Food Chemistry 372 (2022) 131354

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Development and characterization of medium and high internal phase

novel multiple Pickering emulsions stabilized by hordein nanoparticles
Sareh Boostani a, b, Masoud Riazi c, Ali Marefati b, Marilyn Rayner b, Seyed Mohammad
Hashem Hosseini a, *
a
Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran
b
Department of Food Technology, Engineering and Nutrition, Faculty of Engineering, Lund University, Lund, Sweden
c
Enhanced Oil Recovery (EOR) Research Center, School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iran

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

Keyword: Medium and high internal phase W1/O/W2 multiple Pickering emulsions (MPEs) were fabricated by physically-
Double Pickering emulsion modified hordein nanoparticles. A triphasic system was developed at dispersed phase volume fraction (Φ) of 0.5
Encapsulation with an overrun value of ~40%. No overrun was detected in high internal phase MPEs (Φ 0.8). Optical and
Hordein
confocal laser scanning microscopy confirmed the formation of MPEs. Monomodal droplet size distribution with
MLS
a mean diameter of 32.90 and 21.48 μm was observed for MPEs at Φ 0.5 and Φ 0.8, respectively. Static multiple
Vitamin B12
light scattering confirmed that creaming was the main mechanism behind the instability of MPEs. Both MPEs
revealed pseudo-plastic behavior and predominant storage modulus (G′ ) over the applied frequency range. The
encapsulation efficiency of vitamin B12 in MPEs was 98.3% and remained relatively constant during 28 d. These
results suggested the excellent potential of hordein nanoparticles as appropriate candidate for designing multi-
structural colloidal systems using plant proteins.

1. Introduction
The innovative design of multi-purpose functional foods is an
ongoing research area in the food industry to fulfill consumer demands
and diversify food products. In this regard, colloidal delivery systems
(CDSs) are an ingenious option for delivery by design purposes. Among
various colloidal entities, emulsion-based delivery systems are the best
candidates for developing new food products. Recent developments in
the field of complex colloids have led to a renewed interest in multiple
emulsions systems (McClements, 2018; Muschiolik & Dickinson, 2017).
The most common type of multiple emulsion (also known as W1/O/W2
double emulsion) is the system in which the oil droplets (O) containing
internal aqueous phase droplets (W1) are dispersed within an external
water phase (W2). A hydrophobic emulsifier is usually applied to sta­
bilize the primary water-in-oil emulsion (W1/O); while, a hydrophilic
one should be used to stabilize the W1/O emulsion in the W2
(Muschiolik, 2007; Muschiolik & Dickinson, 2017). These systems have
the potential for simultaneous delivery of both hydrophobic and hy­
drophilic bioactive components. W1/O/W2 systems are applied when
controlled release and targeted delivery of bioactive compounds at the
desired site of gastrointestinal tract (GIT) are required (Garti, 1997). The
other potential advantages of multiple emulsions in food systems
include the ability to develop taste contrast properties (e.g., decreasing
the total amount of salt while keeping optimum saltiness perception in
the mouth), and masking undesirable flavors (e.g., bitterness and
metallic aftertaste) (Muschiolik & Dickinson, 2017). Interestingly, W1/
O/W2 multiple emulsions have also been suggested for formulating low-
calorie and low-fat products such as reduced-fat white cheese with
similar rheological behavior and appearance to full-fat counterpart
through embedding tiny water droplets into the oil droplets (Lobato-
Calleros, Rodriguez, Sandoval-Castilla, Vernon-Carter, & Alvarez-
Ramirez, 2006).
Polyglycerol polyricinoleate (PGPR) and Tweens, as hydrophobic
and hydrophilic emulsifiers, respectively, are the most preferred choices
for long-term stability of double emulsions (McClements & Jafari,
2018). There has been a focus on assembling food-grade stabilizers from
proteins, carbohydrates, and lipids, in order to develop functional foods
containing all natural ingredients (Jafari, Sedaghat Doost, Nikbakht
Nasrabadi, Boostani, & Van der Meeren, 2020). Pickering emulsions,
also known as surfactant-free emulsions, have adsorbed nano/micro-

* Corresponding author.
E-mail address: hhosseini@shirazu.ac.ir (S.M.H. Hosseini).

https://doi.org/10.1016/j.foodchem.2021.131354
Received 13 May 2021; Received in revised form 29 September 2021; Accepted 4 October 2021
Available online 7 October 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
S. Boostani et al.
particles at the oil/water interface (Melle, Lask, & Fuller, 2005). The
exclusion volume effect and steric hindrances of particles, accumulated
at the interface, provide stabilization against coalescence. Investigating
appropriate food-grade Pickering candidates is a live area of research in
Pickering emulsions (Dickinson, 2017; Xiao, Li, & Huang, 2016).
Plant-based ingredients exhibit some advantages over animal-based
ones due to the low risk of infection and contamination, no re­
strictions on cultural eating habits and vegetarian consumers, as well as
more significant economic benefits and sustainability (Samborska et al.,
2021). There have been some reports on the successful application of
plant-based ingredients as Pickering stabilizers in double emulsions.
Octenyl succinate derivatives of quinoa starch granules (Lin et al., 2019;
Marefati, Sjöö, Timgren, Dejmek, & Rayner, 2015), gliadin (Chen et al.,
2018), and kafirin (Xiao, Lu, & Huang, 2017) particles were used as
external interface stabilizer of W1/O/W2 multiple Pickering emulsions
(MPEs), and carnauba wax particles as the stabilizer of both interfaces in
O1/W/O2 MPEs (Szumała & Luty, 2016). A mixture of zein nanoparticles
and soybean lecithin has recently been used for the stabilization of W1/
O/W2 high internal phase (HIP) Pickering emulsions (Jiang et al., 2021).
Various mixtures of colloidal microcrystalline cellulose and surfactant
were utilized to stabilize MPEs (Spyropoulos, Duffus, Smith, & Norton,
2019). The adsorption of soluble and insoluble complexes of whey
protein concentrate/gum Arabic at both interfaces of O1/W/O2 MPEs
was reported by Estrada-Fernández et al. (2018). Recently, a
maltodextrin-in-gelatin-in-maltodextrin W/W/W Pickering emulsion
was fabricated by cooling down of simple gelatin-in-maltodextrin W/W
emulsion with the aid of mucin glycoprotein particles (Beldengrün et al.,
2020).
Among plant-based materials, prolamins (as a group of plant storage
proteins mainly found in cereals) can be exploited as appropriate can­
didates for the fabrication of micro- and nano-particles. The limited
solubility of these proteins in aqueous and oil systems as well as their
highly hydrophobic nature make prolamins suitable for Pickering sta­
bilization (Samborska et al., 2021). β-Hordein is the main fraction of
barley prolamins. It shows high surface hydrophobicity and thus ag­
gregation behavior, which result in adsorbing to oil/water and air/water
interfaces and forming viscoelastic films around the dispersed phases
(Boostani et al., 2020). Recently, we reported that hordein particles are
able to develop emulsion-foam (triphasic) Pickering structures with
higher stability against coalescence and aqueous phase separation
compared to zein- and secalin-based particles (Boostani et al., 2019).
Such a property can be applied for mimicking the desirability and sen­
sory properties of whipped cream-like products. In another work, the
ability of hordein particles to stabilize HIP Pickering emulsions was also
confirmed (Boostani et al., 2020). Fabricating stable HIP emulsion,
removing water, and then shearing is a common procedure applied for
converting low viscosity liquid vegetable oils into solid-like structured
oils without the need to hydrogenation or mixing with saturated fats
(Zhou et al., 2018). Structured oils can minimize inappropriate nutri­
tional effects of formulations containing saturated fats, while keeping
the original texture and even developing innovative organoleptic
perception. Additionally, forming an interfacial layer of biopolymer
particles around the oil droplets might lead to designing anti-obesity
formulations via retarding the digestion of triacylglycerols (Xiao et al.,
2017).
Vitamins are the essential components of daily diet. The majority of
these microelements cannot be synthesized within the human body. W1/
O/W2 MPEs have the advantages of both multiple and Pickering emul­
sions. They can be applied to encapsulate hydrophilic bioactive com­
pounds in the W1, while providing physicochemical stability during
processing and storage, as well as controlling the release in GIT. To the
best of our knowledge, the design of MPEs stabilized by hordein-based
particles has not yet been studied. Therefore, the aim of this study was
the fabrication and characterization of food-grade medium internal
phase (MIP) and high internal phase (HIP) MPEs based on hordein
particles. Vitamin B12, as a model hydrophilic compound, was
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Food Chemistry 372 (2022) 131354
encapsulated within the W1 of MIP MPE and the ability of delivery
system to control the ejection of Vitamin B12 from W1 was studied over
time.
2. Materials and methods
2.1. Materials
Hordein was isolated from whole barley grain (Boostani et al., 2019).
Purified soybean oil and vitamin B12 were purchased from Sigma-
Aldrich (St. Louis, MO, USA). PGPR was provided from Danisco Co.
(Denmark).
2.2. Fabrication of hordein particles
Hordein (nano)particles were fabricated according to the procedure
previously explained (Boostani et al., 2019) with slight modifications.
Briefly, hordein was hydrated in ethanol–water mixture (65:35) and
thermally treated (75 ◦ C for 5 min). One part (20 mL) of the ethanolic
dispersion of hordein was added to three parts of deionized water in 2
min under intense stirring, followed by immediate pH adjustment to pH
3.0. Afterward, the protein aggregates were subjected to evaporation in
a rotary evaporator to remove the extra solvents and to reach a final
concentration of 2% w/v. The dispersion was then sonicated for 2 min
by a horn sonicator (model UP200st equipped with sonotrode S26d7,
Hielscher Ultrasound Technology, Germany) in the continuous mode.
The frequency, amplitude, and nominal power were 24 kHz, 75%, and
200 W, respectively. Temperature control was performed using an ice
tank during sonication.
After appropriate dilution with deionized water pre-adjusted to pH 3,
the particle size and zeta-potential of hordein dispersion were deter­
mined using a Zetasizer Nano (Malvern Instruments Ltd., UK) at 25 ◦ C.
The three-phase contact angle (θwo) of hordein nanoparticles was also
determined (Boostani et al., 2019). Briefly, lyophilized powder was
pressed into small pellets by a laboratory scale instrument and analyzed
by a drop shape analyzer (DSA 100, KRÜSS GmbH, Hamburg, Germany).
The surface morphology of protein particles was investigated by a
scanning electron microscope (TESCAN-Vega 3, TESCAN Co., Czech
Republic) at an accelerating voltage of 10 kV and magnification of 35.0
kx.
2.3. Fabrication of multiple Pickering emulsions
MPEs were prepared in a total volume of 6 mL in two steps. Firstly,
the primary emulsions (W1/O) were prepared by mixing the internal
aqueous phase (30% v/v) and the oil phase (70% v/v). The W1 phase
consisted of deionized water at pH 3. The oil phase was prepared by
mixing 5% w/v PGPR in soybean oil under stirring at 65 ◦ C for 20 min.
Homogenization was performed at 24,000 rpm for 10 min using a high-
speed homogenizer equipped with a 6 mm dispersing tool (Ystral Ho­
mogenizer X-10/25, Germany). The MPEs were prepared by the emul­
sification of primary emulsion in the external aqueous phase (W2 pre-
adjusted to pH 3) containing 20 mg of hordein nanoparticle per mL of
the oil phase. For the preparation of medium internal phase (MIP) MPEs
with a dispersed volume fraction (φ) of 0.5, equal volumes (i.e., 3 mL) of
primary emulsion (W1/O) and external aqueous phase (W2) were ho­
mogenized at 16000 rpm for 2 min. High internal phase (HIP) MPEs (φ
0.8) were prepared by the dropwise addition of W1/O primary emulsion
(4.8 mL) to W2 (1.2 mL) under homogenization at 16000 rpm for 5 min.
It is known that the shearing conditions in the second step should be
mild enough to avoid destabilization of the internal droplets but at the
same time being able to create a secondary emulsion (Matos et al.,
2013).
S. Boostani et al.
2.4. Characterization of multiple Pickering emulsions
The size of dispersed droplets was measured by laser light scattering
technique using a Malvern Mastersizer 2000 (Malvern Instruments Ltd.,
UK). Before the measurement, the MPEs were diluted with Milli-Q water
pre-adjusted to pH 3.0. The emulsion samples were added to dispersion
unit under stirring at 2000 rpm until an obscuration of 10%–20% was
recorded. The refractive index and absorption of soybean oil droplets
were 1.46 and 0.001, respectively. Moreover, the refractive index of
1.33 was considered for the dispersant and Mie theory was used to
compute the results. An optical microscope (Olympus BX50, Japan)
equipped with a digital camera (DFK 41AF02, Germany) was used to
take optical micrographs. For further analysis of droplet size, the optical
micrographs of at least 100 random droplets were analyzed using
ImageJ software (National Institutes of Health, USA). Confocal laser
scanning microscopy (CLSM, Nikon A1 + confocal microscope, Nikon
Instruments, USA) was used to capture CLSM images of MPEs. Prior to
emulsification, the aqueous and oil phases were stained with Nile blue A
and Nile red, respectively. The samples (10 μL) were then cast on a single
concave glass slide and monitored with a 60x objective. CLSM was
performed using two laser excitation sources (488 and 633 nm) and two
acquisition channels for Nile red and Nile blue A, respectively, and
combined images under two channels were taken through NIS-elements
software (version 4.6). Owing to the ability of hordein nanoparticles in
the formation of triphasic systems (Boostani et al., 2019), the possible
formation of emulsion-foam structures was assessed by measuring the
overrun (i.e., the increase in volume) immediately after the preparation
of emulsions using equation (1):
V1 − V0
Overrun(%) = × 100 (1)
V0
where, V1 is the final volume of MPE; and V0 is the total volume of oil
and water phases in non-emulsified form.
2.5. Static multiple light scattering (MLS)
Static MLS was applied to monitor the stability profile of MPEs using
a Turbiscan Lab Expert (Formulation Co., Toulouse, France), similar to
the method described by Matos, Gutiérrez, Iglesias, Coca, and Pazos
(2015). This instrument works based on the principle of emitting a light
beam through the whole length of a cylindrical glass vial containing 4.0
mL of emulsion samples. The transmitted (TS) and backscattered (BS)
light intensity was monitored as a function of cell height and time (every
30 min for 3 h and after 10 days of storage) at 25 ◦ C. The emulsion index
(EI) was estimated from the BS graphs by dividing the height of the
cream phase by the total height of the sample after 1 h and 10 days.
Turbiscan stability index (TSI) was evaluated according to a scani-to-
scani-1 difference over total sample height (H) using equation (2):
∑ ∑ |scani (h) − scani− 1 (h)|
TSI = h
(2)
H sonicated aggregates due to the disruption of noncovalent interactions
2.6. Rheological properties
A stress-controlled Kinexus rheometer (Malvern Panalytical Ltd,
UK), equipped with a cup and bob geometry (C14), was used to evaluate
the rheological properties of freshly prepared MPEs at 25 ◦ C. Frequency
sweep test was performed at a range of 0.1–10 Hz within the linear
viscoelastic region (LVR) at 1 Pa. The storage (G′ ) and loss (G′′ ) moduli
were then calculated. The steady shear properties of MPEs were also
monitored at a shear rate range of 0.1–100 s− 1.
2.7. Encapsulation efficiency and release properties
In order to evaluate the encapsulation properties of MIP MPEs,
vitamin B12 (2.5 mg/mL of W1) was used as a hydrophilic model. The
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Food Chemistry 372 (2022) 131354
encapsulation properties were assessed similar to the procedure
described by Matos et al. (2015) with some modifications. The encap­
sulation efficiency (EE) was defined as the fraction of vitamin B12
entrapped in W1 during the emulsification process. In this regard, freshly
fabricated W1/O/W2 MPEs loaded with vitamin B12 were centrifuged at
4000 rpm for 10 min and non-encapsulated vitamins were separated
using a 0.2 µm PVDF syringe filter. The absorbance of filtrate was
measured by a UV–visible spectrophotometer (Carry 50, USA) at 361
nm. The vitamin B12 concentration was determined from a standard
curve (Fig. 6A) prepared by vitamin B12 standard solutions. The EE (%)
was then calculated using equation (3).
Mt − Mp
EE(%) = × 100 (3)
Mt
where, Mt is the total amount of vitamin B12 (mg) added to MPE; and Mp
is the amount of non-encapsulated (free) vitamin B12 (mg) determined in
W2. The centrifugal force did not adversely affect the microstructure of
MPEs.
The encapsulation stability (ES%) of vitamin B12 in MPEs was
monitored during storage at 4 ◦ C and dark conditions by measuring the
EE at given time points (1, 7, 14, 21, and 28 d after preparation).
2.8. Statistical analysis
A completely randomized design was used in this study. Mean values
and standard deviations were reported. Tukey’s multiple range post hoc
tests were applied by SPSS software (ver. 22, IBM Corp. USA) at a sig­
nificance level of 0.05. All experiments were done in triplicate.
3. Results and discussion
3.1. Formation and visual inspection of multiple Pickering emulsions
Spherical-shaped modified hordein nanoparticles with an intensity-
weighted average diameter of approximately 200 nm, zeta potential of
about + 30 mV, and three phase contact angle of 64.1◦ were used to
stabilize the outer interface of MPEs (Fig. 1A-D). A relatively thick layer
of hordein nanoparticles around the oil droplets as well as protein ag­
gregates in the continuous phase were observed in simple O/W emul­
sions, confirming the surface activity of hordein nanoparticles and hence
their ability to adsorb to the O/W interfaces (Fig. 1E-F). This observation
in simple emulsion led to a conclusion that these nanoparticles are also
able to form a thick interface around the dispersed multiple W1/O
droplets in MPEs.
Our previous works have shown that the particle size of hordein
nanoparticles is decreased after thermal and subsequent sonication
treatments (Boostani et al., 2019). The sonication treatment was more
effective than the heat treatment in reducing the hydrophobicity of
particles. This was related to the adaptability and reorganization of
and formation of smaller aggregates along with assuming new equilib­
rium structures via exposing more polar groups buried within the larger
aggregates. In the next study, changes in the aggregation behavior of
hordein particles under the influence of pH and ionic strength was
investigated (Boostani et al., 2020). The lower intensity-weighted mean
diameter and narrower distribution of hordein particles at pH 3 and pH 4
were related to the strong electrostatic repulsion among particles.
Nevertheless, an increase in the particle size was observed at higher pH
values close to isoelectric point, and when the ionic strength exceeded a
certain level (e.g., 0.05 M at pH 3). Therefore, in this study, physically
modified hordein nanoparticles were fabricated in the optimum condi­
tions and investigated for their ability to develop MPEs.
The visual appearance of MPEs at various time intervals is shown in
Fig. 1G. Interestingly, an overrun value of 41% was observed in MPEs
prepared at Φ 0.5 (Fig. 1H), indicating the formation of a complex
S. Boostani et al.
structure ((W1/O)/Air-in-W2) after emulsification arising from the
hordein particles ability to adsorb at both air–water and oil–water in­
terfaces. This finding was consistent with our previous finding (Boostani
et al., 2019), in which hordein particles resulted in the formation of a
triphasic oil/air-in-water simple Pickering emulsion at Φ 0.5. In the case
of hordein-stabilized MPEs at Φ 0.5, a separated aqueous phase at the
bottom was observed after about 1 h and gradually increased over 10
days of storage (Fig. 1G and Fig. 3D). The average droplet size of this
sample remained relatively constant during the storage period (Fig. 2A),
indicating the relative stability of the concentrated emulsion. MPEs
prepared at Φ 0.8 had no overrun after emulsification and showed
higher creaming stability than those prepared at Φ 0.5 during storage
(Fig. 1G, and Fig. 3D), which could be related to the higher viscosity and
lower droplet size (discussed later). The absence of overrun was also
attributed to the increase in the viscosity and elastic behavior of MPEs at
higher droplet concentrations (section 3.5). Besides, under certain cir­
cumstances, the oil phase can destabilize the foam systems via spreading
at the air/water interface and triggering the foam rupture (Boostani
et al., 2020). The potential of hordein nanoparticles for the stabilization
of HIP MPEs is notable as other potent Pickering candidates, such as OSA
quinoa starch and kafirin particles, have shown intense oiling off and
droplet collapse at (W1/O):W2 ratio of 7:3 (Lin et al., 2019; Xiao et al.,
2017). Additionally, it has been reported that the surfactant molecules
are unable to develop concentrated W1/O/W2 emulsion systems even
4
Food Chemistry 372 (2022) 131354
Fig. 1. (A): Intensity-weighted particle size
distribution of physically modified hordein
nanoparticles determined by DLS; (B): Three-
phase contact angle of hordein nanoparticles;
(C): Photographs of isolated hordein and
lyophilized hordein nanoparticles; (D): SEM
micrograph of hordein nanoparticles; (E and
F): CLSM micrographs of hordein nano­
particles adsorption at the interfacial layer of
simple O/W emulsions at two different
excitation wavelength; (G): Visual observa­
tion of hordein nanoparticles-stabilized
MPEs prepared at Φ 0.5 (top) and Φ 0.8
(bottom) over time (from left to right: freshly
prepared, after 1 h, 3 h, 1 d and 10 d,
respectively); (H): Visual observation of W2
and W1/O primary emulsion prior to ho­
mogenization (left) and after homogeniza­
tion and formation of MPE at Φ 0.5 (right)
which indicates obvious overrun; (I): photo­
graphs of MPEs at Φ 0.5 (top) and Φ 0.8
(bottom).
with the aid of a thickening agent in the external phase due to the
emulsion inversion phenomenon (Matos, Guti, Martínez-rey, Iglesias, &
Pazos, 2018).
In our previous study, the O/W simple Pickering emulsion (at Φ 0.8)
stabilized by hordein nanoparticles was self-supporting after the vial
inversion and highly stable to coalescence and aqueous phase separa­
tion. This property was associated with the robust particle-based inter­
facial characteristics of hordein around the oil droplets. The three-
dimensional solid-like network, resulted from the close proximity and
tight packing density of oil droplets, led to the formation of high internal
phase Pickering emulsions (HIP PEs) stable against creaming and coa­
lescence (Boostani et al., 2020). Moreover, in HIP PE systems the steric
hindrance and electrostatic repulsion among droplets induce higher
viscosity (Zhu, Gao, Liu, Zou, & McClements, 2019). However, in this
study HIP MPEs did not exhibit such exact solid-like character (Fig. 1I),
which might be due to the differences in droplet size distribution (sec­
tion 3.2) and viscosity (section 3.5) of HIP PEs and HIP MPEs. Basically,
in emulsion systems, the droplets are caged by other ones at Φ 0.58
which causes a large increase in viscosity (known as jamming). At higher
Φ (e.g., 0.64), the droplets may adopt a random close-packed arrange­
ment, and at Φ 0.74, they may adopt a face-centered cubic arrangement,
where they become entirely close-packed and have a solid-like behavior.
Additionally, the viscosity of an emulsion may increase by decreasing
the droplet size, particularly in the case of more concentrated emulsions
S. Boostani et al.
Fig. 2. Changes in volume-weighted droplet size distribution of MPEs stabilized by hordein nanoparticles at Φ 0.5 (A) and Φ 0.8 (B) during storage; Day 1 indicates
freshly prepared samples. (C): Number-weighted droplet size distribution of freshly prepared MPEs stabilized by hordein nanoparticles at Φ 0.5 and Φ 0.8 determined
by ImageJ software.
(Zhu et al., 2019).
Free oil formation was not observed in MPEs prepared at both Φ
values during the storage period (Fig. 1G). Chen et al. (2018) and Jiang
et al. (2021) similarly reported high stability of HIP MPEs prepared by
gliadin and zein nanoparticles/lecithin, respectively. Such behavior can
be related to the formation of prolamin-based rigid interfaces, which
effectively prevents the dispersed droplets from coalescence and Ost­
wald ripening (Chen et al., 2018). Double emulsions are prone to various
instability mechanisms including osmotic and chemical-driven coales­
cence, diffusion of internal aqueous droplets into the external water
phase, transport of hydrophilic emulsifier into the internal water drop­
lets, flocculation, expulsion, gravitational separation, and Ostwald
ripening (Liu et al., 2020; Muschiolik & Dickinson, 2017; Benichou,
Aserin, & Garti, 2007). Compared to small emulsifier molecules,
biopolymer-based particles do not readily pass through the oil droplets
and accordingly the coalescence of the internal water droplets and the
external water phase is reduced (Schuch, Helfenritter, Funck, &
Schuchmann, 2015). Marefati et al. (2015) reported that Pickering
candidates enhance the stability of W1/O/W2 emulsions against coa­
lescence and Ostwald ripening greater than the non-aggregated indi­
vidual macromolecules.
3.2. Size characteristics of multiple Pickering emulsions
Fig. 2A and 2B show the changes in the volume-weighted droplet size
distribution of MPEs obtained by laser diffraction technique over time.
The number-weighted droplet size distribution of freshly prepared
emulsions determined by ImageJ software is also shown in Fig. 2C. Both
the volume mean (D4,3) and number (arithmetic) mean (D1,0) of MPEs
prepared at Φ 0.5 were relatively larger than those of MPEs prepared at
Φ 0.8. In freshly prepared samples, the volume mean values of MPE
droplets prepared at Φ 0.5 and 0.8 were 32.9 ± 1.8 and 21.5 ± 1.9 μm,
respectively. Considering the equations (or numerator and denomina­
tor) utilized in calculating D4,3, and D1,0, the variations in the volume
and number means might be resulted from the higher contribution of
larger droplets than smaller ones to D4,3 values. Moreover, the number
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Food Chemistry 372 (2022) 131354
of droplets analyzed by ImageJ software was significantly lower than
that analyzed by laser diffraction technique and hence less representa­
tive of the system. It should be noted that the scattering signals caused
by the internal aqueous droplets and the external oil droplets might lead
to some unavoidable experimental errors. However, taking into account
the relatively normal distribution of dispersed droplets, appropriate
correlation was observed between the results of volume- and number-
weighted distributions. The droplet size distribution patterns of both
systems were monomodal similar to those previously reported for MPEs
stabilized by plant-based Pickering particles like OSA-modified quinoa
starch granules (Lin et al., 2019; Marefati et al., 2015; Matos, Timgren,
Sjöö, Dejmek, & Rayner, 2013). Fresh samples prepared at Φ 0.5 had
slightly lower span values than those prepared at Φ 0.8. However, the
increase in the distribution width of MPEs at Φ 0.5 was higher than that
of MPEs at Φ 0.8 indicating lower stability. Creaming was the main
instability mechanism of such system. These results are in accordance
with those reported by Matos et al. (2015), and Fechner, Knoth, Scherze,
and Muschiolik (2007). The sample prepared at Φ 0.8 had higher sta­
bility over time.
The high resistance of Pickering emulsions against destabilization
phenomena is associated with the larger size of Pickering particles than
the size of surfactant molecules. In these systems, the free energy of
detachment (ΔGD) is much greater than the thermal energy and, thus the
particles are irreversibly adsorbed (Dickinson, 2010). In such condi­
tions, the system approaches a steady-state (Estrada-Fernández et al.,
2018). A thick layer of particles can sterically hinder the close contact of
droplets when the particles have appropriate contact angle (Section 3.1,
Fig. 1A-F). This phenomenon makes the fabrication of highly stable
concentrated emulsions possible even in the presence of coarse droplets
(Marefati & Rayner, 2020; Marefati et al., 2015). The ΔGD (i.e., the
minimum energy needed to detach a spherical particle from the inter­
face) is a function of particle size, contact angle, and interfacial tension.
It rapidly increases with increasing the particle size (Dickinson, 2017).
Similar to other prolamin-based Pickering candidates such as gliadin
(Hu et al., 2016), kafirin (Xiao et al., 2017) and zein/lecithin (Jiang
et al., 2021), hordein nanoparticles were irreversibly anchored onto the
S. Boostani et al. Food Chemistry 372 (2022) 131354

Fig. 3. Turbiscan profiles of MPEs prepared at Ф 0.5 (A) and Ф 0.8 (B) over 10 d; (C): Turbiscan profile of PGPR-stabilized W1/O primary emulsion during 30 d; (D):
TSI values of MPEs over time. Different colors indicate different time intervals.

6
S. Boostani et al.
Fig. 3. (continued).
interface and provided stability against the coalescence of oil droplets.
The differences in such emulsion systems, reported in literature, is
rooted in the experimental conditions, as well as the characteristics of
the protein aggregates. Hoffmann, and Reger (2014) stated that the
unique properties of the emulsions are a reflection of the properties of
the proteins in the bulk solution.
The emulsifying ability of particles depends not only on the particle
size but also on the contact angle and interfacial tension (Dickinson,
2017). The three-phase contact angle is an important parameter which
indicates the wettability of particles by continuous phase and/or oil
phase (Jiang et al., 2021). Pickering particles remain in the continuous
phase when it contributes more than the oil phase to the wetting of
particles, and thus better steric stabilization is achieved (Dickinson,
2017). The contact angle of hordein nanoparticle was about 64.1◦
indicating the preferential wetting by water. Chen et al. (2018) reported
that the water–air-gliadin contact angle was about 60◦ and gliadin
nanoparticles were able to stabilize W1/O/W2 emulsion gels against
phase separation. Practically, conventional double emulsions stabilized
by surfactants have large and polydisperse droplets that are unstable
against coalescence, flocculation, and creaming phenomena. Therefore,
the increase in the droplet size is common during the storage. Addi­
tionally, the span values are increased with time (Giroux et al., 2013;
Benichou et al., 2007). The osmotic pressure difference between two
aqueous phases is also another mechanism behind the instability of
double emulsions and is responsible for the changes in the size of
dispersed droplets owing to the diffusion of water molecules from the
external phase to the internal phase and vice versa. Therefore, control­
ling the osmotic balance by appropriate salt solutions can maintain the
stability of W1/O/W2 emulsions over time (Matos et al., 2018). In the
current study, the ionic strength (I) of both aqueous phases were not
adjusted as the values above 0.05 M caused large aggregation of hordein
particles mainly due to the charge screening effects (Boostani et al.,
2020). The consequence of small protein particles’ affinity to either
adsorb at the interface or form large aggregates with other particles
determines the efficacy of particulate entities in the Pickering stabili­
zation (Fig. 1E-F).
3.3. Turbiscan stability profile
Monitoring the profiles of backscattering (BS) and transmission (TS)
was performed for further studying the stability of MPEs (Fig. 3). Since
the average size of colloidal particles was larger than the wavelength of
the incident light, a turbid appearance was observed both in the
creamed layer and at the bottom of the measuring cell (Fig. 1G). In such
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Food Chemistry 372 (2022) 131354
systems, the BS profile is more representative than the TS profile for the
stability analysis owing to the ability of oil droplets to scatter 880-nm
near-infrared light beam. Since the BS intensity of W1/O primary
emulsion remained constant over the whole monitoring time (Fig. 3C), it
can be concluded that PGPR was able to stabilize water droplets within
the continuous oil phase. Based on the BS profile, Matos et al. (2015)
similarly reported high stability of W1/O emulsion stabilized by PGPR as
well as no significant changes in the water droplet size.
The BS intensity of both MPEs at time zero exhibited a flat line along
the measuring cell (Fig. 3A and B). A decrease in the BS intensity of
MPEs at Φ 0.5 was observed at the height range of 10–19 mm during the
measuring time (Fig. 3A), which implied creaming and clarification
instability mechanisms. The TS intensity was kept constant along the
measuring cell during the monitoring period. The movement of W1/O
droplets toward the top of the measuring cell results in the clarification
process. This causes an increase in the BS intensity (also known as
creaming phenomena) (Matos et al., 2018). Zero BS signal was not
detected at the top of the BS graph, which was interpreted as the absence
of free oil in the creamed layer (Fig. 3A). As mentioned previously, the
size of dispersed droplets was larger than the wavelength of the light
source; nevertheless, the BS variation in the middle part of the
measuring cell probably indicated the growth in the emulsion droplet
size. The existence of air bubbles could also have some influences on the
results. A slight reduction in the BS intensity accompanied by the fluc­
tuations in the BS line was possibly due to the foam destruction in the
case of MPEs at Φ 0.5 during the test period (Fig. 3A), even though the
collapse of bubbles was not significant. A decrease in the BS intensity
along the whole measuring cell was attributed to the increase in the
droplet size due to coalescence (Matos et al., 2015).
MPEs at Φ 0.8 revealed remarkable stability since the BS signal was
almost steady over time (Fig. 3B). After 1 h, the emulsion index (EI) of
MPEs prepared at Φ 0.8, and 0.5 was 100%, and 83.9%, and after 10
d reached to 96.1%, and 76.7%, respectively. TSI is inversely propor­
tional to the stability of emulsions. After 1 h, this value was 0.90 and
11.86 in the emulsions prepared at Φ 0.8 and 0.5 and reached to 6.56
and 28.13 after 10 d, respectively. The TSI values further confirmed the
higher stability of MPEs prepared at Φ 0.8 (Fig. 3D). These results are in
accordance with the results of droplet size (section 3.2) and rheological
properties (section 3.5). Similar to our findings, Matos et al. (2018) re­
ported that by increasing the ratio of W1/O to W2, the creaming rate was
decreased. This observation can be explained by the larger volume
occupied by the W1/O droplets and hence having less space for free
movement within the network (i.e., higher stability). Matos et al. (2018)
also reported that the clarification occurred in a higher rate in double
S. Boostani et al.
emulsions with larger droplet sizes and higher BS variations (Matos
et al., 2018). The BS intensity of MPEs prepared at Φ 0.5 was lower than
that of emulsions prepared at Φ 0.8 due to the less compact structure
resulting from the presence of air bubbles (Fig. 1H). MPEs at Φ 0.8
showed a slight decrease of BS at the top of the measuring cell after 10
d of storage, which indicated minor creaming. Instability mechanisms
derived from bridging or depletion flocculation are not typically a
matter of emulsions with high Φ value, since the droplets are packed
tightly together. A highly viscous or gelled continuous phase also pre­
vents the flocculated droplets from moving (Zhou et al., 2018).
3.4. Microstructure of multiple Pickering emulsions
The formation of MPEs was also confirmed by microscopic evalua­
tion. In both samples, relatively large oil droplets containing numerous
tiny water droplets were present (Fig. 4A). The optical micrographs also
revealed that the adjacent droplets were packed with each other to form
a network. The higher packing density of W1/O droplets in MPEs pre­
pared at Φ 0.8 led to the formation of irregular polyhedral morphology
(Fig. 4A) which increased the viscoelastic properties and developed
structured emulsion system (Fig. 5B). The visual observation of these
emulsions are in good agreement with the experimental data reported in
Fig. 2 and Fig. 3. Confocal laser scanning microscopy (CLSM) was also
performed (Fig. 4B). Due to the complexity of the system, the samples
were highly diluted with W2 to capture separate droplets and for better
illustration, CLSM 3-D images were taken at different depths. The CLSM
Fig. 4. (A): Optical micrographs (magnification 100 x; scale bar = 10 μm) of MPEs at different Ф values; (B): 3D CLSM images of scanned droplets (magnification 60
x) at three layers from bottom to top at different Ф values.
8
Food Chemistry 372 (2022) 131354
micrographs also confirmed that the water droplets were dispersed
within the oil (red) droplets. In MPE systems stabilized by zein nano­
particles and lecithin, the W1 droplets tend to be located near the O/W
interface, which was attributed to the density difference and hydro­
phobic interactions (Jiang et al., 2021). Chen et al. (2018) reported a
high degree of oil droplets flocculation which was related to a decrease
in the electrical charge on gliadin nanoparticles near their isoelectric
point.
3.5. Rheological properties
3.5.1. Apparent viscosity
Both samples exhibited shear thinning behavior (Fig. 5A), which
indicates the disruption of network structure or deflocculation of
droplets by increasing the shear rate (Boostani et al., 2020; Xi, Liu,
McClements, & Zou, 2019). Higher droplet concentration increases the
friction forces within the network. Therefore, the sample prepared at Φ
0.8 had higher apparent viscosity (Fig. 5A). In the emulsions with high
oil content, there is an increase of droplet flocculation owing to the
higher volume fraction occupied by dispersed droplets and possibly a
higher sensitivity to increasing shear rate (Xi et al., 2019).
3.5.2. Frequency sweep test
The viscoelastic properties of MPEs prepared at Φ 0.5 and 0.8 are
illustrated in Fig. 5B. In both samples, the elastic character was domi­
nant (G′ > G′′ ) over the studied frequency range. A minor dependency of
S. Boostani et al.
Fig. 5. (A): Apparent viscosity vs. shear rate plots of MPEs at Ф 0.5 and Ф 0.8; (B): Frequency sweep test of MPEs at a frequency range of 0.1–10 Hz.
both moduli on the applied frequency (0.1–10 Hz) was also noted. In
concentrated emulsions, the droplets are tightly packed together and
become trapped in “cages” by their neighbors and thus elastic-like fea­
tures are observed (Zhu et al., 2019). As shown in Fig. 5B, the storage
modulus of MPEs at Φ 0.8 was significantly higher than that of emul­
sions prepared at Φ 0.5 which can be related to the higher degree of
inter-droplet interactions of hordein-stabilized droplets. Additionally,
sharing hordein nanoparticles at the interface might result in the floc­
culation of tightly packed oil droplets and increase the viscoelastic
properties (Boostani et al., 2020).
3.6. Encapsulation properties
The standard curve of vitamin B12 is shown in Fig. 6A and the
encapsulation efficiency (EE) of vitamin B12 in W1 during 28 d is shown
in Fig. 6C. In freshly prepared samples, the EE was 98.3% and reached to
97.1% at the end of storage. Changes in the EE over time were not
9
Food Chemistry 372 (2022) 131354
significant (p > 0.05), which demonstrated appropriate encapsulation
stability (ES) or very low ejection of vitamin B12 from W1 to W2 during
the time period. The decrease in the EE vs. time was well fitted with a
logarithmic regression (Fig. 6C). The measurement of EE% over a
defined time is an indicator of losing internal water droplets by coa­
lescence (Schuch et al., 2015). From ES point of view, a double emulsion
system can be considered stable if the initial EE is around 95% and
reaches to 70–80% after some weeks of storage (Xiao et al., 2017). Type
of bioactive component, encapsulant matrix, and surrounding medium
are the key parameters that affect the release of bioactive compounds
from the encapsulating materials. Owing to the molecular dimension as
well as the concentration gradient between W1 and W2, vitamin B12 can
rapidly diffuse from W1, pass through the oil medium and ultimately
reach to W2 during homogenization or storage. The main challenges
associated with common W1/O/W2 double emulsions are controlling the
diffusion of water and emulsifier molecules across different phases
during preparation and storage. Hydrophilic surfactants tend to migrate
S. Boostani et al.
Fig. 6. (A): Standard curve of vitamin B12; (B): Photographs of multiple Pickering emulsion loaded with vitamin B12 (from left to right: W1/O primary emulsion (top)
and W2 phase (bottom) prior to the second emulsification step; Vitamin B12-loaded MPE; and centrifuged MPE for calculating encapsulation efficiency (EE)); (C):
Logarithmic regression of EE over time.
from the external water phase to the oil medium and aggregate as
reverse micelles (Bou et al., 2014). Such challenges may be overcome
using macromolecules instead of surfactants; although, biopolymer
stabilized double emulsions may themselves undergo some instability
mechanisms. As reported by Fechner et al. (2007), the initial release of
vitamin B12 from W1 to W2 in double emulsions was remarkably
decreased by changing the outer interface stabilizer from sodium
caseinate to sodium caseinate-dextran conjugate (Fechner et al., 2007).
The EE% of vitamin B12 in freshly prepared W1/O/W2 emulsion stabi­
lized by sodium caseinate was about 16.6% and increased to about
27.4% when sodium caseinate-maltodextrin conjugates were used as an
emulsifier (O’Regan & Mulvihill, 2010). A total reduction in the EE over
7 d (ranging from 39.2% to 27.7%) was observed in all types of emul­
sions. The ES over 7 d was better in the presence of conjugates (O’Regan
& Mulvihill, 2010). Giroux et al. (2013) reported high EE of vitamin B12
(98.4%) in W1/O/W2 emulsions prepared in skim milk or sodium
caseinate dispersion (as W2). In conclusion, the individual biopolymer
molecules have advantages over small surfactant molecules; however,
some instability phenomena such as the leakage of W1 into W2 may still
occur. Pickering particles-stabilized multiple emulsions containing
water-soluble markers generally show better EE and ES compared to
macromolecules- or surfactant-stabilized systems. MPEs stabilized by
10
Food Chemistry 372 (2022) 131354
hordein nanoparticles revealed appropriate encapsulation properties in
terms of both EE and ES (Fig. 6). The interfacial film, created by the
adsorbed particles, developed a physical barrier. This mechanism is not
principally different from that provided by the individual protein and
polysaccharide molecules (Estrada-Fernández et al., 2018). However,
considering the theory of Pickering stabilization, the free energy of
spontaneous desorption of a solid particle is several orders larger than
that of small surfactant molecules; therefore, the particles are assumed
to be irreversibly anchored at the interface and stable against coales­
cence (Dickinson, 2017; Dickinson, 2010). Additionally, it seems that
the stability of W1 can be directly associated with the EE (Dias et al.,
2018). Similar results regarding the high EE and ES of MPEs stabilized
by plant-based particles were reported by other researchers for various
cargoes. The EE of carmine was 98.5% and 90% in freshly prepared and
21-d old MPEs stabilized by OSA-modified starch granules, respectively
(Matos et al., 2013). The EE values of 96.6% and 98.5% for liquid and
solid shea nut oil, respectively, and the ES value of 97% were reported
by Marefati et al. (2015) in MPEs stabilized by OSA-modified quinoa
starch particles. The EE of anthocyanin in freshly prepared MPEs sta­
bilized by kafirin particles was 85.1% and reached to 75.9% after 2 w
(Xiao et al., 2017). HIP Pickering double emulsion/gels stabilized by
gliadin particles showed an EE of 65.5% for epigallocatechin gallate and
S. Boostani et al.
97.2% for quercetin (Chen et al., 2018). The lower stability of some
systems might be associated with the mass transport, driven by the
gradient of osmotic pressure and chemical potential difference between
W1 and W2 (Muschiolik & Dickinson, 2017; Dickinson, 2017), which
increases the oil droplet size owing to the diffusion of W1 to W2. As
reported by Xiao et al. (2017) in MPEs stabilized by kafirin particles, the
growth in oil droplets size was concomitant with a decrease in the
packing density of nanoparticles at the interface which led to the
collapse of emulsion droplets. In some systems, it is not crucial to pre­
cisely balance the osmotic pressure difference to obtain high stability
(Matos et al., 2013).
4. Conclusion
In this study, the fabrication of concentrated (Φ 0.5) and high in­
ternal phase (Φ 0.8) MPEs stabilized by physically-modified hordein
nanoparticles was investigated. The microstructure, and physical sta­
bility of emulsions were affected by the droplet size distribution, dis­
tribution width, and rheology of the systems. Increasing the amount of
dispersed phase (i.e., W1/O primary emulsion) led to an increase in the
apparent viscosity and the elastic character. Creaming and clarification
phenomena occurred during storage but not oiling off. Excellent
encapsulation efficiency and stability were observed after the entrap­
ment of vitamin B12 (as a model hydrophilic component) in W1. These
observations indicated the great potential of hordein nanoparticles in
stabilizing MPEs. Hordein nanoparticles can therefore be utilized as
green Pickering candidates for developing complex multi-purpose
colloidal systems with innovative techno-functional applications.
CRediT authorship contribution statement
Sareh Boostani: Formal analysis, Investigation, Writing - original
draft. Masoud Riazi: Validation, Writing - review & editing. Ali Mar­
efati: Validation, Writing - original draft. Marilyn Rayner: Validation,
Resources, Supervision. Seyed Mohammad Hashem Hosseini:
Conceptualization, Resources, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was financially supported by Shiraz University (Grant
number: 95GCU2M194065). Authors are very thankful to Lund Uni­
versity for technical support.
References
Beldengrün, Y., Dallaris, V., Jaén, C., Protat, R., Miras, J., Calvo, M., … Esquena, J.
(2020). Formation and stabilization of multiple water-in-water-in-water (W/W/W)
emulsions. Food Hydrocolloids, 102, 105588. https://doi.org/10.1016/j.
foodhyd.2019.105588
Benichou, A., Aserin, A., & Garti, N. (2007). W/O/W double emulsions stabilized with
WPI-polysaccharide complexes. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 294(1-3), 20–32.
Boostani, S., Hosseini, S. M. H., Golmakani, M.-T., Marefati, A., Abdul Hadi, N. B., &
Rayner, M. (2020). The influence of emulsion parameters on physical stability and
rheological properties of Pickering emulsions stabilized by hordein nanoparticles.
Food Hydrocolloids, 101, 105520. https://doi.org/10.1016/j.foodhyd.2019.105520
Boostani, S., Hosseini, S. M. H., Yousefi, G., Riazi, M., Tamaddon, A.-M., & Van der
Meeren, P. (2019). The stability of triphasic oil-in-water Pickering emulsions can be
improved by physical modification of hordein- and secalin-based submicron
particles. Food Hydrocolloids, 89, 649–660.
Bou, R., Cofrades, S., & Jiménez-Colmenero, F. (2014). Physicochemical properties and
riboflavin encapsulation in double emulsions with different lipid sources. LWT –
Food Science and Technology, 59(2), 621–628.
11
Food Chemistry 372 (2022) 131354
Chen, X., McClements, D. J., Wang, J., Zou, L., Deng, S., Liu, W., … Liu, C. (2018).
Coencapsulation of (-)-Epigallocatechin-3-gallate and Quercetin in particle-
stabilized W/O/W emulsion gels: Controlled release and bioaccessibility. Journal of
Agricultural and Food Chemistry, 66(14), 3691–3699.
Dias, S. V. E., Züge, L. C. B., Santos, A. F., & Scheer, A.de P. (2018). Effect of surfactants
and gelatin on the stability, rheology, and encapsulation efficiency of W1/O/W2
multiple emulsions containing avocado oil. Journal of Food Process Engineering, 41,
e12684.
Dickinson, Eric (2010). Food emulsions and foams: Stabilization by particles. Current
Opinion in Colloid and Interface Science, 15(1-2), 40–49.
Dickinson, Eric (2017). Biopolymer-based particles as stabilizing agents for emulsions
and foams. Food Hydrocolloids, 68, 219–231.
Estrada-Fernández, A. G., Román-Guerrero, A., Jiménez-Alvarado, R., Lobato-
Calleros, C., Alvarez-Ramirez, J., & Vernon-Carter, E. J. (2018). Stabilization of oil-
in-water-in-oil (O1/W/O2) Pickering double emulsions by soluble and insoluble
whey protein concentrate-gum Arabic complexes used as inner and outer interfaces.
Journal of Food Engineering, 221, 35–44.
Fechner, A., Knoth, A., Scherze, I., & Muschiolik, G. (2007). Stability and release
properties of double-emulsions stabilised by caseinate–dextran conjugates. Food
Hydrocolloids, 21(5-6), 943–952.
Garti, Nissim (1997). Double emulsions—scope, limitations and new achievements.
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 123-124, 233–246.
Giroux, Hélène J., Constantineau, Stéphane, Fustier, Patrick, Champagne, Claude P., St-
Gelais, Daniel, Lacroix, Monique, & Britten, Michel (2013). Cheese fortification using
water-in-oil-in-water double emulsions as carrier for water soluble nutrients.
International Dairy Journal, 29(2), 107–114.
Hoffmann, H., & Reger, M. (2014). Emulsions with unique properties from proteins as
emulsifiers. Advances in Colloid and Interface Science, 205, 94–104.
Hu, Ya-Qiong, Yin, Shou-Wei, Zhu, Jian-Hua, Qi, Jun-Ru, Guo, Jian, Wu, Lei-Yan, …
Yang, Xiao-Quan (2016). Fabrication and characterization of novel Pickering
emulsions and Pickering high internal emulsions stabilized by gliadin colloidal
particles. Food Hydrocolloids, 61, 300–310.
Jafari, Seid Mahdi, Sedaghat Doost, Ali, Nikbakht Nasrabadi, Maryam, Boostani, Sareh, &
Van der Meeren, Paul (2020). Phytoparticles for the stabilization of Pickering
emulsions in the formulation of novel food colloidal dispersions. Trends in Food
Science & Technology., 98, 117–128.
Jiang, Hang, Zhang, Tong, Smits, Joeri, Huang, Xiaonan, Maas, Michael, Yin, Shouwei, &
Ngai, To (2021). Edible high internal phase pickering emulsion with double-
emulsion morphology. Food Hydrocolloids, 111, 106405. https://doi.org/10.1016/j.
foodhyd.2020.106405
Lin, Xiaoying, Li, Songnan, Yin, Juhua, Chang, Fengdan, Wang, Chan, He, Xiaowei, …
Zhang, Bin (2020). Anthocyanin-loaded double Pickering emulsion stabilized by
octenylsuccinate quinoa starch: Preparation, stability and in vitro gastrointestinal
digestion. International Journal of Biological Macromolecules, 152, 1233–1241.
Liu, Jinning, Zhou, Hualu, Muriel Mundo, Jorge L., Tan, Yunbing, Pham, Hung, &
McClements, David Julian (2020). Fabrication and characterization of W/O/W
emulsions with crystalline lipid phase. Journal of Food Engineering, 273, 109826.
https://doi.org/10.1016/j.jfoodeng.2019.109826
Lobato-Calleros, C., Rodriguez, E., Sandoval-Castilla, O., Vernon-Carter, E. J., & Alvarez-
Ramirez, J. (2006). Reduced-fat white fresh cheese-like products obtained from W1/
O/W2 multiple emulsions: Viscoelastic and high-resolution image analyses. Food
Research International, 39(6), 678–685.
Marefati, A., & Rayner, M. (2020). Starch granule stabilized Pickering emulsions: An
eight-year stability study. Journal of the Science of Food and Agriculture., 100,
2807–2811.
Marefati, Ali, Sjöö, Malin, Timgren, Anna, Dejmek, Petr, & Rayner, Marilyn (2015).
Fabrication of encapsulated oil powders from starch granule stabilized W/O/W
Pickering emulsions by freeze-drying. Food Hydrocolloids, 51, 261–271.
Matos, María, Timgren, Anna, Sjöö, Malin, Dejmek, Petr, & Rayner, Marilyn (2013).
Preparation and encapsulation properties of double Pickering emulsions stabilized
by quinoa starch granules. Colloids and Surfaces A: Physicochemical and Engineering
Aspects, 423, 147–153.
Matos, M., Gutiérrez, G., Iglesias, O., Coca, J., & Pazos, C. (2015). Enhancing
encapsulation efficiency of food-grade double emulsions containing resveratrol or
vitamin B12 by membrane emulsification. Journal of Food Engineering, 166, 212–220.
Matos, M., Guti, G., Martínez-rey, L., Iglesias, O., & Pazos, C. (2018). Encapsulation of
resveratrol using food-grade concentrated double emulsions : Emulsion
characterization and rheological behaviour. Journal of Food Engineering, 226, 73–81.
McClements, David Julian (2018). Recent developments in encapsulation and release of
functional food ingredients: Delivery by design. Current Opinion in Food Science, 23,
80–84.
McClements, David Julian, & Jafari, Seid Mahdi (2018). Improving emulsion formation,
stability and performance using mixed emulsifiers: A review. Advances in Colloid and
Interface Science, 251, 55–79.
Melle, Sonia, Lask, Mauricio, & Fuller, Gerald G. (2005). Pickering emulsions with
controllable stability. Langmuir, 21(6), 2158–2162.
Muschiolik, Gerald (2007). Multiple emulsions for food use. Current Opinion in Colloid &
Interface Science, 12(4-5), 213–220.
Muschiolik, Gerald, & Dickinson, Eric (2017). Double emulsions relevant to food systems:
Preparation, stability, and applications. Comprehensive Reviews in Food Science and
Food Safety, 16(3), 532–555.
O’Regan, Jonathan, & Mulvihill, Daniel M. (2010). Sodium caseinate–maltodextrin
conjugate stabilized double emulsions: Encapsulation and stability. Food Research
International, 43(1), 224–231.
Samborska, Katarzyna, Boostani, Sareh, Geranpour, Mansoureh, Hosseini, Hamed,
Dima, Cristian, Khoshnoudi-Nia, Sara, … Jafari, Seid Mahdi (2021). Green
S. Boostani et al.
biopolymers from by-products as wall materials for spray drying microencapsulation
of phytochemicals. Trends in Food Science & Technology, 108, 297–325.
Schuch, A., Helfenritter, C., Funck, M., & Schuchmann, H. P. (2015). Observations on the
influence of different biopolymers on coalescence of inner water droplets in W/O/W
(water-in-oil-in-water) double emulsions. Colloids and Surfaces A: Physicochemical
and Engineering Aspects, 475, 2–8.
Spyropoulos, Fotis, Duffus, Laudina J., Smith, Paul, & Norton, Ian T. (2019). Impact of
Pickering intervention on the stability of W1/O/W2 double emulsions of relevance to
foods. Langmuir, 35(47), 15137–15150.
Szumała, Patrycja, & Luty, Natalia (2016). Effect of different crystalline structures on W/
O and O/W/O wax emulsion stability. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 499, 131–140.
Xi, Zewen, Liu, Wei, McClements, David Julian, & Zou, Liqiang (2019). Rheological,
structural, and microstructural properties of ethanol induced cold-set whey protein
emulsion gels: Effect of oil content. Food Chemistry, 291, 22–29.
12
Food Chemistry 372 (2022) 131354
Xiao, Jie, Li, Yunqi, & Huang, Qingrong (2016). Recent advances on food-grade particles
stabilized Pickering emulsions: Fabrication, characterization and research trends.
Trends in Food Science & Technology, 55, 48–60.
Xiao, Jie, Lu, Xuanxuan, & Huang, Qingrong (2017). Double emulsion derived from
kafirin nanoparticles stabilized Pickering emulsion: Fabrication, microstructure,
stability and in vitro digestion profile. Food Hydrocolloids, 62, 230–238.
Zhou, F. Z., Zeng, T., Yin, S. W., Tang, C. H., Yuan, D. B., & Yang, X. Q. (2018).
Development of antioxidant gliadin particle stabilized Pickering high internal phase
emulsions (HIPEs) as oral delivery systems and the: In vitro digestion fate. Food and
Function, 9, 959–970.
Zhu, Y., Gao, H., Liu, W., Zou, L., & McClements, D. J. (2019). A review of the rheological
properties of dilute and concentrated food emulsions. Journal of Texture Studies, 51,
45-44.