Physical and chemical stability of b carotene

Physical and chemical stability of b-carotene-enriched nanoemulsions: Influence
of 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 2011
Received in revised form 11 October 2011
Accepted 16 November 2011
Available online 2 December 2011
Keywords:
Nanoemulsion
b-Lactoglobulin
Protein
b-Carotene
Carotenoids
Degradation
Stability
Functional foods
Nutraceuticals
a b s t r a c t
The enrichment of foods and beverages with carotenoids may reduce the incidences of certain chronic
diseases. 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 and
protecting b-carotene was examined. The influence of temperature, pH, ionic strength, and emulsifier
type 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 mM
of 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 elevated
storage 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.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Carotenoids are a class of natural pigments mainly found in fruits
and 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) carotenes
comprised 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 potential
health 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 of
carotenoids, 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
nutraceutical ingredients within foods is currently limited because
of their poor water-solubility, high melting point, chemical instabil-
ity, and low bioavailability.
The relatively low bioavailability of carotenoids from natural
sources has been attributed to the fact that they exist as either crys-
tals or within protein complexes in fruit and vegetables that are not
fully released during digestion within the gastrointestinal tract
(Williams, Boileau, & Erdman, 1998). Carotenoids can be isolated
from natural sources and used as nutraceutical ingredients, but
there 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 at
ambient temperature, which usually means they have to be dis-
solved in oils or dispersed in other suitable matrices before they
can be utilised in foods. A number of studies have shown that carot-
enoid bioavailability depends strongly on the composition and
structure 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 during
food 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).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.11.091
⇑ Corresponding author.
E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).
Food Chemistry 132 (2012) 1221–1229
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Attempts have therefore been made to develop effective delivery
systems to improve the utilisation, bioavailability, and stability of
carotenoids in foods (Mao, Yang, Xu, Yuan, & Gao, 2010; Silva
et 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 phase
prior 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 oil
phase should be kept below the saturation level at the homogenisa-
tion temperature. This limits the maximum amount of lipophilic
materials that can be incorporated into an emulsion-based system,
and sometimes means that the emulsion must be homogenised at
an elevated temperature, which can promote chemical instability
of labile ingredients. A number of previous studies have investigated
the formation, properties, and stability of oil-in-water emulsions
enriched with carotenoids. A high-pressure homogenisation meth-
od was used to prepare lycopene-enriched O/W emulsions stabilised
by 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 emulsions
stabilised by phospholipids (Losso, Khachatryan, Ogawa, Godber, &
Shih, 2005) and proteins (Batista, Raymundo, Sousa, & Empis,
2006). Recently, a high pressure homogenisation method was used
to prepare b-carotene enriched O/W emulsions stabilised by small
molecule 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/W
emulsions 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 have
also shown that the bioavailability of carotenoids is increased when
they are incorporated into O/W emulsions (Grolier, Agoudavi, &
Azaisbraesco, 1995; Parker, 1997; Ribeiro et al., 2006), which may
enhance their health-promoting activities.
Recently, there has been great interest in utilising nanoemulsions
to encapsulate bioactive components for applications in food and
beverage products (McClements, 2011b; McClements & Rao,
2011). Oil-in-water nanoemulsions consist of small lipid droplets
(r < 100 nm) dispersed within an aqueous continuous phase. Similar
to conventional emulsions, nanoemulsions are thermodynamically
unstable 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 lipophilic
substances; they scatter light weakly and so can be incorporated
into optically transparent products; and they have a high stability
to particle aggregation and gravitational separation (Acosta, 2009;
McClements, 2011a). Nanoemulsions containing carotenoids have
previously 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 are
typically stabilised against chemical degradation by adding antiox-
idants, reducing oxygen levels, and minimising exposure to light and
pro-oxidants. However, once a sealed product is opened and
exposed to the atmosphere some of these protective measures
may be lost, and so it is important to understand the major factors
that influence b-carotene stability.
In the present study, a high pressure homogenisation method
was used to prepare nanoemulsions containing b-carotene, and
then test their stability to environmental stresses that might be
encountered in typical food and beverage applications (pH, ionic
strength, and temperature). A primary goal of this study is to
formulate nanoemulsions entirely from food-grade ingredients
that are perceived to be safe and label friendly. We therefore
utilised orange oil as the carrier oil phase since this is widely used
for this purpose in commercial beverage emulsions, and utilised a
globular protein (b-lactoglobulin) as the emulsifier since whey pro-
teins are already widely used for this purpose in food and beverage
products (McClements, 2005). In addition, previous studies have
shown that certain types of food protein are effective at reducing
the 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 lipid
droplets with a protein layer may improve the chemical stability of
the encapsulated b-carotene. For this reason, the rate of chemical
degradation of b-carotene in protein-stabilised and surfactant-
stabilised nanoemulsions was compared. The results of this study
will 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 grade
b-lactoglobulin was obtained from Davisco Foods International
Inc. (Le Sueur, MN). Beta-carotene (Type I, C9750) and Tween 20
were 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 emulsions
An 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 ensure
full dissolution (i.e., the sample became completely transparent
with no evidence of crystals). The samples were flushed with nitro-
gen during this process to inhibit degradation of the b-carotene. An
aqueous 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 nanoemulsions
were 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 passed
through 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, or
5 °C incubators. In one study, Tween 20 (1.5% (w/w)) was used as
an emulsifier rather than b-lactoglobulin, but otherwise the nano-
emulsions were prepared and characterised using the same
procedures.
2.2.2. Measurement of emulsion stability
2.2.2.1. Particle size. The particle size distribution and mean parti-
cle radius (Z-average) of diluted emulsions were measured by a
commercial dynamic light-scattering device (Nano-ZS, Malvern
Instruments, Worcestershire, UK). Samples were diluted (1:100)
with buffer solution prior to analysis to avoid multiple scattering
effects to reach an instrument attenuation factor 66. The buffers
1222 C. Qian et al. / Food Chemistry 132 (2012) 1221–1229

used for dilution had the same pH and ionic composition as the
samples being analysed.
2.2.2.2. b-Carotene degradation. The chemical degradation of
b-carotene during storage was measured using two different
approaches: solvent extraction and colourimetry.
Solvent extraction: In this destructive approach, the b-carotene
was isolated from a nanoemulsion using solvent extraction,
and then quantified using a UV–visible spectroscopy method.
b-Carotene enriched nanoemulsions completely separated into an
aqueous phase and an organic phase after addition of a solvent
containing methanol and methylene chloride (volume fraction
1:2). The transparent lower organic phase (orange coloured) con-
taining the b-carotene was removed, transferred to a cuvette, and
then its absorbance was measured at 450 nm using a UV–visible
spectrometer. A pure methanol and methylene chloride solution
was used as a blank. An orange oil nanoemulsion containing no
added b-carotene was also analysed as a control. The b-carotene
content 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 chemical
degradation of b-carotene was monitored in situ by measuring
changes in emulsion colour: as the b-carotene degraded, the colour
became less intense. The tristimulus colour coordinates (L⁄
a⁄
b⁄
) of
the nanoemulsions were measured using a hand-held colourimeter
(ColourMunki, X-Rite, Grand Rapids, MI). L⁄
values are a measure of
lightness (higher value indicates a lighter colour); a⁄
values are a
measure of redness (higher positive values indicate a redder col-
our, higher negative values indicate a greener colour); b⁄
values
are a measure of yellowness (higher positive values indicate a
more yellow colour, higher negative values indicate a more blue
colour). Emulsions were placed into a transparent flat-faced cuv-
ette, the measuring device of the colourimeter was pressed against
the 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 stability
The physical and chemical stability of b-carotene enriched
nanoemulsions to environmental stresses typically encountered
by food products were tested:
Temperature: 20-ml emulsion samples (pH 7.0) were transferred
into glass tubes and stored in the dark at 5, 20, 37, and 55 °C for
15 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 in
a 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 the
same droplet concentration, but different salt concentrations
(0–500 mM NaCl). The emulsions were stirred for 30 min and
then transferred into glass tubes and stored in a dark place at
ambient 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 was
78 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 particles
being 100 nm in radius (Fig. 1). The mean particle radius
(r = 79 nm) and particle size distribution (Fig. 1) did not change
appreciably from the initial values after the nanoemulsions were
stored 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 temperature
Initially, 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 an
intense orange-red colour, but this colour tends to fade when it
undergoes chemical degradation (Patras, Brunton, Da Pieve, Butler,
Downey, 2009). We therefore monitored the chemical stability of
b-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 solvent
extraction method is shown in Fig. 2. The b-carotene concentration
fell 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 highly
unstable to chemical degradation when stored at elevated temper-
atures in nanoemulsions. The influence of storage temperature on
the colour of b-carotene enriched nanoemulsions measured using a
colourimeter is shown in Fig. 3. In general, the lightness (L⁄
) and
colour intensity (a⁄
and b⁄
) of the nanoemulsions progressively de-
creased during storage, which is indicative of colour fading. The
rate 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 is
indicative 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 higher
fraction of light is reflected from its surface (McClements, 2002).
In practice, a slight decrease in the lightness of the nanoemulsions
during storage (Fig. 3a) was observed, which may have been due to
Fig. 1. Particle size distribution of b-carotene enriched nanoemulsions stabilised by
b-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

changes in the intensity of light scattering, e.g., due to changes in
particle 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):
DEÃ
¼ ½ðLÃ
À LÃ
0Þ2
þ ðaÃ
À aÃ
0Þ2
þ ðb
Ã
À b
Ã
0Þ2
Š1=2
ð1Þ
Here L⁄
, a⁄
, and b⁄
are the measured colour coordinates of the
nanoemulsion at storage time t, and LÃ
0, aÃ
0, and b
Ã
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, which
clearly highlights the rapid acceleration in colour fading when the
storage temperature is increased from 20 to 55 °C. An indication
of the relative rate of b-carotene degradation at different storage
temperatures was obtained by determining the slope of the initial
linear region of the DE⁄
versus time plots using linear regression
analysis (Fig. 5). Previous studies have also found a relatively rapid
loss 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 of
bioactivity.
Fig. 2. Influence of storage temperature on the chemical degradation of b-carotene
encapsulated within nanoemulsions stabilised by b-lactoglobulin measured using a
solvent 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 nanoemulsions stabilised by b-lactoglobulin measured using a colourimeter (0.025% b-
carotene, 10% orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, pH 7): (a) lightness (L⁄
-value); (b) ‘‘redness’’ (a⁄
-value), (c) ‘‘yellowness’’ (b⁄
-value).

From a practical viewpoint, it is important that nanoemulsions
also remain physically stable during storage. Therefore changes in
the mean particle radius over time when the nanoemulsions were
stored 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 storage
was relatively small (5% after 15 days) for all the samples, indicat-
ing that they were fairly stable. The most likely reason for the slight
increase in particle size during storage in globular-protein stabilised
emulsions 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 originally
located in their hydrophobic interior (Kim, Decker, McClements,
2002a; Kim et al., 2002b). As a consequence there is an increase in
the hydrophobic attraction between lipid droplets, which can pro-
mote droplet flocculation. The reason that the observed particle
aggregation rate was relatively slow in these systems can be attrib-
uted to the presence of a high activation energy associated with the
electrostatic repulsion between the droplets (Kim et al., 2002b). At
neutral pH, lipid droplets coated by b-lactoglobulin have a relatively
high negative charge, which generates a substantial electrostatic
repulsion between the droplets when the ionic strength is not too
high, e.g., 100 mM (Guzey McClements, 2007).
3.2.2. pH
The pH of the aqueous phase in food and beverage emulsions
may vary considerably, ranging from acidic in soft drinks to slightly
basic in some nutritional beverages. We therefore examined the
influence 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⁄
) of
the nanoemulsions was measured periodically during storage
(Fig. 7). The overall change in colour during storage was relatively
small in all of the samples (DE⁄
10), however, the rate of colour
degradation 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 acidic
pH values (Boon, McClements, Weiss, Decker, 2009). Studies car-
ried out to determine the mechanism of carotenoid instability in
the 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 implications
for their commercial application in food and beverage products.
The mean particle radius was measured after the nanoemulsions
were 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
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-
Fig. 6. Influence of storage temperature on droplet aggregation in b-carotene-

indicated by the fact that the particle size remained constant and
there was no visible evidence of phase separation. On the other
hand, the nanoemulsions stored at pH 4 and 5 were highly unsta-
ble to droplet aggregation, exhibiting a large increase in mean
particle radius (Fig. 8) and visible evidence of phase separation
due to droplet creaming (data not shown). These effects can be
attributed 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 (high
positive charge) and pH 6–8 (high negative charge) the adsorbed
protein layer is a long way from its isoelectric point and so there
is 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 isoelectric
point and so the droplets have little or no net charge. Conse-
quently, the electrostatic repulsion is insufficient to overcome
the van der Waals and hydrophobic attraction, which leads to
droplet aggregation. This result suggests that b-carotene-enriched
lipid droplets cannot be incorporated into low viscosity products
that 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-enriched
lipid droplets were to be incorporated into highly viscous or
solid-like products, such as dressings, yogurts, sauces, and desserts.
It is interesting to note that we did not see an appreciable change
in the colour of the nanoemulsions even though they became
highly aggregated at intermediate pH values (Fig. 7). Previous
studies have also shown that droplet flocculation does not have a
major impact on the colour of O/W emulsions (Chantrapornchai,
Clydesdale, McClements, 2001).
3.2.3. Ionic strength
The ionic strength of emulsified foods and beverages may also
vary considerably depending on the nature of the food products
in which the oil droplets are present. We therefore examined the
influence of ionic strength (0–500 mM NaCl) on the chemical and
physical stability of b-carotene-enriched nanoemulsions (pH 7,
%25 °C). The salt concentration had little influence on the rate of
colour fading in the nanoemulsions (Fig. 9), with the overall
changes in total colour difference being rather small (DE⁄
6). On
the 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 the
mean particle radius during storage, but addition of higher levels
promoted extensive droplet aggregation (Fig. 10). Similar results
have 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 of
the nanoemulsions at high salt concentrations can be attributed
to screening of the electrostatic repulsion between the protein-
coated droplets by the salt ions (McClements, 2005). At relatively
low salt levels the electrostatic repulsion is still sufficiently strong
to overcome the van der Waals and hydrophobic attraction, but
above a critical salt level it is no longer strong enough so that the
attractive forces dominate, leading to droplet aggregation.
Fig. 7. Influence of pH on total colour change (DE⁄
) during storage of b-carotene-
orange oil, 2% b-lactoglobulin, 10 mM phosphate buffer, 25 °C).
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% orange
oil, 2% b-lactoglobulin, 10 mM phosphate buffer, 25 °C).
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 phosphate
buffer, 25 °C).

3.3. Impact of emulsifier type
A variety of different emulsifiers are available to stabilise food
and beverage emulsions, and therefore the influence of emulsifier
type 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 by
a globular protein (b-lactoglobulin) was compared. There was a
slight increase in the particle size of the Tween 20 stabilised nano-
emulsions during storage, with the mean radius increasing from
55 nm immediately after homogenisation to 60 nm after 15 days
storage (data not shown). This effect can be attributed to some
coalescence when the non-ionic surfactant-coated droplets are
maintained at temperatures approaching the surfactants phase
inversion temperature (PIT) (Rao McClements, 2010). The cloud
point (which is related to the PIT) of Tween 20 has been reported
to be around 76 °C (Mahajan, Chawla, Bakshi, 2004; Saveyn
et al., 2009), but the droplet coalescence rate is known to increase
as one gets closer to the PIT (Minana-Perez, Gutron, Zundel, Anderez,
Salager, 1999). There was little change in the mean particle size of
the b-lactoglobulin-stabilised nanoemulsions during storage, with
the 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 flocculation
resulting 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 a
pronounced influence on the chemical stability of the b-carotene
nanoemulsions (Fig. 11). The rate of b-carotene degradation was
appreciably faster in the nanoemulsion stabilised by Tween 20
than in the one stabilised by b-lactoglobulin. There are a number
of possible reasons for this phenomenon. First, many types of pro-
teins are known to be effective antioxidants, either by chelating
transition metals or by acting as free radical scavengers (Berton
et al., 2011; Hu et al., 2003; McClements Decker, 2000). For
example, b-lactoglobulin contains cysteyl residues, disulphide
bonds and thiol functional groups that can inhibit lipid oxidation
by scavenging free radicals at the oil–water interface or in the
aqueous 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), which
may help protect the carotenoids from degradation. Third, the
layer 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-carotene
present within the droplets (McClements Decker, 2000). Fourth,
the size of the droplets was larger in the b-lactoglobulin-stabilised
nanoemulsions, which means that the oil–water interfacial was
smaller (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 reaction
rate. Previous studies have also found that whey proteins (WPI)
are more effective at inhibiting the degradation of emulsified
b-carotene than non-ionic surfactants (Mao et al., 2009). Further
work is clearly needed to identify the physicochemical origin of
the differences between the ability of the non-ionic surfactant
and 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 that
influence the chemical and physical stability of these nanoemul-
sions were identified. During storage, encapsulated b-carotene had
a tendency to chemically degrade, which led to colour fading over
time. The rate of colour fading increased with increasing storage
temperature, was fastest at the most acidic pH value (pH 3), and
was largely independent of salt concentration (0–500 mM NaCl).
Our results also demonstrated that b-carotene encapsulated within
protein-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 chemical
stability of b-carotene in nanoemulsion-based delivery systems.
Nevertheless, the physical stability of any delivery system must also
be considered before selecting it for incorporation into a particular
product. 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
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 phosphate
buffer, 25 °C).
Fig. 11. Influence of emulsifier type (2% b-lactoglobulin or 1.5% Tween 20) on total
colour change (DE⁄
) of b-carotene-enriched nanoemulsions during storage (0.025%
b-carotene, 10% orange oil, pH 7, 10 mM phosphate buffer, 25 °C).

elevated temperatures (37 °C), which may limit their application in
some commercial products. The information obtained from this
study 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 Cooperative
State Research, Extension, Education Service, United State Depart-
ment of Agriculture, Massachusetts Agricultural Experiment Sta-
tion and a United States Department of Agriculture, CREES, NRI
and AFRI Grants. We greatly thank Davisco Foods International
for donating the b-lactoglobulin.
References
Abdel-Aal, E. S. M., Akhtar, M. H. (2006). Recent advances in the analyses of
carotenoids and their role in human health. Current Pharmaceutical Analysis,
2(2), 195–204.
Acosta, E. (2009). Bioavailability of nanoparticles in nutrient and nutraceutical
delivery. Current Opinion in Colloid and Interface Science, 14(1), 3–15.
Batista, A. P., Raymundo, A., Sousa, I., Empis, J. (2006). Rheological
characterization of coloured oil-in-water food emulsions with lutein and
phycocyanin added to the oil and aqueous phases. Food Hydrocolloids, 20(1),
44–52.
Berton, C., Ropers, M. H., Viau, M., Genot, C. (2011). Contribution of the interfacial
layer to the protection of emulsified lipids against oxidation. Journal of
Agricultural and Food Chemistry, 59(9), 5052–5061.
Boon, C. S., McClements, D. J., Weiss, J., Decker, E. A. (2009). Role of iron and
hydroperoxides in the degradation of lycopene in oil-in-water emulsions.
Journal of Agricultural and Food Chemistry, 57(7), 2993–2998.
Chantrapornchai, W., Clydesdale, F. M., McClements, D. J. (2001). Influence of
flocculation on optical properties of emulsions. Journal of Food Science, 66(3),
464–469.
Demetriades, K., Coupland, J. N., McClements, D. J. (1997). Physical properties of
whey protein stabilized emulsions as related to pH and NaCl. Journal of Food
Science, 62(2), 342–347.
Failla, M. L., Chitchumroonchokchai, C., Ishida, B. K. (2008). In vitro
micellarization and intestinal cell uptake of cis isomers of lycopene exceed
those of all-trans lycopene. Journal of Nutrition, 138(3), 482–486.
Failla, M. L., Huo, T., Thakkar, S. K. (2007). In vitro screening of relative
bioaccessibility of carotenoids from foods. In 10th Asian Congress of Nutrition,
(pp. 200–203). Taipei, Taiwan: HEC Press, Healthy Eating Club Pty Ltd.
Gerster, H. (1993). Anticarcinogenic effect of common carotenoids. International
Journal for Vitamin and Nutrition Research, 63(2), 93–121.
Grolier, P., Agoudavi, S., Azaisbraesco, V. (1995). Comparative bioavailability of
diet-based, oil-based and emulsion-based preparations of vitamin-A and beta-
carotene in rat. Nutrition Research, 15(10), 1507–1516.
Guzey, D., McClements, D. J. (2007). Impact of electrostatic interactions on
formation and stability of emulsions containing oil droplets coated by beta-
lactoglobulin–pectin complexes. Journal of Agricultural and Food Chemistry,
55(2), 475–485.
Higuera-Ciapara, I., Felix-Valenzuela, L., Goycoolea, F. M. (2006). Astaxanthin: A
review of its chemistry and applications. Critical Reviews in Food Science and
Nutrition, 46(2), 185–196.
Hu, M., McClements, D. J., Decker, E. A. (2003). Lipid oxidation in corn oil-in-water
emulsions stabilized by casein, whey protein isolate, and soy protein isolate.
Journal of Agricultural and Food Chemistry, 51(6), 1696–1700.
Khoo, H. E., Prasad, K. N., Kong, K. W., Jiang, Y. M., Ismail, A. (2011). Carotenoids
and their isomers: Colour pigments in fruits and vegetables. Molecules, 16(2),
1710–1738.
Kim, H. J., Decker, E. A., McClements, D. J. (2002a). Impact of protein surface
denaturation on droplet flocculation in hexadecane oil-in-water emulsions
stabilized by beta-lactoglobulin. Journal of Agricultural and Food Chemistry,
50(24), 7131–7137.
Kim, H. J., Decker, E. A., McClements, D. J. (2002b). Role of postadsorption
conformation changes of beta-lactoglobulin on its ability to stabilize oil
droplets against flocculation during heating at neutral pH. Langmuir, 18(20),
7577–7583.
Losso, J. N., Khachatryan, A., Ogawa, M., Godber, J. S., Shih, F. (2005). Random
centroid optimization of phosphatidylglycerol stabilized lutein-enriched oil-in-
water emulsions at acidic pH. Food Chemistry, 92(4), 737–744.
Mahajan, R. K., Chawla, J., Bakshi, M. S. (2004). Depression in the cloud point of
Tween in the presence of glycol additives and triblock polymers. Colloid and
Polymer Science, 282(10), 1165–1168.
Mao, L. K., Xu, D. X., Yang, J., Yuan, F., Gao, Y. X., Zhao, J. (2009). Effects of small and
large molecule emulsifiers on the characteristics of beta-carotene
nanoemulsions prepared by high pressure homogenization. Food Technology
and Biotechnology, 47(3), 336–342.
Mao, L. K., Yang, J., Xu, D. X., Yuan, F., Gao, Y. X. (2010). Effects of homogenization
models and emulsifiers on the physicochemical properties of -carotene
nanoemulsions. Journal of Dispersion Science and Technology, 31(7), 986–993.
McClements, D. J. (2002). Theoretical prediction of emulsion colour. Advances in
Colloid and Interface Science, 97(1–3), 63–89.
McClements, D. J. (2005). Food emulsions: Principles, practice, and techniques (2nd
ed.). Boca Raton: CRC Press.
McClements, D. J. (2010). Emulsion design to improve the delivery of functional
lipophilic components. Annual review of food science and technology, 1, 241–269.
McClements, D. J. (2011a). Edible nanoemulsions: Fabrication, properties, and
functional performance. Soft Matter, 7(6), 2297–2316.
McClements, D. J. (2011b). Edible nanoemulsions: Fabrication, properties, and
functional performance. Soft Matter.
McClements, D. J., Decker, E. A. (2000). Lipid oxidation in oil-in-water emulsions:
Impact of molecular environment on chemical reactions in heterogeneous food
systems. Journal of Food Science, 65(8), 1270–1282.
McClements, D. J., Decker, E. A., Park, Y., Weiss, J. (2009). Structural design
principles for delivery of bioactive components in nutraceuticals and functional
foods. Critical Reviews in Food Science and Nutrition, 49(6), 577–606.
McClements, D. J., Li, Y. (2010). Structured emulsion-based delivery systems:
Controlling the digestion and release of lipophilic food components. Advances in
Colloid and Interface Science, 159(2), 213–228.
McClements, D. J., Rao, J. (2011). Food-grade nanoemulsions: Formulation,
fabrication, properties, performance, biological fate, and potential toxicity.
Critical Reviews in Food Science and Nutrition, 51(4), 285–330.
Mcguire, R. G. (1992). Reporting of objective colour measurements. Hortscience,
27(12), 1254–1255.
Minana-Perez, M., Gutron, C., Zundel, C., Anderez, J. M., Salager, J. L. (1999).
Miniemulsion formation by transitional inversion. Journal of Dispersion Science
and Technology, 20(3), 893–905.
Mortensen, A., Skibsted, L. H. (2000). Kinetics and mechanism of the primary steps
of degradation of carotenoids by acid in homogeneous solution. Journal of
Mortensen, A., Skibsted, L. H., Sampson, J., RiceEvans, C., Everett, S. A. (1997).
Comparative mechanisms and rates of free radical scavenging by carotenoid
antioxidants. FEBS Letters, 418(1–2), 91–97.
Nguyen, M. L., Schwartz, S. J. (1998). Lycopene stability during food
processing. Proceedings of the Society for Experimental Biology and Medicine,
218(2), 101–105.
Parker, R. S. (1997). Bioavailability of carotenoids. European Journal of Clinical
Nutrition, 51, S86–S90.
Patras, A., Brunton, N., Da Pieve, S., Butler, F., Downey, G. (2009). Effect of thermal
and high pressure processing on antioxidant activity and instrumental colour of
tomato and carrot purees. Innovative Food Science and Emerging Technologies,
10(1), 16–22.
Qian, C., Decker, E. A., Xiao, H., McClements, D. J. (2011). Comparison of
biopolymer emulsifier performance in formation and stabilization of orange
oil-in-water emulsions. Journal of the American Oil Chemists Society, 88(1),
47–55.
Rao, J. J., McClements, D. J. (2010). Stabilization of phase inversion temperature
nanoemulsions by surfactant displacement. Journal of Agricultural and Food
Chemistry, 58(11), 7059–7066.
Ribeiro, H. S., Ax, K., Schubert, H. (2003). Stability of lycopene emulsions in food
systems. Journal of Food Science, 68(9), 2730–2734.
Ribeiro, H. S., Chu, B. S., Ichikawa, S., Nakajima, M. (2008). Preparation of
nanodispersions containing [beta]-carotene by solvent displacement method.
Food Hydrocolloids, 22(1), 12–17.
Ribeiro, H. S., Guerrero, J. M. M., Briviba, K., Rechkemmer, G., Schuchmann, H. P.,
Schubert, H. (2006). Cellular uptake of carotenoid-loaded oil-in-water
emulsions in colon carcinoma cells in vitro. Journal of Agricultural and Food
Chemistry, 54(25), 9366–9369.
Ribeiro, H. S., Rico, L. G., Badolato, G. G., Schubert, H. (2005). Production of O/W
emulsions containing astaxanthin by repeated Premix membrane
emulsification. Journal of Food Science, 70(2), E117–E123.
Saveyn, P., Cocquyt, E., Zhu, W. X., Sinnaeve, D., Haustraete, K., Martins, J. C., et al.
(2009). Solubilization of flurbiprofen within non-ionic Tween 20 surfactant
micelles: A F-19 and H-1 NMR study. Physical Chemistry Chemical Physics,
11(26), 5462–5468.
Silva, H., Cerqueira, M., Souza, B., Ribeiro, C., Avides, M., Quintas, M. et al. (2010).
Nanoemulsions of [beta]-carotene using a high-energy emulsification-
evaporation technique. Journal of Food Engineering.
Singh, P., Goyal, G. K. (2008). Dietary lycopene: Its properties and
anticarcinogenic effects. Comprehensive Reviews in Food Science and Food
Safety, 7(3), 255–270.
Sun, C., Gunasekaran, S., Richard, M. P. (2007). Effect of xanthan gum on
physicochemical properties of whey protein isolate stabilized oil-in-water
emulsions. Food Hydrocolloids, 21, 555–564.
Tai, C. Y., Chen, B. H. (2000). Analysis and stability of carotenoids in the flowers of
daylily (Hemerocallis disticha) as affected by various treatments. Journal of
Tan, C. P., Nakajima, M. (2005a). Beta-Carotene nanodispersions: Preparation,
characterization and stability evaluation. Food Chemistry, 92(4), 661–671.
Tan, C. P., Nakajima, M. (2005b). Effect of polyglycerol esters of fatty acids on
physicochemical properties and stability of beta-carotene nanodispersions
prepared by emulsification/evaporation method. Journal of the Science of Food
and Agriculture, 85(1), 121–126.

Thakkar, S. K., Maziya-Dixon, B., Dixon, A. G. O., Failla, M. L. (2007). Beta-Carotene
micellarization during in vitro digestion and uptake by Caco-2 cells is directly
proportional to beta-Carotene content in different genotypes of cassava. Journal
of Nutrition, 137(10), 2229–2233.
Tong, L. M., Sasaki, S., McClements, D. J., Decker, E. A. (2000). Mechanisms of the
antioxidant activity of a high molecular weight fraction of whey. Journal of
Tyssandier, V., Lyan, B., Borel, P. (2001). Main factors governing the transfer of
carotenoids from emulsion lipid droplets to micelles. Biochimica Et Biophysica
Acta-Molecular and Cell Biology of Lipids, 1533(3), 285–292.
Tyssandier, V., Reboul, E., Dumas, J. F., Bougteloup-Demange, C., Armand, M.,
Marcand, J., et al. (2003). Processing of vegetable-borne carotenoids in the
human stomach and duodenum. American Journal of Physiology –
Gastrointestinal and Liver Physiology, 284(6), G913–G923.
von Lintig, J. (2010). Colours with functions: Elucidating the biochemical and
molecular basis of carotenoid metabolism. In Annual Review of Nutrition, (Vol.
30) (pp. 35–56).
Wackerbarth, H., Stoll, T., Gebken, S., Pelters, C., Bindrich, U. (2009). Carotenoid-
protein interaction as an approach for the formulation of functional food
emulsions. Food Research International, 42(9), 1254–1258.
Waraho, T., McClements, D. J., Decker, E. A. (2011). Mechanisms of lipid oxidation
in food dispersions. Trends in Food Science and Technology, 22(1), 3–13.
Williams, A. W., Boileau, T. W. M., Erdman, J. W. (1998). Factors inﬂuencing the
uptake and absorption of carotenoids. Proceedings of the Society for Experimental
Biology and Medicine, 218(2), 106–108.
Xianquan, S., Shi, J., Kakuda, Y., Yueming, J. (2005). Stability of lycopene during
food processing and storage. Journal of Medicinal Food, 8(4), 413–422.

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