saito
and Taira [17] found no correlation between surface hydrophobicity of plasma
proteins and the emulsifying activity index. Poor correlation was found between
EC and surface tension. Some proteins with low surface hydrophobicity exhibited
good emulsifying properties.
An important property of proteins is sensitivity to surface denaturation at the
oil-water and air-water interface. This sensitivity to denaturation may influence the
functional properties of proteins. Since the surface hydrophobicity of protein
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increases with denaturation at the interface, the emulsifying properties may be

improved. The sensitivity of flexible proteins to the action of protease was utilized
as a method to detect the role of protein flexibility. Kato et al. [18] reported good
correlations between the emulsifying activity (r = 0.93), and the digestion velocity
of proteins determined by a-chymotrypsin. The flexibility of the protein structure
detected by protease digestion was correlated with the emulsifying properties.
Protein solubility is an important factor determining emulsifying properties of
proteins. As protein solubility increased the capacity of a protein to form and
stabilize emulsion was improved. Undissolved proteins are poor stabilizers
because proteins must dissolve and migrate to the interface. The emulsifying
capacity of com germ proteins was influenced by solubility especially in diluted
emulsions. Correlation was established between EC and protein solubility and the
effects of pH on emulsion formation [19]. Minimum EC was found in the
isoelectric range.
A significant number of plant proteins are underutilized in food preparation
because of their emulsifying properties. Utilization of plant proteins as emulsion
stabilizers is limited by their poor solubility. Solubility and functionality might be
improved by protein modification. Solubility and surface activity of modified
proteins are improved by partial hydrolysis during enzymic modification.
Increased concentration of soluble protein may cause the formation of smaller
particles during emulsification and may increase the stability of the emulsion to
creaming and drainage [1]. However, data related to correlation between EC and
protein aqueous solubility are controversial. The positive correlation between
solubility of proteins and EC and ES was reported. However, other data showed
evidence that emulsifying properties and protein solubility are not well correlated
[20,21]. At the same time, solubility of proteins appears to contribute more to the
quality of emulsions formed than to quantities of oil emulsified. Paulson et al. [22]
suggested that insoluble proteins participate in emulsion stabilization. Insoluble
protein particles incorporated during emulsification can increase stability of
emulsions. Solubility and hydrophobicity of proteins have been suggested as
indicators of EC of proteins [7]. Combinations of effective hydrophobicity and
solubility were more effective predictors of emulsifying properties than
hydrophobicity and solubility alone. The importance of surface hydrophobicity
(So) and solubility to the EC and ES of proteins was reported by Shimizu et al.
[23].
Changes in protein conformation during heat treatment affected solubility and
hydrophobicity [7]. Heating of meat proteins to 70 °C resulted in significant two-
to threefold increase in So of proteins and decrease in solubility values to 25%.
Consequently, heating effectively exposed the hydrophobic residues of the
proteins. Results of this study indicated that both hydrophobicity and solubility
influence the emulsifying properties of meat proteins. Solubility was the more
important parameter for predicting emulsifying activity index (EAI) and EC of
protein samples with low solubility, whereas hydrophobicity was critical for
predicting EAI and EC of samples with high solubility. Combination of high
hydrophobicity and high solubility values resulted in good emulsifying
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functionality. Capacities of proteins to emulsify fat were improved with increasing

hydrophobicity values at solubility values above 30%, and with increasing
solubility values at any given hydrophobicity value. Heating at 75°C slightly
improved EAI of myosin, however, the emulsifying stability index decreased [24].
Emulsifying properties of globular proteins were improved by heating due to
unfolding of protein polypeptide chains. Nakai et al. [3] demonstrated the
importance of protein surface hydrophobicity and reported a correlation between
bound linoleate and dispersibility of surfactant treated soy, sunflower and rapeseed
proteins. Treatment with surfactants affected solubilization of these proteins. The
dispersibility was improved with increase bound linoleate. High protein
dispersibility was associated with high effective hydrophobicity. The data that the
So of proteins increased with dispersibility is contrary to the known mechanism of
hydrophobicity-solubility relationship of proteins.
Partial hydrolysis with proteinase-treated soy, sunflower, and rapeseed protein
isolates showed that these proteins did not change their So, whereas protein
solubility increased from 55 to 95% [3]. This evidence demonstrated the more
significant effect of So on emulsifying properties than solubility of proteins.

3.3 Interfacial Film Formation and Properties

The interfacial films of proteins at an oil-water interface have been thoroughly

studied. However, there is no extended information related to thickness, structure,
textural properties of films and protein conformation in films. The surface activity
of proteins is expressed as the protein ability to migrate, adsorb, unfold and form a
layer at the interface as a result of rearrangement [25]. Proteins form membranes
or films around the fat droplets and lower the interfacial tension between water and
oil. As a result proteins retard coalescence of fat droplets. Surface film formation
is a result of diffusion and adsorption of protein molecules at the interface.
Formation of the protein film is facilitated if protein is solubilized, and is
influenced by protein capacity to diffuse at the interface. The property of proteins
to form films is a subsequent result of protein diffusion and adsorption at the
interface, partial unfolding and reorientation at the interface [26]. These processes
are affected by intrinsic and extrinsic factors. The important property of proteins
for their surface activity is molecular flexibility and conformation. Molecular
properties of the proteins influence the formation of an interfacial film which
occurs in sequential steps. Formation of the interfacial film is a three-step process
and includes:
1. Diffusion of proteins to the interface,
2. Protein adsorption at the interface,
3. Changes of protein conformation resulted from unfolding and reorientation
of protein molecules.