Food Chemistry 132 (2012) 1221–1229

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Food Chemistry

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Physical and chemical stability of b-carotene-enriched nanoemulsions: Influenceof pH, ionic strength, temperature, and emulsifier type

Cheng Qian, Eric Andrew Decker, Hang Xiao, David Julian McClements ⇑Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States

a r t i c l e i n f o

Article history:Received 24 June 2011Received in revised form 11 October 2011Accepted 16 November 2011Available online 2 December 2011

Keywords:Nanoemulsionb-LactoglobulinProteinb-CaroteneCarotenoidsDegradationStabilityFunctional foodsNutraceuticals

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.11.091

⇑ Corresponding author.E-mail address: mcclements@foodsci.umass.edu (D

a b s t r a c t

The enrichment of foods and beverages with carotenoids may reduce the incidences of certain chronicdiseases. However, the use of carotenoids in foods is currently limited because of their poor water-solu-bility, high melting point, low bioavailability, and chemical instability. The potential of utilising oil-in-water (O/W) nanoemulsions stabilised by a globular protein (b-lactoglobulin) for encapsulating andprotecting b-carotene was examined. The influence of temperature, pH, ionic strength, and emulsifiertype on the physical and chemical stability of b-carotene enriched nanoemulsions was investigated.The rate of colour fading due to b-carotene degradation increased with increasing storage temperature(5–55 �C), was faster at pH 3 than pH 4–8, and was largely independent of ionic strength (0–500 mMof NaCl). b-Lactoglobulin-coated lipid droplets were unstable to aggregation at pH values close to the iso-electric point of the protein (pH 4 and 5), at high ionic strengths (NaCl >200 mM, pH 7), and at elevatedstorage temperatures (55 �C). b-Carotene degradation was considerably slower in b-lactoglobulin-stabi-lised nanoemulsions than in Tween 20-stabilised ones. These results provide useful information for facil-itating the design of delivery systems to encapsulate and stabilise b-carotene for application within food,beverage, and pharmaceutical products.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Carotenoids are a class of natural pigments mainly found in fruitsand vegetables that typically have 40-carbon molecules and multi-ple conjugated double bonds (Failla, Huo, & Thakkar, 2007). Carote-noids are usually divided into two categories: (i) carotenescomprised entirely of carbon and hydrogen, e.g., a-carotene, b-caro-tene, and lycopene; and (ii) xanthophylls comprised of carbon,hydrogen, and oxygen, e.g., lutein and zeaxanthin (Failla et al.,2007). Carotenoids may be beneficial to human health when con-sumed at appropriate levels (Khoo, Prasad, Kong, Jiang, & Ismail,2011). Epidemiological studies have identified a number of potentialhealth benefits of carotenoids, e.g., an increased intake of caroten-oid-rich food was correlated with a decreased risk for some cancers,cardiovascular disease, age-related macular degeneration, and cata-racts (Gerster, 1993; von Lintig, 2010). Various physiological mech-anisms have been proposed to account for the health benefits ofcarotenoids, including preventing oxidative damage, quenching sin-glet oxygen, altering transcriptional activity, and serving as precur-sors for vitamin A (Abdel-Aal & Akhtar, 2006; Failla et al., 2007;Higuera-Ciapara, Felix-Valenzuela, & Goycoolea, 2006; Singh &Goyal, 2008; von Lintig, 2010). Nevertheless, their utilisation as

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.J. McClements).

nutraceutical ingredients within foods is currently limited becauseof their poor water-solubility, high melting point, chemical instabil-ity, and low bioavailability.

The relatively low bioavailability of carotenoids from naturalsources has been attributed to the fact that they exist as either crys-tals or within protein complexes in fruit and vegetables that are notfully released during digestion within the gastrointestinal tract(Williams, Boileau, & Erdman, 1998). Carotenoids can be isolatedfrom natural sources and used as nutraceutical ingredients, butthere are a number of challenges associated with successfully incor-porating them into a wide range of food and beverage products.Carotenoids have very low water-solubilities and are crystalline atambient temperature, which usually means they have to be dis-solved in oils or dispersed in other suitable matrices before theycan be utilised in foods. A number of studies have shown that carot-enoid bioavailability depends strongly on the composition andstructure of the food matrix in which they are dispersed (Failla, Chit-chumroonchokchai, & Ishida, 2008; Thakkar, Maziya-Dixon, Dixon,& Failla, 2007; Tyssandier, Lyan, & Borel, 2001; Tyssandier et al.,2003). Carotenoids are also strongly coloured (red/orange/yellow),which limits the types of foods that they can be incorporated into.Finally, carotenoids are highly prone to chemical degradation duringfood processing and storage due to the effects of chemical, mechan-ical, and thermal stresses (Mao et al., 2009; Nguyen & Schwartz,1998; Tai & Chen, 2000; Xianquan, Shi, Kakuda, & Yueming, 2005).

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Attempts have therefore been made to develop effective deliverysystems to improve the utilisation, bioavailability, and stability ofcarotenoids in foods (Mao, Yang, Xu, Yuan, & Gao, 2010; Silvaet al., 2010).

Emulsion-based systems are particularly suitable for encapsulat-ing and delivering lipophilic bioactive components (McClements,2010; McClements, Decker, Park, & Weiss, 2009; McClements & Li,2010). The lipophilic components are incorporated into the oil phaseprior to formation of an oil-in-water emulsion by homogenisation.The oil phase should remain liquid during the homogenisation pro-cess, and so the concentration of the lipophilic component in the oilphase should be kept below the saturation level at the homogenisa-tion temperature. This limits the maximum amount of lipophilicmaterials that can be incorporated into an emulsion-based system,and sometimes means that the emulsion must be homogenised atan elevated temperature, which can promote chemical instabilityof labile ingredients. A number of previous studies have investigatedthe formation, properties, and stability of oil-in-water emulsionsenriched with carotenoids. A high-pressure homogenisation meth-od was used to prepare lycopene-enriched O/W emulsions stabilisedby globular proteins or non-ionic surfactants (Ribeiro, Ax, &Schubert, 2003). High pressure homogenisation has also been inves-tigated as a means of preparing lutein-enriched O/W emulsionsstabilised by phospholipids (Losso, Khachatryan, Ogawa, Godber, &Shih, 2005) and proteins (Batista, Raymundo, Sousa, & Empis,2006). Recently, a high pressure homogenisation method was usedto prepare b-carotene enriched O/W emulsions stabilised by smallmolecule surfactants (Tween 20 and decaglycerol monolaurate)and biopolymers (WPI and modified starch) (Mao et al., 2009). Mem-brane homogenisation methods have been investigated as an alter-native means of encapsulating carotenoids (astaxanthin) in O/Wemulsions due to their ability to produce narrow particle size distri-butions, low energy requirements, and mild processing conditions(Ribeiro, Rico, Badolato, & Schubert, 2005). A number of studies havealso shown that the bioavailability of carotenoids is increased whenthey are incorporated into O/W emulsions (Grolier, Agoudavi, &Azaisbraesco, 1995; Parker, 1997; Ribeiro et al., 2006), which mayenhance their health-promoting activities.

Recently, there has been great interest in utilising nanoemulsionsto encapsulate bioactive components for applications in food andbeverage products (McClements, 2011b; McClements & Rao,2011). Oil-in-water nanoemulsions consist of small lipid droplets(r < 100 nm) dispersed within an aqueous continuous phase. Similarto conventional emulsions, nanoemulsions are thermodynamicallyunstable systems that tend to breakdown over time. Nevertheless,they do have some potential advantages over conventional emul-sions: they can greatly increase the bioavailability of lipophilicsubstances; they scatter light weakly and so can be incorporatedinto optically transparent products; and they have a high stabilityto particle aggregation and gravitational separation (Acosta, 2009;McClements, 2011a). Nanoemulsions containing carotenoids havepreviously been prepared using high pressure homogenisation(Mao et al., 2009, 2010) and combined homogenisation/solvent dis-placement (Silva et al., 2010; Tan & Nakajima, 2005a, 2005b) meth-ods. Commercially, colloidal dispersions containing b-carotene aretypically stabilised against chemical degradation by adding antiox-idants, reducing oxygen levels, and minimising exposure to light andpro-oxidants. However, once a sealed product is opened andexposed to the atmosphere some of these protective measuresmay be lost, and so it is important to understand the major factorsthat influence b-carotene stability.

In the present study, a high pressure homogenisation methodwas used to prepare nanoemulsions containing b-carotene, andthen test their stability to environmental stresses that might beencountered in typical food and beverage applications (pH, ionicstrength, and temperature). A primary goal of this study is to

formulate nanoemulsions entirely from food-grade ingredientsthat are perceived to be safe and label friendly. We thereforeutilised orange oil as the carrier oil phase since this is widely usedfor this purpose in commercial beverage emulsions, and utilised aglobular protein (b-lactoglobulin) as the emulsifier since whey pro-teins are already widely used for this purpose in food and beverageproducts (McClements, 2005). In addition, previous studies haveshown that certain types of food protein are effective at reducingthe oxidation rate of emulsified lipids, such as polyunsaturated oils(Berton, Ropers, Viau, & Genot, 2011; Hu, McClements, & Decker,2003; McClements & Decker, 2000; Waraho, McClements, &Decker, 2011). It was therefore hypothesised that coating the lipiddroplets with a protein layer may improve the chemical stability ofthe encapsulated b-carotene. For this reason, the rate of chemicaldegradation of b-carotene in protein-stabilised and surfactant-stabilised nanoemulsions was compared. The results of this studywill be useful for designing effective delivery systems to encapsu-late and stabilise b-carotene for application within food, beverage,and pharmaceutical products.

2. Materials and methods

2.1. Materials

Orange oil was supplied by a food ingredient manufacturer(Givaudan Flavors Corporation, Cincinnati, OH). Food gradeb-lactoglobulin was obtained from Davisco Foods InternationalInc. (Le Sueur, MN). Beta-carotene (Type I, C9750) and Tween 20were purchased from the Sigma Chemical Company (St. Louis,MO). All other chemicals used were of analytical grade. Double-distiled water was used to prepare all solutions and emulsions.

2.2. Methods

2.2.1. Preparation of the b-carotene O/W emulsionsAn oil phase was prepared by dispersing 0.25% (w/w) of crystal-

line b-carotene in orange oil with mild heating (<5 min, �50 �C),and then stirring at ambient temperature for about 1 h to ensurefull dissolution (i.e., the sample became completely transparentwith no evidence of crystals). The samples were flushed with nitro-gen during this process to inhibit degradation of the b-carotene. Anaqueous phase was prepared by dispersing 2% (w/w) b-lactoglobu-lin (b-Lg) in aqueous buffer solution (10.0 mM phosphate buffer,0.01% (w/w) sodium azide, pH 7.0). Oil-in-water nanoemulsionswere prepared by homogenising 10% (w/w) oil phase with 90%(w/w) aqueous phase at ambient temperature (�25 �C). An emul-sion pre-mix was prepared using a high-speed blender (2 min, Bio-spec Products Inc., Bartlesville, OK), which was then passedthrough a high pressure microfluidiser (Model 101, Microfluidics,Newton, MA) three times at 9000 psi. The freshly prepared emul-sions were then divided into different aliquots and sealed in alu-minium foil covered glass tubes before storing in 55, 37, 20, or5 �C incubators. In one study, Tween 20 (1.5% (w/w)) was used asan emulsifier rather than b-lactoglobulin, but otherwise the nano-emulsions were prepared and characterised using the sameprocedures.

2.2.2. Measurement of emulsion stability2.2.2.1. Particle size. The particle size distribution and mean parti-cle radius (Z-average) of diluted emulsions were measured by acommercial dynamic light-scattering device (Nano-ZS, MalvernInstruments, Worcestershire, UK). Samples were diluted (1:100)with buffer solution prior to analysis to avoid multiple scatteringeffects to reach an instrument attenuation factor 66. The buffers

Fig. 1. Particle size distribution of b-carotene enriched nanoemulsions stabilised byb-lactoglobulin measured after 0 and 20 days storage at 20 �C (0.025% b-carotene,10% orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, pH 7).

C. Qian et al. / Food Chemistry 132 (2012) 1221–1229 1223

used for dilution had the same pH and ionic composition as thesamples being analysed.

2.2.2.2. b-Carotene degradation. The chemical degradation ofb-carotene during storage was measured using two differentapproaches: solvent extraction and colourimetry.

Solvent extraction: In this destructive approach, the b-carotenewas isolated from a nanoemulsion using solvent extraction,and then quantified using a UV–visible spectroscopy method.b-Carotene enriched nanoemulsions completely separated into anaqueous phase and an organic phase after addition of a solventcontaining methanol and methylene chloride (volume fraction1:2). The transparent lower organic phase (orange coloured) con-taining the b-carotene was removed, transferred to a cuvette, andthen its absorbance was measured at 450 nm using a UV–visiblespectrometer. A pure methanol and methylene chloride solutionwas used as a blank. An orange oil nanoemulsion containing noadded b-carotene was also analysed as a control. The b-carotenecontent was determined using a standard curve created from solu-tions with varying amounts of known b-carotene. All measure-ments were repeated three times.

Colourimetry: In this non-destructive approach, the chemicaldegradation of b-carotene was monitored in situ by measuringchanges in emulsion colour: as the b-carotene degraded, the colourbecame less intense. The tristimulus colour coordinates (L⁄a⁄b⁄) ofthe nanoemulsions were measured using a hand-held colourimeter(ColourMunki, X-Rite, Grand Rapids, MI). L⁄ values are a measure oflightness (higher value indicates a lighter colour); a⁄ values are ameasure of redness (higher positive values indicate a redder col-our, higher negative values indicate a greener colour); b⁄ valuesare a measure of yellowness (higher positive values indicate amore yellow colour, higher negative values indicate a more bluecolour). Emulsions were placed into a transparent flat-faced cuv-ette, the measuring device of the colourimeter was pressed againstthe cuvette surface, and then the colour was recorded. All measure-ments were repeated three times.

2.2.3. The influence of environmental stresses on emulsion stabilityThe physical and chemical stability of b-carotene enriched

nanoemulsions to environmental stresses typically encounteredby food products were tested:

� Temperature: 20-ml emulsion samples (pH 7.0) were transferredinto glass tubes and stored in the dark at 5, 20, 37, and 55 �C for15 days.� pH: Emulsion samples were prepared in aqueous buffer solu-

tions, and then the pH was adjusted to the desired final value(pH 3–8) using either NaOH and/or HCl solution. Emulsion sam-ples (20 ml) were then transferred into glass tubes and stored ina dark place at ambient temperature (�25 �C) for 5 days.� Salt: Emulsions (pH 7.0) were diluted with different amounts of

NaCl and buffer solution to form a series of samples with thesame droplet concentration, but different salt concentrations(0–500 mM NaCl). The emulsions were stirred for 30 min andthen transferred into glass tubes and stored in a dark place atambient temperature for 5 days.

3. Results and discussion

3.1. Nanoemulsion formation and physical stability

Nanoemulsions were prepared by homogenising 10% oil phase(0.25% b-carotene in orange oil) with 90% aqueous phase (2% b-Lg in buffer solution). The mean particle radius obtained was78 nm, confirming that nanoemulsions were formed (i.e.,r < 100 nm). A monomodal particle size distribution was obtained

immediately after homogenisation, with the majority of particlesbeing <100 nm in radius (Fig. 1). The mean particle radius(r = 79 nm) and particle size distribution (Fig. 1) did not changeappreciably from the initial values after the nanoemulsions werestored at 20 �C for 15 days, indicating that they were relatively sta-ble to particle aggregation under these conditions.

3.2. Impact of environmental conditions on b-carotene degradation

3.2.1. Storage temperatureInitially, the influence of storage temperature on the chemical

and physical stability of b-carotene-enriched nanoemulsions stabi-lised by b-lactoglobulin was examined. b-Carotene normally has anintense orange-red colour, but this colour tends to fade when itundergoes chemical degradation (Patras, Brunton, Da Pieve, Butler,& Downey, 2009). We therefore monitored the chemical stability ofb-carotene by determining colour fading using both a destructive(solvent extraction) method and a non-destructive (colourimeter)method.

The influence of storage temperature on the b-carotene concen-tration remaining in the nanoemulsions determined by the solventextraction method is shown in Fig. 2. The b-carotene concentrationfell from an initial value of 252 lg/ml after preparation to 143, 111,17, and 0 lg/ml after storage at 5, 20, 37, and 55 �C for 14 days,respectively. These results show that the carotenoid was highlyunstable to chemical degradation when stored at elevated temper-atures in nanoemulsions. The influence of storage temperature onthe colour of b-carotene enriched nanoemulsions measured using acolourimeter is shown in Fig. 3. In general, the lightness (L⁄) andcolour intensity (a⁄ and b⁄) of the nanoemulsions progressively de-creased during storage, which is indicative of colour fading. Therate of colour fading increased with increasing storage tempera-ture, in agreement with the results of the solvent extraction meth-od (Fig. 2). A decrease in the magnitude of the positive a⁄ value isindicative of a reduction in the redness of the nanoemulsions,whereas a decrease in the positive b⁄ value is indicative of a reduc-tion in the yellowness. The lightness of an emulsion usually in-creases when the colour intensity decreases, since then a higherfraction of light is reflected from its surface (McClements, 2002).In practice, a slight decrease in the lightness of the nanoemulsionsduring storage (Fig. 3a) was observed, which may have been due to

Fig. 2. Influence of storage temperature on the chemical degradation of b-caroteneencapsulated within nanoemulsions stabilised by b-lactoglobulin measured using asolvent extraction and spectroscopy method (0.025% b-carotene, 10% orange oil, 2%b-lactoglobulin, 10 mM phosphate buffer, pH 7).

Fig. 3. Influence of storage temperature on colour fading of b-carotene-enriched nanocarotene, 10% orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, pH 7): (a) lightne

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changes in the intensity of light scattering, e.g., due to changes inparticle size or spatial organisation.

It is convenient to use a single parameter to compare colour fad-ing in different nanoemulsions. Consequently, we calculated the to-tal colour difference (DE⁄) from the tristimulus values (Mcguire,1992):

DE� ¼ ½ðL� � L�0Þ2 þ ða� � a�0Þ

2 þ ðb� � b�0Þ2�1=2 ð1Þ

Here L⁄, a⁄, and b⁄ are the measured colour coordinates of thenanoemulsion at storage time t, and L�0, a�0, and b�0 are the initial col-our coordinates of the nanoemulsion. The influence of storage tem-perature on the total colour difference is shown in Fig. 4, whichclearly highlights the rapid acceleration in colour fading when thestorage temperature is increased from 20 to 55 �C. An indicationof the relative rate of b-carotene degradation at different storagetemperatures was obtained by determining the slope of the initiallinear region of the DE⁄ versus time plots using linear regressionanalysis (Fig. 5). Previous studies have also found a relatively rapidloss of b-carotene in nanoemulsions stored at elevated tempera-tures (Mao et al., 2009, 2010; Ribeiro, Chu, Ichikawa, & Nakajima,2008). These results highlight the importance of preparing, trans-porting and storing b-carotene-enriched nanoemulsions under rel-atively cool conditions to avoid colour fading and potential loss ofbioactivity.

emulsions stabilised by b-lactoglobulin measured using a colourimeter (0.025% b-ss (L⁄-value); (b) ‘‘redness’’ (a⁄-value), (c) ‘‘yellowness’’ (b⁄-value).

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10 12 14 16

ΔΔE*

Incubation Time (days)

5

20

37

55

Tween 20

Fig. 4. Influence of storage temperature on total colour change (DE⁄) of b-carotene-enriched nanoemulsions stabilised by b-lactoglobulin (0.025% b-carotene, 10%orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, pH 7).

Fig. 5. Influence of storage temperature on colour degradation rate of b-carotene-enriched nanoemulsions stabilised by b-lactoglobulin (0.025% b-carotene, 10%orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, pH 7).

Fig. 6. Influence of storage temperature on droplet aggregation in b-carotene-enriched nanoemulsions stabilised by b-lactoglobulin (0.025% b-carotene, 10%orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, pH 7).

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From a practical viewpoint, it is important that nanoemulsionsalso remain physically stable during storage. Therefore changes inthe mean particle radius over time when the nanoemulsions werestored at different temperatures (Fig. 6) were measured. In general,there was a slight increase in mean particle radius during storage,with the rate of particle growth increasing with storage tempera-ture. Nevertheless, the overall change in particle size during storagewas relatively small (<5% after 15 days) for all the samples, indicat-ing that they were fairly stable. The most likely reason for the slightincrease in particle size during storage in globular-protein stabilisedemulsions is flocculation (Kim, Decker, & McClements, 2002b).When adsorbed globular proteins are held at elevated temperatures

they tend to undergo conformational transitions (‘‘surface denatur-ation’’), thereby exposing some of the non-polar groups originallylocated in their hydrophobic interior (Kim, Decker, & McClements,2002a; Kim et al., 2002b). As a consequence there is an increase inthe hydrophobic attraction between lipid droplets, which can pro-mote droplet flocculation. The reason that the observed particleaggregation rate was relatively slow in these systems can be attrib-uted to the presence of a high activation energy associated with theelectrostatic repulsion between the droplets (Kim et al., 2002b). Atneutral pH, lipid droplets coated by b-lactoglobulin have a relativelyhigh negative charge, which generates a substantial electrostaticrepulsion between the droplets when the ionic strength is not toohigh, e.g., <100 mM (Guzey & McClements, 2007).

3.2.2. pHThe pH of the aqueous phase in food and beverage emulsions

may vary considerably, ranging from acidic in soft drinks to slightlybasic in some nutritional beverages. We therefore examined theinfluence of pH on the chemical and physical stability of the b-car-otene-enriched nanoemulsions. Nanoemulsions were prepared,adjusted to different pH values, and then stored at ambient tem-perature (�25 �C) for 5 days. The total colour difference (DE⁄) ofthe nanoemulsions was measured periodically during storage(Fig. 7). The overall change in colour during storage was relativelysmall in all of the samples (DE⁄ < 10), however, the rate of colourdegradation was appreciably faster at pH 3 than at higher pH val-ues. Previous studies have also found that the rate of carotenoid(lycopene) degradation in O/W emulsions was higher at acidicpH values (Boon, McClements, Weiss, & Decker, 2009). Studies car-ried out to determine the mechanism of carotenoid instability inthe presence of acids have shown that carotenoids are protonated,and then undergo cis–trans isomerisation and additional degrada-tion reactions (Mortensen & Skibsted, 2000; Mortensen, Skibsted,Sampson, RiceEvans, & Everett, 1997).

The influence of pH on the physical stability of the nanoemul-sions was also examined since this has important implicationsfor their commercial application in food and beverage products.The mean particle radius was measured after the nanoemulsionswere stored for 5 days at ambient temperature (Fig. 8). The nano-emulsions were stable to droplet aggregation at pH 3, 6, 7, and 8 as

Fig. 7. Influence of pH on total colour change (DE⁄) during storage of b-carotene-enriched nanoemulsions stabilised by b-lactoglobulin (0.025% b-carotene, 10%orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, 25 �C).

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indicated by the fact that the particle size remained constant andthere was no visible evidence of phase separation. On the otherhand, the nanoemulsions stored at pH 4 and 5 were highly unsta-ble to droplet aggregation, exhibiting a large increase in meanparticle radius (Fig. 8) and visible evidence of phase separationdue to droplet creaming (data not shown). These effects can beattributed to the influence of pH on the electrostatic repulsion be-tween globular protein-coated lipid droplets (Demetriades, Coup-land, & McClements, 1997; McClements, 2005). At pH 3 (highpositive charge) and pH 6–8 (high negative charge) the adsorbedprotein layer is a long way from its isoelectric point and so thereis a large electrostatic repulsion between the protein-coated drop-lets that prevents them from coming into close proximity (McCle-ments, 2005). At pH 4 and 5, the protein is close to its isoelectricpoint and so the droplets have little or no net charge. Conse-quently, the electrostatic repulsion is insufficient to overcome

Fig. 8. Influence of pH on droplet aggregation in b-carotene-enriched nanoemul-sions stabilised by b-lactoglobulin stored for 5 days (0.025% b-carotene, 10% orangeoil, 2% b-lactoglobulin, 10 mM phosphate buffer, 25 �C).

the van der Waals and hydrophobic attraction, which leads todroplet aggregation. This result suggests that b-carotene-enrichedlipid droplets cannot be incorporated into low viscosity productsthat have intermediate pH values (pH 4–6) since droplet aggrega-tion and gravitational separation would be a problem. Neverthe-less, this would not be a limitation if the b-carotene-enrichedlipid droplets were to be incorporated into highly viscous orsolid-like products, such as dressings, yogurts, sauces, and desserts.It is interesting to note that we did not see an appreciable changein the colour of the nanoemulsions even though they becamehighly aggregated at intermediate pH values (Fig. 7). Previousstudies have also shown that droplet flocculation does not have amajor impact on the colour of O/W emulsions (Chantrapornchai,Clydesdale, & McClements, 2001).

3.2.3. Ionic strengthThe ionic strength of emulsified foods and beverages may also

vary considerably depending on the nature of the food productsin which the oil droplets are present. We therefore examined theinfluence of ionic strength (0–500 mM NaCl) on the chemical andphysical stability of b-carotene-enriched nanoemulsions (pH 7,�25 �C). The salt concentration had little influence on the rate ofcolour fading in the nanoemulsions (Fig. 9), with the overallchanges in total colour difference being rather small (DE⁄ < 6). Onthe other hand, the ionic strength had an appreciable effect on par-ticle aggregation during storage. Addition of low levels of salt(<200 mM NaCl) to the nanoemulsions caused little change in themean particle radius during storage, but addition of higher levelspromoted extensive droplet aggregation (Fig. 10). Similar resultshave also been reported upon addition of NaCl to b-lactoglobulin-stabilised emulsions in other studies (Kim et al., 2002a, 2002b;Qian, Decker, Xiao, & McClements, 2011). The destabilisation ofthe nanoemulsions at high salt concentrations can be attributedto screening of the electrostatic repulsion between the protein-coated droplets by the salt ions (McClements, 2005). At relativelylow salt levels the electrostatic repulsion is still sufficiently strongto overcome the van der Waals and hydrophobic attraction, butabove a critical salt level it is no longer strong enough so that theattractive forces dominate, leading to droplet aggregation.

Fig. 9. Influence of ionic strength (0–500 mM NaCl) on total colour change (DE⁄)during storage of b-carotene-enriched nanoemulsions stabilised by b-lactoglobulin(0.025% b-carotene, 10% orange oil, 2% b-lactoglobulin, pH 7, 10 mM phosphatebuffer, 25 �C).

Fig. 10. Influence of ionic strength (0–500 mM NaCl) on droplet aggregation of b-carotene-enriched nanoemulsions stabilised by b-lactoglobulin during storage(0.025% b-carotene, 10% orange oil, 2% b-lactoglobulin, pH 7, 10 mM phosphatebuffer, 25 �C).

Fig. 11. Influence of emulsifier type (2% b-lactoglobulin or 1.5% Tween 20) on totalcolour change (DE⁄) of b-carotene-enriched nanoemulsions during storage (0.025%b-carotene, 10% orange oil, pH 7, 10 mM phosphate buffer, 25 �C).

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3.3. Impact of emulsifier type

A variety of different emulsifiers are available to stabilise foodand beverage emulsions, and therefore the influence of emulsifiertype on the stability of b-carotene-enriched nanoemulsions (pH 7,37 �C) was examined. In particular, the stability of droplets stabi-lised by a non-ionic surfactant (Tween 20) with those stabilised bya globular protein (b-lactoglobulin) was compared. There was aslight increase in the particle size of the Tween 20 stabilised nano-emulsions during storage, with the mean radius increasing from55 nm immediately after homogenisation to 60 nm after 15 daysstorage (data not shown). This effect can be attributed to somecoalescence when the non-ionic surfactant-coated droplets aremaintained at temperatures approaching the surfactants phaseinversion temperature (PIT) (Rao & McClements, 2010). The cloudpoint (which is related to the PIT) of Tween 20 has been reportedto be around 76 �C (Mahajan, Chawla, & Bakshi, 2004; Saveynet al., 2009), but the droplet coalescence rate is known to increaseas one gets closer to the PIT (Minana-Perez, Gutron, Zundel, Anderez,& Salager, 1999). There was little change in the mean particle size ofthe b-lactoglobulin-stabilised nanoemulsions during storage, withthe radius only increasing from 78 nm immediately after homogeni-sation to 80 nm after 15 days storage (Fig. 6). As discussed earlier,this slight increase in size may be attributed to droplet flocculationresulting from the increased surface hydrophobicity of protein-coated droplets when the globular proteins unfold.

The nature of the emulsifier used to stabilise the system had apronounced influence on the chemical stability of the b-carotenenanoemulsions (Fig. 11). The rate of b-carotene degradation wasappreciably faster in the nanoemulsion stabilised by Tween 20than in the one stabilised by b-lactoglobulin. There are a numberof possible reasons for this phenomenon. First, many types of pro-teins are known to be effective antioxidants, either by chelatingtransition metals or by acting as free radical scavengers (Bertonet al., 2011; Hu et al., 2003; McClements & Decker, 2000). Forexample, b-lactoglobulin contains cysteyl residues, disulphidebonds and thiol functional groups that can inhibit lipid oxidationby scavenging free radicals at the oil–water interface or in theaqueous phase (Sun, Gunasekaran, & Richard, 2007; Tong, Sasaki,McClements, & Decker, 2000). Second, proteins can form molecular

complexes with carotenoids through hydrophobic interactions(Wackerbarth, Stoll, Gebken, Pelters, & Bindrich, 2009), whichmay help protect the carotenoids from degradation. Third, thelayer of adsorbed b-lactoglobulin molecules at the oil–water inter-face may have acted as a physical barrier that prevented any pro-oxidants in the aqueous phase from contacting the b-carotenepresent within the droplets (McClements & Decker, 2000). Fourth,the size of the droplets was larger in the b-lactoglobulin-stabilisednanoemulsions, which means that the oil–water interfacial wassmaller (Mao et al., 2009). If carotenoid degradation is a surface-mediated chemical reaction that occurs at the oil–water interface,then having a smaller surface area may lead to a slower reactionrate. Previous studies have also found that whey proteins (WPI)are more effective at inhibiting the degradation of emulsifiedb-carotene than non-ionic surfactants (Mao et al., 2009). Furtherwork is clearly needed to identify the physicochemical origin ofthe differences between the ability of the non-ionic surfactantand globular protein at protecting carotenoids from degradation.

4. Conclusions

This study has shown that b-carotene can be effectively encapsu-lated within food-grade nanoemulsions stabilised by globular pro-teins or non-ionic surfactants. A number of important factors thatinfluence the chemical and physical stability of these nanoemul-sions were identified. During storage, encapsulated b-carotene hada tendency to chemically degrade, which led to colour fading overtime. The rate of colour fading increased with increasing storagetemperature, was fastest at the most acidic pH value (pH 3), andwas largely independent of salt concentration (0–500 mM NaCl).Our results also demonstrated that b-carotene encapsulated withinprotein-coated lipid droplets was more stable to chemical degrada-tion than that encapsulated within non-ionic surfactant (Tween 20)-coated droplets. This result suggests that the globular protein used(b-lactoglobulin) may be an effective means of increasing chemicalstability of b-carotene in nanoemulsion-based delivery systems.Nevertheless, the physical stability of any delivery system must alsobe considered before selecting it for incorporation into a particularproduct. b-Carotene-enriched nanoemulsions (b-lactoglobulin-coated) have been shown to be prone to droplet aggregation at inter-mediate pH values (4–6), high ionic strengths (>200 mM NaCl) and

1228 C. Qian et al. / Food Chemistry 132 (2012) 1221–1229

elevated temperatures (>37 �C), which may limit their application insome commercial products. The information obtained from thisstudy is important for designing effective delivery systems to encap-sulate and stabilise b-carotene for application within food, beverage,and pharmaceutical products.

Acknowledgements

This material is based upon work supported by the CooperativeState Research, Extension, Education Service, United State Depart-ment of Agriculture, Massachusetts Agricultural Experiment Sta-tion and a United States Department of Agriculture, CREES, NRIand AFRI Grants. We greatly thank Davisco Foods Internationalfor donating the b-lactoglobulin.

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