Food Hydrocolloids 61 (2016) 1e10

Contents lists available at ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Encapsulation of b-carotene in alginate-based hydrogel beads: Impact

on physicochemical stability and bioaccessibility
Zipei Zhang a, Ruojie Zhang a, David Julian McClements a, b, *
a
Department of Food Science, University of Massachusetts Amherst, Amherst, 01003, MA, USA
b
Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah, 21589, Saudi Arabia

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

Article history: Delivery systems are needed to protect carotenoids in foods, but release them at an appropriate location
Received 19 February 2016 within the gastrointestinal tract (GIT). In this study, b-carotene was incorporated into three different
Received in revised form delivery systems: free lipid droplets; filled hydrogel beads formed using 0.5% alginate (“0.5% beads”);
5 April 2016
and, filled hydrogel beads formed using 1% alginate (“1% beads”). Hydrogel beads were fabricated by
Accepted 26 April 2016
Available online 27 April 2016
injecting an alginate solution into a calcium ion solution using an extrusion device (Encapsulator). Light
scattering and confocal microscopy measurements indicated that the 0.5% beads had much smaller
diameter (285 mm) than the 1% beads (660 mm). b-carotene encapsulated in free lipid droplets (nano-
Keywords:
b-carotene emulsions) was highly unstable to chemical degradation when stored at elevated temperatures.
Alginate Conversely, incorporation of the b-carotene-loaded lipid droplets into hydrogel beads greatly improved
Hydrogel beads its chemical stability. Simulated GIT studies indicated that the rate and extent of lipid digestion decreased
Stability in the following order: free lipid droplets >0.5% beads >1% beads. The encapsulated b-carotene had a
Digestion higher bioaccessibility in free lipid droplets than in hydrogel beads, whereas its chemical stability within
Bioaccessibility the GIT was higher in the hydrogel beads, with the 1% beads giving better protection against degradation
than the 0.5% beads, which was attributed to differences in hydrogel pore size. Overall, our results
provide valuable information for the rational design and development of nutraceutical delivery systems
for utilization in functional food products.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction hydrocarbon backbone and high degree of unsaturation of b-caro-

Carotenoids are natural pigments present in certain fruits and
vegetables where they play key roles in photosynthesis and pho-
toeprotection reactions (Cazzonelli, 2011; DemmigAdams,
Gilmore, & Adams, 1996; Namitha & Negi, 2010). b-carotene, one
of the major types of carotenoid, is a natural precursor of vitamin A
(Maiani et al., 2009; Rao & Rao, 2007). b-carotene also demon-
strates antioxidant and non-antioxidant biological activities, which
may account for its ability to reduce the risk of certain chronic
diseases including cardiovascular disease, eye disease, and certain
cancers (Rao & Rao, 2007). There is therefore interest in fortifying
food and beverage products with this potentially beneficial nutra-
ceutical to create products designed to promote human health and
wellness (van den Berg et al., 2000). However, the extended
* Corresponding author. Department of Food Science, University of Massachu-
setts Amherst, Amherst, 01003, MA, USA.
E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).
tene restrict its incorporation into many foods and beverages
because these factors lead to low water-solubility, poor chemical
instability and low bioaccessibility (Fernandez-Garcia et al., 2012;
Qian, Decker, Xiao, & McClements, 2012b; Yi, Li, Zhong, &
Yokoyama, 2014). Moreover, b-carotene can be chemically trans-
formed within the gastrointestinal tract (GIT), which may alter its
potential health benefits (Sy, Dangles, Borel, & Caris-Veyrat, 2015).
Consequently, there is a need to develop effective carotenoid de-
livery systems that can overcome these challenges (Donhowe &
Kong, 2014; Lopes, Martins-Lopes, & Souto, 2010).
Colloidal delivery systems are one of the most convenient
methods of incorporating lipophilic bioactives (such as caroten-
oids) into foods (McClements, 2012; Patel & Velikov, 2011; Velikov
& Pelan, 2008). These systems consist of small colloidal particles,
typically fabricated from lipids, phospholipids, surfactants, and/or
biopolymers, which encapsulate the lipophilic bioactives. These
colloidal particles are designed to facilitate the incorporation of
bioactives into food products, improve their chemical/biochemical
stability, and control their GIT fate (McClements, 2014). A typical

http://dx.doi.org/10.1016/j.foodhyd.2016.04.036
0268-005X/© 2016 Elsevier Ltd. All rights reserved.
2 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10
bioactive-loaded colloidal system can be simply prepared by solu-
bilizing the lipophilic bioactive components within an oil phase
and then homogenizing it with an aqueous phase containing an
emulsifier to form oil-in-water emulsions or nanoemulsions
(Donsi, Sessa, Mediouni, Mgaidi, & Ferrari, 2011; Marze, 2015;
McClements, Decker, & Weiss, 2007). Previous studies indicate
that emulsion-based systems are particularly suitable for encap-
sulation and delivery of carotenoids such as lutein (Losso,
Khachatryan, Ogawa, Godber, & Shih, 2005), lycopene (Boon,
McClements, Weiss, & Decker, 2009) and b-carotene (Qian,
Decker, Xiao, & McClements, 2012a; Salvia-Trujillo, Qian, Martín-
Belloso, & McClements, 2013; Silva et al., 2011). In addition, the
bioaccessibility and bioavailability of carotenoids may increase
considerably after encapsulation in emulsion-based systems
(Ribeiro et al., 2006; Salvia-Trujillo et al., 2013).
One limitation of using emulsions or nanoemulsions as delivery
systems is they have limited scope for controlling the chemical
stability of encapsulated carotenoids. This is because only a thin
layer of emulsifier molecules coats the lipid droplets, and so any
carotenoids located near the droplet surfaces are prone to chemical
degradation promoted by hydrophilic components in the aqueous
phase (such as acids, transition metals, or enzymes). In particular,
carotenoids have a conjugated polyunsaturated hydrocarbon chain,
which makes them highly prone to degradation due to autoxidation
promoted by light, heat, singlet oxygen, transition metals, free
radicals and highly acidic conditions (Qian et al., 2012a). This
problem could be overcome by trapping the carotenoid-loaded
lipid droplets inside hydrogel beads (“microgels”). The hydrogel
matrix surrounding the carotenoids could then be designed to
provide a local environment that protects them from degradation.
Hydrogel beads suitable for utilization in foods are usually
constructed from food-grade biopolymers such as proteins and/or
polysaccharides using a variety of approaches (Chen, Remondetto,
& Subirade, 2006; Joye & McClements, 2014; Shewan & Stokes,
2013). The injection-gelation method is one of the simplest ap-
proaches for the encapsulation, protection, and delivery of nutra-
ceuticals and other active agents (Shewan & Stokes, 2013). In this
method, a biopolymer solution containing the bioactive is injected
into another ‘‘hardening’’ solution under conditions that promote
biopolymer gelation. This procedure results in the formation of a
hydrogel bead with the nutraceuticals trapped inside. The nature of
the hydrogel matrix surrounding the bioactive can be designed to
improve its physical and chemical stability, as well as to control its
GIT fate. Typically, hydrogel beads with a particle diameter greater
than about 1 mm are prepared using a syringe with a needle or a
pipette (Li, Hu, Du, Xiao, & McClements, 2011). Smaller beads
(<1 mm) can be fabricated using an extrusion device with a
vibrating nozzle (Encapsulator), which may be an advantage for
certain applications. The dimensions of hydrogel beads can be
further adjusted by altering several factors including biopolymer
and cross-linking ion concentration, instrument parameters, and
hardening time (Gombotz & Wee, 2012; Li et al., 2011).
In the present study, microfluidization was used to prepare oil-
in-water nanoemulsions containing b-carotene. The b-carotene-
loaded lipid droplets were then used as delivery systems them-
selves, or they were incorporated into hydrogel beads by injecting
them and an alginate mixture into a gelling solution (Ca2þ). Cal-
cium alginate beads formed using this approach are widely used in
the pharmaceutical industry to encapsulate and protect bioactive
agents (Giri et al., 2012; Lee & Mooney, 2012). Hydrogel beads with
different structural, physical, and functional attributes were pre-
pared by using two different alginate concentrations (0.5% and 1%):
typically bead hardness increases with increasing alginate, but
bead pore size decreases. The chemical stability and simulated GIT
fate of b-carotene encapsulated within either nanoemulsions or
filled hydrogel beads were compared. The information obtained
from this study may facilitate the production of delivery systems
that improve the stability and bioavailability of lipophilic
bioactives.
2. Materials and methods
2.1. Materials
Whey protein isolate (WPI) was provided by Davisco Foods In-
ternational Inc. (Le Sueur MN). The WPI was reported to contain
97.9 wt.% protein and 0.2 wt.% fat. Corn oil was purchased from a
local supermarket. The following chemicals were purchased from
the Sigma Chemical Company (St. Louis, MO): b-carotene; alginic
acid (sodium salt); mucin from porcine stomach; pepsin from
porcine gastric mucosa; lipase from porcine pancreas; porcine bile
extract; and Nile Red. All chemicals used were analytical grade.
Double distilled water was used to make all solutions.
2.2. Preparation of b-carotene-loaded emulsions
Initially, an aqueous phase was prepared by mixing 1% (w/w)
WPI with a buffer solution (5 mM phosphate buffer, pH 7) and
stirring for at least 2 h. The emulsifier solution was stored over-
nighted at 4  C and filtered before use. The b-carotene-loaded lipid
droplets were prepared by dispersing b-carotene (0.1%, w/w) in
corn oil by heating (55  C, 10 min) and then ultrasonic processing
(2 min) with vibrational frequency of 40 kHz. This procedure was
repeated several times to ensure the complete dissolution of b-
carotene in the oil phase. A coarse emulsion was prepared by ho-
mogenizing 10% (w/w) corn oil with 90% (w/w) aqueous emulsifier
solution using a high-speed blender for 2 min (M133/1281-0, Bio-
spec Products, Inc., ESGC, Switzerland) with the speed of
10,000 rpm. The droplet size in this coarse emulsion was further
reduced by passing it through a microfluidizer (Microfluidizer,
M110Y, Microfluidics, Newton, MA) with a 75 mm interaction
chamber (F20Y) at an operational pressure of 12,000 psi for 3
passes. The resulting b-carotene-loaded emulsions were stored in a
refrigerator at 4  C before use.
2.3. Encapsulation of b-carotene in alginate beads
Aqueous solutions containing different amounts of alginate
were prepared by dissolving powdered alginic acid (1% or 2%, w/w)
in distilled water and stirring continuously at 60  C for an hour, and
then reducing the temperature to 35  C. Alginate solutions and b-
carotene-loaded emulsions were then mixed together (1:1, mass
ratio) for 2 h with continuous stirring to form a mixture that con-
tained 5% oil (w/w) and either 0.5% or 1% alginate (w/w). The b-
carotene-loaded hydrogel beads were prepared using a commercial
encapsulation unit (Encapsulator B-390, BUCHI, Switzerland) with
a 120 mm vibrating nozzle to inject the b-carotene/alginate solution
into 10% calcium chloride solution. The encapsulation device was
operated under fixed conditions: frequency 800 Hz; electrode
800 V; and pressure 450 mbar. The hydrogel beads were held in the
Ca2þ solution for 1 h at ambient temperature to promote cross-
linking. The beads were then collected by filtration and subse-
quently washed with distilled water and phosphate buffer to
remove any excess ions from their surfaces. The formed beads were
then storage in a refrigerator so that any residual external water
was removed, and then their total weight was determined.
2.4. Measurement of b-carotene stability
The physicochemical stability of b-carotene encapsulated in the
 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10
nanoemulsions and in the filled hydrogel particles was tested: 4 mL
of nanoemulsions (5% oil) or 4 mL filled hydrogel beads (5% oil)
were transferred into glass tubes and stored in the dark at 55  C for
12 days (pH 7). The b-carotene was isolated from the samples using
a solvent extraction method: each sample was extracted by chlo-
roform and alcohol (volume fraction 1:1) solution at least twice. For
the filled hydrogel beads, saturated ethylenediaminetetraacetic
acid (EDTA) solution was used to dissolve the beads and release the
encapsulated b-carotene. The transparent lower organic phase
containing the b-carotene was collected and transferred to a
cuvette, and then its absorbance was measured at 450 nm using a
UVevisible spectrometer. A pure chloroform and alcohol solution
was used as a blank. A nanoemulsion containing no added b-
carotene was also analyzed as a control. The b-carotene content was
determined using a standard curve created from solutions with
varying amounts of known b-carotene. All measurements were
repeated three times.
2.5. Gastrointestinal tract model
The potential gastrointestinal fate of the delivery systems was
monitored using a simulated gastrointestinal tract model based on
previously reported standardized in vitro methods (McClements &
Li, 2010; Minekus et al., 2014). b-carotene delivery systems (free
emulsion and filled hydrogel beads) with the same total lipid
concentration (2%) and total volume (7.5 mL) were prepared by
diluting the original systems with buffer solution (5 mM phosphate
buffer, pH 7). The samples were then passed through a static GIT
model that simulated the mouth, stomach, and small intestine
phases. This method has been described in detail in our previous
study (Zhang, Zhang, Zhang, Decker, & McClements, 2015a), and so
is only briefly summarized below.
Initial System: 7.5 mL of the initial samples were placed into a
glass beaker in an incubator shaker at a rotation speed of 100 rpm
for 15 min at 37  C for preheating (Innova Incubator Shaker, Model
4080, New Brunswick Scientific, Edison, NJ). Three different initial
b-carotene-loaded delivery systems were tested: (i) lipid droplets;
(ii) filled alginate beads (0.5% alginate); and, (iii) filled alginate
beads (1% alginate).
Mouth phase: 7.5 mL of simulated saliva fluid (SSF) containing
0.03 g/mL mucin was preheated to 37  C and then mixed with the
initial samples. After being adjusted to pH 6.8, the mixture was
incubated in an incubator shaker at a rotation speed of 100 rpm for
10 min at 37  C to mimic agitation in the mouth.
Stomach phase: 15 mL of the “bolus” sample resulting from the
mouth phase was mixed with 15 mL of simulated gastric fluid
containing 0.0032 g/mL pepsin preheated to 37  C and then the pH
was adjusted to 2.5. This mixture was incubated in the incubator
shaker for 2 h at 37  C to mimic stomach conditions.
Small intestine phase: 30 mL of “chyme” sample from the
stomach phase was placed into a 150 mL glass beaker that was
placed into a water bath at 37  C and then adjusted to pH 7.00.
1.5 mL of simulated intestinal fluid was added to the reaction vessel,
followed by 3.5 mL of bile salt solution with constant stirring. The
pH of the reaction system was adjusted back to 7.00. Then, 2.5 mL of
lipase solution was added to the sample and an automatic titration
unit (Metrohm, USA Inc.) was used to monitor the pH and maintain
it at pH 7.0 by titrating 0.25 N NaOH solution into the reaction
vessel for 2 h at 37  C. The amount of free fatty acids released was
calculated from the titration curves as described previously (Jones
& McClements, 2010).
2.6. Determination of particle characterization
The particle size distribution of the samples after passing each
3
digestion process was measured using a static light scattering de-
vice (Mastersizer 2000, Malvern Instruments Ltd., Malvern, Wor-
cestershire, UK). Initial, mouth, and small intestine samples were
diluted with phosphate buffer (5 mM, pH 7.0) and stomach samples
were diluted with acidified water (pH 2.5) to avoid multiple scat-
tering effects. The refractive indices of the oil and water phases
used in the calculations were 1.472 and 1.333, respectively. The
average particle sizes are reported as volume-weighted mean
diameter (d43) for all the samples.
2.7. z-potential measurements
The electrical charge (z-potential) of the samples after passing
each digestion process was measured using a particle electropho-
resis instrument (Zetasizer Nano ZA series, Malvern Instruments
Ltd. Worcestershire, UK). Initial, mouth, and small intestine sam-
ples were diluted with phosphate buffer (5 mM, pH 7.0) and
stomach samples were diluted with acidified water (pH 2.5) prior to
analysis to keep the instrument attenuation value between 5 and
10.
2.8. Microstructure analysis
The microstructure of samples after passing through each stage
of the GIT model was studied using optical microscopy. The
microstructure of the all the samples was examined using confocal
scanning laser microscopy with a 20  objective lens (Nikon D-
Eclipse C1 80i, Nikon, Melville, NY, U.S.). For the confocal micro-
scopy, each sample was dyed prior to confocal microscopy obser-
vation. The corn oil was dyed with Nile Red solution (1 mg/mL
ethanol) by adding 0.1 mL of Nile Red dye solution to 2 mL of
sample and storage at 5  C overnight. The excitation and emission
spectrum for Nile Red were 543 nm and 605 nm, respectively. The
microstructure images for confocal microscopy were taken and
analyzed using image analysis software (NIS-Elements, Nikon,
Melville, NY).
2.9. Determination of b-carotene concentration, bioaccessibility,
and stability
After the samples had passed through the simulated GIT, 20 g of
digesta from the small intestine phase was collected and centri-
fuged (18,000 rpm, Thermo Scientific, Waltham, MA) at 4  C for
30 min. The b-carotene in the digesta and micelle phase was
extracted based on the methods described in Section 2.4. EDTA
solution was used to dissociate the hydrogel beads and release the
encapsulated b-carotene prior to analysis. The clear supernatant
after extraction was collected and assumed to be the “micelle
fraction” in which the b-carotene was solubilized. Aliquots of 2 mL
of micelle fraction were mixed with 2 mL of chloroform and 2 mL of
alcohol, vortexed several times, and centrifuged at 3500 rpm for
5 min at ambient temperature. The collected chloroform layers
were diluted to an appropriate level, and then analyzed using a UV-
visible spectrophotometer at 450 nm. The concentration of b-
carotene was calculated from the absorbance using a standard
curve.
The bioaccessibility of b-carotene (i.e., the fraction in the small
intestine that was solubilized in the mixed micelle phase) was
calculated from this data: B* ¼ 100  CMicelle/CDigesta, where CMicelle
and CDigesta are the b-carotene concentrations in the micelle faction
and in the total digesta collected after the small intestine phase,
respectively. The stability of b-carotene (i.e., the fraction remaining
in a non-transformed state in the small intestine) was calculated:
S* ¼ 100  CDigesta/CInitial, where CInitial is the b-carotene concen-
tration in the small intestine phase calculated based on the initial
4 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10

amount added. The effective bioavailability (i.e., the fraction of Table 1

ingested b-carotene present in the mixed micelle phase) was also The mean particle diameter of three b-carotene delivery systems (nanoemulsions
and filled alginate beads) measured after each digestion stage.
calculated: BA ¼ B*  S* ¼ 100  CMicelle/CInitial.
Mean Particle diameter (mm)

2.10. Statistical analysis Initial Mouth Stomach Intestine

Nanoemulsion 0.24 ± 0.01 4.59 ± 0.77 73.4 ± 11 10.1 ± 1.73

All measurements were made on at least two freshly prepared 0.5% Alginate beads 285 ± 5 470 ± 28 364 ± 18 405 ± 9
1% Alginate beads 661 ± 26 880 ± 34 718 ± 23 837 ± 32
samples and each sample was measured in duplicate. The results
are reported as averages and standard deviations, and the differ-
ences among treatments were calculated based on an analysis of
variance (ANOVA) and a post-hoc Duncan test with a confidence Kispert, 2003; Krinsky, 1998). Consequently, the impact of encap-
level of 95%. These analyses were carried out using statistical sulating b-carotene-loaded lipid droplets in filled hydrogel beads
analysis software (SPSS, IBM Corporation, Armonk, NY, USA). on the chemical stability of b-carotene was examined. The decrease
in b-carotene concentration during storage at 55  C compared to
the initial value (C/C0) was measured for the different delivery
3. Results and discussion
systems (Fig. 2). The relative b-carotene content fell from an initial
value of 100% immediately after preparation to around 0.2%, 38%
3.1. Filled hydrogel beads formation and b-carotene stability
and 55% for the nanoemulsions, 1% beads and 0.5% beads respec-
tively, after storage for 12 days. These results indicated that the b-
Filled hydrogel beads were formed by injection of a mixture of
carotene in the nanoemulsions was highly unstable to degradation,
nanoemulsion and alginate solution into a Ca2þ solution using an
and that encapsulation of the lipid droplets in the hydrogel beads
extrusion device with a vibrating nozzle. Two different alginate
could significantly improve its stability. Encapsulation of the lipid
concentrations (0.5% and 1%) were chosen to fabricate hydrogel
droplets may have improved b-carotene stability by providing a
beads with different properties. Beads formed using different
physical barrier that limited the diffusion of pro-oxidants or free
alginate concentrations had different sizes and appearances (Fig. 1
radicals into the hydrogel bead core where they could interact with
and Table 1). The 0.5% alginate beads had mean diameters around
the carotenoids. Alternatively, encapsulation may have improved b-
285 mm and tended to cream to the top of the test tubes, whereas
carotene stability because the alginate in the hydrogel beads was
the 1% alginate beads had diameters around 660 mm and tended to
able to bind and inactivate pro-oxidants, e.g., anionic alginate
sediment to the bottom of the test tubes. These results indicate that
molecules may have bound cationic transition metals (such as Fe2þ
both the physical properties and dimensions of the beads depend
or Fe3þ). Surprisingly, the b-carotene appeared to be slightly more
on initial alginate concentration. The smaller size of the 0.5% algi-
stable when it was encapsulated in 0.5% alginate beads than in 1%
nate beads may have been because this alginate solution had a
alginate beads. One might have expected that beads that were
lower viscosity, and therefore bead formation was facilitated. The
larger and had smaller pores (1% alginate) would provide better
fact that the 0.5% alginate beads moved upwards suggests that the
protection because they should be capable of restricting molecular
reduction in bead density caused by the oil droplets (low density)
diffusion more effectively, and of binding more transition methods.
was greater than the increase in density caused by the alginate
The physicochemical origin of this phenomenon is currently un-
molecules (high density) (Matalanis & McClements, 2013).
known, and certainly warrants further study.
Conversely, the 1% alginate beads moved downwards because the
The visual appearance of the various b-carotene delivery sys-
increase in bead density caused by the high level of alginate mol-
tems before and after storage was also measured (Fig. 3). Initially,
ecules was greater than the decrease in density caused by the oil
all the delivery systems had a strong yellow (in the web version)
droplets.
b-carotene is known to be highly sensitive to oxidation under
certain conditions because of its high degree of unsaturation (Gao &

100 Nanoemulsion
1% alginate bead
0.5% alginate bead
80
C/C0 (%)

60

40

20

0
0 2 4 6 8 10 12
Storage Time (days)
Fig. 1. Appearance of filled hydrogel beads (b-carotene-loaded nanoemulsions trapped
in alginate beads) after a few hours storage: 1% alginate beads (left) and 0.5% alginate Fig. 2. Degradation rates of b-carotene loaded in different delivery systems (nano-
beads (right). emulsions and hydrogel beads) during storage at 55  C.
 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10 5

color, which was attributed to selective absorption of light by the b-

carotene molecules. However, after storage there was a pronounced
decrease in the intensity of the yellow color, which was evidence of
color fading due to b-carotene degradation. Overall, these results
suggest encapsulation of b-carotene-loaded lipid droplets within
alginate beads may improve their chemical stability during storage.

3.2. Influence of simulated GIT conditions on particle properties

The encapsulation of nutraceuticals within hydrogel beads may

impact their gastrointestinal fate, and so it is important to establish
how they behave under gastrointestinal conditions. The charge,
size, and microstructure of the nanoemulsions and filled hydrogel
beads was therefore measured as they passed through the various
stages of a simulated GIT to provide information about their po-
tential gastrointestinal fate.

3.2.1. Particle charge

The z-potentials of the initial nanoemulsions, 0.5% alginate
beads, and 1% alginate beads were 47.6, 9.0, and 9.5 mV,
respectively (Fig. 4). The strong negative charge on the lipid drop-
lets in the nanoemulsions can be attributed to the presence of a
layer of adsorbed whey protein molecules. The pH of the initial
solution (pH 7) was appreciably higher than the isoelectric point of
the whey proteins (pI z 5), which therefore accounts for their high
negative charge. The alginate beads had a moderate negative
charge, which can be attributed to the presence of anionic car-
boxylic (eCOO) groups from the alginate molecules. Presumably,
these anionic carboxylic acid groups were located at the surface of
the hydrogel beads and had not formed cross-links with the
cationic calcium ions during bead formation. The alginate con-
centration in the beads had little impact on the magnitude of the
electric charge. These results are in accordance with another recent
study of the electrical properties of alginate beads (Zeeb, Saberi,
Weiss, & McClements, 2015).
After being subjected to simulated mouth conditions, there was
only a slight decrease in the magnitude of the negative charge on
the nanoemulsion droplets, while there was only a slight increase
in the negative charge on the alginate beads (Fig. 4). The reason that
there was not an appreciable change in the electrical charge of the
particles can be attributed to the fact that the pH of the simulated
saliva was similar to that of the initial samples. Nevertheless, there
would still have been some changes in particle charge due to
electrostatic screening by mineral ions present within the simu-
lated saliva, or due to interactions of the mucin molecules with the
surfaces of the particles, which may account for the observed dif-
ferences in z-potential.
There was a pronounced decrease in the magnitude of the
Fig. 4. The effective electrical potential of different delivery systems as they pass
including free nanoemulsion, nanoemulsion in 0.5% alginate beads, and nanoemulsion
in 1% alginate beads after each digestion stage.
negative charge of all the samples after being exposed to the
stomach phase (Fig. 4). These changes were probably a result of the
low pH and high ionic strength of the simulated gastric fluids,
which would result in an alteration in the ionization of certain
charge groups and to electrostatic screening. Interestingly, the
droplets in the nanoemulsions had a slight negative charge under
simulated gastric conditions when they might have been expected
to have an appreciable positive charge because the pH was well
below their isoelectric point. This effect probably occurred because
anionic mucin molecules from the simulated saliva adsorbed to the
surfaces of the cationic lipid droplets (Zhang, Zhang, Zhang, Decker,
& McClements, 2015b). The relatively low negative charge on the
alginate beads in the gastric fluids can be attributed to protonation
of the anionic carboxylic (eCOO 0 COOH, pKa 3.5) on the bead
surfaces in the acidic gastrointestinal fluids.
After exposure to the small intestine phase, all the samples had
relatively high negative charges (around 23 mV). Previous studies
have attributed this effect to the presence of various types of
anionic colloidal particles in the mixture resulting from lipid
digestion, including micelles, vesicles, calcium salts, undigested
lipids and proteins (Zhang et al., 2015b). These anionic species may
have come from the original emulsions (e.g., peptides or free fatty

Fig. 3. The appearance of different b-carotene delivery systems (nanoemulsions and filled hydrogel beads) before and after storage at storage at 55  C for 12 days.
6 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10

acids) or from the gastrointestinal fluids (e.g., bile salts and
phospholipids).
3.2.2. Particle size and microstructure
b-carotene-loaded nanoemulsions: The particle size distribution
of the lipid droplets in the initial nanoemulsions had a single peak
around 0.15 mm diameter (Table 1, Fig. 5). In addition, the lipid
droplets in the nanoemulsions were evenly distributed throughout
the system as shown in the confocal microscopy images (Fig. 6).
These measurements suggest that the WPI-coated lipid droplets
were initially relatively small and stable to aggregation. The good
aggregation stability of the droplets can be attributed to the strong
electrostatic repulsion between the highly charged droplets. After
passing through the mouth phase, the particle size distributions of
these emulsions became bimodal, which led to an increase in the
mean particle diameter (d32 z 0.29 mm). The confocal microscopy
images indicated that extensive droplet flocculation occurred in the
mouth phase, which can be attributed to bridging or depletion
flocculation induced by the presence of mucin in the simulated
saliva, as reported in previous studies (Ritzoulis et al., 2012; Sarkar,
Goh, & Singh, 2009; Vingerhoeds, Blijdenstein, Zoet, & van Aken,
2005). Bridging flocculation occurs due to the binding of mucin
molecules to the surfaces of two or more lipid droplets, whereas
depletion flocculation occurs due to the increase in the osmotic
attraction between the lipid droplets generated by non-adsorbed
mucin molecules (Zhang et al., 2015b). After exposure to the
stomach phase, these emulsions showed an appreciable increase in
mean particle diameter (d32 z 33 mm), and extensive droplet

Fig. 5. Particle size distributions of b-carotene delivery systems after exposure to different regions of the GIT model: (a) nanoemulsions; (b) nanoemulsions in 0.5% alginate beads;
and, (c) nanoemulsions in 1% alginate beads. It should be noted that the volume fraction data has been incremented by different amounts so that the curves can be more easily
compared on the same figure.
 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10 7

Chen, Tong, & McClements, 2015e). The shrinking of the beads

Fig. 6. Microstructures of b-carotene delivery systems after exposure to different re-
gions of the GIT model: (a) nanoemulsions; (b) nanoemulsions in 0.5% alginate beads;
and, (c) nanoemulsions in 1% alginate beads. (Scale bar is 20 mm).
under gastric conditions may help stabilize encapsulated compo-
nents, since then the pore size of the hydrogel beads should
decrease, which may restrict molecular diffusion processes.
After exposure to small intestine conditions, there was a
decrease in the fluorescence intensity of the interior of hydrogel
beads (Fig. 6), which is indicative of a reduction in the amount of
lipids present inside them. Presumably, the lipid droplets had been
digested by lipase molecules that had diffused through the
hydrogel matrix, resulting in the formation of free fatty acids that
diffused out of the hydrogel beads and into the surrounding
aqueous phase. Indeed, there was evidence of large irregular sha-
ped lipid-rich particles outside of the alginate beads after digestion,
which may have been mixed micelles formed from free fatty acids,
monoacylglycerols, and bile salts. In summary, these results suggest
that the alginate beads were not able to completely inhibit the
digestion of the encapsulated lipid droplets.
3.3. Influence of delivery system type on lipid digestion

aggregation could be observed in the confocal microscopy images The effect of the different delivery systems on the rate and
(Fig. 6). It has been reported that protein-stabilized emulsions are extent of lipid digestion was evaluated using an automatic titration
prone to aggregation under gastric conditions because of hydrolysis (pH stat) method. The fraction of free fatty acids (FFAs) released
of adsorbed proteins, weakening of electrostatic repulsion, and from the samples after addition of lipase was calculated from
depletion or bridging flocculation induced by mucin (Kenmogne- measurements of the volume of alkaline solution required to keep
Domguia, Meynier, Viau, Llamas, & Genot, 2012; Shani-Levi, Levi- the pH constant at 7.0. The nanoemulsions were rapidly digested
Tal, & Lesmes, 2013; Zhang et al., 2015a). After passing through the during the first 5 min with over 76% of the FFAs being released,
small intestinal stage, the nanoemulsion samples still contained followed by a more gradual digestion until a relatively constant
relatively large particles (d32 z 7 mm) as detected by both light final value (96%) was attained (Fig. 7). The rapid digestion of the
scattering and confocal microscopy methods. It is difficult to nanoemulsions can be attributed to the large surface area associ-
identify the nature of these particles from light scattering experi- ated with small droplet sizes (Golding & Wooster, 2010).
ments, but previous studies suggest they are a mixture of undi- Conversely, the FFA release rate was much slower for the filled
gested lipid droplets, undigested protein aggregates, micelles, hydrogel beads and depended on alginate concentration. After
vesicles, and insoluble calcium salts. 5 min of digestion, around 22% and 37% of the FFAs were released
Filled hydrogel beads. The particle size and microstructure of the for the 1% and 0.5% alginate beads, respectively, and around 56%
filled hydrogel beads depended on the alginate concentration used and 83% of the FFAs were released after 120 min of digestion (see
in their formation. The filled hydrogel beads fabricated using 1% Fig. 7).
alginate were larger than those fabricated using 0.5% alginate These results indicate that encapsulation of the lipid droplets
(Table 1, Fig. 6). This difference in bead dimensions may be a result
of the differences in the viscosities of the alginate solutions. The
0.5% alginate solution has a lower viscosity than the 1% alginate
solution and therefore flows through the nozzle of the injector
more rapidly, leading to the formation of smaller droplets.
For both types of alginate beads, the general shape of the par-
ticle size distribution (Fig. 5) and the overall microstructure (Fig. 6)
of the beads remained fairly similar throughout the entire simu-
lated gastrointestinal tract, which suggests that they maintained
their overall integrity when exposed to upper GIT conditions.
Nevertheless, there were some changes in the dimensions of the
alginate beads in different regions of the GIT. The size of the beads
increased when they moved from the initial phase to the mouth
phase (Table 1), suggesting that they became swollen. This swelling
may have occurred due to a reduction in the electrostatic attraction
between oppositely charged components within the hydrogel
beads (such as calcium ions and alginate) or due to an increase in
the electrostatic repulsion between similarly charged components
(such as alginate molecules). On the other hand, the size of the
beads decreased when they moved from the mouth to the stomach,
which suggests that they shrank somewhat. This shrinkage may be
the result of a decrease in the electrostatic repulsion between
alginate molecules due to protonation of the carboxyl groups at low
pH. Previous studies have also reported that calcium alginate beads
tend to swell in high pH solutions, but shrink in low pH solutions Fig. 7. Amount of fatty acids released from b-carotene delivery systems measured
brard, Hoffart, Cardot, Subirade, & Beyssac, 2013; Zhang, Zhang, using a pH-stat in the small intestine phase of the GIT model: (a) nanoemulsions; (b)
(He
nanoemulsions in 0.5% alginate beads; and, (c) nanoemulsions in 1% alginate beads.
8 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10
within the hydrogel beads reduced the rate and extent of lipid
digestion within the simulated small intestine phase. The observed
decrease in digestion rate for the encapsulated lipid droplets may
have occurred for a number of reasons. Firstly, lipid digestion can
only happen once lipase has adsorbed to the surfaces of the lipid
droplets and come into close contact to the underlying lipid mol-
ecules (Reis, Holmberg, Watzke, Leser, & Miller, 2009). For the
nanoemulsion, lipase molecules can quickly move from the
aqueous phase to the free lipid droplet surfaces and promote lipid
digestion. Conversely, for the filled hydrogel beads, the lipase must
first penetrate through the hydrogel beads before it can reach the
lipid droplet surfaces. The penetration of small molecules into the
center of hydrogel beads tends to occur more rapidly for beads with
larger pores or smaller diameters (Li et al., 2011). One would
therefore expect the lipase molecules to move through the 1%
alginate beads more slowly because they are bigger and would be
expected to contain smaller pore sizes. Secondly, long chain FFAs
generated by the lipid digestion process must be removed from the
droplet surfaces otherwise lipolysis will be retarded (Li et al., 2011).
FFAs are normally removed from the droplet surfaces by being
incorporated into mixed micelles with bile salts and phospholipids,
or by forming insoluble salts with calcium ions (Devraj et al., 2013).
In the case of hydrogel beads, the removal of FFAs from the lipid
droplet surfaces may be inhibited because bile salts, phospholipids,
and calcium ions have to penetrate into the hydrogel beads.
Furthermore, the mixed micelles and calcium salts formed may
have to diffuse out of the beads, which would also be inhibited by
the hydrogel beads.
3.4. Influence of delivery system type on b-carotene bioaccessibility
As mentioned earlier, b-carotene typically has a low oral
bioavailability because of its poor chemical stability and low solu-
bility in gastrointestinal fluids. We therefore measured the impact
of the different delivery systems on the stability and bio-
accessibility of the encapsulated b-carotene. The bioaccessibility
(B*) of the encapsulated b-carotene was appreciably higher for the
nanoemulsions than for the hydrogel beads (Fig. 8). The higher B*
for the nanoemulsions can be attributed to the fact that a greater
Fig. 8. Bioaccessibility (B*) and effective bioavailability (BA) of b-carotene in different
delivery systems measured after exposure to simulated small intestine conditions: (a)
nanoemulsions; (b) nanoemulsions in 0.5% alginate beads; and, (c) nanoemulsions in
1% alginate beads.
fraction of the lipid phase was digested, thereby leading to the
formation of more mixed micelles capable of solubilizing the b-
carotene molecules. Previous studies in our laboratory have also
reported that nanoemulsions can increase the bioaccessibility of
lipophilic bioactive agents through this mechanism (Zhang et al.,
2015d, 2015c). The relatively low B* of the b-carotene encapsu-
lated in the filled hydrogel beads can be attributed to several rea-
sons. Firstly, some of the b-carotene molecules remained within the
undigested lipid droplets trapped inside the hydrogel beads. Sec-
ondly, there were fewer free fatty acids and monoacylglycerols
available to form mixed micelles, which reduced the solubilization
capacity of the intestinal phase for the b-carotene molecules.
The chemical stability (S*) of the b-carotene was also calculated
to determine the extent of any carotenoid degradation within the
simulated GIT (Fig. 9). In general, the b-carotene remained rela-
tively stable to degradation with over 87% of the initial carotenoid
remaining at the end of the small intestine phase. The extent of b-
carotene degradation was slightly higher when it was encapsulated
in nanoemulsions than when it was encapsulated in filled hydrogel
beads, i.e., less remained after the small intestine phase. b-carotene
is known to be unstable to acid-degradation (Mortensen &
Skibsted, 2000), and therefore the slight loss of b-carotene
observed in our study is most likely to have occurred within the
highly acidic conditions of the gastric phase. The reason that the b-
carotene was less stable to degradation in the nanoemulsions may
have been because more of it was at the surface of the lipid droplets
where it was exposed to the aqueous phase. Despite the fact that
the hydrogel beads could protect the b-carotene from degradation,
the magnitude of this effect was not large enough to overcome the
reduction in b-carotene bioaccessibility that was observed in
hydrogel beads. Consequently, the effective bioavailability (BA ¼
B*S*) of the b-carotene was still lower for the hydrogel beads than
for the nanoemulsions.
4. Conclusions
Filled hydrogel beads were fabricated by injecting a mixture of
b-carotene-loaded lipid droplets and alginate molecules into a
calcium solution. Storage studies indicated that the hydrogel beads
partially protected the b-carotene from chemical degradation, with
the extent of the effect depending on the alginate level within the
Fig. 9. Chemical stability (%) of b-carotene in different delivery systems measured after
exposure to simulated small intestine conditions: (a) nanoemulsions; (b) nano-
emulsions in 0.5% alginate beads; and, (c) nanoemulsions in 1% alginate beads.
 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10
beads. Simulated gastrointestinal studies indicated that free lipid
droplets were digested more rapidly and completely than those
encapsulated within hydrogel beads. The rate and extent of lipid
digestion was lower when the hydrogel beads contained a higher
alginate concentration, which was attributed to an increase in bead
diameter and decrease in bead pore size. The bioaccessibility of b-
carotene was higher when it was encapsulated within free lipid
droplets than within hydrogel beads. This effect was attributed to
the fact that some of the b-carotene remained trapped within un-
digested lipid droplets inside the hydrogel beads, and that fewer
mixed micelles were generated to solubilize any b-carotene mole-
cules released from the beads. Thus, this kind of hydrogel bead may
be useful for inhibiting the chemical degradation of b-carotene
during food storage or within the gastrointestinal tract, but one
must be aware that the bioaccessibility of b-carotene may be
compromised. Nevertheless, it should be noted that in reality filled
hydrogel beads would continue to move along the gastrointestinal
tract, and would eventually reach the colon where they would be
broken down by digestive enzymes produced by colonic bacteria.
Consequently, alginate beads may be a useful means of delivering
lipophilic bioactives to the large intestine.
Acknowledgements
This material was partly based upon work supported by USDA
(2013-03795), NRI Grants (2014-67021).
References
van den Berg, H., Faulks, R., Granado, H. F., Hirschberg, J., Olmedilla, B.,
Sandmann, G., et al. (2000). The potential for the improvement of carotenoid
levels in foods and the likely systemic effects. Journal of the Science of Food and
Agriculture, 80(7), 880e912.
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), 2993e2998.
Cazzonelli, C. I. (2011). Carotenoids in nature: insights from plants and beyond.
Functional Plant Biology, 38(11), 833e847.
Chen, L. Y., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials
as nutraceutical delivery systems. Trends in Food Science & Technology, 17(5),
272e283.
DemmigAdams, B., Gilmore, A. M., & Adams, W. W. (1996). Carotenoids .3. In vivo
functions of carotenoids in higher plants. Faseb Journal, 10(4), 403e412.
Devraj, R., Williams, H. D., Warren, D. B., Mullertz, A., Porter, C. J. H., & Pouton, C. W.
(2013). In vitro digestion testing of lipid-based delivery systems: calcium ions
combine with fatty acids liberated from triglyceride rich lipid solutions to form
soaps and reduce the solubilization capacity of colloidal digestion products.
International Journal of Pharmaceutics, 441(1), 323e333.
Donhowe, E. G., & Kong, F. B. (2014). Beta-carotene: digestion, microencapsulation,
and in vitro bioavailability. Food and Bioprocess Technology, 7(2), 338e354.
Donsi, F., Sessa, M., Mediouni, H., Mgaidi, A., & Ferrari, G. (2011). Encapsulation of
bioactive compounds in nanoemulsion-based delivery systems. In G. Saravacos,
P. Taoukis, M. Krokida, V. Karathanos, H. Lazarides, N. Stoforos, et al. (Eds.), 11th
international congress on engineering and food (Vol. 1, pp. 1666e1671).
Fernandez-Garcia, E., Carvajal-Lerida, I., Jaren-Galan, M., Garrido-Fernandez, J.,
Perez-Galvez, A., & Hornero-Mendez, D. (2012). Carotenoids bioavailability from
foods: from plant pigments to efficient biological activities. Food Research In-
ternational, 46(2), 438e450.
Gao, Y., & Kispert, L. D. (2003). Reaction of carotenoids and ferric chloride: equi-
libria, isomerization, and products. The Journal of Physical Chemistry B, 107(22),
5333e5338.
Giri, T. K., Thakur, D., Alexander, A., Ajazuddin, Badwaik, H., & Tripathi, D. K. (2012).
Alginate based hydrogel as a potential biopolymeric carrier for drug delivery
and cell delivery systems: present status and applications. Current Drug De-
livery, 9(6), 539e555.
Golding, M., & Wooster, T. J. (2010). The influence of emulsion structure and stability
on lipid digestion. Current Opinion in Colloid & Interface Science, 15(1e2),
90e101.
Gombotz, W. R., & Wee, S. F. (2012). Protein release from alginate matrices.
Advanced Drug Delivery Reviews, 64, 194e205.
Hebrard, G., Hoffart, V., Cardot, J.-M., Subirade, M., & Beyssac, E. (2013). Develop-
ment and characterization of coated-microparticles based on whey protein/
alginate using the encapsulator device. Drug development and Industrial Phar-
macy, 39(1), 128e137.
Jones, O. G., & McClements, D. J. (2010). Functional biopolymer particles: design,
fabrication, and applications. Comprehensive Reviews in Food Science and Food
9
Safety, 9(4), 374e397.
Joye, I. J., & McClements, D. J. (2014). Biopolymer-based nanoparticles and micro-
particles: fabrication, characterization, and application. Current Opinion in
Colloid & Interface Science, 19(5), 417e427.
Kenmogne-Domguia, H. B., Meynier, A., Viau, M., Llamas, G., & Genot, C. (2012).
Gastric conditions control both the evolution of the organization of protein-
stabilized emulsions and the kinetic of lipolysis during in vitro digestion.
Food & Function, 3(12), 1302e1309.
Krinsky, N. I. (1998). The antioxidant and biological properties of the carotenoidsa.
Annals of the New York Academy of Sciences, 854(1), 443e447.
Lee, K. Y., & Mooney, D. J. (2012). Alginate: properties and biomedical applications.
Progress in Polymer Science, 37(1), 106e126.
Li, Y., Hu, M., Du, Y., Xiao, H., & McClements, D. J. (2011). Control of lipase di-
gestibility of emulsified lipids by encapsulation within calcium alginate beads.
Food Hydrocolloids, 25(1), 122e130.
Lopes, C. M., Martins-Lopes, P., & Souto, E. B. (2010). Nanoparticulate carriers (NPC)
for oral pharmaceutics and nutraceutics. Pharmazie, 65(2), 75e82.
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), 737e744.
Maiani, G., Caston, M. J. P., Catasta, G., Toti, E., Cambrodon, I. G., Bysted, A., et al.
(2009). Carotenoids: actual knowledge on food sources, intakes, stability and
bioavailability and their protective role in humans. Molecular Nutrition & Food
Research, 53, S194eS218.
Marze, S. (2015). Bioaccessibility of lipophilic micro-constituents from a lipid
emulsion. Food & Function, 6(10), 3218e3227.
Matalanis, A., & McClements, D. J. (2013). Hydrogel microspheres for encapsulation
of lipophilic components: optimization of fabrication & performance. Food
Hydrocolloids, 31(1), 15e25.
McClements, D. J. (2012). Advances in fabrication of emulsions with enhanced
functionality using structural design principles. Current Opinion in Colloid &
Interface Science, 17(5), 235e245.
McClements, D. J. (2014). Nanoparticle- and microparticle-based delivery systems:
Encapsulation, protection and release of active components. Boca Raton, FL: CRC
Press.
McClements, D. J., Decker, E. A., & Weiss, J. (2007). Emulsion-based delivery systems
for lipophilioc bioactive components. Journal of Food Science, 72(8), R109eR124.
McClements, D. J., & Li, Y. (2010). Review of in vitro digestion models for rapid
screening of emulsion-based systems. Food & Function, 1(1), 32e59.
Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., et al. (2014).
A standardised static in vitro digestion method suitable for food e an inter-
national consensus. Food & Function, 5(6), 1113e1124.
Mortensen, A., & Skibsted, L. H. (2000). Kinetics and mechanism of the primary
steps of degradation of carotenoids by acid in homogeneous solution. Journal of
Agricultural and Food Chemistry, 48(2), 279e286.
Namitha, K. K., & Negi, P. S. (2010). Chemistry and biotechnology of carotenoids.
Critical Reviews in Food Science and Nutrition, 50(8), 728e760.
Patel, A. R., & Velikov, K. P. (2011). Colloidal delivery systems in foods: a general
comparison with oral drug delivery. Lwt-Food Science and Technology, 44(9),
1958e1964.
Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2012a). Inhibition of b-carotene
degradation in oil-in-water nanoemulsions: influence of oil-soluble and water-
soluble antioxidants. Food Chemistry, 135(3), 1036e1043.
Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2012b). Nanoemulsion delivery
systems: influence of carrier oil on b-carotene bioaccessibility. Food Chemistry,
135(3), 1440e1447.
Rao, A. V., & Rao, L. G. (2007). Carotenoids and human health. Pharmacological
Research, 55(3), 207e216.
Reis, P., Holmberg, K., Watzke, H., Leser, M. E., & Miller, R. (2009). Lipases at in-
terfaces: a review. Advances in Colloid and Interface Science, 147, 237e250.
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 emul-
sions in colon carcinoma cells in vitro. Journal of Agricultural and Food Chemistry,
54(25), 9366e9369.
Ritzoulis, C., Siasios, S., Melikidou, K. D., Koukiotis, C., Vasiliadou, C., & Lolakos, S.
(2012). Interactions between pig gastric mucin and sodium caseinate in solu-
tions and in emulsions. Food Hydrocolloids, 29(2), 382e388.
Salvia-Trujillo, L., Qian, C., Martín-Belloso, O., & McClements, D. J. (2013). Modu-
lating b-carotene bioaccessibility by controlling oil composition and concen-
tration in edible nanoemulsions. Food Chemistry, 139(1), 878e884.
Sarkar, A., Goh, K. K. T., & Singh, H. (2009). Colloidal stability and interactions of
milk-protein-stabilized emulsions in an artificial saliva. Food Hydrocolloids,
23(5), 1270e1278.
Shani-Levi, C., Levi-Tal, S., & Lesmes, U. (2013). Comparative performance of milk
proteins and their emulsions under dynamic in vitro adult and infant gastric
digestion. Food Hydrocolloids, 32(2), 349e357.
Shewan, H. M., & Stokes, J. R. (2013). Review of techniques to manufacture micro-
hydrogel particles for the food industry and their applications. Journal of Food
Engineering, 119(4), 781e792.
Silva, H. D., Cerqueira, M. A., Souza, B. W. S., Ribeiro, C., Avides, M. C.,
Quintas, M. A. C., et al. (2011). Nanoemulsions of b-carotene using a high-energy
emulsificationeevaporation technique. Journal of Food Engineering, 102(2),
130e135.
Sy, C., Dangles, O., Borel, P., & Caris-Veyrat, C. (2015). Stability of bacterial carot-
enoids in the presence of iron in a model of the gastric compartment e
10 Z. Zhang et al. / Food Hydrocolloids 61 (2016) 1e10
comparison with dietary reference carotenoids. Archives of Biochemistry and
Biophysics, 572, 89e100.
Velikov, K. P., & Pelan, E. (2008). Colloidal delivery systems for micronutrients and
nutraceuticals. Soft Matter, 4(10), 1964e1980.
Vingerhoeds, M. H., Blijdenstein, T. B. J., Zoet, F. D., & van Aken, G. A. (2005).
Emulsion flocculation induced by saliva and mucin. Food Hydrocolloids, 19(5),
915e922.
Yi, J., Li, Y., Zhong, F., & Yokoyama, W. (2014). The physicochemical stability and
in vitro bioaccessibility of beta-carotene in oil-in-water sodium caseinate
emulsions. Food Hydrocolloids, 35, 19e27.
Zeeb, B., Saberi, A. H., Weiss, J., & McClements, D. J. (2015). Retention and release of
oil-in-water emulsions from filled hydrogel beads composed of calcium algi-
nate: impact of emulsifier type and pH. Soft Matter, 11(11), 2228e2236.
Zhang, Z., Zhang, R., Chen, L., Tong, Q., & McClements, D. J. (2015e). Designing
hydrogel particles for controlled or targeted release of lipophilic bioactive
agents in the gastrointestinal tract. European Polymer Journal, 72, 698e716.
Zhang, R., Zhang, Z., Zhang, H., Decker, E. A., & McClements, D. J. (2015a). Influence
of emulsifier type on gastrointestinal fate of oil-in-water emulsions containing
anionic dietary fiber (pectin). Food Hydrocolloids, 45, 175e185.
Zhang, R., Zhang, Z., Zhang, H., Decker, E. A., & McClements, D. J. (2015b). Influence
of lipid type on gastrointestinal fate of oil-in-water emulsions: in vitro diges-
tion study. Food Research International, 75, 71e78.
Zhang, R., Zhang, Z., Zou, L., Xiao, H., Zhang, G., Decker, E. A., et al. (2015c).
Enhancing nutraceutical bioavailability from raw and cooked vegetables using
excipient emulsions: influence of lipid type on carotenoid bioaccessibility from
carrots. Journal of Agricultural and Food Chemistry, 63(48), 10508e10517.
Zhang, R., Zhang, Z., Zou, L., Xiao, H., Zhang, G., Decker, E. A., et al. (2015d). Impact of
lipid content on the ability of excipient emulsions to increase carotenoid bio-
accessibility from natural sources (raw and cooked carrots). Food Biophysics,
1e10.