Nanoencapsulation Systems Based on Milk Proteins and Phospholipids

7/29/2019 Nanoencapsulation Systems Based on Milk Proteins and Phospholipids

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Chapter 8NanoencapsulationSystemsBasedonMilk Proteins

andPhospholipidsHarjinder Singh1, Aiqain Ye1,2, and Abby Thompson

1Riddet Centre, MasseyUniversity, PrivateBag 11 222, PalmerstonNorth,New Zealand

2Current address:Fonterra Co-operativeLimted, PalmerstonNorth,New Zealand

M i l k contains several components thatcan beutilized to makenanoparticles for encapsulation and delivery of bioactivecompounds. Caseins in milk are essentially naturalnanoparticles, designed to deliver essential nutrients, inparticular calcium. Similarly, whey proteins, particularly -lactoglobulin, have been designed by nature to bind andtransport hydrophobic molecules. Theability of milk proteinsto interact strongly with charged polysaccharides opens upfurther possibilities for making novel hybrid nanoparticles.Phospholipid-rich fractions, extracted from fat globulemembranes, can be used to form liposomes. Due to their highsphingomyelin content, these liposomes have some uniquestabilityand entrapment characteristics.

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132Sincethe end of the 20 h century, therehas been a growing realization of the

pivotal link between diet and human health. Consequently, the food industry hascreated a new category of foods, the so-called functional foods. To fully realizethis opportunity the food industry must address several critical challenges,including discovering the potential bioactivity of beneficial compounds,establishing optimal intake levels, and developing adequate food deliveringmatrix and product formulations.

Traditionally, microencapsulation can be used for many applications in thefood industry including stabilizing the core material, controlling the oxidativereaction, providing sustained or controlled release, masking flavors, colors orodors, to extend shelf life or protect components against nutritional loss. Inrecent years, there is considerable interest in developing high performancedelivery vehicles for encapsulation and protection of biologically activesubstancesof food origin. Nanosciences, which investigates how tobuild matteron nanometer scale, usually between 1 and 100 nm, by manipulating individualmolecules or atoms, has the potential to provide new solutions in many of thesefronts. Certainly nanoparticles may seem attractive as delivery vehicles. Bycarefully choosing the molecular components, it seems possible to designparticles with different surface properties. These nanoparticles are able toencapsulate and deliver the active compounds directly to appropriate sites,maintain their concentration at suitable levels for long periods of time, andprevent their prematuredegradation. Research efforts are already beingmade todevelop food-based delivery vehicles, such as protein-polysaccharidecoacervates, multiple emulsions, liposomes and cochleates.

M i l k contains several components thatcan beutilized to make nanoparticlesfor encapsulation and delivery of bioactive compounds. Caseins in milk areessentially natural nanoparticles, designed to deliver essential nutrients, inparticular calcium. Similarly, whey proteins, particularly -lactoglobulin, havebeen designed by nature to bind and transport hydrophobic molecules. M i l kproteins interact strongly with charged polysaccharides, creating possibilities fornovel hybrid nanoparticles. Phospholipid-rich fractions, extracted from fatglobule membranes, can be used to form liposomes. These liposomes have someunique stability and entrapment characteristics for both hydrophobic andhydrophilicmolecules.

This paper provides an overview of potential nanoparticle-based deliverysystems, based on milk proteins and phospholipids. A particular focus is placedon recentwork in theareaof protein-polysaccharide nanoparticles and liposomescarried out in our laboratory at Massey University in New Zealand.

Milk ProteinsasPotential Nano-encapsulationSystemsNormal bovinemilk contains about 3.5% protein which can be separated

into caseins and whey proteins (/). Caseins can be fractionated into four distinct


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133proteins, otsr, as2-, - and - caseins; theserepresent approximately 38%, 10%,36%and 12% of whole caseins, respectively. The structures and properties ofcaseins have been extensively studied (2). In comparisonwith typical globularproteins, the structures of caseins are quite unique. The most unusual feature isthe amphiphilicity of their primary structure. The hydrophobic residues andmany of the charged residues, particularly the phosphoserine residues, in thecaseins are notuniformy distributed along the polypeptide chain. Therefore, allfour caseins have a distinctly amphpathc character withseparatehydrophobicand hydrophilic domains, with relatively open and unordered secondarystructures. As an example, the distribution of charged residues andhydrophobicityas a function of sequence position of -casein is shown in Figure1. -Casein has two large hydrophobic regions (55-90 and 130-209). The N-termnal 21-residue sequence has a net charge of -12, while the rest of themoleculehas no net charge.

Because the casein monomers cannot sufficiently remove their hydrophobicsurfaces fromcontact withwater, the caseins tend to associatewiththemselvesand witheach other. In addition, all caseins are able to bind calcium with theextent ofbindingbeing proportional to the number of phosphoserine residues inthe molecule.asr and a2-caseins are most sensitive tocalcium followed by -caseinwhile-casein is insensitive tocalcium. -Casein is capable ofstabilisingother caseins against calcium-inducedprecipitation and allows the formation ofcolloidal size aggregates.The unique physio-chemcal properties of caseins have been traditionallyexploited to modify and enhance textural and sensory characteristics of foods(J). Casein and casenates can bind water, stabilise foams, emulsify fat andcontrol viscosity in formulated foods, in addition to providing high nutritionalvalue. Caseins also possess many of the properties required of a good wallmaterial for encapsulation (4). The ability of caseins to self-assemble intoparticles of varying sizes with different stability characteristics offersopportunities for nanoencapsulation for the delivery of bioactivecompounds. Arecent study (5) showedthathydrophobic compounds, such as vitamnD2, canbe incorporated into casein particles, formed by the re-assembly of caseins.These reassembled casein particles can provide partial protection against U V -light-induced degradation of vitamn D2 entrapped in them. Furtherunderstanding of "surfactant-like" self-assembly properties of caseins wouldallow us to create novel nanometer-scale structures suitable for delivery ofbioactivecompounds.

Theprincipal fractions of whey proteins are-lactoglobulin, bovine serumalbumn, ct-lactalbumn and immunoglobulins(I). In contrast to caseins, thewhey proteins possess high levels of secondary, tertiary and, in most cases,quarternary structures. -Lg is built up of two -sheets, formed fromninestrands converging at one end to form a hydrophobiccalyx or pocket, and aflanking three-turn -helix (6).Thispocket serves as a binding locus for apolar


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135molecules such as retinol (7) and long-chain fatty acids (8). Similarly, bovineserum albumn binds a large variety of compounds, includingretinol and long-chain fatty acids (P). The concept of using ligand-selective whey proteins fordelivery and protection of activeagents is relatively new, and such systems needfurther development.

The ability of whey proteins to aggregate and form gels during heattreatment is of considerable importance (1,3). By controlling the assembly ofprotein molecules during the aggregation process, hydrogels, mcro- andnanoparticles suitable for the delivery of bioactivecompounds can be produced.Forexample, a monodisperse dispersionof 40 nm whey proteinnanosphereswasobtained by Chen et al. (10)by heating whey proteins at relatively low proteinconcentration and ionic strength and atemperaturearound 55C. The potentialof thesenanospheresas carriers of nutraceutical agentswas studied invitro; itappears that protein nanoparticles could be internalized by cells and degradedtherein to release nutraceutical compounds.

Partial hydrolysis of whey protein, -lactalbumn, by a proteasefromBacillus lichenifroms has been shown to produce peptides that self-assembleinto nanometer-sized tubular structures under certain conditions (11). Thesemcrometer longhollow tubes, with a diameter of only 20 nm, have potentialapplications in thedeliveryof nutraceuticals.

Milk Protens-Polysaccharide CompositeSystemsProtein structure can be modified through processingtreatmentsor changeof solutionconditions to allow formation of complexeswithpolysaccharides. A

widevariety of nutrients can be incorporated intothesecomplexes by relativelynon-specific means. Specific binding of a nutrient to amnoacidside chains canalso be achieved in some cases.A t pH values below their isoelectric points (pi), proteins carry positivecharges and can interact withpolysaccharides bearingcarboxylic, phosphate, orsulfate groups. This inter-biopolymer complexation of positively chargedproteins and anionic polysaccharides can lead to the formation of soluble andinsolublecomplexes (12). Interbiopolymer complexes can be regarded as a newtype of food biopolymer whose functional properties differ strongly fromthoseof the macromolecular reactants.The complex coacervation ofglobular proteinsand polyelectrolytes, e.g., gelatin, -lactoglobulin, bovine serum albumn, eggalbumn,and soy protein, has been extensively studied(13-14).

Recently, we have observed the formation of soluble and stable complexesonthemxingof gumarabic and sodium caseinate at a wide pH range(pH 4 to5.4); the complexes resulted in stable dispersionswithparticle size between 100to 200 nm(15).


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136Mixtures containing 0.1% sodium caseinate and 0.5% gum arabic were

acidifiedby dropwise addition of HC1 frompH 7.0 to pH 2.0 and samples werestored for 24 h at20C. The absorbance profiles of mxtures are shown in Figure2. The absorbance values of 0.1%sodium caseinate solution abruptly increasedat pH 5.4 and reached amaximumat pH 5.0 (Figure 2). Further decreasein pHcaused adecrease in absorbance due to large-scale aggregation andsubsequentprecipitationof the caseins around their pi. In contrast, the absorbance of sodiumcaseinate/gum arabic mxtures increased slightly at pH 5.4 (pHc) but remanedalmost constant between pH 5.4 and pH 3.0. Nophaseseparation occurred inthis pH range (see photographs of 0.1% sodium caseinate/0.5% gum arabicmxtures at different pH values inFigure 2).On decreasing thepH belowpH 3.0,the absorbance values increased andphaseseparation took place subsequently.A t pH 2.0, the absorbance values of the sodium caseinate/gum arabic mxturedecreased.

Particlesizes of thesestable dispersions in thepH rangefrom5.4 topH 3.0,measured using dynamc light scattering, showedthatthe average diameter of theparticles in the sodium caseinate/gum arabic mxtures remaned stable atapproximately 110 nm as the pH was decreased frompH 5.4 to pH 3.0 andincreased to very large values (> 10 when the pH was reduced further.Electron mcroscopy confirmed the presence of these composite nanoparticlesbetweenpH 5.4 topH 3.0(15).

The mechanism of the formation of thesenanoparticles based around theself-aggregation of casein and the electrostatic interaction between theaggregated particles of caseinand gum arabic molecules has been proposed(15,Figure 2). As the pH of the mxture decreases below pH 5.4, the caseinatemolecules tend to undergo small-scale aggregation prior to large-scaleaggregation and precipitation at pH values closer to their pi (pH 4.6). In thiscase, the gum arabic molecules may attach to the outside of thesesmall-scaleaggregates in the early stages of aggregation through electrostatic interactionsbetween negatively charged gum arabic and exposed positive patches on thesurface of the caseinate aggregates. The presence of hydrophilic gum arabicmoleculeson the outside of the caseinate aggregatemay be enough to stericallystabilise thesenano-particles and consequently prevent self-aggregation. As thecharge on the nano-particles is quite low, for example,~15mV at pH 4.0, stericstabilisation is probably important.

Effectof NaCI andCaCI 2 on thepropertiesofnanoparticlesDifferent amounts of NaCI or CaCl 2 were added to the stable nanoparticledispersionof caseinate/gum arabic at pH 4.6. The changes in the absorbance and

average particle size of mxtures, whichwere measured after storage for 24 h,are shown in Figure 3. The absorbance value of the dispersions increasedwith


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137increasingNaCI concentration upon to 45mM but decreased abruptly at higherNaCI concentrations (Figure3A). The average particle sizeof particles increasedfrom -105 nm to -450 nm at 40 mM added NaCI. It also was noted that thedispersions containing 0 to 40 mM added NaCI were stable (no phaseseparation) after 7days, whereas precipitation occurred at addedNaCI >40mM(Figure3A ). The analysisof theseprecipitated samples showedthattherewas noprotein and nearly 100% of gum arabic remained at the top clear layer (data notshown). This indicated that the precipitation was due to self-association ofcaseinate molecules, and the association of gum arabic with the caseinaggregates was prevented by the added NaCI. This is consistent with ourprevious work (15)that showed that the complexes can not be formed betweenthe sodium caseinate and gum arabic in the presence of NaCI >50mM .

A t addedNaCI


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Figure2.AbsorbanceasafunctionofpH off).1%sodiumcaseinate solution(O)andmxtures ( ) of0.1%sodiumcaseinateand0.5%gumarabicat20.ThemxtureswereacidifiedusingHCl andthenstoredat4X1for 24h. Theproposedmodel ofYeetal (15)for theformationofnanoparticles isalsodepicted. Picturesof0.1%sodiumcaseinateand0.5%gumarabicmxtures

atdifferentpH valuesarealsoshown.


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Nanoencapsulation Systems Based on Milk Proteins and Phospholipids