2012-EL-SALAM - Formation and Potential Uses of Milk Proteins as Nano Delivery Vehicles for Nutraceuticals - A Review

REVIEWFormation and potential uses of milk proteins as nanodelivery vehicles for nutraceuticals: A review

MOHAMED H ABD EL-SALAM* and SAFINAZ EL-SHIBINYDairy Department, National Research Centre, Dokki, Cairo, Egypt

*Author forcorrespondence. E-mail:[email protected]

� 2011 Society ofDairy Technology

The aim of this review was to highlight the progress achieved in the use of milk protein as nano vehiclesfor nutraceuticals. Reassembled casein and b-casein micelles and core ⁄ shell nanoparticles from caseinwith other biopolymers have been prepared. Also, cross-linking of casein micelles has developed stablenanoparticles. Nanogels of whey proteins (WP), b-lactoglobulin (b-LG) and lactoferrin (Lf) have beenprepared by controlled thermal treatment, and several core ⁄ shell nanoparticles have been developedfrom WP or b-LG with several polysaccharides. The developed caseins and WP nanoparticles have beenused as carriers for several nutraceuticals. Examples have been presented and discussed.

Keywords Nanoparticles, Nutraceuticals, Casein, Whey proteins, Polysaccharides, Bioavailability.

INTRODUCT ION

Development of functional foodsThere is a growing awareness nowadays of thehealth benefits of a category of bioactive food con-stituents known as nutraceuticals and probioticmicro-organisms. This has created society demandfor products (functional foods) and preparationsrich in these constituents to improve public health.Several groups of minor food constituents andmicro-organisms including antioxidants, probio-tics, prebiotics, fatty acids, vitamins, etc. have beencharacterised by their health-promoting activities.Nutraceuticals either provide an overall improve-ment of health or they can reduce the risk of spe-cific degenerative diseases, for example cancer,cardiovascular diseases and osteoporosis (Huanget al. 2004; Lee et al. 2004). The mechanisms ofnutraceuticals action are not fully explored. How-ever, now it is well documented that their7consumption produces physiological benefits orreduce the long-term risk of developing degenera-tive diseases.The delivery of nutraceuticals is a major chal-

lenge as their effectiveness depends mainly on theirstability and bioavailability. For this purpose, twogroups of factors should be considered: (i) effect ofprocessing of the food matrix and storage condi-tions on nutraceuticals, for example heating condi-tions, oxygen and light, which results in significantlosses of the loaded bioactive constituents; (ii) pro-tection of bioactive molecules from degradation

during their passage through the gut, whichdepends on gastric residence time, permeabilityand ⁄or solubility, pH and compatibility with otherfood constituents. Generally, a small proportion ofnutraceuticals consumed in the free form remainsavailable. A study on the bioavailability of severalanthocyanins from grapes showed that they wereunavailable in their free form (Mozafari et al.2006).On the other hand, the added bioactive constitu-

ents may have some undesirable effects on the tasteand odour of the food matrix used as many of theseconstituents have undesirable taste and odour.Therefore, the objectives of food manufacturers

and nutritionists have been to maximise theavailability of administered nutraceuticals withoutcompromising consumer acceptability. Severalstrategies have been developed to maintain the sta-bility of the active principle throughout processing,handling and passage through the gut until it reachestheir target in the body. Also, the used approachshould minimise the deleterious effect of the addedfree nutraceutical molecules on the quality of thefoodmatrix.

Encapsulation of bioactive constituentsEncapsulation technology alleviates many of theproblems encountered with the direct utilisation ofnutraceuticals (Augustin and Sanguansri 2007) asthey entrapped ⁄or encased in the capsulate material.Also, encapsulation has proved effectiveness in pro-tecting the active ingredients during processing and

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storage and possible interaction with other food constituents.Further, a real benefit of encapsulation is the ability to controlthe release of the incorporated ingredients and to deliver them toa specific target at the required time and condition.The capsule size and material affect greatly the degree of

protection and bioavailability of the trapped materials. With theadvent of nanotechnology, several nano systems became avail-able for the delivery of active ingredients. The nano deliverysystems include nanoemulsions, nanoliposomes, nanoparticlesand nanotubes (Moraru et al. 2003). All these systems are char-acterised by enhancing markedly the bioavailability of theincorporated ingredients (Acosta 2009; Huang et al. 2010).However, each of these systems has its advantages and limita-tions (Moraru et al. 2003; Huang et al. 2010).Nanoparticles are structured with a dense polymeric network

in which active molecules can be dispersed (Augustin andSanguansri 2007). Many synthetic polymers have been usedsuccessfully as delivery systems in biomedical and pharmaceu-tical sector (Reis et al. 2006). However, these polymers cannotbe used in food applications that require food grade, that isGenerally Recognised As Safe (GRAS) ingredients.Proteins are GRAS food ingredients widely used in formu-

lated foods because of their diversified functional propertiesand high nutritive value. The functional properties, particularlythe gel-forming properties, are important for the suitability ofproteins as a carrier for bioactive ingredients. The diversemechanical and microstructural protein gels offer the possibilityof GRAS biocompatible delivery systems for nutraceuticals infood products. However, there is always the need to minimisethe size of protein gel particles to avoid any deleterious effectthe coarse particles on the mouth feeling of foods. In thisrespect, protein nanoparticles satisfy this need.The protein-based nanoparticles are particularly interesting

because they are relatively easy to prepare and their size distri-bution can be monitored (Chen et al. 2006). Various modifica-tions in the protein matrix allow them to form complexes withother biopolymers, particularly polysaccharides as a base forseveral nanoparticles. Also, protein-based nanoparticles canconjugate nutrients via either primary amino groups or ionicand hydrophobic binding.Milk contains several proteins of unique and diversified func-

tional properties. The structures and functional properties ofmilk proteins have been the subject of recent reviews (Wonget al. 1996; Abd El-Salam et al. 2009). The ease of their prepa-ration and fractionation on industrial scale and their uniquefunctional properties resulted in large number of milk proteinproducts are now commercially available for use as functionalprotein ingredients in formulated foods.The use of milk proteins as delivery vehicle for bioactive

materials is a new trend (Livney 2010) that received muchattention. This subject has been part of several publications andreview article (Chen et al. 2006; Graveland-Bikker and de Kru-if 2006; Weiss et al. 2006; Semo et al. 2007; Huppertz and deKruif 2008; Santipanichwong et al. 2008; Jones et al. 2009).

The aim of the present article was to compile and discussavailable information about the potential uses of milk proteinsas a base of nanoparticles for the delivery of bioactive com-pounds in functional foods.

M ILK PROTE INS AS NANO CARRIERS

Caseins

Caseins and casein micellesCasein is present in milk mainly (�94%) as a naturally assem-bled micelles of sizes ranging from 50 to 500 nm (Fox andMcSweeney 2003). Casein micelles can be regarded as natu-rally designed nano vehicle for delivery of minerals particularlyCa to neonate. However, casein micelles have no fixed struc-tures. Changes in temperature, pH, ionic strength and wateractivity and high hydrostatic pressure treatment lead to changesin size distribution of casein micelles (de Kruif 1999) becauseof the absence of a rigid three dimensional tertiary structure(McMahon and Ommen 2008).Caseins have the following features that can support their use

in designing food grade nanoparticles for the delivery of bioac-tive compounds:Caseins are a family of open-structured, proline-rich phos-

phoproteins. The main four caseins in bovine milk are a-s1-CN, a-s2-CN, b-CN and j-CN (Farrell et al. 2004). They differin the number and sequence of amino acids, number of phos-phorus atoms, and proline and carbohydrate contents.The caseins have distinct hydrophilic and hydrophobic

domains, which favour conformational changes in solutionsdepending on environmental conditions. b-casein is unique intemperature-controlled conformation being monomeric at 0–8 �C and form micelles with hydrodynamic radius of 12 nm at15–30 �C.The most characteristic feature of caseins is their ability of

self-assembly into natural or artificially simulated micelles. Themicelles are stabilised by the colloidal calcium phosphate andsurface coverage with the hydrophilic domain of j-CN (Horne2009).The phosphorus atoms are present in clusters in a-s1-CN,

a-s2-CN, and b-CN, which allow the chelation of minerals aspreferred vehicle for these ingredients.The formed micelles are assembled in the way that they have

hydrophobic core and hydrophilic shell. Also, micelles haveporous structure that retains 2 g water ⁄g protein.The casein micelles can stand processing treatments, for

example heat treatment.However, the colloidal instability of casein micelles can be

induced by treatment with milk clotting enzyme, addition ofacids. Therefore, stabilisation of casein micelles under gastroin-testinal environment would improve their use as nano vehiclefor delivery for bioactive compounds.Recently, natural casein micelles have been used for the

preparation of nanogel particles by enzymatic cross-linking and

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subsequent removal of the calcium and calcium phosphate fromthe modified micelles. Because of their low level of secondaryand tertiary structures, caseins are excellent substrates for cross-linking by transglutaminase (TGase), which can covalently linkglutamine residues in the peptide chain with the lysine residue.Treatment of casein micelles with TGase has been reported toinduce intra-micelle cross-linking with minimum cross-linkingbetween micelles (Huppertz and de Kruif 2008). This can beseen from particle size measurements of TGase-treated caseinmicelles. The average particle size of TGase-treated caseinmicelles (�213 nm) was not markedly different from theuntreated casein micelles (195 nm) indicating very little inter-cross-linking (Vasbinder et al. 2003; Mounsey et al. 2005).Cross-linking has progressively increased the intra-micellar sta-bility (Smiddy et al. 2006) and cross-linking of all caseins con-verted casein micelles to nanogel particles (Huppertz et al.2007). Micellar calcium phosphate (MCP) can be removedfrom the TGase-treated micelles by cooling to 5 �C, adjustmentof pH (�4.5) and than dialysed against skim milk at 5 �C for48 h (Huppertz and de Kruif 2008). Also, the MCP can be par-tially or completely removed from the cross-linked caseinmicelles with high-pressure (HP) treatment without disruptingthe micellar structure (Huppertz and de Kruif 2007b), and HPof �400 MPa was needed for complete removal of MCP fromcross-linked micelles. Treatment of casein micelles with TGasefor 4–24 h at 30 �C inhibited rennet-induced gelation of thecross-linked micelles (Huppertz and de Kruif 2007a). Also,MCP-free casein nanogel was more heat stable, but less acidstable than natural micelles (Huppertz and de Kruif 2008). Ithas been suggested that the prepared MCP-free casein nanogelcan be used as a nano carrier for mineral and vitamins(Huppertz and de Kruif 2008), but no study has been cited forthis application.

Reassembled casein micellesReassembled casein micelles (rCM) can be prepared from acidcasein (Mounsey et al. 2005) or sodium caseinate (Semo et al.2007). However, successful preparation of rCM by these meth-ods requires control of the critical parameters (pH, temperature,ionic strength) involved.The flow sheet (Figure 1) summarises steps followed for the

preparation of rCM from acid casein and sodium caseinate,respectively. The size of rCM was slightly larger than the nativecasein micelles, and the properties of the native and reassem-bled casein micelles were slightly different (Mounsey et al.2005).Also, casein nanoparticles have been prepared by pH manip-

ulation of casein solution (Aimi et al. 2009; Kanazawa 2010).The preparation and use of acid casein as a nano carrier for bio-active compounds have been described in a three-step proce-dure (Aimi et al. 2009). The first step is to dissolve acid caseinin a basic aqueous medium pH > 8–<11, followed by the 2ndstep where a solution of an active substance is added to thecasein solution to form a complex between casein and the

bioactive compound. In the 3rd step, the mixture from step 2 ismicro-injected in acidic solution �1 pH higher than the pI ofthe casein. The patent claims that the formed casein nanoparti-cles have sizes that range from 10 to 300 nm. A second patent(Kanazawa 2010) has described the preparation of casein nano-particles of positive zeta potential (+3–30 mV) and sizes thatrange from 10 nm to less than 300 nm by mixing casein inacidic solution (pH > 0.5–<7.0) of several organic and inor-ganic acids followed by increasing the pH of the solution to pHbelow the isoelectric point of casein. The prepared nanoparti-cles are then stirred vigorously with a solution of the bioactivecompound to form a complex between them.The use of rCM as nano delivery system is based on the for-

mation of caseinate–bioactive compound complex followed byremicellisation (Semo et al. 2007) of the complex in a similarway to sodium caseinate. Using vitamin D2 as an example forbioactive food compounds (BFC), the D2–caseinate complexhas been prepared by dropwise addition of D2 solution in abso-lute ethanol to sodium caseinate solution (5 g ⁄kg) while stir-ring. The obtained rCM and D2CM had an average diameter of�125 and 128 nm, respectively. The rCM can provide partialprotection against light-induced degradation to entrappedvitamin D2.A patent (Livney and Dalglish 2007) has been issued for the

preparation of rCM by several methods and their use for nanocapsulation of a wide range of hydrophobic compounds.Successful preparation of rCM nanoparticles without the use

of calcium and phosphate has been described (Zimet et al.2011) and used for the delivery of Docosahexaenoic acid(DHA). However, the size of DHA-loaded casein nanoparticles(288.9 ± 9.5 nm) has been larger than the DHA-loaded rCM.

b-Caseinb-casein (b-CN) is intrinsically unstructured amphiphilic pro-tein that self-assemble into micelles. The dimensions and

Method (Mounsey et al,2005) Method (Semo et al, 2007)

Acid casein (88 g) 200 mL sodium caseinate (5 g/kg)Dispersed in + 4 mL K3citrate (1 M) Deionised water (912 g) + 24 mL K2HPO4 (0.2 M)Mixed 5000 g/5 min + 20 mL CaCl2 (0.2 M)

Casein dispersion (88 g/kg) Caseinate solution + Ca(OH)2slurry (100 g/kg) 8 successive (at 15 min intervals)To pH 7.1 (after 15 min) addition of 2.5 mL K2HPO4 0.2 M

+ 5 mL CaCl2 (0.2 M) Stirred at 37°C

Calcium caseinate micelles (18 mg Ca/g protein) re-assembled casein micelles Titrate simultaneously with adjust to 400 mL and pH 6.7 CaCl2 (2H2O) (113 g/kg) stirred for 1 hr and Na2HPO4 (116 g/kg) ultracentrifuged

Re-assembled Ca caseinate phosphate Precipitate (36 mg Ca, 18 mg P/g protein) suspended in water/heated at 60°C/20 min simulated milk serum

homogenised (185 MPa)

Re-assembled casein micelles Re-assembled casein micelles

Figure 1 Preparation of re-assembled casein micelles.

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shape of these micelles are strongly affected by pH (Portnayaet al. 2008). At pH below the pI of b-CN (pH 2.6) and24 �C, b-CN has been reported to form uniform disc-likeshape with a round cross-section of 20–25 nm in diameterand a width of 3–4 nm independent of the concentration inthe range 10–40 mg ⁄mL and anion type. At neutral pH,b-CN has been found in a monomeric form at 0–8 �C andforms nonuniform oblate micelles with hydrodynamic radiusof about 12 nm at 15–30 �C. Also, considerable differenceshave been found in the charge distribution along the back-bone of b-CN in acidic and neutral pH.Most of the hydrophobic residues of b-CN form the core

whereas the most of the hydrophilic residues form the shell ofthe micelles (de Kruif and Grinberg 2002). b-casein has highaffinity to bind hydrophobic compounds. Vitamin D3, as amodel for bioactive hydrophobic compounds, has been reportedto interact with b-CN in a molar ratio of 1.16–2.05 ⁄mole pro-tein depending on the solution conditions (Forrest et al. 2005).Nano encapsulation of vitamin D in b-casein micelles has beendescribed recently (Bargarum et al. 2009). Based on this work,a patent has been issued (Danino et al. 2009) in which b-caseinhas been used instead of the whole casein in the preparationnanoparticles for the delivery for hydrophobic additives at pHas low as 2.0.

Casein ⁄biopolymer complexesLinear b-CN and globular lysozyme have been used to pre-pare nanoparticles with a simple process (Pan et al. 2007b).The two proteins form polydisperse electrostatic complexmicelles in the pH range of 3.0–12.0 at a molar ratio ofb-CN to lysozyme 0.4. b-casein ⁄ lysozyme nanoparticles wereformed after heating the mixed micelles solution at 80 �Cwhere lysozyme gelated and b-CN was trapped mainly in thenanoparticles. Small-sized nanoparticles (about 100 nm) havebeen formed at pH 10 and larger particles (about 300 nm) inacidic pH. The differences in the particle size in alkaline andacidic pH can be explained on the basis of the percentage ofb-CN and lysozyme located on the surface. For nanoparticlesprepared at pH 10, more b-CN was located on the surface,which increases the surface area leading to smaller-sizednanoparticles, while for nanoparticles produced at pH 5.0more globular lysozyme molecules were found on the sur-face, which decreases the surface area resulting in larger size.The obtained nanoparticles have been stable and relativelyhydrophobic at pH 5 and 10.Formation and properties of chitosan–caseinate complexes

are controlled by the concentration of the two biopolymers aswell as by environmental conditions (pH and ionic strength).Nano complexes with diameter between 250 and 350 nm havebeen formed between chitosan and sodium caseinate at pH 5.0–6.0 and low protein to polysaccharide ratio (Anal et al. 2008).The complex remained constant in particle size, stable andsoluble over a defined pH range depending on the ratio of thebiopolymer in the mixture and on the ionic strength.

Stable nanoparticles have been prepared by electrostaticcomplexation of sodium caseinate and gum arabic using slowacidification to a specified pH values (Flanagan and Singh2008), and the size of the formed nanoparticles ranged from100 to 150 nm. It has been postulated that these particles com-prise an aggregated casein core protected from further aggrega-tion by steric repulsion of the electrostatically attached gumArabic shell.

Whey proteinsWhey proteins are a mixture of globular proteins of variablecomposition and functional properties. Several whey proteinproducts, for example whey protein concentrates (WPC) andwhey protein isolates (WPI), in their native form are industri-ally produced as food protein ingredients. The functional prop-erties of these products are largely controlled by the majorwhey protein b-lactoglobulin. The whey protein and b-lacto-globulin preparations have been used as a vehicle for the deliv-ery of bioactive compounds.The use of whey proteins and specifically b-lactoglobulin as

carrier for bioactive compounds is based mainly on the entrap-ment of these components in whey protein hydrogels. Hydro-gels are water-swollen network that can hold large amount ofwater while maintaining a network structure (Qui and Park2001).

b-Lactoglobulin (b-LG) is a suitable candidate for the prepa-ration of nano delivery systems for lipophilic bioactivecompounds as a stable system and its capability to bind hydro-phobic constituents.Native b-LG is stable in acid condition and quite resistant to

digestion by gastric proteases (Wang et al. 1997a). b-Lacto-globulin is a lipophilic binding protein similar to retinol-bind-ing protein. However, b-LG showed high affinity to vitaminD2 (10 times higher than rotenoids and some other lipophiliccompounds) (Wang et al. 1997b). The structure of b-LG ischaracterised by the presence of three possible legend bindingsites; the solvent conical b-barrel as the main site, a second sitenear the a-helix on the external surface of the b-barrel and thethird site at the dimmer interface (Jameson et al. 2002). How-ever, the bound bioactive compounds are poorly protectedbecause of the solvent accessibility of the binding sites.An important functional property of whey proteins and b-LG

is their ability to form cold-induced gel matrices by addition ofcations to preheated denatured b-LG suspension (Barbut andFoegeding 1993; Remondetto and Subirade 2003), whichresults in the formation of a network hydrogel cross-linked viathe added cation with carboxyl groups on denatured b-LG.Modulation of gel microstructure and functional propertiesshould allow tailoring of water soluble delivery systems for bio-active compounds (Chen et al. 2006). However, controllingand maintaining the size of the formed hydrogels have beendifficult to achieve.A novel method has been developed for the preparation of

whey protein nanoparticles based on the use of microemulsions



as nanoreactors (Zhang and Zhong 2009). Whey protein isolate(WPI) solution (5%) was treated with TGase at the ratio of 5%of WPI mass and pH 7.5 and 50 �C for 10 h. The WPI solutionwas then mixed with an oil surfactant mixture (limonine ⁄n-butanol ⁄Tween 60) at a ratio of 0.3 mL ⁄10 g mixture, heatedat 90 �C ⁄20 min and immediately cooled and centrifuged. Thesupernatant was discarded and the pellet (thermally aggregatedwhey proteins) was washed thoroughly with ethanol. Theformed nanoparticles have been found to be heat stable withparticle diameter >100 nm.Whey protein nanoparticles have been prepared by thermal

denaturation, pH cycling and Ca+2 cross-linking (Giroux et al.2010). Soluble denatured whey protein polymers (SDWPP)were first produced by heating (80 �C ⁄15 min) WPI dispersion(8%) in deionised water at pH 7.0. The diluted SDWPP solu-tion (2%) was acidified to aggregation pH (5.0–6.0), and CaCl2 (0–5 mM) was then added. The mixture was aged at 4 �Cup to 75 h to allow the formation of disulphide bonds betweenprotein molecules, pH was adjusted to 7.0 and homogenised(20 MPa 1st pass and 3.5 MPa 2nd pass). The formed nanopar-ticles had diameters of 100 300 nm depending on the prepara-tion conditions. The volumenosity of the particles decreasedwith increasing Ca concentration during pH cycling. The pre-pared nanoparticles showed good stability in the presence ofdissociating agents.Recently, the formation of lactoferrin nanoparticles by con-

trolled thermal treatment has been described (Bengoechea et al.2011). Lactoferrin (Lf) has been found to have two thermaldenaturation temperatures (61 and 93 �C), and the formationand size of Lf nanoparticles depended on the temperature andduration of the thermal treatment. The obtained nanoparticleswere resistant to subsequent change in pH (3–11) and ionicstrength (0–200 mM NaCl. Lf nanoparticles can be used toencapsulate and deliver bioactive compounds or to deliver Lfnanoparticle rather than individual molecules when Lf is usedas functional component.

Core ⁄ shell whey protein nanoparticlesTo avoid the drawbacks of using whey proteins nano hydrogelsfor the delivery of nutraceuticals, core ⁄ shell nanoparticles havebeen developed, which are finding wide applications nowadays.The core ⁄ shell nanoparticles are nanostructures that have coremade from a material coated with another material. The chosenshell material should prevent agglomeration of particles. Thecore ⁄ shell structures enhance the thermal and chemical stabilityof the nanoparticles and improve their solubility. The shell canalso prevent the oxidation of the core material (Sounderya andZhang 2008).The formation of core ⁄ shell whey protein nanoparticles is

based on complex coacervates between proteins and ionic poly-saccharides under conditions where there is a moderately strongattraction between the protein and polysaccharide molecules.This offers a relatively simple method for creating core ⁄ shellnanoparticles that can be utilised for encapsulation purposes.

Several polysaccharides have been used to prepare core ⁄ shellnanoparticles mainly with b-LG.

Chitosan ⁄b-lactoglobulin nanoparticles. Chitosan (CS) is acopolymer widely used in numerous food and biomedicalapplications because it is biodegradable, biocompatible andmuco-adhesive (Hejazi and Amiji 2003). Recently, much atten-tion has been paid to CS nanoparticles because of their featureto adhere to the mucosal surface and transiently opening thetight junctions between epithelial cells. However, CS matricesare not stable at low pH, which could lead to destruction of thesensitive nutraceuticals. To overcome this drawback, the sur-faces of CS nanoparticles need to be coated with an acid resis-tant biomaterial. Also, CS can be used only as a vehicle forhydrophilic nutraceuticals.The formation of CS ⁄b-LG core ⁄ shell nanoparticles for the

delivery of bioactive compounds is attractive in combining theadvantages of the two biopolymers and to avoid the drawbacksfrom their use individually. The following methods have beendeveloped for the preparation of CS ⁄b-LG nanoparticles:

Cold gelation using tripolyphosphate. Native and thermallydenatured (80 �C ⁄30 min) b-LG solutions were added to CSsolution (2.0 mg ⁄mL) in aqueous acetic acid (0.1%) to formCS ⁄b-LG complex (Chen and Subirade 2005). Tripolyphos-phate (TPP) solution was then added with stirring until an opal-escent suspension was formed spontaneously. The formednanoparticles consisted of a CS and TPP core and b-LG shellwith its hydrophobic portion lying close to the core to allowmaximum b-LG loading. The nanoparticles prepared withnative b-LG had favourable properties to resist acid and pepsindegradation in simulated gastric conditions, but b-LG shell ofthe nanoparticles degraded under simulated intestinal condi-tions. Optimal nanoparticles with size of about 100 nm wereobtained at pH 6.1 for native b-LG and at pH 6.5 for denaturedb-LG at b-LG concentration of 2.0 mg ⁄mL. The preparednanoparticles have been designed to encapsulate negativelycharged nutraceuticals in the CS-TPP core. It has the advantagethat heat-sensitive bioactive compounds can be encapsulatedwithout any loss in their activity.

Thermal treatments. The formation b-LG–CS coacervate washighly dependent on pH and achieved maximum value at a pHof 6.5 (Lee and Hong 2009). Also, the denaturation temperatureof b-LG was reduced in the presence of CS. Mixed solutions ofb-LG (0.5%) and CS (0.1%) form soluble complex at pH 4.5through electrostatic interaction between the two biopolymers(Hong and McClement 2007). After heating (80 �C ⁄20 min),relatively small size (about 140 nm) and cationic (zeta poten-tial > +0.20 mV) hydrogel particles were formed. These parti-cles consisted of aggregated b-LG molecules with chitosanmolecules trapped inside. The prepared material had the advan-tage that only natural biopolymers have been used in its prepa-ration. However, the application of heat treatment in their



preparation may limit their use for encapsulation of heat labilenutraceuticals.

Heat-denatured b-Lactoglobulin ⁄pectin nanoparticles. Pectinis a family of anionic polysaccharides with a predominantstructure of a (1 fi 4) linked D galacturonic acid. Also, pectincontains arabinose, galactose and rhamnose (Yapo et al. 2007).Pectins are identified by two parameters: the degree of methylesterification of the carboxyl groups (DM) and the distributionof methyl esters along pectin backbone (DB), that is randomdistribution (low DB) and in block wise (high DB). Also, pectinfrom different sources differs markedly in their mineral con-tents. Beet sugar pectin has been used frequently in the prepara-tion of b-LG ⁄pectin nanoparticles (Jones et al. 2009). Thispectin often contains much more and diverse minerals, organiccompounds and protein composition than citrus and applepectin. In addition, beet pectin contains appreciable amount offerulic acid side chains, which give it some hydrophobiccharacter.Two methods have been used to prepare nanoparticles from

heat-denatured b-LG–pectin complexes. The first method isbased on formation of b-LG nanoparticles and then coatingthem with pectin, and the second is based on the formation byheating b-LG–pectin complexes together (Jones et al. 2010a).Comparing the first and second methods for the preparation ofb-LG–pectin nanoparticles (Jones et al. 2010b) suggested thatparticles from the second method have higher surface coveragewith pectin, thereby reducing their tendency to aggregate. Thetwo methods are briefly described in the following:

Electrostatic deposition. b-LG solution (0.5%), pH 5.8, washeated (80 �C ⁄15 min) where a stable sub-micron sized(d = 100–300 nm) protein aggregates were formed (Santipa-nichwong et al. 2008). The heated b-LG aggregates suspensionwasmixedwith a beet pectin solution at pH 7.0 and then adjustedto pH < 6 where beet pectin was adsorbed on the core b-LGaggregates. The formed particles had low zeta potential and werestable to aggregation at pH 4.0–6 and had good salt stability.

Heat treatment of b-LG ⁄pectin complex. Initially, biopolymercomplexes in the sub-micron sizes (d = 100–300 nm) wereprepared by mixing 0.5% b-LG solution and 0.1% beet pectinat pH 5.0. These particles were then heated (83 �C ⁄15 min).The formed biopolymer particles were stable to aggregationover a range of pH (3–7) (Jones et al. 2009).The charge of the used pectin affects greatly the formation

and properties of the core ⁄ shell b-LG–pectin nano structures(Jones et al. 2010b). The pH stability was greater for b-LG-high methoxyl pectin (HMP) than for b-LG-low methoxyl pec-tin (LMP). The addition of 200 mM sodium chloride improvedgreatly the stability of b-LG-HMP particles. The formedbiopolymer nanoparticles were spheroid in shape. Neutralco-solvent, for example sorbitol, can be used to modulate theproperties of biopolymer nanoparticles prepared by thermal

treatment of protein–polysaccharide electrostatic complexes(Chanasattru et al. 2009).

Lactoferrin ⁄pectin complexes. Lactoferrin (Lf) is a minor glob-ular protein found in whey, which has considerable potentialuses as a functional food ingredient. Lactoferrin has beenreported to have two denaturation temperatures (61 and 93 �C)associated each with one of the two lobes of the protein (Ben-goechea et al. 2011). Thermal treatment of Lf solution led tothe formation of nanoparticles whose size depended on theheating conditions (temperature ⁄duration). The obtained Lfnanoparticles were resistant to changes in pH (from 3 to 11)and to addition of salt (0–200 mM NaCl). The Lf nanoparticlescan be used to encapsulate and deliver bioactive ingredient butno work has been carried out in this area.

APPL ICAT IONS OF MILK PROTE IN NANOVEHICLES IN DEL IVERY OFNUTRACEUT ICALS

Polyphenols(-)-Epigallocatechin-3-gallate (EGCG), the major catchin ingreen tea, is a potent antioxidant with numerous health benefits.However, the use of EGCG for enrichment of foods is limitedbecause of its poor oxidative stability. Preheated (75–85 �C ⁄20 min) b-LG interacted with EGCG to form nanoparti-cles of diameter smaller than 50 nm during cooling andvortexing (Shpigelman et al. 2010). The prepared nanostructureoffered considerable protection to EGCG against oxidative de-gradation. The prepared b-LG-EGCG nanostructure had excel-lent transparency that can be used to enrich clear beverages.Resveratrol (3,5,4¢-trihydroxystilbene), a natural polypheno-

lic compound found in grapes, exhibits many physiologicaleffects associated with health benefits. Resveratrol interactedwith b-LG to form 1:1 complexes, with binding constant thatranges between 104 and 106 ⁄M (Liang et al. 2008). Complex-ing with b-LG increased slightly the photo-stability of resvera-trol but increased significantly its hydrosolubility.Encapsulation of b-LG–resveratrol complex in b-LG–pectinnanoparticles can also increase the bioavailability of resveratroland its use in clear beverages.

Water soluble vitaminsOptimum thiamine entrapment with both pre- and post-blendsof whey protein isolates-low methoxyl pectin occurs at pH 3.5(Bedie et al. 2008), which is appropriate for acidic foods. Max-imum entrapment efficiency was approximately 7% for pre-blending acidification and 4.5% for post-blending acidification.However, the protective effect of the complexes on theentrapped vitamin has not been determined.

Hydrophobic nutraceuticalsThe formation of nanoparticle and encapsulation of b-carotenewere carried out simultaneously by hydrophobic interaction of



casein-grafted dextran and b-carotene (Pan et al. 2007a).Casein and dextran solutions were mixed, lyophilised and thefreeze-dried powder was then reacted at 60 �C and at a relativehumidity of 78.9%. b-carotene suspension was sonicated, andequal volume of this solution and the aqueous solution ofcasein-grafted dextran (1 mg ⁄mL) were mixed and stirred for24 h. The mixture was then dried under vacuum at 0 �C, dis-solved in water and redried. The formed particles had sphericalshape of a core of casein and entrapped b-carotene and a shellof dextran molecules. They were stable in aqueous solutionagainst dilution, pH change, ionic strength, FeCl3 oxidation andlong storage. The encapsulated b-carotene can be released bypepsin and trypsin hydrolysis.Docosahexaenoic acid is the main representative of omega-3

fatty acids and has many health benefits (Lopez-Huertas 2010).However, DHA is highly hydrophobic of low oxidative stabilityleading to undesirable off-flavours. DHA has high affinity tobind to casein (3–4molecules DHA ⁄mole casein). rCM has beenused as nano vehicle for DHA, which showed remarkable pro-tective effect against DHA oxidation (Zimet et al. 2011). Sim-ply, the DHA ⁄casein complex can be prepared by titratingsodium caseinate solution with ethanolic solution of DHA, andDHA-loaded rCM can be prepared from the DHA–caseinatecomplex in the same way followed in preparing rCM (Semoet al. 2007).Reassembled b-CN micelles at low pH (Danino et al. 2009)

have been used as nano vehicle for hydrophobic nutraceuticals.It has been claimed that the obtained nano micelles of b-CN-loaded with hydrophobic nutraceuticals can be used in clearbeverages at low pH.The formation of b-LG–bioactive compound complexes and

subsequent preparation of the nanoparticle have been used(Zemit and Livney 2009; Ron et al. 2010) to prepare and entrap-ment of hydrophobic bioactive materials in b-LG–pectin nano-particles. Ethanolic solution of vitamin D2 or DHA was addedto 0.2% b-LG solution (molar ratio of 1:2 DHA: b-LG) at pH7.0 while stirring vigorously. Pectin solution (0–0.6%) was thenadded, and the mixed solution was diluted four times to b-LGconcentration of 0.05% and pectin (0–0.15% and pH 4.5). Asprotein: pectin ratio increased, sub-micron protein–pectin com-plexes started to form with pectin gradually neutralising thepositive charge of protein. Further increase in the pectinconcentration to about 0.05% for 0.05% b-LG gave particles ofminimummean diameter of about 100 nm.

CONCLUS IONS

Milk proteins are excellent material to develop nano vehiclefor the delivery of bioactive compounds. They have theadvantage of being easy to prepare with diversified composi-tion and functional properties. Several methods have beendeveloped to prepare nanoparticles from milk proteins andfrom their complexes with other biopolymers mainly polysac-charides and their use for the entrapment of several types of

nutraceuticals. However, the use of the milk protein nanopar-ticles containing the entrapped nutraceuticals in foods has notbeen thoroughly investigated. The effect of processing andstorage on the stability and bioavailability of the encapsulatednutraceuticals requires further studies in animal and humanexperiments.

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2012-EL-SALAM - Formation and Potential Uses of Milk Proteins as Nano Delivery Vehicles for Nutraceuticals - A Review