Food Hydrocolloids 139 (2023) 108499

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

Effect of internal and external gelation on the physical properties, water

distribution, and lycopene encapsulation properties of alginate-based
emulsion gels
Jingxiang Shu a, David Julian McClements b, Shunjing Luo a, Jiangping Ye a, *, Chengmei Liu a
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China
b
Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts Amherst, Amherst, MA, 01003, USA

A R T I C L E I N F O A B S T R A C T

Keywords: It is challenging to incorporate lycopene, a strongly hydrophobic nutraceutical, into functional foods because of
Emulsion gels its low water-solubility and poor bioaccessibility. In this study, we therefore examined the potential of alginate-
Lycopene based emulsion gels to encapsulate and deliver lycopene. Alginate can be gelled using either internal or external
Gelation mechanism
gelation mechanisms, but few studies have compared the advantages and disadvantages of these two approaches
Digestion
Bioaccessibility
for producing emulsion gels. For this reason, we compared the structure, water distribution, water mobility, and
simulated digestion of lycopene-loaded emulsion gels formed by gelling alginate within the aqueous phase using
either the internal or external methods. The internal gelation method produced emulsion gels that contained
smaller oil droplets, and had a higher hardness, water holding capacity, and freeze-thaw stability. Moreover, the
internal gelation method led to emulsion gels that had a slightly higher lycopene bioaccessibility (36.1%) than
the ones formed by the external gelation method (33.4%). These findings are important for optimizing the design
and fabrication of emulsion gels suitable as delivery systems for nutraceuticals in the food industry.

1. Introduction Several kinds of encapsulation technologies have been developed to

Lycopene is a natural pigment and antioxidant because it contains
multiple conjugated double bonds (Przybylska, 2019). It can scavenge
reactive oxygen species and inhibit the oxidation of lipids, proteins, and
DNA (Jaswir, Noviendri, Hasrini, & Octavianti, 2011). Lycopene can not
be synthesized by the human body and so it must be obtained through
food consumption. In the human gastrointestinal tract (GIT), lycopene is
usually released from the fatty regions in foods when the lipids are
digested, and then, it is solubilized in mixed micelles formed from bile
salts, phospholipids, and lipid digestion products (Liang, Ma, Yan, Liu, &
Liu, 2019). In foods, lycopene is easily isomerized and oxidized under
the influence of oxygen, light, metal ions, and temperature, which could
reduce its bioactivity (Santos, Rubio, da Silva, Pinho, &
Favaro-Trindade, 2021). Moreover, lycopene had limited bio­
accessibility because of its strong hydrophobicity and low intestinal
permeability, which also limit its application as a nutraceutical in food
systems (Desmarchelier & Borel, 2017). Consequently, there is consid­
erable interest in improving the dispersibility, stability, and bio­
accessibility of lycopene.
enhance the performance of lycopene in foods, including emulsification
technique (Liang et al., 2021; Zhao et al., 2020), nanostructured lipid
carriers (Singh, Neupane, Panda, & Kohli, 2017), microencapsulation by
complex coacervation (Neagu et al., 2020), and emulsion gels (Liu et al.,
2022). Oil-in-water type emulsion gels are viscoelastic colloidal mate­
rials containing oil droplets trapped inside a gelled aqueous phase (Lin,
Kelly, & Miao, 2020). Typically, the aqueous phase is gelled using
food-grade biopolymers such as proteins or polysaccharides (Dragan &
Dinu, 2019).
In this study, we used sodium alginate as a gelling agent, because it is
already widely used to form emulsion gels (Chen et al., 2021; Lin, Kelly,
Maidannyk, & Miao, 2020). The anionic polysaccharide chains of algi­
nate can be crosslinked by adding multivalent cationic calcium ions.
These calcium alginate gels can be formed using two different mecha­
nisms: internal and external gelation (Cao, Lu, Mata, Nishinari, & Fang,
2020). The internal gelation mechanism involves dispersing a
non-soluble form of calcium into an aqueous alginate solution (such as
EDTA-Ca or CaCO3), and then adding a weak acid or lactone to slowly
release the calcium ions into the aqueous phase to promote gelation

* Corresponding author.
E-mail address: jpye@ncu.edu.cn (J. Ye).

https://doi.org/10.1016/j.foodhyd.2023.108499
Received 23 September 2022; Received in revised form 9 January 2023; Accepted 19 January 2023
Available online 20 January 2023
0268-005X/© 2023 Elsevier Ltd. All rights reserved.
J. Shu et al.
(Ahmed, El-Rasoul, Auda, & Ibrahim, 2013). Typically, this mechanism
leads to relatively slow gelation and the formation of a more homoge­
neous gel network (Cao et al., 2020; Puguan, Yu, & Kim, 2014). In
contrast, the external gelation mechanism involves adding a soluble
form of calcium into an aqueous alginate solution (such as CaCl2), which
results in rapid gelation and the formation of a less homogeneous gel
network (Lin, Kelly, & Miao, 2022). Due to their different mechanisms of
action, bulk emulsion gels prepared using external or internal gelation
methods may have different structures and functional properties.
A recent study showed that the external gelation mechanism could be
used to form emulsion gel beads suitable for loading probiotics and
phytochemicals (Šipailienė, Šlimaitė, Jeznienė, Venskutonis, &
Leskauskaitė, 2021). This mechanism has also been used to form
lycopene-loaded emulsion gel beads (Lin, Kelly, Maidannyk, & Miao,
2021). The internal gel mechanism has been used to encapsulate
lutein-loaded nanoparticles in alginate hydrogels (Xu et al., 2021). This
mechanism has also been used to create emulsion gels that were used as
fat replacers in meat products (Pintado, Munoz-Gonzalez, Salvador,
Ruiz-Capillas, & Herrero, 2021).
In the current study, we compare the advantages and disadvantages
of the internal and external gelation mechanisms for creating bulk
emulsion gels suitable for encapsulating an important hydrophobic nu­
traceutical. External gelation was achieved by adding soluble calcium
(CaCl2) to oil-in-water emulsions containing alginate in the aqueous
phase, whereas internal gelation was performed by adding insoluble
calcium (CaCO3) and D-(+)-gluconic acid δ-lactone to these emulsions.
The structure and physicochemical properties of the resulting emulsion
gels were then characterized. Moreover, the impact of gelation mecha­
nism on the gastrointestinal fate and bioaccessibility of lycopene-loaded
emulsion gels was investigated using a simulated gastrointestinal tract.
The results of this study should facilitate the design and development of
emulsion gels that can encapsulate, protect, and release hydrophobic
nutraceuticals in functional foods.
2. Materials and methods
2.1. Materials
Corn oil was bought from a local supermarket. Whey protein isolate
(WPI, 90% purity) was purchased from Milk Specialties Global (Norfolk,
NE, USA). Lycopene and Nilo blue A were purchased from Shanghai
Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Sodium alginate
(SA) and D-(+)-gluconic acid δ-lactone (GDL) were purchased from
Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Calcium
carbonate (CaCO3), calcium chloride dehydrate (CaCl2⋅2H2O), and Nile
red were purchased from the Shanghai Aladdin Biochemical Technology
Co., Ltd. (Shanghai, China). Lipase from porcine pancreas, pancreatin
from porcine pancreas, and pepsin from porcine gastric mucosa were
obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents
used were analytical grade.
2.2. Preparation of emulsion gels
The emulsions gels were prepared according to a previous study with
slight modifications (Liu et al., 2022). A WPI stock solution (5.0% w/w)
and SA stock solution (0.6% w/w) were prepared by dispersing whey
protein isolate powder and sodium alginate powder into double-distilled
water, followed by constant stirring at 1000 rpm at room temperature
for 2 h. After that, the WPI stock solution was combined with the SA
solution at a mass ratio of 1:1. Corn oil was then added and sheared at
13,000 rpm for 3 min using a high-shear mixer (ULTRA TURRAX® T25,
IKA, Germany) to obtain an oil-in-water (O/W) emulsion. The final
system consisted of 2 wt% WPI, 0.24 wt% SA, and 20 wt% corn oil.
External emulsion gels: CaCl2 solution and O/W emulsion were mixed
together at a volume ratio of 1:1, and the resulting system was stored in a
refrigerator at 4 ◦ C for 24 h CaCl2 solutions with different concentrations
2
Food Hydrocolloids 139 (2023) 108499
were used to create emulsion gels with final Ca2+ levels of 5, 15, and 25
mM.
Internal emulsion gels: A freshly prepared GDL solution was added to
an O/W emulsion at a volume ratio of 1:1. In this case, however, the
emulsion contained CaCO3 particles at a CaCO3-to-GDL molar ratio of
1:2. After mixing, the resulting system was placed at 4 ◦ C for 24 h to
form a gel. The final Ca2+ concentrations in these internal emulsion gels
was the same as that in the external emulsion gels.
2.3. Textural analysis
A TA-XT-Plus texture analyzer (Stable Micro Systems Ltd, Surrey,
UK) with a P/36R cylindrical probe was used to assess the textural
properties of the external and internal emulsion gels, as described in a
previous study with slight modifications (Gao et al., 2022). Texture
profile analysis (TPA) was used to provide information about gel prop­
erties, with force and height calibration performed before measurement.
The following parameters were used for the measurements: trigger
force, 5.0 g; pre-test speed, 1.0 mm/s; test speed, 0.5 mm/s; and post-test
speed 0.5 mm/s. The hardness, adhesiveness, gumminess, chewiness,
and cohesiveness of the test samples were obtained from the resulting
force-distance profiles after two compression-decompression cycles.
2.4. Rheological characterization of emulsion gels
The emulsion gels were drained with filter paper and then sheared at
1000 rpm using a stirrer for 10 min, as described in a previous study
with slight modifications (Soukoulis, Cambier, Hoffmann, & Bohn,
2016). The rheological tests were performed on a stress-controlled
rheometer (MCR 302, Anton Paar, Australia). A sample (about 1.5 g)
was placed in the center of a parallel plate with a gap distance of 1 mm
and a measuring temperature of 25 ◦ C, and then allowed to stand for 5
min. The sample’s edge was coated with a layer of silicone oil to prevent
moisture from evaporation during the measurements. The apparent
shear viscosity was measured as the shear rate was increased from 0.1 to
100 s− 1 at 25 ◦ C.
2.5. Water holding capacity (WHC)
The WHC of each sample was determined by centrifugation ac­
cording to a modified version of a method described in the literature (Lin
et al., 2021). About 2.5 g of emulsion gels were weighed into a 10 mL
centrifuge tube 24 h after sample preparation. The sample was then
centrifuged at 10,000 rpm for 20 min and any excess moisture was
removed. The WHC was then calculated using Equation (1):
nR0 − (R0 − R1 )
WHC(%) = × 100 (1)
nR0
Where, R0 is the weight of the initial gel (g), R1 is the weight of the gel
after centrifugation (g), and n is the mass fraction of water in the
emulsions.
2.6. Freeze-thaw stability
The freeze-thaw stability of the samples was determined by placing
them in a centrifuge tube that was then frozen and thawed several times
(Zhang, Chen, Zhao, Wan, & Prakash, 2022). Briefly, 1.5 g of emulsion
gel was placed in a centrifuge tube and then frozen at − 20 ◦ C for 22 h
and then thawed in a 25 ◦ C water bath for 2.5 h. The freeze-thaw cycle
was repeated up to 5 cycles. After centrifuging at 1000 rpm for 5 min,
the mass of precipitated water in the emulsion gels was determined after
the freeze-thaw process. The percent of syneresis was then calculated
using Equation (2):
m
Syneresis(%) = × 100 (2)
M
J. Shu et al.
Where, m is the weight of the precipitate water and M is the original
weight of the gel.
2.7. Low field nuclear magnetic resonance (LF-NMR)
The relaxation time T2 of the samples was measured by LF-NMR
using a previously described method (Li et al., 2018). An emulsion gel
(about 2 g) was placed in a glass bottle (20 mm in diameter), which was
then inserted into a LF-NMR glass tube (25 mm in diameter). These
samples were then analyzed using the Carr–Purcell–Meiboom–Gill
(CPMG) sequence using a low-field pulsed NMR analyzer (Niumag Co.,
Ltd., Shanghai, China). The echo time (TE), wait time (TW), echo points
(NECH), and the number of scans (NS) were set to 0.4 ms, 5000 ms,
16000, and 8 scans, respectively. Finally, inversion software was used to
compute the T2 values and the corresponding peak areas.
2.8. Confocal laser scanning microscopy (CLSM)
CLSM (Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Ger­
many) was used to examine the microstructure of the emulsion gels.
Prior to analysis, the oil and water phases were stained with Nile red
(excitation wavelength 488 nm) and Nilo blue A (excitation wavelength
638 nm), respectively.
2.9. Encapsulation of lycopene in emulsion gels
Lycopene (0.03%wt) was added to corn oil and heated in an oil bath
at 120 ◦ C for 10 min to dissolve the carotenoid. The internal and external
emulsion gels were then prepared separately using the methods
described in section 2.2.
2.10. Simulated gastrointestinal study of lycopene-loaded emulsion gels
2.10.1. In-vitro digestion model
A simulated gastrointestinal tract (GIT) was used to monitor the in
vitro digestion of the emulsion gels, which included mouth, gastric, and
small intestine stages. Simulated Salivary Fluid (SSF) (NaCl 1.594 mg/
mL, NH4NO3 0.328 mg/mL, KH2PO4 0.636 mg/mL, KCl 0.202 mg/mL,
potassium citrate 0.308 mg/mL, sodium dihydrogen urate 0.021 mg/
mL, urea 0.198 mg/mL, lactic acid sodium salt 0.146 mg/mL, and dis­
solved porcine mucin 3 mg/mL), Simulated Gastric Fluid (SGF) (NaCl 2
mg/mL, HCl 7 mL/L, and pepsin 3.2 mg/mL), and Simulated Intestinal
Fluid (SIF) (CaCl2⋅H2O 36.7 mg/mL, NaCl 218.7 mg/mL, lipase 24 mg/
mL, trypsin 24 mg/mL, and bile salt 54 mg/mL) prepared in the present
study according to the instructions described previously (Minekus et al.,
2014).
Mouth stage: After 5 min of preheating at 37 ◦ C, 7.5 g of sample and
7.5 mL of SSF were added to a glass container followed by quickly
adjusting the pH to 6.8 using 1 mol/L HCl. The mixtures were then
incubated for 10 min in a shaking water bath (100 r/min) at 37 ◦ C.
Gastric stage: The sample collected at the end of the mouth stage was
mixed with 15 mL of SGF and 1 mol/L NaOH was used to adjust the
mixture to pH 2.5. The resulting mixtures were then incubated for 2 h in
a shaking water bath (100 r/min) at 37 ◦ C.
Small intestine stage: The sample collected at the end of the stomach
phase was mixed with 1.5 mL SIF, 3.5 mL bile salt solution, 2.5 mL lipase
solution, and 2.5 mL pancreatic enzyme solution (all dissolved in 5 mM
phosphate buffer solution, pH 7.0), which were added sequentially.
Then resulting mixtures were the incubated for 2 h in a shaking water
bath (100 r/min) at 37 ◦ C. During this time, the pH of the samples was
constantly adjusted to maintain a constant pH of 7.0.
2.10.2. Microstructure
The microstructure of the samples before and after 2 h of intestinal
digestion were analyzed by fluorescence microscopy. The samples were
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Food Hydrocolloids 139 (2023) 108499
dripped onto glass slides, stained with 10 μL Nile red (0.1% w/w, dis­
solved in DMSO), and then observed using an epifluorescence micro­
scope with a 40 × magnification lens (ECLIPSE Ni–U, Nikon Inc., Tokyo,
Japan) and a filter cube (excitation 465–495 nm, dichroic mirror 505 nm
long pass, emission 512–558 nm).
2.10.3. Lycopene release and retention
Following centrifugation, lycopene was extracted by adding hexane
and ethanol (2:1 v/v) to 1 mL of intestinal digestive juices and the
middle micelle phase was collected after centrifuging the digestion
juice. The upper organic phase was membrane filtered (0.22 μL millipore
nylon filter) and then analyzed by HPLC (Agilent 1260 series, Santa
Clara, CA, USA) using a C18 column (5 μm, 250 mm × 4.6 mm; Waters,
Milford, MA, USA), detection at 472 nm, and a 20 μL injection volume.
The HPLC mobile phase was 75% acetonitrile and 25% ethyl acetate.
Equations (3) and (4) was used to calculate the bioaccessibility and
retention of the lycopene using a method described previously (Lin et al.,
2022; Yan et al., 2021):
Bioaccessibility (%) =
CMicelle
× 100 (3)
CDigestion
CDigesta
Retention (%) = × 100 (4)
COriginal
Where, CMicelle , CDigesta , CDriginal and COriginal are the lycopene concentra­
tions (g/mL) in the micelle fraction, digesta, digestion solution, and
initial emulsion gel, respectively.
2.11. Statistical analysis
All experimental data were expressed as means ± standard de­
viations, which were calculated using SPSS software (version 26) for
one-way ANOVA analysis. A Duncan’s test was used to determine sig­
nificant differences (p < 0.05) between means.
3. Results and discussion
3.1. Appearance and microstructure of emulsion gels
Initially, the impact of internal or external gelation mechanisms on
the overall appearance of the emulsion gels was recorded (Fig. 1a). The
internal gelation method produced emulsion gels that maintained their
shape more easily than the ones formed using the external gelation
method. Moreover, the internal emulsion gels had better formability,
smoother surfaces, and were less prone to collapse. These effects were
mainly attributed to the much more rapid and localized gelation for the
external emulsion gels. When the soluble calcium chloride solution first
comes into contact with the alginate solution, there is a high local
concentration of Ca2+ ions that promotes rapid gelation of the anionic
alginate molecules in the immediate vicinity through salt bridging ef­
fects. The smoother surfaces and better shape-holding properties of the
internal emulsion gels was mainly attributed to the more gradual release
of the Ca2+ ions when the calcium carbonate was slowly acidified. As a
result, there was more even gelation throughout the alginate solution.
The results of the CLSM analysis showed that the external emulsion
gels containing 5 mM calcium (E− 5mM) had larger droplets (Fig. 1b).
The size of the oil droplets decreased significantly with increasing Ca2+
concentration and the gels had a more compact structure (Fig. 1d).
These results suggest that low levels of calcium promoted droplet coa­
lescence, which may be because the oil droplets were brought closer
together because of electrostatic screening and bridging effects.
Conversely, high levels of calcium inhibited droplet coalescence because
the higher gelation rate resulting in the viscous aqueous phase of gels
that the oil droplets could not easily move around and contact each
other (Gao et al., 2022). The confocal microscopy images confirmed that
J. Shu et al. Food Hydrocolloids 139 (2023) 108499

Fig. 1. CLSM of emulsion gels prepared by internal/external gelation methods (a column, macroscopic images; b column, lipids stained with Nile red were green; c
column, protein stained with Nilo blue A were red; d column, images from columns a and b combined). The external (E) and internal (I) gels with calcium ion
concentrations of 5 mM, 15 mM and 25 mM named as E− 5mM, E− 15mM, E− 25mM, I-5mM, I-15mM, and I-25mM, respectively.

there was a more even distribution of the oil droplets in the internal
emulsion gels than the external ones (Fig. 1b). Indeed, the images of the
external emulsion gels contained several large black regions, which
correspond to regions that did not contain oil or protein. Previous
studies suggest that a more even distribution of droplets and bio­
polymers should lead to emulsion gels with better physical properties (Li
et al., 2022). These differences in microstructure may therefore account
for the smoother surfaces and firmer structure of the internal emulsion
gels (Fig. 1c).
3.2. Texture profile analysis (TPA)
The probe used in these experiments had a larger surface area than
the emulsion gels to provide information about the textural character­
istics of the entire gels. The hardness of the emulsion gels was greatest
for the samples containing 25 mM Ca2+, being around 214 g and 165 g
for the I-25mM and E− 25mM samples, respectively. At the same calcium
concentration, the gels prepared by the internal gelation method were
significantly harder than those prepared using the external gelation
method (p < 0.05), and the adhesiveness, gumminess, and chewiness
followed the same trend (Table 1). These changes were due to that
calcium ions were evenly distributed in the emulsion with and therefore
formed a denser network structure, while the external gels formed an
uneven surface resulting in a weaker network structure because of the
faster release of calcium ions. Cohesiveness is the ability of the gel to
maintain the original network structure during two compression-
decompression cycles, which reflects the ability of the gel to recover
its overall structure. The cohesiveness of the internal emulsion gels was
higher than that of the external emulsion gels, but it decreased as the
Ca2+ concentration increased (Table 1). These results suggest that these
gels become more plastic when higher levels of Ca2+ were added,
making them more fragile and less able to regain their previous struc­
ture. Adhesiveness depends on the surface characteristics of a material,
as it is related to the ability of the material to stick to the surfaces of the

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J. Shu et al. Food Hydrocolloids 139 (2023) 108499

Table 1
Textural properties of emulsion gels with different gelation methods and Ca2+ concentration.
Sample Hardness(g) Adhesiveness (g/s) Gumminess(g) Chewiness(g) Cohesiveness
e d e e
E− 5mM 116.4 ± 3.3 13.2 ± 0.9 84.7 ± 1.8 78.0 ± 1.5 0.728 ± 0.005c
E− 15mM 163.1 ± 7.5c 40.3 ± 1.1b 118.0 ± 5.0c 102.5 ± 4.2c 0.724 ± 0.006cd
E− 25mM 164.9 ± 6.0c 23.4 ± 3.2c 117.5 ± 4.5c 94.2 ± 2.2d 0.713 ± 0.007d
I-5mM 131.2 ± 8.6d 15.9 ± 2.6d 100.5 ± 5.8d 92.7 ± 4.8d 0.767 ± 0.012a
I-15mM 193.0 ± 7.5b 45.1 ± 2.9b 144.8 ± 5.0b 130.6 ± 4.6b 0.738 ± 0.009b
I-25mM 214.2 ± 8.7a 60.4 ± 4.9a 158.1 ± 7.0a 140.4 ± 6.1a 0.737 ± 0.019bc

Note: Different superscript letters in each data column indicate significant differences according to LSD and Duncan test (p < 0.05). The external (E) and internal (I)
gels with calcium ion concentrations of 5 mM, 15 mM and 25 mM named as E− 5mM, E− 15mM, E− 25mM, I-5mM, I-15mM, and I-25mM, respectively.

test probe when it is raised (Huang, Kennedy, Li, Xu, & Xie, 2007). The internal emulsion gels at 25 mM Ca2+. The adhesiveness of the external
adhesiveness of the external and internal emulsion gels increased with emulsion gels containing 25 mM Ca2+ was lower than the ones con­
the increasing of calcium ion concentration with exception of the taining 15 mM. This may be caused by the rapid gelation action on the

Fig. 2. G’ (solid symbols) and G’’ (open symbols) of emulsion gels prepared by internal/external gelation methods with different Ca2+ concentrations as a function of
frequency (A). Apparent viscosity of emulsion gels as a function of shear rates (raged from 0.01 s− 1–100 s− 1) (B). The external (E) and internal (I) gels with calcium
ion concentrations of 5 mM, 15 mM and 25 mM named as E− 5mM, E− 15mM, E− 25mM, I-5mM, I-15mM, and I-25mM, respectively.

5
J. Shu et al. Food Hydrocolloids 139 (2023) 108499

surface, resulting in a rough and uneven gel surface when a higher 3.4. Water holding capacity, freeze-thaw stability, and T2 relaxation time
concentration of CaCl2 was added. While the calcium ions in the internal of bulk emulsion gels
gel could be better diffused and not accumulated on the surface
(Ramdhan, Ching, Prakash, & Bhandari, 2019). The water holding capacity of a gel depends on its ability to hold and
retain free water. Water molecules may bind to the surfaces of
biopolymer molecules or they may be trapped within the 3D biopolymer
3.3. Rheological analysis
network structure by holes and capillaries (Babaei, Khodaiyan,
Mohammadian, & Sheikhi, 2021). The WHC of the internal emulsion
The storage modulus (G′ ) and loss modulus (G′′ ) correspond to the
gels was greater than that of the external ones (Fig. 3A). This effect can
elastic and viscous rheological properties of the emulsion gels, respec­
again be attributed to the move even gelation of the alginate molecules
tively. The solidity of the samples was demonstrated by the fact that G’
when calcium ions are slowly released. As a result, a 3D alginate
> G′′ over the frequency range from 0.1 to 10 Hz. The G′ and G′′ values
network is formed that contains smaller pores that bind water more
increased with increasing frequency for all samples, indicating they had
strongly through capillary forces. However, as the Ca2+ concentration
similar frequency dependences. These values increased significantly
increased, the WHC of both the internal and external emulsion gels
over the frequency range from 1 to 10 Hz for the emulsion gels con­
gradually decreased. Thus, the WHC of the emulsion gels containing 5
taining 5 mM Ca2+. This effect was probably because the degree of
mM Ca2+ was significantly higher than those containing 15 or 25 mM
crosslinking in these gels was less than that in the gels formed at higher
(Fig. 3A).
calcium levels, and so they were more affected by frequency changes
The impact of freezing and thawing on the water holding properties
(Fig. 2A). Increasing the Ca2+ concentration from 15 to 25 mM only
of the emulsion gels was also assessed by measuring their degree of
increased the shear modulus slightly. This was probably because most of
syneresis. The internal emulsion gels exhibited less syneresis after
the anionic binding sites on the alginate molecules were already bound
freezing-thawing than the external ones, indicating that they had a
to cationic calcium ions (Cao et al., 2020). Increasing, the calcium
stronger freeze-thaw stability (Fig. 3B). There was an increase in syn­
concentration above this value may weaken the gels because there is less
eresis in the emulsion gels as the calcium ion concentration increased,
bridging – instead of two carboxyl groups being linked by a single cal­
which suggested that the presence of high levels of Ca2+ ions reduced
cium ion, each carboxyl group binds its own calcium ion, thereby
their freeze-thaw stability. It could be also observed that the syneresis
inhibiting bridge formation. The internal emulsion gels had higher G′
values of all emulsion gels increased with increasing the number of
and G′′ values than the external ones. As discussed earlier, this may have
freeze-thaw cycles. The increase of syneresis was ascribed to the ice
been due to a more uniform gelation throughout the alginate solution. In
crystals formed in the emulsion gels after each freeze-thaw cycle,
contrast, in the external emulsion gels there were some regions that were
thereby disrupting the gel network structure (Liang et al., 2020).
highly crosslinked and others that had little crosslinking. The apparent
The three main reasons for the observed decrease in WHC and freeze-
viscosity of the samples was measured by the static rheological test, and
thaw stability within increasing Ca2+ are as follows. First, when Ca2+
it was clear that the viscosity was increased with the increase of calcium
ions bind to alginate molecules and crosslink them, there are fewer
ions concentration, and the viscosity of I-5mM is about twice than
exposed hydrophilic groups for the water molecules to interact with,
E− 5mM at the shear rate of 0.1 s− 1 (Fig. 2B). Moreover, the viscosity of
thereby increasing the amount of free water in the gel network (Babaei
all samples decreased as the shear rate increased, which could be
et al., 2021; Kuhn, Cavallieri, & da Cunha, 2011). Second, the brittleness
explained by shear thinning behavior, which might be due to the in­
of the emulsion gels increased with increasing Ca2+ concentration. As a
crease of shear rate causing droplet aggregation in the shear process
result, they were more likely to collapse during centrifugation and
(Wang et al., 2022).

Fig. 3. Water holding capacity of emulsion gels,

different superscript letters among bars denote sig­
nificant difference (p < 0.05) (A). Freeze-thaw sta­
bility of emulsion gels, different capital letters
indicate a significant difference (p < 0.05) among the
same freeze-thaw cycles of the different emulsion
gels, and different lowercase letters indicate a sig­
nificant difference (p < 0.05) among the same sam­
ples with the number of freeze-thaw cycles (B). The
curves of relaxation time T2 of emulsion gels (C). The
water ratio of emulsion gels (D). The external (E) and
internal (I) gels with calcium ion concentrations of 5
mM, 15 mM and 25 mM named as E− 5mM,
E− 15mM, E− 25mM, I-5mM, I-15mM, and I-25mM,
respectively.

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J. Shu et al. Food Hydrocolloids 139 (2023) 108499

freezing-thawing, thereby releasing more water (Mao, Tang, & Swanson, calcium, which may have been because there was some droplet coales­
2001). Third, extensive protein aggregation may occur in the presence of cence at low calcium levels, as discussed earlier (Lin, Kelly, Maidannyk,
high Ca2+ concentrations, which reduces the number of hydrophilic & Miao, 2020). After digestion, the size of the lipid-rich regions
groups exposed for water to bind to, as well as reducing the number of decreased, which is consistent with hydrolysis of the fat droplets by
pores that can hold water through capillary forces (Pan, Guo, Li, Song, & lipase in the small intestine. These results suggest that the fat droplets
Ren, 2017; Wang, Luo, Liu, Adhikari, & Chen, 2019). were released from the alginate gels and then exposed to lipase, or that
Low-field NMR analysis was used to provide more insights into the the lipase diffused into the alginate gels and then hydrolyzed the fat
distribution of the water molecules within the emulsion gels. Relaxation droplets. Alginate gels are known to swell under gastrointestinal con­
time (T2) measurements provide information about the different mo­ ditions due to exchange of Ca2+ ions for Na + ions, as well as due to
bilities of the water molecules in a gel: the higher T2, the more mobile increases in the electrostatic repulsion between anionic carboxyl groups
the water. In general, the water in hydrogels can be divided into three on the alginate molecules under neutral pH conditions (Rayment et al.,
populations based on their relaxation times: T21, T22, and T23, corre­ 2009). Moreover, some of the proteins in the emulsion gels would have
sponding to bound, immobilized, and free water, respectively. The been digested by gastric and pancreatic proteases.
corresponding P21, P22, and P23 values represent the percentages of The impact of gelation mechanism and calcium concentration on the
water molecules in each of these three populations. retention and bioaccessibility of lycopene encapsulated in the emulsion
According to our results, increasing the Ca2+ concentration reduced gels was also measured (Fig. 5). In general, the retention of the lycopene
P21 and increased P23, suggesting there was less immobilized water and increased with increasing calcium concentration, which may have been
more free water present (Fig. 3D, Table 2). This result is therefore because calcium alginate gels formed at higher crosslinking concentra­
consistent with the explanation given earlier to account for the changes tions were more resistant to disruption under gastrointestinal condi­
in water holding properties. High calcium levels promote greater ag­ tions. At low Ca2+ concentrations, the retention of the carotenoids was
gregation of the biopolymers, thereby reducing the number of hydro­ greater in the internal emulsion gels than in the external ones (Fig. 5a),
philic sites available for water to bind to. Moreover, there may be more which may have been because they had a more uniform gel network that
larger pores formed within the 3D biopolymer gel network, thereby was more resistant to disruption and that inhibited the diffusion of
increasing the amount of free water within the pores (Ramdhan et al., digestive enzymes more effectively. Conversely, at high Ca2+ concen­
2019). trations, there was little difference between the two types of emulsion
gels.
The impact of calcium concentration on the bioaccessibility of the
3.5. Gastrointestinal fate of lycopene-loaded emulsion gels lycopene depended on the gelation mechanism used to form the emul­
sion gels. For the external emulsion gels, the bioaccessibility decreased
Emulsion gels are often used to encapsulate and deliver hydrophobic significantly with increasing calcium concentration, but for the internal
nutraceuticals. For this application, it is important that the nutraceut­ ones there was only a small (but non-signficiant) decrease. At higher
icals are released and solubilized under gastrointestinal conditions. For calcium concentrations, the external emulsion gels had a lower lycopene
this reason, we used a simulated gastrointestinal tract (GIT) to monitor bioaccessibility than the internal ones.
the digestive behavior of emulsion gels containing different calcium The observed differences in bioaccessibility can be attributed to
levels and gelled by different mechanisms. differences in the release and solubilization of the hydrophobic carot­
The microstructures of the emulsion gels were measured before and enoids under gastrointestinal conditions (McClements, Decker, & Park,
after exposure to the simulated GIT (Fig. 4). Before digestion, individual 2008). The fat droplets must be digested by lipase so that the lycopene
fat droplets could be observed in all of the samples. These droplets can be released and then solubilized in mixed micelles formed from bile
appeared to be somewhat larger for the samples containing 5 mM acids, monoacylglycerols, and free fatty acids (Park, Mun, & Kim, 2018).
For the external emulsion gels, high calcium concentrations led to the
Table 2 formation of a heterogeneous gel network containing clumps of fat
T2 relaxation time and water ratio of emulsion gels with different gelation droplets trapped inside a dense alginate gel. It is possible that these
methods and Ca2+ concentration. clumps did not breakdown easily within the gastrointestinal environ­
Sample T21 T22 (ms) T23 (ms) P21 (%) P22 (%) P23 (%) ment, or that the lipase molecules could not easily diffuse through them
(ms) and reach the fat droplets, thereby inhibiting lipid digestion and lyco­
E− 5mM 59.32 634.05 – 14.60 85.40 – pene release. This effect was less important for the internal emulsion gels
± 4.65b ± 11.24a ± 0.80b ± 0.80a because they had a more uniform gel network. Moreover, the internal
E− 15mM 21.54 231.01 1873.82 4.00 ± 70.40 25.60 emulsion gels contained smaller fat droplets than the external ones,
± 0.00bc ± 0.00b 0.57c ± 0.00d ± 0.57a
which should lead to faster and more complete lipid digestion, thereby
±
0.00cd
E− 25mM 25.27 210.95 1873.82 3.80 ± 72.20 24.00 increasing the release and solubilization of lycopene (Park et al., 2018).
± 3.99c ± ± 0.00b 0.44c ± ± 3.56a
17.37cd 3.97cd 4. Conclusions
I-5mM 75.65 613.59 3274.55 17.80 81.60 0.60 ±
± 0.00a ± 0.00a ± 0.00a ± 3.39a ± 0.00b
3.39ab In this study, we compared the microstructure, texture, water dis­
I-15mM 18.92 231.01 1629.75 1.35 ± 76.95 21.70 tribution, and water mobility of bulk emulsion gels prepared using in­
± ± 0.00bc ± 0.00c 1.63c ± ± 0.85a ternal and external gelation mechanisms. Moreover, we examined the in
3.71cd 0.78bc vitro digestion of lycopene-loaded emulsion gels using a simulated
I-25mM 17.82 192.20 1417.47 2.37 ± 73.10 24.53
± 1.33d ± 15.11d ± 0.00d 0.25c ± ± 1.72a
gastrointestinal model. The gels formed using the external gelation
1.51cd mechanism had rougher surfaces, were less moldable, were softer, and
had worse water holding properties than those formed using the internal
Note: Different superscript letters in each data column indicate significant dif­
gelation mechanism. These effects were attributed to the fact that the
ferences according to LSD and Duncan test (p < 0.05). The external gels with
calcium ion concentrations of 5 mM, 15 mM and 25 mM named as E− 5mM, external gelation mechanism involved mixing a soluble form of calcium
E− 15mM, and E− 25mM, and the internal gels corresponding named as I-5mM, with an alginate solution, which led to uneven gelation throughout the
I-15mM, and I-25mM, respectively. T21, T22, and T23 represented the bound system. There were some regions where gelation was strong and other
water, immobilized water and free water, and their corresponding percentages regions where it was weak. In contrast, for the internal gelation mech­
were know as P21, P22, and P23, respectively. anism the calcium ions were slowly released into the alginate solution,

7
J. Shu et al. Food Hydrocolloids 139 (2023) 108499

Fig. 4. Fluorescence microscopy images of initial and small intestine digest juice of emulsion gels prepared by external (a) and internal (b) gelation methods with the
green color of the oil phase stained with Nile Red. The external (E) and internal (I) gels with calcium ion concentrations of 5 mM, 15 mM and 25 mM named as
E− 5mM, E− 15mM, E− 25mM, I-5mM, I-15mM, and I-25mM, respectively.

Fig. 5. Lycopene retention (A) and bioaccessibility (B) of emulsion gels prepared by internal/external gelation methods with Ca2+ concentrations of 5 mM, 15 mM,
and 25 mM.

which led to a much more uniform gel network throughout the system.
There were also some differences in the behavior of the emulsion gels
under simulated gastrointestinal conditions depending on the gelation
mechanism used. The retention and bioaccessibility of lycopene was
higher in the internal emulsion gels than the external ones. This effect
was attributed to faster digestion and release of the lycopene from the
emulsion gels when they had a more even network structure. Overall,
this study has important implications for the selection of suitable
methods for the preparation of emulsion gels for the encapsulation,
protection, and release of hydrophobic bioactive substances, like
carotenoids.
Author statement
Jingxiang Shu: Writing - Original Draft, Experiment. David Julian
McClements: Review & Editing Shunjing Luo: Conceptualization,
Investigation. Jiangping Ye: Review & Editing, Investigation, Project
administration Chengmei Liu: Conceptualization, Funding acquisition.
Declaration of competing interest
The authors state that they have no conflicts of interest related to this
study.

8
J. Shu et al.
Data availability
Data will be made available on request.
Acknowledgments
This study was funded by the fund of National Natural Science
Foundation of China (32101996, 32072257), Science and Technology
Major Project of Jiangxi Province (20203ABC28W015), Central Gov­
ernment Guide Local Special Fund Project for Scientific and Techno­
logical Development of Jiangxi Province (20221ZDD02001).
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