Food Chemistry 158 (2014) 466–472

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Food Chemistry

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Tangeretin-loaded protein nanoparticles fabricated fromzein/b-lactoglobulin: Preparation, characterization,and functional performance

http://dx.doi.org/10.1016/j.foodchem.2014.03.0030308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +1 (413) 545 2281; fax: +1 (413) 545 1262.E-mail address: hangxiao@foodsci.umass.edu (H. Xiao).

Jingjing Chen a, Jinkai Zheng a,b, David Julian McClements a, Hang Xiao a,⇑a Department of Food Science, University of Massachusetts, Amherst, MA 01003, USAb Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China

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

Article history:Received 20 September 2013Received in revised form 19 February 2014Accepted 1 March 2014Available online 12 March 2014

Keywords:TangeretinZeinb-LactoglobulinNanoparticlesSolubilityNutraceuticals

The aim of this study was to design a colloidal delivery system to encapsulate poor water-soluble bioac-tive flavonoid tangeretin so that it could be utilized in various food products as functional ingredient.Tangeretin-loaded protein nanoparticles were produced by mixing an organic phase containing zeinand tangeretin with an aqueous phase containing b-lactoglobulin and then converted into powder byfreeze-drying. This powder formed a colloidal suspension when dispersed in water that is relatively sta-ble to particle aggregation and sedimentation. The influence of temperature, ionic strength, and pH on thestability of the protein nanoparticles was tested. Extensive particle aggregation occurred at high ionicstrength (>100 mM) and intermediate pH (4.5–5.5) due to reduced electrostatic repulsion. Extensiveaggregation also occurred at temperatures exceeding 60 �C, which was presumably due to increasedhydrophobic attraction. Overall, this study shows that protein-based nanoparticles can be used to encap-sulate bioactive tangeretin so that it can be readily dispersed in compatible food products.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Tangeretin (5,6,7,8,40-pentamethoxyflavone, shown in Fig. 1) isa flavonoid (polymethoxyflavone or PMF) found in citrus fruits.There has been considerable interest in the use of PMFs as func-tional ingredients in foods and pharmaceuticals because of the po-tential of their beneficial activities, such as anti-carcinogenicactivities and anti-inflammatory activities (Li et al., 2009; Maket al., 1996; Manthey & Guthrie, 2002). However, the extensiveapplication of PMFs is currently limited because they have lowwater-solubility, which makes them difficult to be incorporatedinto liquid food products (Li et al., 2009). In addition, the lowwater-solubility of PMFs means they may be present in foods ascrystals (rather than soluble form), which may limit their bioavail-ability (Patel, Heussen, Hazekamp, Drost, & Velikov, 2012). Studiesin the pharmaceutical industry suggest that most lipophilic com-pounds must be in the form of a molecular dispersion (solubleform) before they can be absorbed through the biological mem-branes of human intestinal tract (Porter, Trevaskis, & Charman,2007). It is therefore important to develop effective delivery sys-tems to encapsulate, protect and release PMFs to the targeted site.

In this study, we focused on the fabrication of food-grade colloi-dal delivery systems containing protein nanoparticles as a meansof encapsulating and delivering tangeretin, a representative PMF.We chose protein nanoparticles because they are particularly suit-able for commercial applications because they can be preparedfrom food-grade ingredients, the manufacturing process can ofteneasily be scaled up, and they are biodegradable (Elzoghby, Samy, &Elgindy, 2012). In addition, powders could be formed from proteinnanoparticles suspensions by method such as spray drying, whichare already widely used in the food industry (Desobry, Netto, &Labuza, 1997). Many different types of proteins can potentiallybe used to fabricate colloidal delivery systems for bioactive foodcomponents, including soy proteins, caseinate, whey proteins,and zein (Matalanis, Jones, & McClements, 2011). The uniquemolecular structure and physicochemical properties of each ofthese protein types make them suitable for different types ofapplications (Elzoghby, Abo El-Fotoh, & Elgindy, 2011). A varietyof different preparation methods can be used to fabricateprotein nanoparticles, including controlled thermal denaturation,extrusion methods, gel disruption methods, molding methods,and antisolvent precipitation methods (Matalanis et al., 2011).

In this study, we used a hydrophobic protein (zein) as a core forforming protein nanoparticles based on antisolvent precipitationmethod. Zein is a GRAS (generally recognized as safe) food-grade

Fig. 1. Chemical structure of tangeretin (5,6,7,8,40-pentamethoxyflavone) a bioac-tive flavonoid found in citrus fruits.

Fig. 2. Schematic representation for preparing tangeretin loaded zein colloidalsystem.

J. Chen et al. / Food Chemistry 158 (2014) 466–472 467

protein that is mainly found in corn kernels (Lai & Guo, 2011). Zeinis soluble in high concentration alcohol solutions but insoluble inwater, which provides the potential for creating protein particlesthat encapsulate lipophilic bioactive agents (Elzoghby et al.,2012). The lipophilic bioactive agent and zein are initially dissolvedin an alcohol solution, which was then introduced into an aqueoussolution. Zein precipitates when the water-soluble alcoholmoves into the surrounding aqueous phase, leading to the forma-tion of a protein nanoparticle or micro-particle that encapsulatesthe bioactive agent. This method has been successfully used toencapsulate oils, such as fish oil, flax oil, and essentialoils(Quispe-Condori, Saldaña, & Temelli, 2011; Wu, Luo, & Wang,2012; Zhong & Jin, 2009) and lipophilic bioactives, such as curcu-min (Patel, Hu, Tiwari, & Velikov, 2010). We chose b-lactoglobulinas an emulsifier because it is a model globular protein with well-defined properties that is already widely used in the food industry.In this study, we examined the possibility of using zein nanoparti-cles to encapsulate tangeretin (a representative PMF), and to deter-mine its stability to various environmental stresses that it mayexperience in commercial food and beverage products. To theauthor’s knowledge, this is the first time that zein nanoparticleshave been used as a delivery system for tangeretin, which is animportant nutraceutical suitable for incorporation into functionalfoods and beverages. The knowledge gained from this study shouldtherefore be useful for the design of food-grade delivery systemsfor lipophilic bioactive compounds that need to be incorporatedinto functional foods and beverages.

2. Materials and methods

2.1. Materials

Tangeretin (purity 98.4%) was purchased from Bepharm Ltd.(Shanghai, China). Corn oil (Mazola) was purchased from a localsupermarket (ACH Food Companies, Inc., USA). b-lactoglobulin(Lot # JE 002-8-415) was obtained from Daviso Foods International(Le Sueur, MN). The manufacturer reported the composition of thispowder to be 97.4% total protein, 92.5% b-lactoglobulin (b-lg), and2.4% Ash. Zein (purity 92%, w/w) and uranyl acetate was purchasedfrom Sigma–Aldrich (St. Louis, MO). Other chemicals, such as so-dium chloride (NaCl), sodium hydroxide (NaOH), calcium chloride(CaCl2), hydrochloric acid (HCl), HPLC grade methanol, tetrahydro-furan (THF), trifluoroacetic acid (TFA), and acetonitrile (ACN) wereobtained from Fisher Scientific (Fairlawn, NJ, USA). Ammoniumacetate was obtained from EMD Chemicals Inc. (Gibbstwon, NJ,USA). Double distilled water was prepared using a Nanopure waterpurification system (Model D14031, Thermo Scientific, USA).

2.2. Preparation of tangeretin loaded zein nanoparticle

The tangeretin-loaded zein colloidal system was preparedusing a liquid–liquid dispersion method described previously(Parris, Cooke Peter, Moreau Robert, & Hicks Kevin, 2008). Theschematic representation of this procedure is shown in Fig. 2.

2.2.1. Organic phaseZein and tangeretin (mass ratio 25:1, 0.5 g/0.02 g) were dissolved

in 20 ml of ethanol solution (90%, v/v) and stirred (Corning StirrerPC-420, Corning Inc., USA) for at least 2 h to ensure full dissolution.The criteria for choosing this ratio was based on our preliminaryexperiments to ensure there is enough zein to encapsulate tangere-tin and no tangeretin crystals were formed during preparation.

2.2.2. Aqueous phaseb-lactoglobulin was dissolved in phosphate buffer saline

(10 mM, pH 7) solution (3%, w/v) and stirred for at least 2 h to en-sure complete hydration.

2.2.3. Colloidal system preparationThe organic phase was introduced into the aqueous phase drop

by drop under constant stirring at 1000 rpm (organic phase: aque-ous phase = 1:3, v/v). The organic solvent was then removed usinga rotary evaporator (Rotavapor R110, Buchi Crop., Switzerland). Asample containing no b-lactoglobulin was made using the samemethod and was regarded as a control. The resulting tangeretin-loaded zein colloidal suspensions were dried using a freeze dryer(VirTis Genesis Lyophilizer, Virtis genesis company Inc., USA) andkept in a refrigerator before analysis. In order to determine the max-imum amount of tangeretin that could be encapsulated into the zeinnanoparticles, a series of different colloidal systems (organic phasecontaining 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% tangeretin) weremade using the same method. The encapsulation efficiency of tan-geretin was calculated using the following formulation:

Encapsulation efficiency ð%Þ ¼ Ccolloidal

CTotal� 100

where CColloidal is the concentration of tangeretin in the colloidalsuspension, and CTotal is the concentration of tangeretin in the totalsystem. The value of CColloidal was measured by extracting the

468 J. Chen et al. / Food Chemistry 158 (2014) 466–472

tangeretin from the dried powders and measuring its concentra-tion using HPLC, where as the value of CTotal was known from theinitial amount of tangeretin added to the system.

2.3. Tangeretin concentration determination

The content of tangeretin in different systems was determinedusing a HPLC method. For dried powders, samples were first recon-stituted to their initial concentrations using PBS buffer (10 mM, pH7). After dilution, 100 lL of sample was transferred into a 1.5 mLcentrifuge tube. 400 lL of ethyl acetate was added to the tubeand vortexed for 30 s. Then the mixture was centrifuged (1170 g)for 6 min. The ethyl acetate layer was collected and the remainingsample was prepared using the same procedure for a second time.The two ethyl acetate layers were combined and dried on a vac-uum concentrator (Model: SVC 100H, Thermo Fisher ScientificInc., USA). Then 100 lL of mobile phase was added to the tube todissolve the tangeretin extracted by ethyl acetate. The tube wasvortexed and centrifuged (1170 g) for at least 30 s to removenon-dissolved material. A tangeretin standard solution was pre-pared by diluting 50 lM of stock solution (dissolved in DMSO) withmobile phase to a series of desired concentration (5, 10, 20, and50 lM). An aliquot (90 lL) of solution were collected from thetop of the tube and analyzed using a CoulArray HPLC system (Ther-mo Scientific Inc., USA) that contains a Model 584 binary solventdelivery system, an Model 542 auto-sampler, a CoulArray 5600Adetector and a Model 526 UV detector (Waters, USA). A RP-Amidereversed-phase HPLC column (15 cm � 4.6 mm id, 3 lm, AscentisExpress, Sigma–Aldrich Corp., USA) was used on this HPLC system.The mobile phase and detection conditions used were as describedpreviously with some modifications (Dong et al., 2010; Zheng et al.,2013). The 50% (B) mobile phase contained 50 mM ammoniumacetate, 0.05% TFA, 10% THF, 40% ACN and 50% water. The 25%(A) mobile phase was made up of 25 mM ammonium acetate,0.05% TFA, 5%THF, 20% ACN and 75% water. The injection volumewas 50 lL. The running time was 35 min and a linear gradient elu-tion mode was used, i.e., 10%, 100% and 100% mobile phase B attime 0 min, 20 min and 30 min. UV detector was set at 330 nmto detect the signal of tangeretin (y = 0.3228x + 0.14, R2 = 0.997).

2.4. Characterization of tangeretin-loaded zein nanoparticles

2.4.1. Particle size and f-potentialThe particle size and f-potential of the particles in the colloidal

dispersions were determined using a commercial dynamic lightscattering and micro-electrophoresis device (Nano-ZS, MalvernInstruments, UK). Freshly prepared samples were diluted 5 timeswith buffer solution (10 mM phosphate buffer, pH 7) at room tem-perature before measurement. Freeze-dried samples were re-dis-persed in buffer solutions (10 mM phosphate buffer, pH 7),stirred for 5 min, and then diluted 5 times with the same buffer.The particle size data are reported as the intensity-weighted(‘‘Z-average’’) mean particle diameter, while the particle chargedata are reported as the f-potential.

2.4.2. Visual and microstructure observationThe appearance of tangeretin-loaded zein colloidal dispersions,

dried powders, and other samples were recorded with a digitalcamera (Canon PowershotG12, NY). Microstructure of nanoparti-cles in solutions and powders were observed using an opticalmicroscope (Nikon Eclipse E400, Nikon Crop., Japan). For aqueoussamples, 7 lL of solution was placed on a microscope slide andcovered by a cover slip. For dried samples, powders were spreadevenly on the microscope slide and covered by a cover slip.

2.4.3. Transmission electron microscopySamples were prepared with negative staining for TEM observa-

tion. Tangeretin-loaded zein colloidal suspensions were dilutedwith PBS buffer (10 mM, pH 7) until the solution was semi-opaque.A drop of this solution was applied to the grid surface and kept forabout 2 min. Excess sample was removed with a wedge of filter pa-per. Then the grid was immersed in a drop of negative stain solu-tion uranyl acetate (2%, w/w), quickly taken out and left to standfor 45 s. Excess uranyl acetate was removed with a wedge of filterpaper. The grid was then observed under a transmission electronmicroscope (Tecnai 12, FEI Company, USA).

2.5. Stability of tangeretin loaded zein colloidal system

2.5.1. pHThe pH of freshly prepared colloidal suspensions were adjusted

to the desired values (from pH 3 to 7) using NaOH or HCl solution.The mixtures were then transferred to glass tubes and stored atambient temperature overnight.

2.5.2. SaltColloidal suspensions were diluted with the same volume of

concentrated salt solutions to form a series of samples with differ-ent salt concentrations (0–500 mM NaCl). The resulting mixtureswere then stirred for 30 min and stored at ambient temperatureovernight.

2.5.3. TemperatureSamples with no salt and 150 mM salt were incubated in water

bath for 30 min (30–90 �C) and then stored at ambient tempera-ture overnight.

The particle size, f-potential and appearance of the sampleswere then measured.

2.6. Data analysis

All experiments were carried out at least three times and the re-sults were reported as averages and standard deviations of thesemeasurements. Statistical significance was at the level of p < 0.05.

3. Results and discussion

3.1. Characterization

In this study, we first determined the role of emulsifier (b-lacto-globulin) in the formation of stable colloidal suspensions of zein. Inthe absence of emulsifier, we observed that some material coagu-lated on the wall of the test tubes during the preparation proce-dure, which was not observed when b-lactoglobulin was present(Fig. 3). These visual observations were supported by particle sizedistribution measurements (Fig. 3). Colloidal suspensions preparedwithout emulsifier had a bimodal particle size distribution with apopulation of particles >1000 nm. This suggests that some of thezein particles formed clumps that adhered to the sides of the con-tainer, presumably through hydrophobic interactions. It should benoted that dynamic light scattering cannot detect particles greaterthan about 3000 nm, and therefore the large zein particles thatwere visible in this system would not show up in the particle sizedistributions. All the particles in the colloidal suspensions pre-pared in the presence of b-lactoglobulin were relatively small,and the particle size distribution was narrow. Presumably, the b-lactoglobulin molecules adsorbed to any hydrophobic patches onthe surfaces of the zein particles, and therefore prevented precipi-tation. Previous studies have also shown that a protein emulsifier(caseinate) could prevent the aggregation of zein particles (Patel,

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Fig. 3. Particle size distribution and appearance of suspensions of tangeretin-loaded protein nanoparticles made without and with b-lactoglobulin (b-lg) in theinitial aqueous solution.

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Bouwens, & Velikov, 2010). Hence, the emulsifier plays animportant role in the formation and stabilization of zein colloidsuspensions.

The freshly prepared zein particles had a mean particlediameter of 462 ± 24 nm in the aqueous colloidal suspensions.After evaporation, the mean particle diameter decreased to 249 ±

Fig. 4. Structure of suspensions of tangeretin-loaded protein nanoparticles: (a) visual appoptical microscopy image of freeze dried powder; (d) transmission electron microscopstructure of rehydrated powder.

4 nm, which can be attributed to removal of ethanol that was orig-inally present inside the particles. The morphology of tangeretin-loaded nanoparticles depended on the nature of the system(Fig. 4). The freeze-dried samples were white powders (Fig. 4a)that contained irregularly shaped particles when observed usingoptical microscopy (Fig. 4c). After the samples were rehydratedin PBS buffer, the colloidal suspension had a similar appearanceas the freshly prepared samples (Fig. 4b). The TEM image showsthat these particles had well-defined spherical shapes, with diam-eters around 200 nm (Fig. 4d), which was in agreement with thedynamic light scattering measurements (d = 263 ± 13 nm). Nocrystals were observed by optical microscopy after they were rehy-drated (Fig. 4e). The encapsulation efficiency of tangeretin in theprotein nanoparticles was found to be around 73%. In summary,we were able to prepare a homogeneous colloidal suspension con-taining protein nanoparticles with tangeretin encapsulated inside.This system had good water-dispersibility after lyophilisation, thusit has potential to be incorporated into a variety of food and bever-age products.

To determine the maximum amount of tangeretin that can beencapsulated in the zein nanoparticles, we made a series of proteinnanoparticle suspensions that contained different amounts of tan-geretin, while keeping other parameters constant. There was aslight increase in mean particle diameters of these systems withincreasing tangeretin concentration as determined by dynamiclight scattering (Fig. 5). Nevertheless, the mean particle diameterwas relatively insensitive to tangeretin concentration in the initialorganic phase (d = 350 ± 20 nm), which suggests this bioactivecomponent did not have a major influence on protein nanoparticlesformation. However, optical microscopy clearly showed thatthere were large crystals in samples containing relatively hightangeretin concentrations, i.e., >0.4% w/v in the initial organic phase(Fig. 5). We assumed that these crystals were formed due to thesupersaturation of tangeretin in the mixture of organic phase andaqueous phase (David Julian McClements, 2012). The fact that the

earance after freeze drying; (b) visual appearance after rehydration in PBS buffer; (c)y image of rehydrated protein nanoparticles and (e) optical microscopy image of

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Fig. 5. Influence of tangeretin concentration on the mean particle diameter oftangeretin-loaded protein nanoparticles. The insert shows optical microscopyimages of systems made with different tangeretin concentrations: 0.1%; 0.2%; 0.4%;and 0.8%. Notice crystals were formed at higher tangeretin concentrations.

470 J. Chen et al. / Food Chemistry 158 (2014) 466–472

dynamic light scattering did not indicate the presence of these largecrystals was probably because they settled to the bottom of themeasurement cells prior to analysis. In addition, the crystals weretoo large to be detected by dynamic light scattering since this meth-od relies on movement of the particles due to Brownian motion.Based on these findings, we used 0.2% w/v tangeretin in the organicphase in the subsequent experiments to ensure no crystals wereformed during preparation of the protein nanoparticles.

3.2. Stability of tangeretin-loaded zein colloidal suspensions

It is important to test the stability of delivery systems under dif-ferent environmental conditions because this determines the typeof commercial products it can be incorporated into, as well as theirbehaviour in the human gastrointestinal tract (Matalanis et al.,2011). For this reason we examined the influence of a number ofdifferent environmental stresses on the stability of the tangere-tin-loaded protein nanoparticles.

3.2.1. Ionic strengthDelivery systems may be dispersed within aqueous solutions

containing different types and amounts of electrolytes within com-mercial products and within the human gastrointestinal tract, andso it is important to establish the influence of ionic strength ontheir properties. Tangeretin-loaded protein nanoparticles were sta-ble to aggregation at relatively low salt concentrations (<50 mM),but exhibited extensive aggregation at higher levels, as indicatedby an increase in mean particle diameter and the formation of awhite sediment at the bottom of the test tubes (Fig. 6a). As the saltcontent increased, the sediment layer became thicker and thesolution above it became clearer indicating that more extensiveparticle aggregation had occurred. The mean particle diameter in-creased from around 310 nm in the absence of salt to over 4500 nmin the presence of 500 mM NaCl. These effects can be attributed tothe influence of ionic strength on the colloidal interactionsbetween the protein nanoparticles. In the absence of salt, there will

be a relatively strong electrostatic repulsion between the nanopar-ticles, which prevents them from aggregating (Israelachvili, 2011;McClements, 2004). In the presence of salt, the electrostaticinteractions between the nanoparticles are screened and so theattractive interactions (e.g., van der Waals and hydrophobic)dominate the repulsive interactions (e.g., electrostatic and steric),leading to extensive nanoparticle aggregation (Israelachvili, 2011).These results suggest that the protein nanoparticles will remain sta-ble in low-viscosity commercial products with relatively low saltconcentrations (such as fortified waters or soft drinks), but mayaggregate and sediment in products with high levels of salt. It alsosuggests that they may be susceptible to aggregation after ingestionsince human gastrointestinal fluids have relatively high ionicstrengths (approximately 100 mM in stomach and 140 mM in smallintestine) (Fatouros & Mullertz, 2008; Fatouros, Walrand,Bergenstahl, & Mullertz, 2009). On the other hand, they may be suit-able for utilization in high viscosity, gelled or solids foods, since theparticle sedimentation is less of a problem in these products.

3.2.2. pHDelivery systems may experience different pH environments

when they are incorporated into commercial food and beverageproducts, or when they pass through the human gastrointestinaltract, e.g. mouth, stomach, small intestine, and colon. We thereforeinvestigated the influence of pH on the stability and physicochem-ical properties of the tangeretin-loaded protein nanoparticles.

It was postulated that the protein nanoparticles consisted of ahydrophobic zein core coated by an amphiphilic b-lactoglobulinlayer, and so the net charge on the protein nanoparticles was dom-inated by b-lactoglobulin’s electrical characteristics. This hypothe-sis was supported by the pH dependence of the electrical charge onthe protein nanoparticles. The f-potential changed from positive tonegative as the solution was adjusted from pH 3 to 7, with a pointof zero charge around pH 5 (Fig. 6b). This pH is close to the re-ported isoelectric point of b-lactoglobulin (pI � pH 5) (Matalaniset al., 2011), and is considerably lower that the isoelectric pointof zein (pI � pH 6.2) (Patel, Bouwens, & Velikov, 2010). The tan-geretin-loaded zein nanoparticles were stable to aggregation at rel-atively low pH (<4.5) and high pH (>5.5), but were highly unstableat intermediate pH values (Fig. 6c). The good aggregation stabilityat low and high pH values can be attributed to a strong electro-static repulsion between the particles at values far from the iso-electric point, whereas the poor stability at intermediate pH canbe attributed to the weak electrostatic repulsion near the isoelec-tric point (McClements, 2004). Previous studies have shown thatcharge reversal occurs when an amphiphilic protein (caseinate)was added to a suspension of zein particles, which also supportsthe idea of the amphiphilic proteins forming a thin layer aroundthe water-insoluble zein core (Patel, Bouwens, & Velikov, 2010).

3.2.3. TemperatureThe purpose of these experiments was to determine the influ-

ence of temperature on the stability of the tangeretin-loaded pro-tein nanoparticles. This knowledge is important since deliverysystems may experience variations in temperature during themanufacturing, storage, and utilization. In the absence of addedsalt (Fig. 6d), the size of the particles in the colloidal suspension re-mained relatively small from 30 to 60 �C (d < 500 nm), but at high-er temperatures there was an appreciable increase in particle size.Previous differential scanning calorimetry studies have shown thatnative b-lactoglobulin has a transition temperature around 70 �C,which was attributed to thermal denaturation of this protein(Chanasattru, Decker, & McClements, 2007; Seo et al., 2010).Protein denaturation exposes reactive non-polar and sulfhydrylgroups (Dickinson & Matsumura, 1991; McClements, Monahan, &Kinsella, 1993), which increases hydrophobic attraction and disul-

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(c) (d)Fig. 6. (a) Influence of ionic strength on the stability of tangeretin loaded protein nanoparticles. At higher salt concentrations, the mean particle diameter increases andsediments are formed in the bottom of the test tubes. Tube No. 1 to 6 represents samples with 0 mM, 50 mM, 100 mM, 200 mM, 400 mM and 500 mM salt concentration; (b)Influence of pH on the electrical charge of suspensions of tangeretin loaded protein nanoparticles; (c) Influence of pH on the mean particle diameter and appearance ofsuspensions of tangeretin loaded protein nanoparticles. Tube No. 1 to 8 represents samples with pH 2, 3, 4, 4.5, 5, 5.5, 6 and 7 and (d) Influence of temperature and saltaddition on the mean particle diameter and appearance of suspensions of tangeretin-loaded protein nanoparticles. Light scattering measurements could not be made on thesamples containing salt after high temperature processing since they formed gels (inset photograph).

J. Chen et al. / Food Chemistry 158 (2014) 466–472 471

phide bond formation within and between protein-coated particles(Chanasattru et al., 2007).

In the presence of salt (150 mM), the mean particle diameterswere much greater than in its absence even at temperatures wellbelow the thermal denaturation temperature, i.e., 30 �C (Fig. 6d).In addition, gelation was observed in samples that were held attemperatures around and above the thermal denaturationtemperature of b-lactoglobulin, i.e., >70 �C. The addition of salt tothe system will have decreased the electrostatic repulsion betweenthe protein nanoparticles so that the net attractive forces (van derWaals and hydrophobic) dominated the net repulsive forces (elec-trostatic and steric). More extensive aggregation was observedabove the thermal denaturation temperature because of theincrease in the surface hydrophobicity of the b-lactoglobulin-coated particles after they unfold (Vittayanont, Steffe, Flegler, &Smith, 2002). In general, gelation occurs when the concentrationof particles exceeds a specific concentration so that a three-dimensional network can form throughout the system (Baussay,

Bon, Nicolai, Durand, & Busnel, 2004). In summary, the zein nano-particles were prone to extensive aggregation at elevated temper-atures, particularly in the presence of salt.

4. Conclusion

The purpose of this study was to establish an effective methodfor the encapsulation and delivery of a lipophilic bioactivecompound. Tangeretin has low water-solubility at ambienttemperature and is therefore difficult to incorporate into manyaqueous-based food and beverages. We therefore fabricated a col-loidal system that could potentially deliver tangeretin in thesetypes of products. We showed that tangeretin could be incorpo-rated into relatively small protein-nanoparticles that consisted ofa hydrophobic zein core and an amphiphilic b-lactoglobulin shell.These protein-nanoparticles behaved similarly to b-lactoglobulin-coated fat droplets under different environmental conditions: they

472 J. Chen et al. / Food Chemistry 158 (2014) 466–472

were stable at low salt concentrations at pH values far from theisoelectric point, but aggregated at higher salt levels and pH valuesnear the isoelectric point. In addition, they were stable to aggrega-tion at temperatures below the thermal denaturation temperatureof b-lactoglobulin, but aggregated at higher temperatures, particu-larly in the presence of salt. These delivery systems may be usefulfor incorporating bioactive flavonoids into aqueous-based foodproducts. In future research, we intend to focus on the behaviourof tangeretin-loaded protein nanoparticles under simulated gastro-intestinal conditions so as to better understand the factors that af-fect the bioaccessibility and absorption of tangeretin.

Acknowledgements

The authors thank the United States Department of Agriculture,CREES, NRI and AFRI Grants, and Massachusetts Department ofAgricultural Resources for financial support of this work.

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