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 article info Article history: Received 26 April 2013 Received in revised form 24 July 2013 Accepted 21 August 2013 Available online 30 August 2013 Keywords: Nanoemulsions Non-protein surfactants Whey protein and peptide emulsifiers Bioactive peptides Dual-functional peptides Nano-delivery systems abstract Milk proteins are used to make emulsions, and may be used to make nanoemulsions. Nanoemulsions are a nanotechnology with food applications, and possess superior physicochemical and sensorial properties compared to macro- and microemulsions. They are also able to deliver bioactive compounds when consumed. In this review, three aspects of food nanoemulsions will be examined: (1) the production 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- ysates as nanoemulsifiers is discussed, with a particular focus on whey protein, (3) the potential of whey protein derived peptides as both emulsifiers and bioactive compounds in nanoemulsion delivery systems. Lastly, the potential delivery of bioactive peptides and other bioactive compounds within nanoemulsion systems is also discussed. Ó 2013 Elsevier Ltd. All rights reserved. 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 0260-8774/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2013.08.034 ⇑ 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: [email protected](P. Torley). Journal of Food Engineering 122 (2014) 15–27 Contents lists available at ScienceDirect Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Whey protein peptides as components of nanoemulsions: A reviewof emulsifying and biological functionalities
0260-8774/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jfoodeng.2013.08.034
⇑ Corresponding author at: School of Agricultural & Wine Sciences, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australia. Tel.: +61 2 69E-mail address: [email protected] (P. Torley).
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, Australiab National Wine & Grape Industry Centre, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australiac 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
Article history:Received 26 April 2013Received in revised form 24 July 2013Accepted 21 August 2013Available online 30 August 2013
Keywords:NanoemulsionsNon-protein surfactantsWhey protein and peptide emulsifiersBioactive peptidesDual-functional peptidesNano-delivery systems
a b s t r a c t
Milk proteins are used to make emulsions, and may be used to make nanoemulsions. Nanoemulsions area nanotechnology with food applications, and possess superior physicochemical and sensorial propertiescompared to macro- and microemulsions. They are also able to deliver bioactive compounds whenconsumed. In this review, three aspects of food nanoemulsions will be examined: (1) the productionand 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-ysates as nanoemulsifiers is discussed, with a particular focus on whey protein, (3) the potential of wheyprotein derived peptides as both emulsifiers and bioactive compounds in nanoemulsion delivery systems.Lastly, the potential delivery of bioactive peptides and other bioactive compounds within nanoemulsionsystems is also discussed.
16 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
1. Introduction
Milk proteins, and whey proteins in particular, are valued asimportant 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 (Chuet al., 2007; Dissanayake and Vasiljevic, 2009; Lee and McCle-ments, 2010). Recent studies have proven the potential of wheyprotein ingredients as emulsifiers in nanoemulsions that have beentailored for food applications (Lee and McClements, 2010; Relkinet al., 2011; Shah et al., 2012). Nanoemulsions are a specific typeof 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 fattyacids, hydrophobic vitamins, flavour and aroma compounds), andcan 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, opticaltransparency, and physical stability) compared to those of otheremulsion 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 theuse 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 thesafety, toxicity and metabolism of these synthetic emulsifiers inthe human body limit their application to food systems.
The majority of the studies using proteins have reported on thenative protein but not its hydrolysates. While hydrolysates maypossess enhanced interfacial diffusivity and emulsifying capacity,they have shown poor stabilising ability in conventional emulsionsystems, preventing long term storage (Agboola et al., 1998a;Scherze and Muschiolik, 2001). Moreover, studies on emulsionhave often focused on the crude hydrolysate which consists of het-erogeneous mixtures of amino acids, and small, medium to largechain peptides (Scherze and Muschiolik, 2001). The prospect ofstepwise fractionation to enrich sufficiently large peptides maygenerate peptides with adequate surface activities that are capableof 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, mineralcarrier, 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 andPouliot, 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 functionaland nutraceutical agents (Chu et al., 2007; Qian et al., 2012a; Rel-kin et al., 2008) thus, allowing them to more effectively expresstheir 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 wheyprotein hydrolysates and the potential for whey protein peptidesas 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 followingdiscussion.
2. Nanoemulsions
2.1. Properties of nanoemulsions
Nanoemulsions are a technology that has food and pharmaceu-tical applications (Tarver, 2006), and their evolution has paralleledthe development of efficient emulsification technologies (Cortés-Muñoz et al., 2009). An efficient emulsification process is able toform emulsions with small droplet sizes and narrow size distribu-tions. These characteristics are, however, a function of the twoopposing forces; droplet breakup and droplet–droplet coalescence(Jafari et al., 2006). These properties have been identified in severalworks (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/translucentsystems and as a result, they can be incorporated as components offood 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 nanoemulsionsstems from the characteristic physicochemical properties that theirsmall droplet sizes provide (Table 1). Their small droplet size allowsfor efficient delivery, accelerated release and rapid absorption ofhydrophobic 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 thefree energy of the separated oil and aqueous phases is always low-er than that of the formed emulsion and therefore, nanoemulsionsdo not form spontaneously (McClements and Rao, 2011). To breaklarge 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 overcomethe Laplace pressure p (Pa; difference in pressure between the con-vex and concave sides of a curved interface) and to break up theinterface between the oil and water phases (Eq. (1)) (Cortés-Muñozet al., 2009; Walstra, 1993). The Laplace pressure characterises theinterfacial force that acts on droplets to keep them from being dis-rupted (McClements, 2005).
P ¼ 2cr
ð1Þ
where r (m) is the principal radius of curvature of the droplets(assuming droplets are spherical) and c (N m�1) is the interfacialtension between the two phases.
Properties Type of emulsions
Dec
reas
ing
drop
let s
izes
Incr
easi
ng e
nerg
y in
put
Nanoemulsion
- Droplet size: 10–200 nm - Appearance: Transparent/translucent to milky - High/low energy emulsification - Surfactant load: Medium (<1 to >10%) - Does not form spontaneously - Kinetically stable
Microemulsion
- Droplet size: 4–200 nm - Appearance: Transparent - High/low energy emulsification - Surfactant load: Fairly high (10–30%) - Can form spontaneously - Thermodynamically stable
Macroemulsion
- Droplet size: >1 m - Appearance: Formulation dependent - Conventional homogenisation - Surfactant load: Fairly low - Does not form spontaneously - Kinetically stable
Fig. 1. Properties of food macro-, micro-, and nanoemulsions.
Table 1Nanoemulsion properties and applications.
Property Applications References
Stability Gravitationalseparation
The constant Brownian motion of the droplets makes them stable against gravitationally inducedseparation (creaming and sedimentation) and drainage in the manner observed for microscaleand larger emulsion droplets
Graves et al. (2005), Lee andMcClements (2010), Penget al. (2010)
Flocculation Weak flocculation is prevented and this enables the droplets to remain dispersed with noseparation
Qian and McClements(2011), Graves et al. (2005)
Coalescence 1. The significant surfactant film thickness relative to droplet size prevents any thinning ordisruption of the liquid film between the droplets2. 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,phytosterols, and polyunsaturated fatty acids (PUFA)
Yuan et al. (2008), Qianet al. (2012a)
2. Enhances the bioavailability of peptides and proteins carried by nanocapsules due to enhanced surfaceinteraction of nanocarriers with the absorptive epithelium and their protective effect for the associated peptide
Prego et al. (2006)
3. Enhances the encapsulation of lipophilic functional ingredients in nanoemulsion delivery systems and enabletheir controlled release in response to specific environmental triggers
Lee and McClements (2010),Qian et al. (2012a), Tarver(2006)
4. Nanodroplet delivery systems may increase the passive cellular absorption mechanisms and reduce the masstransfer resistances of antimicrobial essential oils (e.g. carvacrol, limonene and cinnamaldehyde, basil oil) againstEscherichia coli, Lactobacillus delbrueckii and Saccharomyces cerevisiae
Donsì et al. (2012), Ghoshet al. (2013)
Opticaltransparency/low turbidity
1. The dimensions of the oil droplets could be much smaller than the wavelength of light, making themtransparent systems suitable for incorporation of active ingredients into many food beverages without loss ofclarity
Lee and McClements (2010),Qian and McClements(2011), Ghosh et al. (2013),Qian et al. (2012a), Kentishet al. (2008)
2. As optically transparent, they are associated with freshness, purity, simplicity, water-like and may lead to alarge variety of products from water-like fluids to ringing gels
Sonneville-Aubrun et al.(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 easilyabsorbed by the skin culminating in a pleasant and aesthetic skin feel
Sonneville-Aubrun et al.(2004)
R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27 17
The high energy emulsification method requires the use of ahigh pressure valve homogeniser (HPVH), a microfluidiser, or anultrasonicator (Jafari et al., 2006; Kentish et al., 2008; Lee andMcClements, 2010; Mao et al., 2010). Droplet disruption withinthe homogeniser is a complex process arising from a combinationof flow dynamics (laminar, turbulent and cavitational) whichdetermines the nature of the disruptive forces (shear/viscous, elon-gational, inertial and cavitational) acting on droplets (McClements,2005; Walstra, 1993). A more in-depth discussion on flow condi-tions within the homogeniser, disruptive forces and energy consid-erations can be obtained from McClements, (2005), Schubert andEngel, (2004) and Walstra, (1993).
The choice of homogenisation technique and homogeniser is vi-tal as it defines the type of flow conditions to which droplets are
subjected in order to cause droplet breakup (McClements, 2005)and determines the smallest droplet size that can be produced dur-ing emulsification (Donsì et al., 2012; Mao et al., 2010). A compar-ison of nanoemulsification techniques applicable to foodnanoemulsion production is given in Table 2. Other methods forproducing nanoemulsions have been reported, such as: the mem-brane emulsification and electrical coaxial liquid jets (Acosta,2009), the aqueous extraction-ultrafiltration method (Nikiforidiset al., 2011) and the typical low energy emulsification approaches,such as the spontaneous emulsification and solvent displacementtechniques (Prego et al., 2006), and the phase inversion or persua-sive technique (Bilbao-Sáinz et al., 2010; Wulff-Pérez et al., 2009).However, these methods have found little application to the foodindustry.
Table 2Comparison between different homogenisation techniques for nanoemulsion formation.
Energy efficiency Low Low High Medium to high Abismaïl et al. (1999), Jafari et al.(2006)
Ease of operation Subject to equipmentcontamination
Easy to operate andclean
Straight forwardoperation and easy toclean
Large quantities of organicsolvent required whichcould be expensive andpose a threat to theenvironment
Abismaïl et al. (1999), Freitaset al. (2006), Lee andMcClements (2010)
HPVH: High pressure valve homogenisation, HEE/SD/E: High energy emulsification/solvent displacement/evaporation.
18 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
3. Emulsifiers/surfactants and co-surfactant systems innanoemulsions
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 designof nanoemulsion systems (McClements and Rao, 2011). Surfactantsinfluence 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 throughelectrostatic repulsive and steric forces (Ghosh et al., 2013; Maoet al., 2010). In addition, the nature and properties of the emulsifiercan affect the interfacial and bulk rheology of the nanoemulsiondroplets, which is vital to the design of nanoemulsion systems tai-lored for a specific application. Surfactant properties also definethe 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 formulatingnanoemulsions and colloidal systems in general. Most studies onnanoemulsions formation have utilised low molecular weight syn-thetic emulsifiers/surfactant/co-surfactant systems (Table 3). Theypossess better interfacial diffusive properties compared to largebiopolymers, such as proteins and polysaccharides. However, con-cerns about their safety, toxicity and metabolism may limit theirapplication 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 astrong and cohesive protective film that helps prevent dropletaggregation (Lee and McClements, 2010). They are also effectiveas emulsifiers in nanoemulsions (Table 4). Whey proteins (a-lact-albumin, b-lactoglobulin, bovine serum albumin, lactoferrins, andimmunoglobulins) constitute about 20% of the total protein in milk(�80% caseins) and have a high nutritional value owing to theirhigh 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 hydrophilicresidues) (Foegeding et al., 2002).
Whey proteins possess globular/rigid structures with buriedhydrophobic residues which tend to negatively affect their func-tionality (Gauthier and Pouliot, 2003). Controlled enzymatichydrolysis 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 characteristicsaccount for their higher rate of diffusion to the oil/water interfaceand their ability to cover a larger area of the interface than the in-tact protein (Davis et al., 2005; O’Regan and Mulvihill, 2010). Theiramphiphilic nature allows them to adsorb onto the surfaces of oildroplets (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 islimited, 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 WPIhydrolysates (with degree of hydrolysis (DH) 8.1% and 18.1%)had smaller droplet sizes (110.3 and 30.4 nm, respectively) thanwhey 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 dropletsurface 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 foodindustry, the potential of hydrolysed protein peptides as emulsifieringredients 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
Table 3An overview of non-protein surfactants/co-surfactants systems stabilised nanoemulsions.
Emulsifier type Homogenisationmethod
Oil phaseconcentration(%)
Emulsifierconcentration(%)
Oil phase Dropletdiameter(nm)
References
Non ionicDecaglycerol monolaurate (DML, ML750) Microfluidisation/
HPVH0.03, 1 1, 10 b-Carotene in sunflower oil 115–279 Mao et al.
(2009, 2010)Polyoxyethylene sorbitan monolaurate
(Tween 20)Microfluidisation/HPVH
0.03, 1 1, 10 b-Carotene in sunflower oil 117–280 Mao et al.(2009, 2010)
Microfluidisation 4 1.5 b-Carotene in corn oil, Miglyol812 and orange oil
20 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
Pouliot, 2003). Protein hydrolysis to produce peptides with desir-able functionalities is usually performed with enzymes due to theirhighly specific mode of action, ease of control, use of milder condi-tions, and tendency not to cause amino acid damage compared tochemical, thermal and microbial hydrolysis (Cheison et al., 2007).Careful enzyme selection means hydrolysates can be producedwhich are tailored specifically for food applications.
3.3.1. Enzyme typeTryptic peptides of heat pre-treated WPI had higher emulsifying
activity and emulsion stability than chymotrypsin, Alcalase andNeutrase peptides (Mutilangi et al., 1996). Trypsin hydrolysatecontained large amphiphilic peptides that favoured emulsion for-mation and stability (Mutilangi et al., 1996). Trypsin hydrolysatesof 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 differentnumbers of hydrophobic regions because they cut in differentplaces. The adsorption of peptide onto droplet interfaces dependson hydrophobic properties of the peptides, especially their surfacehydrophobicity (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 toform 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 bereduced or increased depending on the specificity of the enzymeused, pre-hydrolysis heat treatment, as well as on the extent ofhydrolysis (Adjonu et al., 2013; Mutilangi et al., 1996).
3.3.2. Degree of hydrolysisThe 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 peptidesolubility and emulsifying ability (Lieske and Konrad, 1996), butif 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, theemulsifying capacity of WPC hydrolysates increased to a maximumcapacity at 3% DH, with lower emulsifying capacity at lower andhigher DH (Lieske and Konrad, 1996). In more extensively hydroly-sed whey protein products, the 10% DH showed better emulsioncharacteristics (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 withDH > 20% (Singh and Dalgleish, 1998). As DH was increased from10% to 45%, droplet size increased, and coarse emulsions exhibitingbimodal size distributions were formed. The decreased emulsifyingproperties 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 (Lamand Nickerson, 2013; Miñones Conde and Rodríguez Patino, 2007),and increased peptide–peptide and protein–peptide interactions atthe 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 primarystructure (Foegeding et al., 2002).
3.3.3. Peptide sizeEmulsions 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 21
2001; van der Ven et al., 2001). A minimum peptide size of >2 kDais required for good emulsify properties, as the size of the peptidesdictates the steric stabilisation of emulsion droplets (Gauthier andPouliot, 2003; van der Ven et al., 2001). Also, larger size peptidesare more likely to have both hydrophobic and hydrophilic residueson the same molecule. During emulsion formation, the hydropho-bic side chains of proteins interact with the oil droplets and thehydrophilic residues will favour the aqueous phase and stabilisethe droplets through steric effects (Dalgleish, 1997; Lam and Nick-erson, 2013).
Although small peptides may be sufficiently surface active toform small droplets, their small size may be insufficient to preventdroplet aggregation post-emulsification, as a result of a loss of theirsteric stabilising property (van der Ven et al., 2001). In addition,larger peptides are capable of modifying the internal structure ofan emulsion by forming inter-locking networks and, hence, limitthe tendency to creaming, flocculation and coalescence (Singhand Dalgleish, 1998; Tirok et al., 2001).
3.4. Stability of whey protein hydrolysate stabilised emulsions
Once whey protein hydrolysate emulsions are formed, they aresubjected 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 dropletdestabilisation resulted from the formation of weak interfacialfilms 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 togravitational separation (creaming and sedimentation) as a resultof their constant Brownian motion (Kentish et al., 2008).
Co-surfactants/emulsifiers, stabilisers and texture-modifiers areoften required to improve the stability of whey protein hydrolysateemulsions (Table 3) (McClements and Rao, 2011; Tirok et al., 2001).Co-surfactants such as lecithins and monoglycerides may displacesmall peptides from the droplet interface or act in a synergistic roleto 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 andmay result in less peptides adsorbing at the droplet interface as aresult of the better interfacial properties of lecithin compared tothe peptides making up the hydrolysate. Effectively, smaller sizedroplets may form due to the formation of a thinner adsorbed layeraround 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 interactionsmay lead to an increase in the overall charge and hydration atthe oil droplet surface, resulting in strong interfacial film favouringdroplet stability (Agboola et al., 1998b; McSweeney et al., 2008;Van der Meeren et al., 2005).
Stabilisers and texture modifiers, usually polysaccharides, alsoprovide stability by increasing the continuous phase viscosity aswell as forming three dimensional networks which trap and retarddroplet 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 thecollision frequency of droplets and the rate of drainage from thedroplet surface (Ye et al., 2004; Ye and Singh, 2006). In addition,emulsion droplets were stable against creaming and coalescenceas a result of improved droplet packing that restricted the relativemotion of emulsion droplets (Ye et al., 2004; Ye and Singh, 2006).Protein–polysaccharide interactions can also modify the interfacialrheology, by forming bulkier polymeric layers around emulsiondroplets and provide enhanced stabilisation of droplets throughsteric effects (Akhtar and Dickinson, 2007; O’Regan and Mulvihill,2010).
3.5. Whey protein hydrolysate fractionation
Whey protein hydrolysates are a heterogeneous mixture of freeamino acids, and short to long chain peptides. Large concentrationsof hydrophilic and short chain peptides can limit their interfacialand steric-stabilising properties (Singh and Dalgleish, 1998; Tiroket al., 2001). They can also promote adsorption and desorptionmechanisms, resulting in an uneven, non-continuous and weakmechanical 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 chromatographictechniques 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 andPihlanto, 2006; Scherze and Muschiolik, 2001). A >10 kDa peptidefraction obtained after trypsin, chymotrypsin, Alcalase and Neutr-ase hydrolysis of WPC possessed higher emulsifying activity indexand emulsion stability than the crude unfractionated hydrolysate(Mutilangi et al., 1996). Also, ultrafiltration of WPC hydrolysatesproduced a 1–30 kDa peptide fraction that possessed greater inter-facial adsorption, emulsifying activity and stability than theunfractionated 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 ofstronger interfacial films around emulsion droplets (Gauthier et al.,1993; Turgeon et al., 1996).
While no studies on the emulsifying properties of fractionatedwhey protein hydrolysates in nanoemulsions are known, studiesusing crude hydrolysates have predicted nanoemulsions withdroplet sizes of 30–110 nm (Chu et al., 2007). The stability of suchnanoemulsions has not been investigated, and little informationexists 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, andhomogenisation conditions (e.g. pH, ion concentration) may pro-vide a better understanding about the emulsifying properties ofprotein hydrolysates in food nanoemulsions.
4. Bioactive peptides from whey protein
Aside from modifications to their emulsifying properties andother 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 goodhealth in humans (Table 5). Many of these bioactive peptides havebeen 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 andPihlanto, 2006). These peptides have simple structures and areconsidered safe and healthy compounds which are easily absorbedby 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 bothemulsifiers (technological function) and bioactive compounds(biological function) important for promoting good health (Adjonuet al., 2013). However, studies on the emulsifying and biologicalfunctionalities 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
Table 5Bioactive peptides from whey proteins.
Bioactivity References
Antioxidant Gauthier et al. (2006), Kilara and Panyam(2003), Kim et al. (2007), Kong et al.(2012), Korhonen (2009), Madureira et al.(2010), Théolier et al. (2013)
Mineral bindingAntimicrobial/anti-bacterialAnti-appetisingCytomodulatoryImmunomodulatory
Immunostimulant Hernández-Ledesma et al. (2011), Kilaraand Panyam (2003)Anti-thrombotic
Anti-gastricHypocholesterolemic
Anti-diabetes Silveira et al. (2013)
Opioid Hernández-Ledesma et al. (2011),Korhonen and Pihlanto (2006), Li et al.(2004), Pan et al. (2012), Pihlanto-Leppälä(2000), Teschemacher (2003)
ACE-inhibitoryAnti-hypertensive peptides
22 R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27
emulsifying capacity. The hydrolysates also showed high ACE-inhibitory potencies and were suggested as potential ingredientsfor nutraceutical preparations. Gauthier and Pouliot (2003) alsodemonstrated that some of the peptide sequences responsible forthe emulsifying properties of whey proteins were also related totheir ACE-inhibitory properties. However, the link between peptidebioactivity and their emulsifying properties was not demonstratedby these studies. Gauthier and Pouliot (2003) and Sinha et al.(2007) only reported on the ACE-inhibitory peptides of thesehydrolysates in food emulsion, but not their other bioactivities (Ta-ble 5). Table 6 summarises other studies reporting on the bioactiv-ity and technological functions of peptides from other food proteinsources.
Whey protein bioactive peptides generally have a molecularweight of less than or equal to 10 kDa and sometimes greater
Table 6Protein hydrolysates with dual-functionality (technological and biological).
Protamex™ Emulsifying capacity and waterholding capacity
Yellow stripe trevally(Selaroides leptolepis)
AlcalaseFlavourzyme
Emulsifying activity, foamingcapacity, solubility
a
Bioactive pe
Oil drople
Non-protein surfactant
Fig. 2. Nano-delivery of bioactive peptides (a) nanoencapsulated bioactive peptides stbioactive peptides using bioactive proteins/peptides emulsifiers.
(Pihlanto, 2006). These low molecular weight peptides show poorstabilising abilities in food emulsions (Agboola et al., 1998a,b;van der Ven et al., 2001; Ye et al., 2004). Overcoming these prob-lems may be possible by stepwise fractionation using membraneand chromatographic techniques to generate different bioactivepeptides from crude hydrolysates that are large and surface activeand can stabilise emulsion droplets. These peptides may be effec-tive as sole emulsifiers or in conjunction with other co-emulsifiersto form and stabilise nanoemulsion systems because of the smallersize of nanoemulsion droplets (Fig. 2). The factors affecting thenanoemulsification abilities of these peptides (e.g. dispersion con-ditions, aqueous phase properties, oil mass fractions, surfactant/co-surfactant systems, and ion and salt concentrations) could be elu-cidated in order to better understand their interfacial properties innanoemulsion systems.
In addition, the inclusion of bioactive peptides in nanoemul-sions may extend their application in food and pharmaceuticalindustries because colloidal systems may serve as an excellentmedium to incorporate these bioactive peptides into day-to-dayfoods. Active peptides that show dual-functionality could be iso-lated, characterised, sequenced and possibly synthesised, whichhas occurred for many other bioactive peptides. Also, whey proteinpeptides possess reduced allergenicity and are easy to digest, prop-erties that are vital for formulating infant formulae and sportsnutrition diets (Tirok et al., 2001). Inclusion of whey protein pep-tides in nanoemulsion could result in products with a modificationto many of their macro-scale characteristics, such as texture, taste,sensory attributes, and shelf stability, leading to a variety of prod-ucts with new functionalities (Silva et al., 2012).
Other studies have also looked at the antioxidant activities ofwhey proteins and their peptides to inhibit lipid oxidation andrancidity in emulsions. Hu et al. (2003) studied the oxidativestability of salmon oil/water emulsion formed by whey proteinemulsifiers (whey protein isolate, sweet whey, b-lactoglobulin
Bioactivity Molecular weight References
Antioxidant activity – Tang et al.(2012)
Antioxidant activity – Cumby et al.(2008)
Antioxidant activity,ACE-inhibition
<5, 5–10 and >10 kDa Da byultrafiltration
Aluko andMonu (2003)
Opioid activity,antioxidant activity
0.9–80 kDa by gel filtrationchromatography
Šlizyte et al.(2009)
Antioxidant activity – Klompong et al.(2007)
b
ptide
t
Protein/bioactive peptide emulsifier
abilised by non-protein surfactant systems (e.g. Tween 20), (b) nanoencapsulated
R. Adjonu et al. / Journal of Food Engineering 122 (2014) 15–27 23
and a-lactalbumin). All emulsions, especially those formed by b-lactoglobulin, showed greater oxidative stability with regard tothe formation of hydroperoxide and headspace propanal, particu-larly at pH values below the isoelectric point of the proteins. Tonget al. (2000a, 2000b) and Peña-Ramos et al. (2004) also reportedthe ability of whey protein fractions to inhibit the formation ofthiobarbituric 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) weremore 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 cationiccharges on the surface of emulsion droplets to repel transitionmetals, form protective films around droplets which hinders lipidhydroperoxide–transition interactions, chelate prooxidants andinactivate free radicals through sulphur containing amino acidsand peptides (Hu et al., 2003; Peña-Ramos et al., 2004; Tonget 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 ispossible the mechanisms of their antioxidant activities may differfrom 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 ofhypertension 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 developmentof nutrition tailored functional foods (Phelan et al., 2009; Wangand de Mejia, 2005). However, such applications are limited dueto the lack of appropriate delivery systems capable of protectingbioactive peptides from degradation (e.g. conformational changesand 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 bioactivepeptides are not absorbed from the gastrointestinal tract into theblood, possibly due to poor delivery systems, although their effectshave been proposed to be mediated directly in the gut throughreceptors on the intestinal walls (Korhonen and Pihlanto, 2006;Lee et al., 2007; Phelan et al., 2009).
A suitable delivery system should protect bioactive peptidesfrom 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 mucosato target sites (Balcão et al., 2013; Prego et al., 2006; Yang andMcClements, 2013). Nano-delivery systems (e.g. nanoemulsion,nanoencapsulation, nanovessicles [liposomes]) (Fig. 2) present amechanism for the structural and functional stabilisation of bioac-tive proteins/peptides against denaturation by enzymatic digestionand a way to increase their biopharmaceutical and food applica-tions (Balcão et al., 2013; Prego et al., 2006). In addition, their smalldroplet size may enhance the transport of bioactive peptides car-ried within nanodroplets as droplets may pass across the intestinalwall and facilitate their absorption, bioavailability and bioaccessi-bility (Martins et al., 2007; Watnasirichaikul et al., 2000).
The colloidal delivery of peptide drugs within pharmaceuticalpreparations 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.
Balcão et al. (2013) recently encapsulated lactoferrin (a wheyprotein fraction with bioactivities) within a water–oil–water nano-emulsion as potential antimicrobial formulation. Nanoencapsulat-ed lactoferrin and lactoferrin in solution showed inhibitory effectagainst Staphylococcus aureus, Listeria innocua, Bacillus cereus andCandida albicans, but not Gram negative bacteria such as Salmonellasp., 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 andcaseins have been shown to be effective against Gram-positiveand Gram-negative bacteria (E. coli, Helicobacter, Listeria, Salmo-nella and Staphylococcus, Listeria ivanovii), yeasts and filamentousfungi (Hartmann and Meisel, 2007; Théolier et al., 2013). Nanoen-capsulation of antimicrobial peptides, coupled with the smalldroplet size of nanoemulsion droplets, may extend the applicationsfood protein antimicrobial peptides accross the food productionchain. 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 whichtend to reduce their consumer acceptability (Komai et al., 2007;Pedrosa et al., 2006). Debittering techniques involving the removalof hydrophobic peptides by chromatography, absorption of bitterpeptides on activated carbon or selective extraction with alcoholscould result in a loss of bioactivity, as the majority of bioactiveamino acids and peptides are hydrophobic in nature (FitzGeraldand O’Cuinn, 2006; Leksrisompong et al., 2012). Encapsulation ofbioactive peptides within nanoemulsion delivery systems can beused to inhibit or reduce their bitter taste and off-flavours, as forother bitter and astringent compounds. Micro- and nanoencapsu-lation of polyphenolic compounds and other bitter compounds,such as chloroquine phosphate and trimebutine within polymericcoated 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 colloidaldelivery systems can be shielded from interactions with taste sen-sors until target sites are reached (Hashimoto et al., 2002; Sohiet al., 2004; Sun-Waterhouse and Wadhwa, 2013). Such applica-tions could be extended to encapsulate highly bioactive but bitterpeptides 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 becauseproteins would have to undergo digestion before release at targetsites (He et al., 2011). Pea protein-coated antimicrobial nanoemul-sions were noted to have longer bacteriostatic action againstmicroorganisms, which was beneficial for products requiring longshelf stability, whereas sugar esters and other synthetic surfac-tant-coated nanoemulsions promoted quicker and faster antimi-crobial effects (Donsì et al., 2012). Also, nanoemulsions formedwith 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 emulsifierssuch as Tween 80, Solutol HS 15, Poloxamer 188 and Cremophor(He et al., 2011).
In addition, the actual mode of action of bioactive peptideswhen incorporated into foods are currently not well understood,possibly due to lack of methods available to determine theseactivities when they are included as components of foods, coupledwith the complexity of most food matrices. Nanoemulsion mayserve as a good substrate for use in assessing these bioactivitieswhen peptides are added to foods. The factors that affect thesolubility, loading efficiency, absorption and bioavailability, andcontrol 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 (anatural polyphenolic phytochemical extracted from turmericspice) in nanoemulsions made with short, medium and long chaintriacylglycerol oils (SCT, MCT and LCT, respectively). The averagesolubility was inversely proportional to the molecular weight ofthe carrier oil and increased as the average molecular weight ofthe oil decreased. SCT oils possess more polar groups per unit massthan 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.Miglyol� 812) oils enhanced the bioavailability and bioaccessibilityof bioactive b-carotene and curcumin compared with SCT oils andflavour oils (e.g. orange oil) (Ahmed et al., 2012; Qian et al., 2012a).LCT and MCT oils contain long and medium chain fatty acids thatare 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-inflammatoryactivity of curcumin encapsulated within nanodroplets against12-O-tetradecanoylphorbol-13-acetate-induced edema of mouseear. Nanoemulsions containing 1% curcumin (nanoencapsulate)exhibited greater anti-inflammatory potencies (43% for 618.6 nmor 85% for 79.5 nm droplet sizes, respectively) than a 1% curcuminin 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 efficientdispersibility and absorption. Also, encapsulation of resveratroland curcumin within nanoemulsion delivery systems improvedtheir water disperisbility, and their antioxidant properties werepreserved as a result of protective effects from the nanoemulsionagainst degradation (Donsì et al., 2011a).
The combined delivery of tocotrienols (one form of vitamin E)and simvastatin (a cholesterol lowering drug) by nanoemulsionswas observed to increase their anticancer activity (Alayoubiet al., 2013). Nanoemulsion increased the solubility of tocotrienol(poor water solubility), and when they were encapsulated withsimvastatin, enhanced their anticancer activity against human ade-nocarcenoma cells (Alayoubi et al., 2013). Nanoemulsions havealso 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 Kinawyet 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 tobe 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 ofnanoemulsions depended on the concentration of the active agentloaded and the physicochemical properties of the surfactant/emul-sifier used (Donsì et al., 2012). Also, sunflower nanoemulsions im-proved the shelf stability (microbiological, organoleptic propertiesand sensory qualities) of Indo-Pacific king mackerel (Scomberomo-rus guttatus) steaks stored at 20 �C (Joe et al., 2012). The smalldroplet size of nanoemulsions allowed for efficient penetrationthrough 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 themajority of work concentrating on the use of the native proteinrather than hydrolysates. This likely stems from hydrolysatesapparently possessing poor stabilising ability in conventionalemulsions. With the advent of nanoemulsion, the possibility ofthese peptides being capable of stabilising nanoemulsion dropletssolely or in combination with other emulsifiers has not been ad-dressed. In addition to their interfacial properties, whey peptidespossess bioactive properties. Peptides stabilising emulsion dropletsare consequently responsible for these bioactivities, thus, serving adual-functional role in food systems. Studies that have been under-taken so far have addressed these two functionalities in isolation. Adetailed 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 betterunderstand their novel dual-functionality in foods. With theincreasing awareness of the link between diet and health, peptidespossessing multiple functionalities are prospective additives forthe fast growing functional food industry as nutraceutical andhealth promoting agents.
Acknowledgements
This material is based upon research work supported by theCharles Sturt University International Postgraduate ResearchScholarship (IPRS). The authors would also like to acknowledgethe support of the Graham Centre for Agriculture Innovation to-wards this research work.
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