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Citation: Zhang, J.; Zhao, S.; Li, L.;
Kong, B.; Liu, H. High Internal Phase
Emulsions Stabilized by Pea Protein
Isolate Modified by Ultrasound
Combined with pH-Shifting:
Micromorphology, Rheology, and
Physical Stability. Foods 2023,12,
1433. https://doi.org/10.3390/
foods12071433
Academic Editor: Mathias Porsmose
Clausen
Received: 3 March 2023
Revised: 21 March 2023
Accepted: 24 March 2023
Published: 28 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
foods
Article
High Internal Phase Emulsions Stabilized by Pea Protein
Isolate Modified by Ultrasound Combined with pH-Shifting:
Micromorphology, Rheology, and Physical Stability
Jingnan Zhang, Siqi Zhao, Linte Li, Baohua Kong and Haotian Liu *
College of Food Science, Northeast Agricultural University, Harbin 150030, China
*Correspondence: liuht920@neau.edu.cn
Abstract:
In this study, the interfacial behavior of high internal phase emulsions (HIPEs), stabilized by
ultrasound combined with pH-shifting modified pea protein isolate (MPPI), was investigated, and its
emulsification process and stabilization mechanism were discussed. The effects of MPPI concentration
on the micromorphology, droplet size, rheology, and stability of HIPEs were investigated. As the
MPPI concentration increased, the appearance of HIPEs gradually changed from a relatively fluid
state to a plastic solid-like state with detailed texture. There occurred a gradual decrease in droplet
size, the cohering of an orderly and tight arrangement, in addition to the formation of a bilayer elastic
interface layer. The macro- and microrheological assessments confirmed that the apparent viscosity,
storage modulus, elasticity index, and macroscopic viscosity index increased gradually. Furthermore,
it was demonstrated that 5 wt% MPPI-stabilized HIPEs had the potential to be used as 3D printing
inks. Stability evaluation showed that the TURBISCAN stability index decreased and centrifugal
stability increased. The appearance and microstructure remained highly stable after heating at 80
◦
C
for 30 min and storage at 4
°C
for 90 days. These findings confirm that MPPI improves the rheological
behavior and stability of HIPEs by modulating the interfacial adsorption and network structure.
Keywords:
pea protein isolate; high internal phase emulsions; micromorphology; rheological behavior;
3D printing
1. Introduction
High internal phase emulsions (HIPEs) are highly concentrated emulsion systems with
an internal phase volume fraction (
ϕ
) higher than 74% [
1
]. With ideal solid-like appearance,
gel-like rheological behavior, and high stability, HIPEs have attracted extensive attention
in the fields of food, materials, tissue engineering, medicine, and cosmetics. In the past,
HIPEs have been stabilized with large amounts of surfactants (5–50%) or inorganics, which
have limited applicability in the food industry because of factors such as cost, health,
environmental protection, “clean labelling,” and legal constraints [
2
]. Gradually, the use of
natural biopolymers to stabilize HIPEs has attracted research interest.
Pea protein isolate (PPI), with its high nutritional value and hypoallergenicity, has
aroused considerable interest as a sustainable alternative to animal-based protein [
3
].
However, the highly ordered spherical structure and poor solubility of native PPI inhibit its
ability to emulsify at the oil–water interface [
4
]. Although some studies have demonstrated
that treatment of PPI by glycosylation [
5
], potassium metabisulfite induction [
6
] and
microgel [
7
] formation enabled it to stabilize HIPEs, these strategies are complex and
require precise control of the reaction process. Many studies have used pH shifting, a
simple, mild, and effective chemical modification method, to modify protein in order to
improve its solubility and emulsification [
8
]. Under extreme pH conditions, the charged
functional groups of amino acids create molecular repulsion, which triggers the irreversible
unfolding of the protein structure. If the pH is relocated to a neutral value, the protein
structure will transform into a molten spherical state, and the structure at this time will
Foods 2023,12, 1433. https://doi.org/10.3390/foods12071433 https://www.mdpi.com/journal/foods
Foods 2023,12, 1433 2 of 15
become more flexible, which is conducive to its extension at the oil–water interface [
9
]. For
PPI, however, although pH-shifting drives the formation of molten globules, its naturally
rigid structure still hinders its adsorption and positioning at the oil–water interface and
it cannot provide good stability and support for the system, which is undesirable for
emulsified systems, especially HIPEs.
High-intensity ultrasound (HIU) is a successful physical strategy for disrupting inter-
actions, dispersing aggregates, and modifying the structure of proteins, especially plant
proteins [
10
], to achieve superior functional properties for a variety of formulated foods.
The key to material modification by HIU lies in the acoustic cavitation produced during the
ultrasonic process [
11
]. Bubbles generated by acoustic cavitation collapse upon reaching a
critical size, thereby increasing the local pressure in the cavitation zone and creating high
shear forces and turbulence around the bulk liquid, enabling physical modification of the
material [
12
]. Our group earlier found that HIU treatment can make the conformation
of the molten spherical state PPI undergoing pH-shifting more flexible, endowing better
solubility and emulsification [
4
]. A fascinating hypothesis is that the molten state induced
by pH-shifting promotes the adsorption of proteins at the interface, whereas the flexible
conformation caused by HIU treatment increases the possibility of intertwined interfacial
proteins, providing better stability and support for HIPEs.
Hence, the purpose of this study was to provide a strategy to prepare HIPEs using
ultrasound combined with pH-shifting modified pea protein isolate (MPPI) based on
interfacial emulsification and cross-linked network support. First, various concentrations
of MPPI-stabilized HIPEs were characterized, and the dynamic interfacial adsorption and
regulation and control distribution process were elucidated. Then, the relationship between
the emulsification mechanism and property improvement of MPPI-stabilized HIPE in
terms of appearance, micromorphology, rheological behavior, and stability was discussed.
Thus, this study developed a new approach to preparing PPI-based HIPEs with desirable
viscoelasticity, high plasticity, and high stability and, explored their possible use as a solid
fat mimic, providing insights for the development and utilization of PPIs.
2. Materials and Methods
2.1. Materials
The pea protein isolate (PPI, protein
∼
87.31%) used in this study was provided by
Northeast Agricultural University (Harbin, Heilongjiang, China). Sunflower oil was pur-
chased from Yihai Kerry Co., Ltd. (Shanghai, China). All the chemicals and reagents used
in this study were of analytical grade.
2.2. PPI Modified by High-Intensity Ultrasound and pH-Shifting (MPPI)
Similar to our previous work [
4
], the hydrated overnight PPI (5 wt%) was adjusted to
pH 12 using 2 M NaOH, and then treated at 20 kHz with 500 W ultrasonic power for 10 min.
The solution was stirred at room temperature for 1 h, after which the pH was adjusted
to 7 with 1 M HCl and then the solution diluted to various concentrations (1–5 wt%). If
necessary, the pH was fine-tuned to 7 using 0.1 M HCl.
2.3. Preparation of HIPEs
PPI or MPPI (1–5 wt%) aqueous solution and sunflower seed oil were mixed in a
3:1 ratio (v/v), and 0.02 wt% sodium azide was added to the aqueous phase to prevent
microbial growth before mixing. HIPEs (75% internal phase volume fraction) were then
formed by shearing through a homogenizer (IKA Ultra-Turrax T18, Staufen, Germany)
which operated at 15,000 rpm for 2 min, after which they were stored at 4 ◦C [13].
2.4. Micromorphology
2.4.1. Super-Resolution Microscopy (SRM)
The micromorphology of HIPEs was observed through super-resolution microscopy
(OMX SR, Boston, MA, USA). HIPEs of 0.5 g were stained using 20
µ
L of Nile blue (0.1 wt%
Foods 2023,12, 1433 3 of 15
in water) and 25
µ
L of Nile red (0.1 wt% in ethanol) for 30 min in the dark for the water
and oil phases, respectively [
14
]. The excited wavelengths of Nile blue and Nile red were
633 nm and 488 nm, respectively.
2.4.2. Cryogenic Scanning Electron Microscopy (Cryo-SEM)
The micromorphology of HIPEs was assessed via cryo-SEM (Hitachi, Tokyo, Japan)
according to the method of Yan et al. [
15
]. Briefly, samples were frozen-cut in liquid nitrogen,
and sublimated HIPEs were sputtered with platinum, and these were then inserted into a
cold module chamber for observation.
2.4.3. Optical Microscope
The microscopic morphology of HIPEs was observed using an optical microscope
(BX53, Olympus, Japan). A small amount of each sample was smeared onto the glass slide,
covered with a coverslip, and observed under a 100×oil lens.
2.5. Droplet Size Analysis
The droplet size distribution and mean droplet diameter of the HIPEs were determined
using a static light scattering technique (Malvern 3000, Malvern, UK). The samples were
diluted with deionized water until they reached the appropriate concentration and then
they were dispersed in an automated wet dispersion until the obscuration was between
10–20%. Refractive indices of 1.47 and 1.33 were set for oil droplets and deionized water,
respectively. The mean particle size was expressed as volume mean diameter (D4,3).
2.6. Rheological Behavior Analysis
2.6.1. Macrorheological Behavior
Rheological behavior was measured using a rheometer (DHR-2, Crawley, UK) equipped
with a 40 mm-diameter parallel plate. The apparent viscosities were measured at the shear
rate of 0.1–100 s
−1
. We have selected a more suitable 1% strain in the linear viscoelas-
tic region (LVR). The storage modulus G
0
and loss modulus G
00
were obtained through
frequency sweeps (0.1–10 Hz frequency and 1% strain). In order to evaluate the thermal
stability of the HIPEs, the G
0
and G
00
were obtained (1 Hz frequency and 1% strain) as the
temperature sweep range from 25
◦
C to 80
◦
C at a temperature ramp of 5
◦
C/min, and then
maintained at 80
◦
C for 30 min before being cooled back to 25
◦
C in the same manner [
16
].
2.6.2. Micro-Rheological Behavior
A LAB 6 Microrheometer (Formulaction, Toulouse, France) was used to characterize
the microrheological properties of HIPEs based on diffusive wave spectroscopy (DWS)
technology within 4 h at 25
◦
C [
17
]. The mean-square displacement (MSD), elasticity
index (EI), and macroscopic viscosity index (MVI) were obtained through Rheosoft Mas-
ter 1.4.0 software analysis. The motion of the droplet within the sample was quantified as
the MSD of the scatterer, which was a direct probe used to obtain the dynamic properties
of the droplet embedded in it.
2.7. Back-Scattering Light (BS) and TURBISCAN Stability Index (TSI)
HIPEs destabilization phenomena characterized by the BS and TSI were determined
through vertical scanning by applying 880 nm pulsed near-infrared light from bottom to
top using TURBISCAN LAB Expert (Formulation, Toulouse, France) equipment coupled
with TurbiSoft 2.0 software analysis according to the method of Yue et al. [
18
]. The samples
(20 g) were transferred to a measuring cell and scanned for 24 h at room temperature
(25 ◦C).
2.8. Application of HIPEs in 3D Printing
The HIPEs were printed using a 3D printer (Shinnove-E Pro, Hangzhou, China). The
relevant parameters were as follows. Nozzle height: 0.8 mm; nozzle diameter: 0.84 mm;
Foods 2023,12, 1433 4 of 15
printing speed: 25 mm/s. A cylindrical shape with a diameter of 20 mm and height of
10 mm was printed.
2.9. Processing and Environmental Stress
2.9.1. Centrifugation Stability
Centrifugation at 10,000 rpm for 10 min at 4
◦
C was applied to the HIPEs to observe
the appearance differences and microstructural changes of the samples before and after
treatment to evaluate centrifugation stability.
2.9.2. Storage Stability
Storage at 4
◦
C for 90 days was applied to the HIPEs to observe the appearance
differences and microstructural changes of the samples before and after treatment to
evaluate storage stability.
2.9.3. Thermal Stability
Heating at 80
◦
C for 30 min was applied to the HIPEs to observe the appearance
differences and microstructural changes of the samples before and after treatment in order
to evaluate thermal stability [
19
]. The rheological behavior of HIPEs during the entire
heating and cooling process was also analyzed.
2.9.4. Freeze–Thaw Stability
The HIPEs were frozen in a
−
20
◦
C refrigerator for 24 h, thawed at room temperature
(25
◦
C) for 2 h, and cycled 1–3 times according to the actual situation, and then freeze–thaw
stability was evaluated by observing the appearance of the samples.
2.10. Statistical Analyses
All the measurements were conducted in triplicate, and the data were expressed as
mean ±S.D. of all measurements. All diagrams were created with Origin 2021 software.
3. Results and Discussion
3.1. Appearance and Micromorphology of HIPEs
As shown in Figure 1A, 1–4 wt% natural PPI-stabilized HIPEs undergo drastic demul-
sification during shearing. This phenomenon was improved when the concentration
increased to 5 wt%, but it showed high flow behavior and phase separation also occurred.
However, the HIPEs which were stabilized by MPPI showed a gel-like appearance with
good self-supporting properties and no phase separation, indicating that it had the ability
to stabilize HIPE. The possible explanation for this phenomenon was that the structure of
natural PPI was highly ordered, had a certain rigidity, and presented an agglomeration
state, which meant it could not be adsorbed at the interface [
4
]. As depicted in Figure 1A,
the natural PPI aggregates were scattered in the continuous phase and could not play the
role of stabilizing the oil droplets. A large number of irregularly shaped oil droplets were
free or floating in the system and could not be arranged in order. Under the action of
external force, the system would undergo severe coalescence and demulsification. How-
ever, the hydrophobic groups and polar sites of the MPPI were exposed and the
α
-helical
content was reduced, thus improving the solubility and emulsification [
4
]. MPPI with
flexible structure could effectively expand and rearrange at the oil–water interface during
the shearing process to tightly coat oil droplets and form a compact elastic interface film [
4
].
Thus, HIPEs with the typical hexagonal oil drop structure of mutual extrusion and orderly
arrangement were formed [
1
,
20
], as shown in Figure 1A. In order to evaluate the effect of
MPPI concentration changes on the interfacial behavior and network structure of HIPEs in
more detail, and to reveal the dynamic emulsification process and stabilization mechanism
accordingly, the appearance and microstructure were characterized.
Foods 2023,12, 1433 5 of 15
Foods 2023, 12, x FOR PEER REVIEW 5 of 16
flexible structure could effectively expand and rearrange at the oil–water interface during
the shearing process to tightly coat oil droplets and form a compact elastic interface film
[4]. Thus, HIPEs with the typical hexagonal oil drop structure of mutual extrusion and
orderly arrangement were formed [1,20], as shown in Figure 1A. In order to evaluate the
effect of MPPI concentration changes on the interfacial behavior and network structure of
HIPEs in more detail, and to reveal the dynamic emulsification process and stabilization
mechanism accordingly, the appearance and microstructure were characterized.
Figure 1. (A) Appearance of HIPEs stabilized by natural PPI and modified PPI at different concen-
trations (1–5 wt%). (B) Mechanism schematic diagram of HIPEs stabilized by natural PPI and mod-
ified PPI.
The appearance was an external manifestation of the altered microstructure and
droplet size of the HIPE. In order to observe the surface details more directly, the HIPEs
were spread to a flat surface and photographed from above as in Figure 2A. The surface
of HIPEs stabilized by 1 wt% MPPI shows a macroscopic oily sheen and relatively poor
plasticity. As concentration increased, HIPEs were basically free of oil leakage, and plas-
ticity was enhanced by increase in their surface detail texture, which tends to be stable.
Figure 2. (A) Appearance details, (B) super-resolution microscopy, (C) cryogenic scanning electron
microscopy, (D) droplet size and (E) schematic diagram of emulsification process and stabilization
mechanism HIPEs stabilized by 1–5 wt% MPPI.
Figure 1.
(
A
) Appearance of HIPEs stabilized by natural PPI and modified PPI at different con-
centrations (1–5 wt%). (
B
) Mechanism schematic diagram of HIPEs stabilized by natural PPI and
modified PPI.
The appearance was an external manifestation of the altered microstructure and
droplet size of the HIPE. In order to observe the surface details more directly, the HIPEs
were spread to a flat surface and photographed from above as in Figure 2A. The surface
of HIPEs stabilized by 1 wt% MPPI shows a macroscopic oily sheen and relatively poor
plasticity. As concentration increased, HIPEs were basically free of oil leakage, and plasticity
was enhanced by increase in their surface detail texture, which tends to be stable.
Foods 2023, 12, x FOR PEER REVIEW 5 of 16
flexible structure could effectively expand and rearrange at the oil–water interface during
the shearing process to tightly coat oil droplets and form a compact elastic interface film
[4]. Thus, HIPEs with the typical hexagonal oil drop structure of mutual extrusion and
orderly arrangement were formed [1,20], as shown in Figure 1A. In order to evaluate the
effect of MPPI concentration changes on the interfacial behavior and network structure of
HIPEs in more detail, and to reveal the dynamic emulsification process and stabilization
mechanism accordingly, the appearance and microstructure were characterized.
Figure 1. (A) Appearance of HIPEs stabilized by natural PPI and modified PPI at different concen-
trations (1–5 wt%). (B) Mechanism schematic diagram of HIPEs stabilized by natural PPI and mod-
ified PPI.
The appearance was an external manifestation of the altered microstructure and
droplet size of the HIPE. In order to observe the surface details more directly, the HIPEs
were spread to a flat surface and photographed from above as in Figure 2A. The surface
of HIPEs stabilized by 1 wt% MPPI shows a macroscopic oily sheen and relatively poor
plasticity. As concentration increased, HIPEs were basically free of oil leakage, and plas-
ticity was enhanced by increase in their surface detail texture, which tends to be stable.
Figure 2. (A) Appearance details, (B) super-resolution microscopy, (C) cryogenic scanning electron
microscopy, (D) droplet size and (E) schematic diagram of emulsification process and stabilization
mechanism HIPEs stabilized by 1–5 wt% MPPI.
Figure 2.
(
A
) Appearance details, (
B
) super-resolution microscopy, (
C
) cryogenic scanning electron
microscopy, (
D
) droplet size and (
E
) schematic diagram of emulsification process and stabilization
mechanism HIPEs stabilized by 1–5 wt% MPPI.
Foods 2023,12, 1433 6 of 15
SRM was often used to observe the state of interface distribution and network structure
composition of HIPEs [
15
]. As shown in Figure 2B, the 1 wt% MPPI-stabilized HIPEs
showed a typical irregular hexagonal structure. However, the large size of oil droplets
(green fluorescence) and the thin interfacial layer shared by adjacent droplets were prone to
the occurrence of unstable phenomena such as droplet flocculation and fusion, leading to
oil leakage. In the 2–3 wt% stabilized HIPEs, it was observed that some large-sized droplets
were surrounded by small-sized droplets, which was associated with the thickening of
the local interfacial layer. The droplet size of 4–5 wt% MPPI-stabilized HIPEs was further
reduced and uniformly distributed, and a significant enhancement of red fluorescence was
observed, representing the formation of bilayer interfacial protein membranes [21,22].
Cryo-SEM demonstrated a similar trend (Figure 2C), which was also reflected in the
droplet size results (Figure 2D). In addition to observing dimensional changes, Cryo-SEM
could be used to observe the uniform continuity of the interfacial film and the droplet
accumulation state. As the concentration increases, the droplet surface gradually becomes
smoother and the distance between adjacent droplets seems to increase, which is related to
the formation of bilayer elastic interfacial film and network structure [23].
Based on the information above, the dynamic emulsification and stabilization mecha-
nisms of 1–5 wt% MPPI in the formation of HIPEs were further discussed and elaborated,
as shown in Figure 2E. The low concentration of MPPI could not completely coat the
droplets and form a thin shared interfacial layer, resulting in the formation of unstable
HIPEs systems with large droplet size. However, at high concentrations, MPPI was capable
of producing homogeneous, continuous, and stable encapsulated droplets that even formed
bilayer interfacial films. In addition, unadsorbed MPPI forms a dense 3D network by inter-
or intra-molecular interactions entangled in the continuous phase. Consequently, droplets
did not need to share the interface film to maintain the stability of the system, and this
mechanism was also reflected in the SRM. The double-layer elastic interfacial film and
dense network structure inhibit the free motion of droplets to prevent instability [
19
], and
in addition endow HIPEs with good rheological behavior.
3.2. Rheological Behavior Analysis
3.2.1. Macrorheological Behavior
Macrorheology analyzes the stability and functional properties of HIPEs from an
interface perspective. As shown in Figure 3A, all HIPEs exhibited typical shear-thinning
behavior, with viscosity decreasing with increasing shear rate [
24
]. In general, the viscosity
of HIPEs was related to droplet size, droplet interactions, and structural changes in the
continuous phase [
25
]. The apparent viscosity of HIPEs was positively correlated with
concentration, for the following reasons. Firstly, the increase in MPPI concentration leads
to a decrease in droplet size. Secondly, the thicker interfacial layer increases the friction
between droplets and inhibits the free-flowing behavior of droplets, resulting in increased
viscosity and stability of HIPE. Finally, the unadsorbed MPPI forms a 3D network through
molecular cross-linking, which enhances the filling rate of the voids between the droplets,
increasing in the viscosity of the continuous phase and the formation of steric hindrance [
26
].
Foods 2023, 12, x FOR PEER REVIEW 7 of 16
Figure 3. (A) Strain sweeps and (B) frequency sweep of HIPEs stabilized by 1–5 wt% MPPI.
The frequency sweep reflects the viscoelastic behavior of HIPEs [27]. Figure 3B shows
that the storage modulus (Gʹ) behavior dominates in all samples during the frequency
sweep, confirming the solid elastic behavior of HIPEs. Furthermore, the storage modulus
(Gʹʹ) was almost 10 times higher than the loss modulus (Gʹʹ), indicating the formation of a
strong 3D network structure [28]. Gʹ was almost independent of frequency variation, prov-
ing that HIPEs stabilized by MPPI were strong gel-like emulsions [13]. Gʹ increased with
increasing MPPI concentration. It is possible that, with the increase in MPPI concentration,
the droplet size decreased and became uniformly distributed and closely arranged, which
enhanced the network and formed a more highly viscoelastic gel-like soft structure. Then,
the interfacial MPPI formed a more stable and dense viscoelastic interfacial film through
interaction (hydrophobic interaction) and structural rearrangement. The cross-link bridg-
ing between adsorbed and non-adsorbed MPPI and the development of interconnected
3D network structures were also responsible for the high elasticity of the emulsions [29].
In addition, non-adsorbed protein aggregated, filling the gaps of oil droplets were used
to support HIPE. These factors contribute to the formation of a gel-like stable network
structure with high elasticity.
Macrorheology, too, can be used to evaluate the potential of HIPEs as 3D printing ink
[30]. During the printing process, HIPEs utilize shear thinning properties to allow extru-
sion as well as sufficient viscosity and mechanical strength for supporting layer-by-layer
stacking structures in order to produce high-definition printing products [31].
3.2.2. Microrheological Behavior
Microrheology is a rheology that characterizes the microstructure of a sample and
obtains the viscoelastic information of the sample by tracing the mean-square displace-
ment trajectory of the Brownian motion scaering particles [22]. Therefore, in this study,
microrheology was used to study the interaction relationship between droplet–droplet,
droplet–interface protein, and droplet–continuous phase network.
If the MSD shows a straight line over time, the sample is a free-moving Newtonian
fluid. If the MSD changes with time as a characteristic curve with a plateau region, the
sample is a non-Newtonian fluid that cannot move freely (viscoelastic characteristic) [17].
The MSD curves of HIPEs which had been stabilized by MPPI (1–5 wt%) are shown in
Figure 4A, all showing viscoelastic characteristics. The initial MSD linear region was re-
lated to the viscosity of the solvent. [22]. Then, the droplets were trapped in a “cage mesh”
composed of the viscoelastic material microstructure, and the slope of the MSD curve be-
gan to decrease into a plateau phase. This stage was used to discuss the elastic behavior
of HIPEs by characterizing the structural features of the interfacial film that wraps the
droplets [32,33]. The elasticity ondex (EI) corresponds to the inverse of the height of the
MSD platform, which was the inverse of the distance required for a droplet to touch the
“cage mesh” [18]. A lower MSD plateau height represents a smaller size of the “cage
mesh” or thicker and denser HIPEs, which means less space for the droplet to move freely,
indicating higher elasticity of HIPE. As shown in Figure 4B,C, with the increase in MPPI
Figure 3. (A) Strain sweeps and (B) frequency sweep of HIPEs stabilized by 1–5 wt% MPPI.
Foods 2023,12, 1433 7 of 15
The frequency sweep reflects the viscoelastic behavior of HIPEs [
27
]. Figure 3B shows
that the storage modulus (G
0
) behavior dominates in all samples during the frequency
sweep, confirming the solid elastic behavior of HIPEs. Furthermore, the storage modu-
lus (G
00
) was almost 10 times higher than the loss modulus (G
00
), indicating the formation of
a strong 3D network structure [
28
]. G
0
was almost independent of frequency variation, prov-
ing that HIPEs stabilized by MPPI were strong gel-like emulsions [
13
]. G
0
increased with
increasing MPPI concentration. It is possible that, with the increase in MPPI concentration,
the droplet size decreased and became uniformly distributed and closely arranged, which
enhanced the network and formed a more highly viscoelastic gel-like soft structure. Then,
the interfacial MPPI formed a more stable and dense viscoelastic interfacial film through
interaction (hydrophobic interaction) and structural rearrangement. The cross-link bridg-
ing between adsorbed and non-adsorbed MPPI and the development of interconnected
3D network structures were also responsible for the high elasticity of the emulsions [
29
].
In addition, non-adsorbed protein aggregated, filling the gaps of oil droplets were used
to support HIPE. These factors contribute to the formation of a gel-like stable network
structure with high elasticity.
Macrorheology, too, can be used to evaluate the potential of HIPEs as 3D printing
ink [
30
]. During the printing process, HIPEs utilize shear thinning properties to allow
extrusion as well as sufficient viscosity and mechanical strength for supporting layer-by-
layer stacking structures in order to produce high-definition printing products [31].
3.2.2. Microrheological Behavior
Microrheology is a rheology that characterizes the microstructure of a sample and
obtains the viscoelastic information of the sample by tracing the mean-square displace-
ment trajectory of the Brownian motion scattering particles [
22
]. Therefore, in this study,
microrheology was used to study the interaction relationship between droplet–droplet,
droplet–interface protein, and droplet–continuous phase network.
If the MSD shows a straight line over time, the sample is a free-moving Newtonian
fluid. If the MSD changes with time as a characteristic curve with a plateau region, the
sample is a non-Newtonian fluid that cannot move freely (viscoelastic characteristic) [
17
].
The MSD curves of HIPEs which had been stabilized by MPPI (1–5 wt%) are shown in
Figure 4A, all showing viscoelastic characteristics. The initial MSD linear region was
related to the viscosity of the solvent. [
22
]. Then, the droplets were trapped in a “cage
mesh” composed of the viscoelastic material microstructure, and the slope of the MSD
curve began to decrease into a plateau phase. This stage was used to discuss the elastic
behavior of HIPEs by characterizing the structural features of the interfacial film that wraps
the droplets [
32
,
33
]. The elasticity ondex (EI) corresponds to the inverse of the height of
the MSD platform, which was the inverse of the distance required for a droplet to touch
the “cage mesh” [
18
]. A lower MSD plateau height represents a smaller size of the “cage
mesh” or thicker and denser HIPEs, which means less space for the droplet to move freely,
indicating higher elasticity of HIPE. As shown in Figure 4B,C, with the increase in MPPI
concentration, the MSD platform height decreased and EI increased, indicating that a
double-layer elastic interface protein film was gradually formed, and a sufficient amount
of unadsorbed proteins in the continuous phase were intertwined to form a dense 3D
network that prevents the droplets from merging or deforming, thus endowing HIPEs
with high elasticity. With time, the droplets escape from the “cage net” and the MSD curve
rises linearly again. Interestingly, the plateau phase of HIPEs stabilized at 4 wt% and
5 wt% MPPI seems to be sustained, indicating that the movement of droplets in the system
was very slow, which in turn implies the formation of highly stable HIPEs. The MVI,
which is the inverse of the slope of the MSD curve after the plateau phase, quantifies the
macroscopic viscosity at zero shear and the velocity of the droplet [
34
]. The lower the slope
is, the slower the droplet motion will be, and these phenomena correspond, physically, to
the higher macroscopic viscosity of HIPEs. The same trend was observed for MVI and EI in
Figure 4D. As MPPI concentration increased, droplet size decreased and droplet-to-droplet
Foods 2023,12, 1433 8 of 15
packing was more tightly packed, resulting in the formation of highly stable viscoelastic
HIPEs that were resistant of the deformation network system, which facilitates referencing
in 3D printing. The EI and MVI of HIPEs were consistent with the results obtained for
G
0
(Figure 3B) and the apparent viscosity (Figure 3A) in macrorheology, respectively. In
addition, the curves of EI and MVI values for both 4 wt% and 5 wt% MPPI intersected
throughout the scan, which indicates that their states are similar.
Foods 2023, 12, x FOR PEER REVIEW 8 of 16
concentration, the MSD platform height decreased and EI increased, indicating that a dou-
ble-layer elastic interface protein film was gradually formed, and a sufficient amount of
unadsorbed proteins in the continuous phase were intertwined to form a dense 3D net-
work that prevents the droplets from merging or deforming, thus endowing HIPEs with
high elasticity. With time, the droplets escape from the “cage net” and the MSD curve rises
linearly again. Interestingly, the plateau phase of HIPEs stabilized at 4 wt% and 5 wt%
MPPI seems to be sustained, indicating that the movement of droplets in the system was
very slow, which in turn implies the formation of highly stable HIPEs. The MVI, which is
the inverse of the slope of the MSD curve after the plateau phase, quantifies the macro-
scopic viscosity at zero shear and the velocity of the droplet [34]. The lower the slope is,
the slower the droplet motion will be, and these phenomena correspond, physically, to
the higher macroscopic viscosity of HIPEs. The same trend was observed for MVI and EI
in Figure 4D. As MPPI concentration increased, droplet size decreased and droplet-to-
droplet packing was more tightly packed, resulting in the formation of highly stable vis-
coelastic HIPEs that were resistant of the deformation network system, which facilitates
referencing in 3D printing. The EI and MVI of HIPEs were consistent with the results ob-
tained for G’ (Figure 3B) and the apparent viscosity (Figure 3A) in macrorheology, respec-
tively. In addition, the curves of EI and MVI values for both 4 wt% and 5 wt% MPPI inter-
sected throughout the scan, which indicates that their states are similar.
Figure 4. (A) Mean-square displacement scanning curve and (B) single mean-square displacement
single scanning curve at 1 h of HIPEs stabilized by 1–5 wt% MPPI. (C) Elasticity index and (D)
macroscopic viscosity index of HIPEs stabilized by 1–5 wt% MPPI.
3.3. Physical Stability Evaluation
Within 24 h, the sample was repeatedly scanned from boom to top, and data of
backscaered light and transmied light (i.e., BS and TSI) were collected to obtain a map
Figure 4.
(
A
) Mean-square displacement scanning curve and (
B
) single mean-square displacement
single scanning curve at 1 h of HIPEs stabilized by 1–5 wt% MPPI. (
C
) Elasticity index and (
D
) macro-
scopic viscosity index of HIPEs stabilized by 1–5 wt% MPPI.
3.3. Physical Stability Evaluation
Within 24 h, the sample was repeatedly scanned from bottom to top, and data of
backscattered light and transmitted light (i.e., BS and TSI) were collected to obtain a
map characterizing the stability of the sample [
32
]. According to the highly concentrated
characteristics of the prepared HIPEs, BS was selected to analyze and characterize the
homogeneity, droplet size, and concentration of the system, thereby judging the stability of
the system [35].
The
∆
BS scan patterns of HIPEs stabilized by MPPI (1–5 wt%) are shown in Figure 5A.
The horizontal axis represents the sample height (0 to 40 mm), the left vertical axis repre-
sents the rate of BS change, and the right vertical axis represents the scan time. The BS
value was related to the droplet size. In HIPE systems with high turbidity, the intensity of
the backscattered light follows the Mie scattering theory and decreases with the increase in
droplet size [
36
]. During the entire process, the BS fluctuation of all samples was less than
0.2%, indicating that the internal microstructure and distribution state of HIPEs stabilized
Foods 2023,12, 1433 9 of 15
by MPPI did not change drastically and had high stability. The HIPEs stabilized with
1–3 wt% MPPI had
∆
BS < 0, indicating that a certain degree of contact deformation or even
fusion occurred between the droplets of HIPEs during the test time, resulting in a decrease
in the BS value [
18
]. The BS in the 1–2 wt% groups decreased by 0.09%, indicating that
the MPPI concentration was not sufficient to completely coat the droplets. In addition, it
shows that the oil–water interface was always undergoing dynamic change and that the
adsorption and desorption of proteins continues to occur [
32
]. The BS in the 3 wt% group
decreased by 0.01%, proving that the MPPI adsorbed in the second layer played a protective
role on the MPPI closer to the oil–water interface, that the exchange between adsorbed
and non-adsorbed MPPI was reduced, and that HIPEs developed towards a more stable
direction [
36
]. The
∆
BS > 0 of the 4–5 wt% group indicated that the droplet size of HIPEs
remained stable or even decreased during the test time. As MPPI concentration increased,
the bilayer elastic interfacial film and the continuous phase network structure were grad-
ually formed, the interaction between MPPI was strengthened, the droplets were stably
encapsulated and fixed in the network, and the size and morphology would not change
substantially. Furthermore, a certain period of storage to allow interactions between MPPI
molecules to accomplish tighter adsorption and denser 3D network structure formation
resulted in a droplet size reduction. In Figure 5B, the BS values were obtained by scanning
at the same coordinates. The BS value was proportional to the protein concentration, in-
dicating that the droplet size decreased and the distribution was uniform as the protein
concentration increased.
Foods 2023, 12, x FOR PEER REVIEW 10 of 16
Figure 5. (A) Backscaered light rate (%) of change spectrum, (B) backscaered light (%) at the same
coordinates and (C) TURBISCAN stability index of HIPEs stabilized by 1–5 wt% MPPI.
TSI was used to characterize the overall stability of HIPEs, with high TSI values rep-
resenting system instability [37]. The lowest TSI values occurred at HIPEs stabilized by 5
wt% MPPI, demonstrating that thy had the highest stability. Furthermore, the higher vis-
cosity and elasticity of HIPEs inhibited droplet migration and improved stability, which
was consistent with the results of rheological analysis.
3.4. Application of HIPEs in 3D Printing
Based on the above results on microstructure and rheological properties, the poten-
tial of HIPEs in 3D printing was further discussed. All the HIPEs were smoothly extruded
from the nozzle during printing owing to their shear thinning properties [31].
As illustrated in Figure 6, cylinders printed with HIPEs stabilized by 1–2 wt% MPPI
showed poor shape fidelity, with an obvious collapse at the top, trapezoidal sides, sagging
structures, obvious layer fusion, and even faults. As the protein concentration increased,
the degree of collapse decreased, and the stacked layers were more easily distinguished,
but there were still some minor defects. The 5 wt% group shape had high definition and
high resolution, the collapse largely disappeared, the surface was smooth, and the struc-
ture a certain degree of self-supporting ability. The actual printed shape was close to the
modeled shape, and the details were perfect. This change was associated with an improve-
ment in its rheological behavior, as rheology was a bridge between edible inks and their
3D printability [35].
Figure 5.
(
A
) Backscattered light rate (%) of change spectrum, (
B
) backscattered light (%) at the same
coordinates and (C) TURBISCAN stability index of HIPEs stabilized by 1–5 wt% MPPI.
Foods 2023,12, 1433 10 of 15
TSI was used to characterize the overall stability of HIPEs, with high TSI values
representing system instability [
37
]. The lowest TSI values occurred at HIPEs stabilized
by 5 wt% MPPI, demonstrating that thy had the highest stability. Furthermore, the higher
viscosity and elasticity of HIPEs inhibited droplet migration and improved stability, which
was consistent with the results of rheological analysis.
3.4. Application of HIPEs in 3D Printing
Based on the above results on microstructure and rheological properties, the potential
of HIPEs in 3D printing was further discussed. All the HIPEs were smoothly extruded
from the nozzle during printing owing to their shear thinning properties [31].
As illustrated in Figure 6, cylinders printed with HIPEs stabilized by 1–2 wt% MPPI
showed poor shape fidelity, with an obvious collapse at the top, trapezoidal sides, sagging
structures, obvious layer fusion, and even faults. As the protein concentration increased,
the degree of collapse decreased, and the stacked layers were more easily distinguished, but
there were still some minor defects. The 5 wt% group shape had high definition and high
resolution, the collapse largely disappeared, the surface was smooth, and the structure a
certain degree of self-supporting ability. The actual printed shape was close to the modeled
shape, and the details were perfect. This change was associated with an improvement
in its rheological behavior, as rheology was a bridge between edible inks and their 3D
printability [35].
Foods 2023, 12, x FOR PEER REVIEW 11 of 16
Figure 6. The visual appearance of 3D printed samples of HIPEs stabilized by 1–5 wt% MPPI.
3.5. Processing and Environmental Stress
3.5.1. Centrifugation Stability
The stability of HIPEs was further evaluated by centrifugation, storage, heating, and
freeze–thaw treatments. HIPEs with good centrifugation stability were beneficial to main-
taining structure and properties in practical processing. The appearance of HIPEs stabi-
lized by MPPI after centrifugation is shown in Figure 7A. Some degree of oil leakage oc-
curred in the 1–2 wt% group but was not observed in the 3–5 wt% group. The interfacial
film formed by the low concentration of MPPI was thin and could not resist the forced
collision under the centrifugal action, resulting in the rupture of the interfacial film and
oil being removed (Figure 7D). With the increase in MPPI concentration, the thickness of
the interfacial film was superimposed onto the double layer, and the intermolecular inter-
action was strengthened to form a network structure with a mechanical barrier effect,
which doubles the protection of the droplets to avoid oil breakage. In addition, the high
centrifugation stability enables the manufacture of ultra-high internal phase emulsions by
centrifugation. Zhang et al. [26] used the centrifugation method to produce an ultra-high
internal phase emulsion with good stability under gliadin/sodium carboxymethyl cellu-
lose complex particles (GCCPs) of a relatively low concentration (0.3–1.5 wt%) of an emul-
sifier by adjusting the centrifugal speed (2000–12,000 r/min). Its internal phase could reach
a maximum of 90.28%. Furthermore, it was observed that the water phase at the boom
decreased with the increase in MPPI concentration after centrifugation, indicating that the
HIPEs system had a stronger ability to bind water, which was related to the interaction
between molecules and the formation of a dense network structure. In addition, the in-
creased viscosity of HIPEs may have limited the precipitation of the water layer during
centrifugation [30]. High concentrations of MPPI enhanced the centrifugation stability of
HIPEs. However, it was found that the boom aqueous layer after centrifugation became
gradually turbid with the increasing MPPI concentration. The adsorbed MPPI would un-
dergo a certain degree of unfolding at the interface, resulting in the exposure of hydro-
phobic groups and the interaction between molecules through hydrogen bonding to form
a more elastic interface film. The result of this is that MPPI could be adsorbed tightly and
stably and would not be easily removed from the system [4]. Therefore, the centrifuged
material may comprise more of the unadsorbed protein thrown out of the emulsion sys-
tem. First, it was demonstrated that some unadsorbed proteins were indeed used to form
a network structure in the continuous phase in order to provide steric hindrance and con-
fine oil droplets, thereby enhancing the stability of HIPEs. Second, it was not necessary to
increase the protein concentration on the basis of 5 wt% in order to further improve the
performance and stability of HIPEs because the contribution rate was very low.
Figure 6. The visual appearance of 3D printed samples of HIPEs stabilized by 1–5 wt% MPPI.
3.5. Processing and Environmental Stress
3.5.1. Centrifugation Stability
The stability of HIPEs was further evaluated by centrifugation, storage, heating,
and freeze–thaw treatments. HIPEs with good centrifugation stability were beneficial to
maintaining structure and properties in practical processing. The appearance of HIPEs
stabilized by MPPI after centrifugation is shown in Figure 7A. Some degree of oil leakage
occurred in the 1–2 wt% group but was not observed in the 3–5 wt% group. The interfacial
film formed by the low concentration of MPPI was thin and could not resist the forced
collision under the centrifugal action, resulting in the rupture of the interfacial film and
oil being removed (Figure 7D). With the increase in MPPI concentration, the thickness
of the interfacial film was superimposed onto the double layer, and the intermolecular
interaction was strengthened to form a network structure with a mechanical barrier effect,
which doubles the protection of the droplets to avoid oil breakage. In addition, the high
centrifugation stability enables the manufacture of ultra-high internal phase emulsions
by centrifugation. Zhang et al. [
26
] used the centrifugation method to produce an ultra-
high internal phase emulsion with good stability under gliadin/sodium carboxymethyl
cellulose complex particles (GCCPs) of a relatively low concentration (0.3–1.5 wt%) of
an emulsifier by adjusting the centrifugal speed (2000–12,000 r/min). Its internal phase
Foods 2023,12, 1433 11 of 15
could reach a maximum of 90.28%. Furthermore, it was observed that the water phase
at the bottom decreased with the increase in MPPI concentration after centrifugation,
indicating that the HIPEs system had a stronger ability to bind water, which was related
to the interaction between molecules and the formation of a dense network structure. In
addition, the increased viscosity of HIPEs may have limited the precipitation of the water
layer during centrifugation [
30
]. High concentrations of MPPI enhanced the centrifugation
stability of HIPEs. However, it was found that the bottom aqueous layer after centrifugation
became gradually turbid with the increasing MPPI concentration. The adsorbed MPPI
would undergo a certain degree of unfolding at the interface, resulting in the exposure of
hydrophobic groups and the interaction between molecules through hydrogen bonding to
form a more elastic interface film. The result of this is that MPPI could be adsorbed tightly
and stably and would not be easily removed from the system [
4
]. Therefore, the centrifuged
material may comprise more of the unadsorbed protein thrown out of the emulsion system.
First, it was demonstrated that some unadsorbed proteins were indeed used to form a
network structure in the continuous phase in order to provide steric hindrance and confine
oil droplets, thereby enhancing the stability of HIPEs. Second, it was not necessary to
increase the protein concentration on the basis of 5 wt% in order to further improve the
performance and stability of HIPEs because the contribution rate was very low.
Foods 2023, 12, x FOR PEER REVIEW 12 of 16
Figure 7. Appearance of HIPEs stabilized by 1–5 wt% MPPI after (A) centrifugation, (B) storage,
and (C) heat treatment. (D) Optical microscope of HIPEs stabilized by 1–5 wt% MPPI before and
after centrifugation, storage, and heat treatment.
3.5.2. Storage Stability
No samples showed any significant changes in appearance and microstructure com-
pared with fresh samples after 90 days of storage, as shown in Figure 7B,D, demonstrating
the high storage stability of HIPEs stabilized by MPPI, which has the ability to improve
shelf life in food applications [16]. Although the low concentration of MPPI-stabilized
HIPEs had a single-layer interface film shared by adjacent droplets, limited ability to coat
droplets, and poor centrifugal stability, it could still remain stable for a long time without
the presence of a strong external force. In addition to being able to maintain structural and
property stability, Zhang et al. [38] demonstrated that HIPEs stabilized by quinoa protein
isolate exhibit increased storage modulus and interfacial layer thickness after storage, pos-
sibly because of enhanced protein–oil droplet and protein–protein interactions.
3.5.3. Thermal Stability
Thermal stability may affect the application of HIPEs in food processing, we conse-
quently investigated the effect of heating on the appearance, microstructure, and rheolog-
ical properties of HIPEs. The appearance of the HIPEs remained stable after heating, with
some tiny pores, which were caused by the gas escaping because of heating (Figure 7C).
Droplet size and microstructure did not change significantly in Figure 7D. The response
of G’ and G’’ to temperature change at 1% strain and frequency of 1 Hz are shown in
Figure 8. The G’ of HIPEs stabilized by various MPPI concentrations remained stable dur-
ing heating, indicating its excellent thermal stability. The G’ of all HIPEs increased signif-
icantly during cooling, owing both to the formation of aggregates after denaturation and
the unfolding of MPPI at high temperature, which supported the network structure and
improved the mechanical strength [39]. Similar phenomena had also been observed in
studies of other material-stabilized HIPEs such as gliadin [26] and gelatin [19].
Figure 7.
Appearance of HIPEs stabilized by 1–5 wt% MPPI after (
A
) centrifugation, (
B
) storage, and
(
C
) heat treatment. (
D
) Optical microscope of HIPEs stabilized by 1–5 wt% MPPI before and after
centrifugation, storage, and heat treatment.
3.5.2. Storage Stability
No samples showed any significant changes in appearance and microstructure com-
pared with fresh samples after 90 days of storage, as shown in Figure 7B,D, demonstrating
the high storage stability of HIPEs stabilized by MPPI, which has the ability to improve
shelf life in food applications [
16
]. Although the low concentration of MPPI-stabilized
HIPEs had a single-layer interface film shared by adjacent droplets, limited ability to coat
droplets, and poor centrifugal stability, it could still remain stable for a long time without
Foods 2023,12, 1433 12 of 15
the presence of a strong external force. In addition to being able to maintain structural
and property stability, Zhang et al. [
38
] demonstrated that HIPEs stabilized by quinoa pro-
tein isolate exhibit increased storage modulus and interfacial layer thickness after storage,
possibly because of enhanced protein–oil droplet and protein–protein interactions.
3.5.3. Thermal Stability
Thermal stability may affect the application of HIPEs in food processing, we conse-
quently investigated the effect of heating on the appearance, microstructure, and rheological
properties of HIPEs. The appearance of the HIPEs remained stable after heating, with some
tiny pores, which were caused by the gas escaping because of heating (Figure 7C). Droplet
size and microstructure did not change significantly in Figure 7D. The response of G0and
G
00
to temperature change at 1% strain and frequency of 1 Hz are shown in Figure 8. The
G
0
of HIPEs stabilized by various MPPI concentrations remained stable during heating,
indicating its excellent thermal stability. The G’ of all HIPEs increased significantly during
cooling, owing both to the formation of aggregates after denaturation and the unfolding
of MPPI at high temperature, which supported the network structure and improved the
mechanical strength [
39
]. Similar phenomena had also been observed in studies of other
material-stabilized HIPEs such as gliadin [26] and gelatin [19].
Foods 2023, 12, x FOR PEER REVIEW 13 of 16
Figure 8. The response of storage modulus and loss modulus to temperature change of HIPEs sta-
bilized by 1–5 wt% MPPI.
3.5.4. Freeze–Thaw Stability
As shown in Figure 9, HIPEs stabilized by MPPI were completely demulsified after
one freeze–thaw cycle, demonstrating their poor freeze–thaw stability. First, a large num-
ber of ice crystals formed during the freezing pierced the interfacial film, resulting in se-
vere oil leaching after thawing [16]. Furthermore, the droplets were forced to aggregate in
the unfrozen aqueous phase, causing the droplets to coalesce [19]. According to the exper-
iments in the work of Xu et al. [40], HIPEs stabilized by rapeseed protein isolate were also
strongly demulsified after a single freeze–thaw cycle. Although demulsification occurred
in all HIPEs, the degree of phase separation appeared to decrease as the MPPI concentra-
tion increased. The thicker interfacial film and dense network formed at higher MPPI con-
centrations slightly inhibited droplet coalescence and piercing behavior.
Figure 9. Appearance of HIPE stabilized by 1–5 wt% MPPI before and after one freeze–thaw cycle.
In conclusion, HIPEs stabilized by MPPI of high concentrations had higher viscoe-
lasticity and plasticity, beer stability (centrifugation, storage, and heating), and a wider
range of applications.
4. Conclusions
This study elucidates the emulsification process and stabilization mechanism of
MPPI-stabilized HIPEs by discussing the interfacial distribution state and network struc-
ture composition. It additionally demonstrates that the viscoelasticity and stability
Figure 8.
The response of storage modulus and loss modulus to temperature change of HIPEs
stabilized by 1–5 wt% MPPI.
3.5.4. Freeze–Thaw Stability
As shown in Figure 9, HIPEs stabilized by MPPI were completely demulsified after
one freeze–thaw cycle, demonstrating their poor freeze–thaw stability. First, a large number
of ice crystals formed during the freezing pierced the interfacial film, resulting in severe
oil leaching after thawing [
16
]. Furthermore, the droplets were forced to aggregate in
the unfrozen aqueous phase, causing the droplets to coalesce [
19
]. According to the
experiments in the work of Xu et al. [
40
], HIPEs stabilized by rapeseed protein isolate
were also strongly demulsified after a single freeze–thaw cycle. Although demulsification
occurred in all HIPEs, the degree of phase separation appeared to decrease as the MPPI
concentration increased. The thicker interfacial film and dense network formed at higher
MPPI concentrations slightly inhibited droplet coalescence and piercing behavior.
Foods 2023,12, 1433 13 of 15
Foods 2023, 12, x FOR PEER REVIEW 13 of 16
Figure 8. The response of storage modulus and loss modulus to temperature change of HIPEs sta-
bilized by 1–5 wt% MPPI.
3.5.4. Freeze–Thaw Stability
As shown in Figure 9, HIPEs stabilized by MPPI were completely demulsified after
one freeze–thaw cycle, demonstrating their poor freeze–thaw stability. First, a large num-
ber of ice crystals formed during the freezing pierced the interfacial film, resulting in se-
vere oil leaching after thawing [16]. Furthermore, the droplets were forced to aggregate in
the unfrozen aqueous phase, causing the droplets to coalesce [19]. According to the exper-
iments in the work of Xu et al. [40], HIPEs stabilized by rapeseed protein isolate were also
strongly demulsified after a single freeze–thaw cycle. Although demulsification occurred
in all HIPEs, the degree of phase separation appeared to decrease as the MPPI concentra-
tion increased. The thicker interfacial film and dense network formed at higher MPPI con-
centrations slightly inhibited droplet coalescence and piercing behavior.
Figure 9. Appearance of HIPE stabilized by 1–5 wt% MPPI before and after one freeze–thaw cycle.
In conclusion, HIPEs stabilized by MPPI of high concentrations had higher viscoe-
lasticity and plasticity, beer stability (centrifugation, storage, and heating), and a wider
range of applications.
4. Conclusions
This study elucidates the emulsification process and stabilization mechanism of
MPPI-stabilized HIPEs by discussing the interfacial distribution state and network struc-
ture composition. It additionally demonstrates that the viscoelasticity and stability
Figure 9. Appearance of HIPE stabilized by 1–5 wt% MPPI before and after one freeze–thaw cycle.
In conclusion, HIPEs stabilized by MPPI of high concentrations had higher viscoelas-
ticity and plasticity, better stability (centrifugation, storage, and heating), and a wider range
of applications.
4. Conclusions
This study elucidates the emulsification process and stabilization mechanism of MPPI-
stabilized HIPEs by discussing the interfacial distribution state and network structure
composition. It additionally demonstrates that the viscoelasticity and stability (centrifuga-
tion, storage, and heat stability) of HIPEs improve with the increase in MPPI concentration.
This effect was attributed to the formation of a bilayer elastic interfacial membrane and
three-dimensional network structure, as well as to enhanced protein intra- and intermolec-
ular interactions. The HIPEs constructed in this study may be useful in developing highly
viscous and malleable products (e.g., 3D printing or fat simulants) for applications in the
food industry. However, in view of the poor freeze–thaw stability and the limitations
imposed by the protein’s own structure, further studies are needed to circumvent the dis-
advantages and amplify the advantages of this method in order to expand the application
of HIPEs in a wider range of industries.
Author Contributions:
J.Z.: Conceptualization, Formal analysis, Investigation, Writing—Original
Draft, Visualization. S.Z.: Investigation, Software. L.L.: Formal analysis, Investigation, Visualiza-
tion. B.K.: Supervision, Project administration, Project administration. H.L.: Supervision, Funding
acquisition. All authors have read and agreed to the published version of the manuscript.
Funding:
This study was supported by the National Natural Science Foundation of China (32202088)
and Natural Science Foundation of Heilongjiang Province (YQ2022C023).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available in [high internal phase
emulsions stabilized by ultrasound com-bined with pH-shifting modified pea protein isolate: Micro-
morphology, rheology, and physical stability].
Conflicts of Interest: The authors declare no conflict of interest.
Foods 2023,12, 1433 14 of 15
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