Journal of Food Engineering 122 (2014) 15–27

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Review

Whey protein peptides as components of nanoemulsions: A review

of emulsifying and biological functionalities
Randy Adjonu a,c, Gregory Doran a,c, Peter Torley a,b,c,⇑, Samson Agboola a,c
a
Graham Centre for Agricultural Innovation, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australia
b
National Wine & Grape Industry Centre, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australia
c
School of Agricultural & Wine Sciences, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Milk proteins are used to make emulsions, and may be used to make nanoemulsions. Nanoemulsions are
Received 26 April 2013 a nanotechnology with food applications, and possess superior physicochemical and sensorial properties
Received in revised form 24 July 2013 compared to macro- and microemulsions. They are also able to deliver bioactive compounds when
Accepted 21 August 2013
consumed. In this review, three aspects of food nanoemulsions will be examined: (1) the production
Available online 30 August 2013
and properties of food nanoemulsions, (2) emulsifiers/surfactant (ionic, non-ionic, phospholipid, polysac-
charide, and protein) used in nanoemulsions production. The suitability of proteins and protein hydrol-
Keywords:
ysates as nanoemulsifiers is discussed, with a particular focus on whey protein, (3) the potential of whey
Nanoemulsions
Non-protein surfactants
protein derived peptides as both emulsifiers and bioactive compounds in nanoemulsion delivery systems.
Whey protein and peptide emulsifiers Lastly, the potential delivery of bioactive peptides and other bioactive compounds within nanoemulsion
Bioactive peptides systems is also discussed.
Dual-functional peptides Ó 2013 Elsevier Ltd. All rights reserved.
Nano-delivery systems

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2. Nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.1. Properties of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2. Formation of nanoemulsion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3. Emulsifiers/surfactants and co-surfactant systems in nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.1. Non-protein surfactants/co-surfactants systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2. Whey protein and peptide emulsifiers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.3. Tailoring peptide functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.3.1. Enzyme type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.2. Degree of hydrolysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.3. Peptide size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.4. Stability of whey protein hydrolysate stabilised emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.5. Whey protein hydrolysate fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4. Bioactive peptides from whey protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.1. Whey peptides as dual-functional ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5. Nanoemulsion delivery of bioactive peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
5.1. Delivery of other bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

⇑ Corresponding author at: School of Agricultural & Wine Sciences, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australia. Tel.: +61 2 6933 2283.
E-mail address: ptorley@csu.edu.au (P. Torley).

0260-8774/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2013.08.034
16 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
1. Introduction
Milk proteins, and whey proteins in particular, are valued as
important food ingredients because of their functional and nutri-
tional properties (Christiansen et al., 2004; Sarkar et al., 2009),
and have been extensively used as emulsifiers in foods (Chu
et al., 2007; Dissanayake and Vasiljevic, 2009; Lee and McCle-
ments, 2010). Recent studies have proven the potential of whey
protein ingredients as emulsifiers in nanoemulsions that have been
tailored for food applications (Lee and McClements, 2010; Relkin
et al., 2011; Shah et al., 2012). Nanoemulsions are a specific type
of colloidal dispersion characterised by very small droplet sizes,
usually covering the size range of 10–200 nm (Chu et al., 2007;
Lee and McClements, 2010; Wulff-Pérez et al., 2009). Nanoemul-
sions enhance the solubility, transport, dispersibility, bioavailabil-
ity and bioaccessibility of active food and drug components (e.g.
carotenoids, a-tocopherol, antioxidants, polyunsaturated fatty
acids, hydrophobic vitamins, flavour and aroma compounds), and
can act as excellent encapsulation systems compared to conven-
tional emulsions (Bilbao-Sáinz et al., 2010; Qian et al., 2012a;
Wulff-Pérez et al., 2009).
The advantages of nanoemulsions over other emulsions are de-
rived from the smaller droplet sizes which impart distinct physico-
chemical properties in nanoemulsions (e.g. bulk viscosity, optical
transparency, and physical stability) compared to those of other
emulsion systems (Cortés-Muñoz et al., 2009; Donsì et al., 2012;
Kentish et al., 2008; Peng et al., 2010; Sonneville-Aubrun et al.,
2004). Most studies conducted so far have concentrated on the
use of the synthetic and low molecular weight surfactants (e.g.
the tweens and spans) due to their excellent interfacial diffusivity,
compared to large biopolymers such as proteins and polysaccha-
rides (Donsì et al., 2012; Ghosh et al., 2013; Lee and McClements,
2010; Qian and McClements, 2011). However, concerns about the
safety, toxicity and metabolism of these synthetic emulsifiers in
the human body limit their application to food systems.
The majority of the studies using proteins have reported on the
native protein but not its hydrolysates. While hydrolysates may
possess enhanced interfacial diffusivity and emulsifying capacity,
they have shown poor stabilising ability in conventional emulsion
systems, preventing long term storage (Agboola et al., 1998a;
Scherze and Muschiolik, 2001). Moreover, studies on emulsion
have often focused on the crude hydrolysate which consists of het-
erogeneous mixtures of amino acids, and small, medium to large
chain peptides (Scherze and Muschiolik, 2001). The prospect of
stepwise fractionation to enrich sufficiently large peptides may
generate peptides with adequate surface activities that are capable
of stabilising these nanodroplets, compared to the microdroplets
(Gauthier and Pouliot, 2003).
Aside from enhancing the emulsifying properties of proteins,
the products of enzymatic hydrolysis may also possess bioactivi-
ties (e.g. antioxidant activity, antihypertensive activity, mineral
carrier, immuno-stimulant, anti-thrombotic, and anti-gastric, opi-
oid, antimicrobial, and anti-cancer activities) which can be benefi-
cial for promoting good health (Adjonu et al., 2013; Gauthier and
Pouliot, 2003; Korhonen, 2009; Korhonen and Pihlanto, 2006).
These bioactivities are usually absent or latent in the native unhy-
drolysed protein, but can be released or enhanced upon hydrolysis
(Adjonu et al., 2013). Nanodispersions may serve as efficient deliv-
ery vehicles for incorporating these bioactive peptides into food,
subsequently increasing their utilisation by the body as functional
and nutraceutical agents (Chu et al., 2007; Qian et al., 2012a; Rel-
kin et al., 2008) thus, allowing them to more effectively express
their bioactivities in vivo (Prego et al., 2006).
This review will focus on: (a) the formation of nanoemulsions;
(b) emulsifier (non-protein and protein emulsifiers) effects on
nanoemulsion properties; (c) the emulsifying property of whey
protein hydrolysates and the potential for whey protein peptides
as both nanoemulsifiers and bioactive compounds in foods (i.e.
dual-functionality) and (d) the potential applications for nano-
emulsions for bioactive peptide delivery. The terminologies emul-
sifiers and surfactants are used interchangeably in the following
discussion.
2. Nanoemulsions
2.1. Properties of nanoemulsions
Nanoemulsions are a technology that has food and pharmaceu-
tical applications (Tarver, 2006), and their evolution has paralleled
the development of efficient emulsification technologies (Cortés-
Muñoz et al., 2009). An efficient emulsification process is able to
form emulsions with small droplet sizes and narrow size distribu-
tions. These characteristics are, however, a function of the two
opposing forces; droplet breakup and droplet–droplet coalescence
(Jafari et al., 2006). These properties have been identified in several
works (Donsì et al., 2012; Jafari et al., 2006; Qian and McClements,
2011) as being dependent upon several processes including:
 Homogenising mechanism.
 Type, concentration and interfacial properties of surfactant/
emulsifier.
 Dispersed phase volume/mass fraction and viscosity.
 Timescale of surfactant adsorption onto the surfaces of
newly created droplet.
 Frequency and timescale of inter droplet–droplet collision.
Nanoemulsions, like microemulsions are transparent/translucent
systems and as a result, they can be incorporated as components of
food beverages and gels, nutraceuticals and pharmaceutical prepara-
tions without a loss of clarity (Fig. 1) (Chu et al., 2007; Kentish et al.,
2008; Wulff-Pérez et al., 2009). Increasing interest in nanoemulsions
stems from the characteristic physicochemical properties that their
small droplet sizes provide (Table 1). Their small droplet size allows
for efficient delivery, accelerated release and rapid absorption of
hydrophobic bioactive drug and food agents such as vitamin E, ome-
ga 3 fatty acids, flavonoids and various phyto-polyphenolic com-
pounds (Balcão et al., 2013; Lee and McClements, 2010; Qian et al.,
2012a; Tarver, 2006; Yang and McClements, 2013).
2.2. Formation of nanoemulsion
Nanoemulsion droplets are only kinetically stable, in that the
free energy of the separated oil and aqueous phases is always low-
er than that of the formed emulsion and therefore, nanoemulsions
do not form spontaneously (McClements and Rao, 2011). To break
large emulsion droplets into nanodroplets, a large external force
(homogenisation pressure, Pa; energy applied per volume of li-
quid) must be applied during homogenisation in order to overcome
the Laplace pressure p (Pa; difference in pressure between the con-
vex and concave sides of a curved interface) and to break up the
interface between the oil and water phases (Eq. (1)) (Cortés-Muñoz
et al., 2009; Walstra, 1993). The Laplace pressure characterises the
interfacial force that acts on droplets to keep them from being dis-
rupted (McClements, 2005).
2c
P¼ ð1Þ
r
where r (m) is the principal radius of curvature of the droplets
(assuming droplets are spherical) and c (N m1) is the interfacial
tension between the two phases.
 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27 17

Type of emulsions Properties

- Droplet size: >1 m

- Appearance: Formulation dependent
Macroemulsion - Conventional homogenisation
- Surfactant load: Fairly low
- Does not form spontaneously
- Kinetically stable
Decreasing droplet sizes

Increasing energy input

- Droplet size: 4–200 nm
- Appearance: Transparent
Microemulsion - High/low energy emulsification
- Surfactant load: Fairly high (10–30%)
- Can form spontaneously
- Thermodynamically stable

- Droplet size: 10–200 nm

- Appearance: Transparent/translucent to milky
- High/low energy emulsification
Nanoemulsion
- Surfactant load: Medium (<1 to >10%)
- Does not form spontaneously
- Kinetically stable

Fig. 1. Properties of food macro-, micro-, and nanoemulsions.

Table 1
Nanoemulsion properties and applications.

Property Applications References

Stability Gravitational The constant Brownian motion of the droplets makes them stable against gravitationally induced Graves et al. (2005), Lee and
separation separation (creaming and sedimentation) and drainage in the manner observed for microscale McClements (2010), Peng
and larger emulsion droplets et al. (2010)
Flocculation Weak flocculation is prevented and this enables the droplets to remain dispersed with no Qian and McClements
separation (2011), Graves et al. (2005)
Coalescence 1. The significant surfactant film thickness relative to droplet size prevents any thinning or
disruption of the liquid film between the droplets
2. The small droplet sizes reduce the range of attractive forces acting between the droplets
Large surface area 1. Improves the solubility, bioavailability and bioaccessibility of many functional ingredients such as carotenoids, Yuan et al. (2008), Qian
phytosterols, and polyunsaturated fatty acids (PUFA) et al. (2012a)
2. Enhances the bioavailability of peptides and proteins carried by nanocapsules due to enhanced surface Prego et al. (2006)
interaction of nanocarriers with the absorptive epithelium and their protective effect for the associated peptide
3. Enhances the encapsulation of lipophilic functional ingredients in nanoemulsion delivery systems and enable Lee and McClements (2010),
their controlled release in response to specific environmental triggers Qian et al. (2012a), Tarver
(2006)
4. Nanodroplet delivery systems may increase the passive cellular absorption mechanisms and reduce the mass Donsì et al. (2012), Ghosh
transfer resistances of antimicrobial essential oils (e.g. carvacrol, limonene and cinnamaldehyde, basil oil) against et al. (2013)
Escherichia coli, Lactobacillus delbrueckii and Saccharomyces cerevisiae
Optical 1. The dimensions of the oil droplets could be much smaller than the wavelength of light, making them Lee and McClements (2010),
transparency/ transparent systems suitable for incorporation of active ingredients into many food beverages without loss of Qian and McClements
low turbidity clarity (2011), Ghosh et al. (2013),
Qian et al. (2012a), Kentish
et al. (2008)
2. As optically transparent, they are associated with freshness, purity, simplicity, water-like and may lead to a Sonneville-Aubrun et al.
large variety of products from water-like fluids to ringing gels (2004)
Fluidity 1. This may enhance spreading and interactions with taste sensory cells in the mouth Kentish et al. (2008)
2. At reasonable oil concentrations they are basically fluids and the absence of thickeners may give products easily Sonneville-Aubrun et al.
absorbed by the skin culminating in a pleasant and aesthetic skin feel (2004)

The high energy emulsification method requires the use of a subjected in order to cause droplet breakup (McClements, 2005)
high pressure valve homogeniser (HPVH), a microfluidiser, or an and determines the smallest droplet size that can be produced dur-
ultrasonicator (Jafari et al., 2006; Kentish et al., 2008; Lee and ing emulsification (Donsì et al., 2012; Mao et al., 2010). A compar-
McClements, 2010; Mao et al., 2010). Droplet disruption within ison of nanoemulsification techniques applicable to food
the homogeniser is a complex process arising from a combination nanoemulsion production is given in Table 2. Other methods for
of flow dynamics (laminar, turbulent and cavitational) which producing nanoemulsions have been reported, such as: the mem-
determines the nature of the disruptive forces (shear/viscous, elon- brane emulsification and electrical coaxial liquid jets (Acosta,
gational, inertial and cavitational) acting on droplets (McClements, 2009), the aqueous extraction-ultrafiltration method (Nikiforidis
2005; Walstra, 1993). A more in-depth discussion on flow condi- et al., 2011) and the typical low energy emulsification approaches,
tions within the homogeniser, disruptive forces and energy consid- such as the spontaneous emulsification and solvent displacement
erations can be obtained from McClements, (2005), Schubert and techniques (Prego et al., 2006), and the phase inversion or persua-
Engel, (2004) and Walstra, (1993). sive technique (Bilbao-Sáinz et al., 2010; Wulff-Pérez et al., 2009).
The choice of homogenisation technique and homogeniser is vi- However, these methods have found little application to the food
tal as it defines the type of flow conditions to which droplets are industry.
18 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27

Table 2
Comparison between different homogenisation techniques for nanoemulsion formation.

Property Microfluidisation HPVH Ultrasonication HEE/SD/E References

Smaller droplet High High at high disperse High at higher oil High with increasing Jafari et al. (2006), Mao et al.
size phase viscosities and concentrations organic solvent content (2010), Troncoso et al. (2012),
depends on the Stang et al. (2001)
geometry of the
homogenisation nozzle
Narrow size High Moderate Moderate High with increasing Jafari et al. (2006), Mao et al.
distribution organic solvent content (2010), Pinnamaneni et al.
(2003): Troncoso et al. (2012):
Lee and McClements (2010)
Surfactant usage Moderate Moderate Low Low to moderate Lee and McClements (2010),
Pinnamaneni et al. (2003)
Droplet stability High Moderate High Small droplets are stable to Pinnamaneni et al. (2003)
gravitationally-induced
separation
‘‘Over Present Present Absent Present Jafari et al. (2006), Pinnamaneni
processing’’ et al. (2003)
Energy efficiency Low Low High Medium to high Abismaïl et al. (1999), Jafari et al.
(2006)
Ease of operation Subject to equipment Easy to operate and Straight forward Large quantities of organic Abismaïl et al. (1999), Freitas
contamination clean operation and easy to solvent required which et al. (2006), Lee and
clean could be expensive and McClements (2010)
pose a threat to the
environment

HPVH: High pressure valve homogenisation, HEE/SD/E: High energy emulsification/solvent displacement/evaporation.

3. Emulsifiers/surfactants and co-surfactant systems in
nanoemulsions
Nanoemulsion droplets exhibit an appreciable interfacial ten-
sion (Mao et al., 2010) and hence the choice of emulsifier/surfac-
tant/co-surfactant systems is paramount for the efficient design
of nanoemulsion systems (McClements and Rao, 2011). Surfactants
influence the efficiency of droplet breakup by reducing the interfa-
cial tension between the oil and water phases, adsorb onto the sur-
faces of newly created droplets and stabilise them through
electrostatic repulsive and steric forces (Ghosh et al., 2013; Mao
et al., 2010). In addition, the nature and properties of the emulsifier
can affect the interfacial and bulk rheology of the nanoemulsion
droplets, which is vital to the design of nanoemulsion systems tai-
lored for a specific application. Surfactant properties also define
the physicochemical, sensorial and functionality of the nanoemul-
sion produced.
3.1. Non-protein surfactants/co-surfactants systems
A diverse range of non-protein surfactants exist for formulating
nanoemulsions and colloidal systems in general. Most studies on
nanoemulsions formation have utilised low molecular weight syn-
thetic emulsifiers/surfactant/co-surfactant systems (Table 3). They
possess better interfacial diffusive properties compared to large
biopolymers, such as proteins and polysaccharides. However, con-
cerns about their safety, toxicity and metabolism may limit their
application in food systems.
3.2. Whey protein and peptide emulsifiers
Dairy and plant proteins have been extensively used as emulsi-
fiers in foods as they adsorb to the oil droplet interface, forming a
strong and cohesive protective film that helps prevent droplet
aggregation (Lee and McClements, 2010). They are also effective
as emulsifiers in nanoemulsions (Table 4). Whey proteins (a-lact-
albumin, b-lactoglobulin, bovine serum albumin, lactoferrins, and
immunoglobulins) constitute about 20% of the total protein in milk
(80% caseins) and have a high nutritional value owing to their
high essential amino acid content (Custódio et al., 2009). They
are valued as important emulsifiers in food due to their amphi-
philic properties (possessing both hydrophobic and hydrophilic
residues) (Foegeding et al., 2002).
Whey proteins possess globular/rigid structures with buried
hydrophobic residues which tend to negatively affect their func-
tionality (Gauthier and Pouliot, 2003). Controlled enzymatic
hydrolysis of whey proteins produces peptides that are smaller,
possess fewer secondary and tertiary structures, and have a par-
tially exposed hydrophobic core (Christiansen et al., 2004; Gauthi-
er and Pouliot, 2003; Tirok et al., 2001). These characteristics
account for their higher rate of diffusion to the oil/water interface
and their ability to cover a larger area of the interface than the in-
tact protein (Davis et al., 2005; O’Regan and Mulvihill, 2010). Their
amphiphilic nature allows them to adsorb onto the surfaces of oil
droplets (Tirok et al., 2001), and stabilise the newly created emul-
sion droplets against destabilisation (van der Ven et al., 2001).
The performance of peptides derived from whey and other pro-
teins is well known in conventional emulsions (Christiansen et al.,
2004; Gauthier and Pouliot, 2003; Scherze and Muschiolik, 2001;
Tirok et al., 2001), and is also seen in nanoemulsions. The utilisa-
tion of peptides from food proteins as nanoemulsifying agents is
limited, however, the potential of whey protein isolate (WPI)
hydrolysates as emulsifiers in nanoemulsions has been reported
(Chu et al., 2007). b-carotene nanoemulsions formed by two WPI
hydrolysates (with degree of hydrolysis (DH) 8.1% and 18.1%)
had smaller droplet sizes (110.3 and 30.4 nm, respectively) than
whey protein concentrate (WPC) and soy protein isolate (SPI) sta-
bilised nanoemulsions (145.3 and 196.3 nm, respectively). Zeta po-
tential measurement showed the WPI hydrolysates had droplet
surface charge comparable to unhydrolysed SC and SPI but signif-
icantly higher than unhydrolysed WPI and WPC. With the increas-
ing knowledge of the creation of nanoemulsions in the food
industry, the potential of hydrolysed protein peptides as emulsifier
ingredients in nanoemulsions warrants further investigation.
3.3. Tailoring peptide functionality
The emulsifying properties of peptides depend upon their char-
acteristics (e.g. chain length/molecular size, conformation, hydro-
philicity and hydrophobicity) (Doucet et al., 2003; Gauthier and
 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27 19

Table 3
An overview of non-protein surfactants/co-surfactants systems stabilised nanoemulsions.

Emulsifier type Homogenisation Oil phase Emulsifier Oil phase Droplet References
method concentration concentration diameter
(%) (%) (nm)
Non ionic
Decaglycerol monolaurate (DML, ML750) Microfluidisation/ 0.03, 1 1, 10 b-Carotene in sunflower oil 115–279 Mao et al.
HPVH (2009, 2010)
Polyoxyethylene sorbitan monolaurate Microfluidisation/ 0.03, 1 1, 10 b-Carotene in sunflower oil 117–280 Mao et al.
(Tween 20) HPVH (2009, 2010)
Microfluidisation 4 1.5 b-Carotene in corn oil, Miglyol 140–170 Qian et al.
812 and orange oil (2012a)
Microfluidisation 5 1–10 Corn oil 113–143 Qian and
McClements
(2011)
Microfluidisation/ 0.3 0.5 b-Carotene in hexane 40–260 Tan and
solvent Nakajima
evaporation (2005)
Microfluidisation 10 1 Thyme oil/Miglyol 812 oil 160–176 Chang et al.
Thyme oil/corn oil 170–196 (2012)
Polyoxyethylene sorbitan monopalmitate Sonication 15 5.6 Flaxseed oil 135 Kentish et al.
(Tween 40) (2008)
HPVH 3 4–12 b-Carotene in MCT oil 160–184 Yuan et al.
(2008)
Polyoxyethylene sorbitan monostearate Catastrophic 20 10–20 Acetem 90–50 K 100–200 Bilbao-Sáinz
(Tween 60) phase inversion et al. (2010)
HPVH 3 4–12 b-Carotene in MCT oil 161–174 Yuan et al.
(2008)
Microfluidisation 5 0.5 Thyme oil/corn oil 164–196 Ziani et al.
(2011)
Polyoxyethylene sorbitan monooleate Ultrasonication 6 6–24 Basil oil 29–41 Ghosh et al.
(Tween 80) (2013)
HPVH 20/4/1 1 PCL-liquid/Lipoid S-75/a- 170 Hoeller et al.
tocopherol (2009)
HPVH 3 4–12 b-Carotene in MCT oil 157–178 Yuan et al.
(2008)
Microfluidisation 10 1 Lemon oil 217–296 Rao and
McClements
(2011)
Polyoxyethylene lauryl ether (W/O) Low energy 40–80 4–10 Isohexadecane 26–1277 Peng et al.
emulsification (2010)
Sucrose palmitate Ultra high 8/2, 10 1 D-limonene, trans- 130–168 Donsì et al.
pressure cinnamaldehyde, carvacrol in (2012)
homogenisation sunflower oil
Sucrose laureate HPVH 20/4/1 1 PCL-liquid/lipoid S-75 /a- 161 Hoeller et al.
tocopherol (2009)
Sucrose monopalmitate Microfluidisation 10 1–20 Lemon oil 15–120 Rao and
McClements
(2011)
Ionic
Pluronic F-68 Ultrasonication 25 1–2.5 Olive oil 379 Wulff-Pérez
Sesame oil 368 et al. (2009)
Soybean oil 380
Sodium dodecyl sulphate Microfluidisation Silicone oil 150 Graves et al.
(2005)
Microfluidisation 5 1–10 Corn oil/octadecane 92–131 Qian and
McClements
(2011)
Zwitterionic
Phospholipids e.g. Lecithins HPVH 10 1–5 Neobee 1053a 120 Donsì et al.
Neobee 1095a 110 (2011b)
HPVH 20 1.5 a-Tocopherol in palm oil 200–500 Relkin et al.
(2011)
Polysaccharide
Low-methoxyl pectin, amidated low- Ultra-Turrax 20 0.5–3 Itraconazole in chloroform 200–900 Burapapadh
methoxyl pectin, high-methoxyl pectin Itraconazole in MiglyolÒ 812 >2000 et al. (2010)
Succinylated waxy maize starch/octenyl HPVH 10 15 Neobee 1053 140 Donsì et al.
succinate starch (Purity Gum 2000/OSA/ Neobee 1095 130 (2011b)
Hi-Cap) Microfluidisation/ 1 10 b-Carotene in sunflower oil 262–674 Mao et al.
HPVH (2010)
HPVH 12 12 Peppermint oil/MCT oil 184–228 Liang et al.
(2012)
Maltodextrin/H-Cap Microfluidisation/ 5, 10, 15 30/10 (40) Fish oil 174–274 Jafari et al.
sonication D-limonene 518–919 (2007a,b)

a
Neobee 1053 is a low melting temperature lipid and Neobee 1095 is a high temperature melting lipid (Donsì et al., 2011b).
20 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27

Table 4
An overview of protein stabilised nanoemulsions.

Emulsifier type Homogenisation method Oil phase Emulsifier Oil phase Droplet References
concentration concentration (%) diameter
(%) (nm)
Whey protein HPVH 15, 30, 45 4.3 Pea nut oil 146–236 Cortés-Muñoz et al.
isolate (WPI) (2009)
High energy emulsification/ 10 1 Corn oil 75–121 Lee and McClements
solvent evaporation (2010)
HPVH 0.03, 1 1, 10 b-Carotene in sunflower 160–373 Mao et al. (2009),
oil Mao et al. (2010)
HPVH 20 4.5 a-Tocopherol in palm oil 200–500 Relkin et al. (2011)
WPI-maltodextrin High energy emulsification/ 10, 15, 20, 30 1 Thymol in hexane 67–420 Shah et al. (2012)
conjugate evaporation
Whey protein Microfluidisation 0.1 1 b-Carotene in hexane 145 Chu et al. (2007)
concentrate Microfluidisation/sonication 20, 25 10 D-limonene 125–387 Jafari et al. (2006)
(WPC)
b-Lactoglobulin HPVH 20 1 Soy oil 350 Sarkar et al. (2009)
(b-lg) Microfluidisation 5 1–10 Corn oil/octadecane 162 Qian and McClements
(2011)
Microfluidisation 10 1 Corn oil 181 Ahmed et al. (2012)
MiglyolÒ 812 174
Tributyrin 1981
Sodium caseinate HPVH 40 3.6 a-Tocopherol/low melting 293–304 Relkin et al. (2009,
(SC) triacylglycerols 2008)
a-Tocopherol/high 255–416
melting triacylglycerols
Microfluidisation 0.05–0.3 0.5–5 b-Carotene in hexane 17 Chu et al. (2007)
Microfluidisation 5 1–10 Corn oil/octadecane 179 Qian and McClements
(2011)
Pea protein HPVH 8, 10 3 Sunflower oil 184–218 Donsì et al. (2012)
Soy protein (SPI) Microfluidisation 0.1 1 b-Carotene in hexane 196 Chu et al. (2007)
Maize germ protein Combined aqueous extraction– 5 3 Maize germ oil bodies 155 Nikiforidis et al.
ultrafiltration method (2011)

Pouliot, 2003). Protein hydrolysis to produce peptides with desir-
able functionalities is usually performed with enzymes due to their
highly specific mode of action, ease of control, use of milder condi-
tions, and tendency not to cause amino acid damage compared to
chemical, thermal and microbial hydrolysis (Cheison et al., 2007).
Careful enzyme selection means hydrolysates can be produced
which are tailored specifically for food applications.
3.3.1. Enzyme type
Tryptic peptides of heat pre-treated WPI had higher emulsifying
activity and emulsion stability than chymotrypsin, Alcalase and
Neutrase peptides (Mutilangi et al., 1996). Trypsin hydrolysate
contained large amphiphilic peptides that favoured emulsion for-
mation and stability (Mutilangi et al., 1996). Trypsin hydrolysates
of WPC were also found to have higher interfacial adsorption,
emulsifying capacity and better storage stability compared to chy-
motrypsin WPC peptides (Gauthier et al., 1993; Turgeon et al.,
1992, 1996). Different enzymes produced peptides with different
numbers of hydrophobic regions because they cut in different
places. The adsorption of peptide onto droplet interfaces depends
on hydrophobic properties of the peptides, especially their surface
hydrophobicity (Lam and Nickerson, 2013; Singh and Dalgleish,
1998; Tirok et al., 2001; Turgeon et al., 1992, 1996). Adequate sur-
face hydrophobicity of whey protein hydrolysates is required to
form strong and cohesive films around droplets, and allow hydrol-
ysates to function as good emulsifiers (Lam and Nickerson, 2013).
The surface hydrophobicity of whey protein hydrolysates may be
reduced or increased depending on the specificity of the enzyme
used, pre-hydrolysis heat treatment, as well as on the extent of
hydrolysis (Adjonu et al., 2013; Mutilangi et al., 1996).
3.3.2. Degree of hydrolysis
The capacity of whey protein peptides to form and stabilise
emulsion droplets is influenced by their DH (the percent of peptide
bonds cleaved during hydrolysis). Increased DH increases peptide
solubility and emulsifying ability (Lieske and Konrad, 1996), but
if the DH becomes too high, it ultimately results in reduced emul-
sion formation and stability, and foam stability (Agboola et al.,
1998a; Lam and Nickerson, 2013; Scherze and Muschiolik, 2001;
Singh and Dalgleish, 1998; Tirok et al., 2001). For example, the
emulsifying capacity of WPC hydrolysates increased to a maximum
capacity at 3% DH, with lower emulsifying capacity at lower and
higher DH (Lieske and Konrad, 1996). In more extensively hydroly-
sed whey protein products, the 10% DH showed better emulsion
characteristics (e.g. smaller droplet sizes) followed by the 20%
DH and then the 27% DH (Scherze and Muschiolik, 2001). Commer-
cial whey protein hydrolysates with DH between 10 and 20% pos-
sessed better emulsifying capacity than hydrolysates with
DH > 20% (Singh and Dalgleish, 1998). As DH was increased from
10% to 45%, droplet size increased, and coarse emulsions exhibiting
bimodal size distributions were formed. The decreased emulsifying
properties when proteins are extensively hydrolysed can be attrib-
uted to a greater proportion of the peptides remaining in the con-
tinuous phase rather than adhering to the oil–water interface (Lam
and Nickerson, 2013; Miñones Conde and Rodríguez Patino, 2007),
and increased peptide–peptide and protein–peptide interactions at
the expense of peptide–oil interactions (Creusot et al., 2006). Thus,
for good emulsifying properties, whey proteins must undergo lim-
ited hydrolysis in order to partially unfold their secondary and ter-
tiary structures while minimising the degradation of their primary
structure (Foegeding et al., 2002).
3.3.3. Peptide size
Emulsions formed by hydrolysate with increased small peptide
content tended to show larger droplet sizes, multimodal size distri-
butions, increased creaming and coalescence, with extensive oil-
ing-off compared to the unhydrolysed protein (Agboola et al.,
1998a; Singh and Dalgleish, 1998; Sinha et al., 2007; Tirok et al.,
 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
2001; van der Ven et al., 2001). A minimum peptide size of >2 kDa
is required for good emulsify properties, as the size of the peptides
dictates the steric stabilisation of emulsion droplets (Gauthier and
Pouliot, 2003; van der Ven et al., 2001). Also, larger size peptides
are more likely to have both hydrophobic and hydrophilic residues
on the same molecule. During emulsion formation, the hydropho-
bic side chains of proteins interact with the oil droplets and the
hydrophilic residues will favour the aqueous phase and stabilise
the droplets through steric effects (Dalgleish, 1997; Lam and Nick-
erson, 2013).
Although small peptides may be sufficiently surface active to
form small droplets, their small size may be insufficient to prevent
droplet aggregation post-emulsification, as a result of a loss of their
steric stabilising property (van der Ven et al., 2001). In addition,
larger peptides are capable of modifying the internal structure of
an emulsion by forming inter-locking networks and, hence, limit
the tendency to creaming, flocculation and coalescence (Singh
and Dalgleish, 1998; Tirok et al., 2001).
3.4. Stability of whey protein hydrolysate stabilised emulsions
Once whey protein hydrolysate emulsions are formed, they are
subjected to various forms of instability, including creaming, sedi-
mentation, coalescence, flocculation, aggregation and oiling-off
(Agboola et al., 1998a; van der Ven et al., 2001). Rapid droplet
destabilisation resulted from the formation of weak interfacial
films around droplets that promoted the formation of larger emul-
sion droplets that creamed and coalesced faster (Agboola et al.,
1998a; van Aken, 2003). Small droplets are generally stable to
gravitational separation (creaming and sedimentation) as a result
of their constant Brownian motion (Kentish et al., 2008).
Co-surfactants/emulsifiers, stabilisers and texture-modifiers are
often required to improve the stability of whey protein hydrolysate
emulsions (Table 3) (McClements and Rao, 2011; Tirok et al., 2001).
Co-surfactants such as lecithins and monoglycerides may displace
small peptides from the droplet interface or act in a synergistic role
to compensate for the poor emulsion stability of protein hydroly-
sates (O’Regan and Mulvihill, 2009; Tirok et al., 2001). For example,
phospholipids compete with peptides for available interfaces and
may result in less peptides adsorbing at the droplet interface as a
result of the better interfacial properties of lecithin compared to
the peptides making up the hydrolysate. Effectively, smaller size
droplets may form due to the formation of a thinner adsorbed layer
around droplets (McSweeney et al., 2008; Van der Meeren et al.,
2005), making them highly stable to creaming and sedimentation
(Kentish et al., 2008). In addition, peptide–lecithin interactions
may lead to an increase in the overall charge and hydration at
the oil droplet surface, resulting in strong interfacial film favouring
droplet stability (Agboola et al., 1998b; McSweeney et al., 2008;
Van der Meeren et al., 2005).
Stabilisers and texture modifiers, usually polysaccharides, also
provide stability by increasing the continuous phase viscosity as
well as forming three dimensional networks which trap and retard
droplet movement, and limit droplet coalescence and creaming
(McClements and Rao, 2011). Increasing the concentration of poly-
saccharides (e.g. guar and xantham gum) was found to reduce the
collision frequency of droplets and the rate of drainage from the
droplet surface (Ye et al., 2004; Ye and Singh, 2006). In addition,
emulsion droplets were stable against creaming and coalescence
as a result of improved droplet packing that restricted the relative
motion of emulsion droplets (Ye et al., 2004; Ye and Singh, 2006).
Protein–polysaccharide interactions can also modify the interfacial
rheology, by forming bulkier polymeric layers around emulsion
droplets and provide enhanced stabilisation of droplets through
steric effects (Akhtar and Dickinson, 2007; O’Regan and Mulvihill,
2010).
21
3.5. Whey protein hydrolysate fractionation
Whey protein hydrolysates are a heterogeneous mixture of free
amino acids, and short to long chain peptides. Large concentrations
of hydrophilic and short chain peptides can limit their interfacial
and steric-stabilising properties (Singh and Dalgleish, 1998; Tirok
et al., 2001). They can also promote adsorption and desorption
mechanisms, resulting in an uneven, non-continuous and weak
mechanical stability of the adsorbed interfacial film, while increas-
ing the rate of re-coalescence and oiling-off (Tirok et al., 2001).
Peptide fractionation using membranes or chromatographic
techniques have been used to enrich large and surface active pep-
tides from crude hydrolysates with enhanced food functionalities
(Gauthier and Pouliot, 2003; Gauthier et al., 1993; Korhonen and
Pihlanto, 2006; Scherze and Muschiolik, 2001). A >10 kDa peptide
fraction obtained after trypsin, chymotrypsin, Alcalase and Neutr-
ase hydrolysis of WPC possessed higher emulsifying activity index
and emulsion stability than the crude unfractionated hydrolysate
(Mutilangi et al., 1996). Also, ultrafiltration of WPC hydrolysates
produced a 1–30 kDa peptide fraction that possessed greater inter-
facial adsorption, emulsifying activity and stability than the
unfractionated WPC hydrolysate (Gauthier and Pouliot, 2003; Gau-
thier et al., 1993; Turgeon et al., 1996). These fractions were com-
posed of large amphiphilic peptides that favoured the formation of
stronger interfacial films around emulsion droplets (Gauthier et al.,
1993; Turgeon et al., 1996).
While no studies on the emulsifying properties of fractionated
whey protein hydrolysates in nanoemulsions are known, studies
using crude hydrolysates have predicted nanoemulsions with
droplet sizes of 30–110 nm (Chu et al., 2007). The stability of such
nanoemulsions has not been investigated, and little information
exists on hydrolysate or peptide stabilised nanoemulsion. More-
over, elucidation of the factors that affect the nanoemulsifying po-
tential of whey protein hydrolysates, such as DH, hydrolysate/
peptide properties, lecithin and polysaccharide addition, and
homogenisation conditions (e.g. pH, ion concentration) may pro-
vide a better understanding about the emulsifying properties of
protein hydrolysates in food nanoemulsions.
4. Bioactive peptides from whey protein
Aside from modifications to their emulsifying properties and
other functionalities, whey protein hydrolysates/peptides also pos-
sess bioactive properties (Hernández-Ledesma et al., 2011;
Madureira et al., 2010). Bioactive peptides derived from enzymat-
ically hydrolysed whey protein have the ability to promote good
health in humans (Table 5). Many of these bioactive peptides have
been isolated, purified, characterised and synthesised, and are cur-
rently being marketed as specialty and functional ingredients
(Gauthier and Pouliot, 2003; Gauthier et al., 2006; Korhonen and
Pihlanto, 2006). These peptides have simple structures and are
considered safe and healthy compounds which are easily absorbed
by the human body (Li et al., 2004).
4.1. Whey peptides as dual-functional ingredients
Whey peptides perform a dual-functional role in foods as both
emulsifiers (technological function) and bioactive compounds
(biological function) important for promoting good health (Adjonu
et al., 2013). However, studies on the emulsifying and biological
functionalities of whey proteins, and proteins in general, have usu-
ally been conducted in isolation, although some studies have dem-
onstrated these combined functionalities. Sinha et al. (2007)
reported that a papain and a fungal protease treated WPC pos-
sessed high water solubility, increased foam overrun and had low
22 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27

Table 5 (Pihlanto, 2006). These low molecular weight peptides show poor
Bioactive peptides from whey proteins. stabilising abilities in food emulsions (Agboola et al., 1998a,b;
Bioactivity References van der Ven et al., 2001; Ye et al., 2004). Overcoming these prob-
Antioxidant Gauthier et al. (2006), Kilara and Panyam lems may be possible by stepwise fractionation using membrane
Mineral binding (2003), Kim et al. (2007), Kong et al. and chromatographic techniques to generate different bioactive
Antimicrobial/anti-bacterial (2012), Korhonen (2009), Madureira et al. peptides from crude hydrolysates that are large and surface active
Anti-appetising (2010), Théolier et al. (2013) and can stabilise emulsion droplets. These peptides may be effec-
Cytomodulatory
Immunomodulatory
tive as sole emulsifiers or in conjunction with other co-emulsifiers
to form and stabilise nanoemulsion systems because of the smaller
Immunostimulant Hernández-Ledesma et al. (2011), Kilara
Anti-thrombotic and Panyam (2003)
size of nanoemulsion droplets (Fig. 2). The factors affecting the
Anti-gastric nanoemulsification abilities of these peptides (e.g. dispersion con-
Hypocholesterolemic ditions, aqueous phase properties, oil mass fractions, surfactant/co-
Anti-diabetes Silveira et al. (2013) surfactant systems, and ion and salt concentrations) could be elu-
Opioid Hernández-Ledesma et al. (2011),
cidated in order to better understand their interfacial properties in
ACE-inhibitory Korhonen and Pihlanto (2006), Li et al. nanoemulsion systems.
Anti-hypertensive peptides (2004), Pan et al. (2012), Pihlanto-Leppälä In addition, the inclusion of bioactive peptides in nanoemul-
(2000), Teschemacher (2003) sions may extend their application in food and pharmaceutical
industries because colloidal systems may serve as an excellent
medium to incorporate these bioactive peptides into day-to-day
emulsifying capacity. The hydrolysates also showed high ACE- foods. Active peptides that show dual-functionality could be iso-
inhibitory potencies and were suggested as potential ingredients lated, characterised, sequenced and possibly synthesised, which
for nutraceutical preparations. Gauthier and Pouliot (2003) also has occurred for many other bioactive peptides. Also, whey protein
demonstrated that some of the peptide sequences responsible for peptides possess reduced allergenicity and are easy to digest, prop-
the emulsifying properties of whey proteins were also related to erties that are vital for formulating infant formulae and sports
their ACE-inhibitory properties. However, the link between peptide nutrition diets (Tirok et al., 2001). Inclusion of whey protein pep-
bioactivity and their emulsifying properties was not demonstrated tides in nanoemulsion could result in products with a modification
by these studies. Gauthier and Pouliot (2003) and Sinha et al. to many of their macro-scale characteristics, such as texture, taste,
(2007) only reported on the ACE-inhibitory peptides of these sensory attributes, and shelf stability, leading to a variety of prod-
hydrolysates in food emulsion, but not their other bioactivities (Ta- ucts with new functionalities (Silva et al., 2012).
ble 5). Table 6 summarises other studies reporting on the bioactiv- Other studies have also looked at the antioxidant activities of
ity and technological functions of peptides from other food protein whey proteins and their peptides to inhibit lipid oxidation and
sources. rancidity in emulsions. Hu et al. (2003) studied the oxidative
Whey protein bioactive peptides generally have a molecular stability of salmon oil/water emulsion formed by whey protein
weight of less than or equal to 10 kDa and sometimes greater emulsifiers (whey protein isolate, sweet whey, b-lactoglobulin

Table 6
Protein hydrolysates with dual-functionality (technological and biological).

Protein source Enzyme used Technological Bioactivity Molecular weight References

Peanut Papain Emulsifying capacity, foaming Antioxidant activity – Tang et al.
capacity, solubility (2012)
Canola Alcalase Water holding capacity Antioxidant activity – Cumby et al.
Flavourzyme (2008)
Quinoa Alcalase Solubility, emulsifying capacity and Antioxidant activity, <5, 5–10 and >10 kDa Da by Aluko and
foaming capacity ACE-inhibition ultrafiltration Monu (2003)
Atlantic cod (Gadus morhua) Protamex™ Emulsifying capacity and water Opioid activity, 0.9–80 kDa by gel filtration Šližytė et al.
backbone holding capacity antioxidant activity chromatography (2009)
Yellow stripe trevally Alcalase Emulsifying activity, foaming Antioxidant activity – Klompong et al.
(Selaroides leptolepis) Flavourzyme capacity, solubility (2007)

a b
Oil droplet

Bioactive peptide

Non-protein surfactant Protein/bioactive peptide emulsifier

Fig. 2. Nano-delivery of bioactive peptides (a) nanoencapsulated bioactive peptides stabilised by non-protein surfactant systems (e.g. Tween 20), (b) nanoencapsulated
bioactive peptides using bioactive proteins/peptides emulsifiers.
 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
and a-lactalbumin). All emulsions, especially those formed by b-
lactoglobulin, showed greater oxidative stability with regard to
the formation of hydroperoxide and headspace propanal, particu-
larly at pH values below the isoelectric point of the proteins. Tong
et al. (2000a, 2000b) and Peña-Ramos et al. (2004) also reported
the ability of whey protein fractions to inhibit the formation of
thiobarbituric acid reactive substances (TBARS) in a salmon oil/
water emulsion and a liposomal-oxidising system, respectively,
and showed that larger molecular weight peptides (>3 kDa) were
more effective as antioxidants than smaller molecular weight pep-
tides (<3 kDa). The greater oxidative stability of emulsions oc-
curred as a result of the ability of proteins to form cationic
charges on the surface of emulsion droplets to repel transition
metals, form protective films around droplets which hinders lipid
hydroperoxide–transition interactions, chelate prooxidants and
inactivate free radicals through sulphur containing amino acids
and peptides (Hu et al., 2003; Peña-Ramos et al., 2004; Tong
et al., 2000b). These studies, however, have looked at the antioxi-
dant activities of whey peptides in food emulsions from a techno-
logical perspective rather than a biological perspective. It is
possible the mechanisms of their antioxidant activities may differ
from the technological and biological perspectives.
5. Nanoemulsion delivery of bioactive peptides
Bioactive peptides are ideal supplemental compounds to pre-
vent or reduce oxidative damage to body organs, and the risk of
hypertension and cardiovascular health diseases. Bioactive pep-
tides have been identified in various food proteins such as caseins,
soy, canola, and are finding great applications in the development
of nutrition tailored functional foods (Phelan et al., 2009; Wang
and de Mejia, 2005). However, such applications are limited due
to the lack of appropriate delivery systems capable of protecting
bioactive peptides from degradation (e.g. conformational changes
and denaturation) during processing as well as during administra-
tion, because the structure and functionality of food proteins/pep-
tide are positively correlated. In addition, the majority of bioactive
peptides are not absorbed from the gastrointestinal tract into the
blood, possibly due to poor delivery systems, although their effects
have been proposed to be mediated directly in the gut through
receptors on the intestinal walls (Korhonen and Pihlanto, 2006;
Lee et al., 2007; Phelan et al., 2009).
A suitable delivery system should protect bioactive peptides
from interactions with other food components, stabilise the pep-
tides during processing and administration and should also en-
hance their absorption and transport across the intestinal mucosa
to target sites (Balcão et al., 2013; Prego et al., 2006; Yang and
McClements, 2013). Nano-delivery systems (e.g. nanoemulsion,
nanoencapsulation, nanovessicles [liposomes]) (Fig. 2) present a
mechanism for the structural and functional stabilisation of bioac-
tive proteins/peptides against denaturation by enzymatic digestion
and a way to increase their biopharmaceutical and food applica-
tions (Balcão et al., 2013; Prego et al., 2006). In addition, their small
droplet size may enhance the transport of bioactive peptides car-
ried within nanodroplets as droplets may pass across the intestinal
wall and facilitate their absorption, bioavailability and bioaccessi-
bility (Martins et al., 2007; Watnasirichaikul et al., 2000).
The colloidal delivery of peptide drugs within pharmaceutical
preparations is well known, whereas the delivery of bioactive pep-
tides as part of food formulations has received little attention
(Flanagan and Singh, 2006; Martins et al., 2007). Prego et al.
(2006) demonstrated that when salmon calcitonin (a linear poly-
peptide hormone responsible for controlling blood calcium levels)
was contained within nanoemulsion carriers, enhanced and pro-
longed intestinal absorption occurred.
23
Balcão et al. (2013) recently encapsulated lactoferrin (a whey
protein fraction with bioactivities) within a water–oil–water nano-
emulsion as potential antimicrobial formulation. Nanoencapsulat-
ed lactoferrin and lactoferrin in solution showed inhibitory effect
against Staphylococcus aureus, Listeria innocua, Bacillus cereus and
Candida albicans, but not Gram negative bacteria such as Salmonella
sp., Escherichia coli (E. coli) and Pseudomonas fluorescens. Nanoen-
capsulation of antimicrobial proteins could be extended to antimi-
crobial peptides derived from other food protein sources.
Antimicrobial peptides from a-lactalbumin, b-lactoglobulin and
caseins have been shown to be effective against Gram-positive
and Gram-negative bacteria (E. coli, Helicobacter, Listeria, Salmo-
nella and Staphylococcus, Listeria ivanovii), yeasts and filamentous
fungi (Hartmann and Meisel, 2007; Théolier et al., 2013). Nanoen-
capsulation of antimicrobial peptides, coupled with the small
droplet size of nanoemulsion droplets, may extend the applications
food protein antimicrobial peptides accross the food production
chain. For example, they may be used for decomtamination pur-
poses and for extending the shelf life of food products.
Bioactive peptide products have inherent bitter tastes which
tend to reduce their consumer acceptability (Komai et al., 2007;
Pedrosa et al., 2006). Debittering techniques involving the removal
of hydrophobic peptides by chromatography, absorption of bitter
peptides on activated carbon or selective extraction with alcohols
could result in a loss of bioactivity, as the majority of bioactive
amino acids and peptides are hydrophobic in nature (FitzGerald
and O’Cuinn, 2006; Leksrisompong et al., 2012). Encapsulation of
bioactive peptides within nanoemulsion delivery systems can be
used to inhibit or reduce their bitter taste and off-flavours, as for
other bitter and astringent compounds. Micro- and nanoencapsu-
lation of polyphenolic compounds and other bitter compounds,
such as chloroquine phosphate and trimebutine within polymeric
coated multiple emulsions, reduced their off-flavours, astringency,
bitterness, smell and increased their loading efficiency and bio-
availability (Hashimoto et al., 2002; Munin and Edwards-Lévy,
2011; Sohi et al., 2004; Sun-Waterhouse and Wadhwa, 2013). Bit-
ter tasting compounds dissolved in the internal phase of colloidal
delivery systems can be shielded from interactions with taste sen-
sors until target sites are reached (Hashimoto et al., 2002; Sohi
et al., 2004; Sun-Waterhouse and Wadhwa, 2013). Such applica-
tions could be extended to encapsulate highly bioactive but bitter
peptides within nanoemulsion system for fortification and supple-
mentation purposes in foods.
Protein-coated nanoemulsions containing hydrophobic bioac-
tive agents allow for the controlled release of active agents because
proteins would have to undergo digestion before release at target
sites (He et al., 2011). Pea protein-coated antimicrobial nanoemul-
sions were noted to have longer bacteriostatic action against
microorganisms, which was beneficial for products requiring long
shelf stability, whereas sugar esters and other synthetic surfac-
tant-coated nanoemulsions promoted quicker and faster antimi-
crobial effects (Donsì et al., 2012). Also, nanoemulsions formed
with proteins, such as whey proteins, possess high biocompatibil-
ity with cells when applied as delivery systems, showing over 85%
increase in cell viability compared to other synthetic emulsifiers
such as Tween 80, Solutol HS 15, Poloxamer 188 and Cremophor
(He et al., 2011).
In addition, the actual mode of action of bioactive peptides
when incorporated into foods are currently not well understood,
possibly due to lack of methods available to determine these
activities when they are included as components of foods, coupled
with the complexity of most food matrices. Nanoemulsion may
serve as a good substrate for use in assessing these bioactivities
when peptides are added to foods. The factors that affect the
solubility, loading efficiency, absorption and bioavailability, and
control release of bioactive peptides could also be determined in
24 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
nanoemulsion carrier systems. Furthermore, the pharmacokinetics,
safety and biological fate of bioactive peptides could also be inves-
tigated because nano-delivery systems are model systems for con-
trol delivery studies (Li et al., 2012).
5.1. Delivery of other bioactive compounds
Ahmed et al. (2012) reported the high solubility of curcumin (a
natural polyphenolic phytochemical extracted from turmeric
spice) in nanoemulsions made with short, medium and long chain
triacylglycerol oils (SCT, MCT and LCT, respectively). The average
solubility was inversely proportional to the molecular weight of
the carrier oil and increased as the average molecular weight of
the oil decreased. SCT oils possess more polar groups per unit mass
than MCT and LCT oils which may favour more dipole–dipole inter-
actions with the curcumin molecules and enhance solubilisation
(Ahmed et al., 2012). Conversely, LCT (e.g. corn oil) and MCT (e.g.
MiglyolÒ 812) oils enhanced the bioavailability and bioaccessibility
of bioactive b-carotene and curcumin compared with SCT oils and
flavour oils (e.g. orange oil) (Ahmed et al., 2012; Qian et al., 2012a).
LCT and MCT oils contain long and medium chain fatty acids that
are capable of forming mixed micelles possessing a large hydro-
phobic core to accommodate b-carotene and curcumin molecules
(Qian et al., 2012a).
Wang et al. (2008) demonstrated the high anti-inflammatory
activity of curcumin encapsulated within nanodroplets against
12-O-tetradecanoylphorbol-13-acetate-induced edema of mouse
ear. Nanoemulsions containing 1% curcumin (nanoencapsulate)
exhibited greater anti-inflammatory potencies (43% for 618.6 nm
or 85% for 79.5 nm droplet sizes, respectively) than a 1% curcumin
in 10% tween 20/water solution. The increased activity was attrib-
uted to the enhanced structure stability of nanoencapsulated cur-
cumin against intestinal degradation, resulting in more efficient
dispersibility and absorption. Also, encapsulation of resveratrol
and curcumin within nanoemulsion delivery systems improved
their water disperisbility, and their antioxidant properties were
preserved as a result of protective effects from the nanoemulsion
against degradation (Donsì et al., 2011a).
The combined delivery of tocotrienols (one form of vitamin E)
and simvastatin (a cholesterol lowering drug) by nanoemulsions
was observed to increase their anticancer activity (Alayoubi
et al., 2013). Nanoemulsion increased the solubility of tocotrienol
(poor water solubility), and when they were encapsulated with
simvastatin, enhanced their anticancer activity against human ade-
nocarcenoma cells (Alayoubi et al., 2013). Nanoemulsions have
also been made to encapsulate and deliver other hydrophobic bio-
active food components in order to increase their food value
(McClements and Rao, 2011). These include vitamin E (El Kinawy
et al., 2012; Relkin et al., 2011; Yang and McClements, 2013), fla-
vour oils (Ziani et al., 2012), b-carotene (Qian et al., 2012a,
2012b) and Coenzyme Q10 (Belhaj et al., 2012).
Antimicrobial nanoemulsions have also been demonstrated to
be effective against various food-borne pathogens (Donsì et al.,
2012; Sugumar et al., 2012). Nanoemulsions containing antimicro-
bial essential oils such as carvacrol, cinnamaldehyde, limonene,
eucalyptus oils, were effective against E. coli, Lactobacillus del-
brueckii, Saccharomyces cerevisiae, Bacillus cereus and Staphylococ-
cus aureus (Donsì et al., 2012). The antimicrobial effects of
nanoemulsions depended on the concentration of the active agent
loaded and the physicochemical properties of the surfactant/emul-
sifier used (Donsì et al., 2012). Also, sunflower nanoemulsions im-
proved the shelf stability (microbiological, organoleptic properties
and sensory qualities) of Indo-Pacific king mackerel (Scomberomo-
rus guttatus) steaks stored at 20 °C (Joe et al., 2012). The small
droplet size of nanoemulsions allowed for efficient penetration
through the cell walls of microorganism and exerted bactericidal
effects against H2S-producing and lactic acid bacteria (Joe et al.,
2012).
6. Summary
The utilisation of whey proteins (WPI, WPC and b-lg) as nano-
emulsifying agents has received considerable attention, with the
majority of work concentrating on the use of the native protein
rather than hydrolysates. This likely stems from hydrolysates
apparently possessing poor stabilising ability in conventional
emulsions. With the advent of nanoemulsion, the possibility of
these peptides being capable of stabilising nanoemulsion droplets
solely or in combination with other emulsifiers has not been ad-
dressed. In addition to their interfacial properties, whey peptides
possess bioactive properties. Peptides stabilising emulsion droplets
are consequently responsible for these bioactivities, thus, serving a
dual-functional role in food systems. Studies that have been under-
taken so far have addressed these two functionalities in isolation. A
detailed study addressing the two themes of emulsifying and bio-
logical functionalities of hydrolysed whey proteins, and milk pro-
teins in general, as a single entity is needed in order to better
understand their novel dual-functionality in foods. With the
increasing awareness of the link between diet and health, peptides
possessing multiple functionalities are prospective additives for
the fast growing functional food industry as nutraceutical and
health promoting agents.
Acknowledgements
This material is based upon research work supported by the
Charles Sturt University International Postgraduate Research
Scholarship (IPRS). The authors would also like to acknowledge
the support of the Graham Centre for Agriculture Innovation to-
wards this research work.
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