LWT - Food Science and Technology 167 (2022) 113820

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LWT
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Yellow horn as an alternative source of plant-based protein: The effects of

high-intensity ultrasonication treatment on its physicochemical properties
and emulsifying properties
Cuiping Yu a, b, *, Sihui Li b, Shuang Sun b, Huijia Yan b, Henan Zou b
a
College of Wildlife and Protected Area, Northeast Forestry University, Harbin, 150040, China
b
School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, 116034, China

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

Keywords: In this study, the impacts of high-intensity ultrasonication treatment (0, 150, 300, 450, 600 W) on the physi­
Ultrasonication cochemical and functional properties of yellow horn protein (YHP) were investigated. YHP dispersion (10 mg/
Emulsifying properties mL) was treated with ultrasonication (20 kHz) at 0, 150, 300, 450, and 600 W for 10 min. The results showed
Physicochemical properties
that the physicochemical properties of YHP were improved with the increase of ultrasonic power, manifested by
Functional properties
Yellow horn proteins
the increased surface hydrophobicity, together with the reduced interfacial tension. After ultrasonication, the
solubility of YHP increased from 43.1% to 73.0%. In addition, with the increase of ultrasonic power, the
emulsifying properties of YHP were also improved, including emulsifying activity index, emulsifying stability
index, and percentage of adsorbed protein. After ultrasonication treatment, the particle size and turbidity of YHP
dispersion were reduced, so the emulsion particles stabilized by YHP became smaller and the distribution was
more uniform. As a result, emulsions stabilized by ultrasonicated YHP had higer storage stability. The results
indicated that ultrasonication is a valuable method for improving the physicochemical and emulsifying prop­
erties of YHP, which provided a theoretical basis for YHP as a natural plant-based functional component in the
food processing.

1. Introduction protein for humans.

Yellow horn (Xanthoceras sorbifolium Bunge) is a peculiar tree species
in China, belongs to the family Sapindaceae, and the monotypic genus
Xanthoceras (Yu et al., 2012). It is a woody deciduous shrub native to the
northern part of China (Guo et al., 2013). The trees can survive in a
desert, semi-arid and arid environment, even at extremely low temper­
atures below − 40 ◦ C. Yellow horn trees are traditionally cultivated for
soil conservation and their oilseeds. The trees bear pods with seeds rich
in oil containing unsaturated fatty acids such as oleic and linoleic acid
(Zhang et al., 2010). The oil has been applied for food, cosmetics and
biodiesel production. However, Yellow horn seed oil manufacturing
produces large amounts of by-products, including proteins, phospho­
lipids and saponins. The defatted seed meals are rich in protein (35%)
with high essential amino acid ratios, especially lysine and isoleucine
(Venegas-Calerón, Ruíz-Méndez, Martínez-Force, Garcés, & Salas,
2017). The meals are mainly used as animal feed, fertilizer or directly
discarded. However, this protein source has the potential to be a dietary
High-intensity ultrasonication (HIUS, 16–100 kHz, intensity in the
range of 10–1000 W cm− 2) is considered a clean, environment-friendly
technology which has been applied from laboratory to industrial field in
pharmaceutical, cosmetics and food industry. The effects of HIUS
treatment on the protein system is primarily due to the acoustic cavi­
tation phenomenon. Ultrasonic oscillations at the probe tip to produce
periodic compression and expansion in a liquid system, thereby forming
cavitation bubbles, which periodically increase rapidly and then
collapse violently. The violent collapse of cavitation bubbles will
generate extreme temperature and pressure, and further generate
intense shear forces, shock waves, turbulence and cavitation forces,
thereby changing physicochemical and functional properties of treated
protein solution (Soria & Villamiel, 2010).
Generally, whether food proteins can be used as food ingredients
mainly depends on their functional properties. As for food protein
modification, HIUS treatment has been applied to modify the structural
and physicochemical characteristics of plant and animal proteins to

* Corresponding author. College of Wildlife and Protected Area, Northeast Forestry University, Harbin, 150040, China.
E-mail address: yucuiping1987@sina.com (C. Yu).

https://doi.org/10.1016/j.lwt.2022.113820
Received 13 August 2021; Received in revised form 20 July 2022; Accepted 25 July 2022
Available online 10 August 2022
0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
C. Yu et al.
obtain the desired functional properties which are lacking in native
proteins. It was reported that HIUS treatment improved foaming prop­
erties, solubility and emulsifying properties of whey protein. HIUS
treatment can modify the physicochemical properties of edible animal
and plant proteins (O’Sullivan, Murray, Flynn, & Norton, 2016). Gelling
characteristics of quinoa seed protein together with the emulsifying
capacity and stability, water and oil binding capacities have also been
improved by the application of HIUS treatment (Mir, Riar, & Singh,
2019). Similarly, foaming, emulsifying and gel properties of chickpea
protein improved after sonication which were probably due to the
structural changes and decreased particle size (Wang et al., 2020). HIUS
treatment caused partial unfolding of pea protein isolate, resulting in
reduced particle size and improved foaming properties (Xiong et al.,
2018). As for the ultrasonic modification of oilseed proteins, soybean,
peanut and rapeseed proteins have been studied (Li, Cheng, et al., 2020;
O’Sullivan, Park, & Beevers, 2016; Zhang et al., 2014). However, little
information is available about preparation and modification of proteins
from the by-products of yellow horn seed oil processing.
In this study, the authors prepared proteins from the defatted yellow
horn seed meals, and investigated the impacts of HIUS treatment on
physicochemical and functional properties of YHP, which are very
important for industrial processing. This research would result in better
understanding of ultrasonic physical effects on YHP and highlight the
potential source of proteins from yellow horn seed oil processing by-
products for functional food ingredients.
2. Materials and methods
2.1. Preparation of YHP
YHP was prepared as previously reported (Wu, Hua, Chen, Kong, &
Zhang, 2015). Defatted YHP flour was mixed with distilled water (1:10,
w/v) and the suspension was adjusted to pH 7.0 using 2 M NaOH. The
mixture was stirred at 25 ◦ C for 2 h, and then centrifuged at 10,000g for
30 min at 4 ◦ C.The supernatant was adjusted to pH 4.5 (isoelectric point
of YHP) with 1 M HCl, and then centrifuged at 10,000g for 20 min. After
centrifugation, the precipitate was dissolved in deionized water, and the
pH was adjusted to neutral with 2 M NaOH. The protein concentration
was determined using the biuret method, and then YHP dispersion was
diluted to 10 mg/mL with deionized water. The prepared YHP was
stored at 4 ◦ C.
2.2. HIUS treatment
YHP dispersion was ultrasonicated at 20 kHz using an ultrasonic
generator (Scientz-IID, Ningbo Scientz Biotechnology Co. Ltd., Ningbo,
China) with a 0.6 mm diameter titanium probe and a temperature
measuring probe. YHP dispersion (10.0 mg/mL, 100 mL) was placed in a
150 mL flask, the titanium probe was immersed in the dispersion 2 cm.
The flask was maintained in an ice-water bath to ensure the temperature
below 20 ◦ C during ultrasonication (Nazari, Mohammadifar,
Shojaee-Aliabadi, Feizollahi, & Mirmoghtadaie, 2018). YHP dispersion
was ultrasonicated at 0, 150, 300, 450, and 600 W for 10 min (pulse
duration: on-time, 2 s; off-time, 2 s). After ultrasonication treatment, all
the samples were stored at 4 ◦ C for future use.
2.3. Circular dichroism (CD) measurements
The circular dichroism (CD) was recorded in the far UV range
(190–260 nm) by using Jasco-810 spectropolarimeter (Jasco Interna­
tional, Tokyo, Japan) with a spectral resolution of 0.5 nm at room
temperature (Hu et al., 2013). YHP dispersion was diluted to 0.01
mg/mL with deionized water. The response, scan rate, and bandwidth
were set to 0.25 s, 20 nm/min and 1.0 nm, respectively.
2
LWT 167 (2022) 113820
2.4. Surface hydrophobicity (H0) measurements
YHP dispersion (4 mL) was mixed with 40 μL of 1-anilino-8-naphtha­
lene-sulfonate (8 mM) solution. The prepared dispersion was kept in the
dark. The H0 of YHP dispersion was measured by a fluorescence
photometer (F-2700, Hitachi, Japan) with emission and excitation slits
of 5 nm. The excitation wavelength was set to 390 nm, and the emission
spectrum was measured from 400 to 650 nm. The value of the H0 was
calculated based on the initial slope of the FI relative to the protein
concentration.
2.5. Fluorescence spectra measurements
YHP dispersion (0.1 mg/mL) was analyzed at 280 nm excitation
wavelength and 327–360 nm emission range using a fluorescence
spectrophotometer (F-2700, Hitachi, Japan) with a constant 5 nm slit for
both excitation and emission (Bonomi, Mora, Pagani, & Iametti, 2004).
The scanning speed was 1500 nm/min.
2.6. Particle size and zeta potential measurements
The HIUS-treated YHP dispersion was diluted to 1 mg/mL and stirred
at 25 ◦ C for 1 h and centrifuged at 2,000g for 1min. The particle size and
zeta potential of supernatant were determined by photon correlation
spectroscopy (Zetasizer 3000, Malvern Instruments, Southboro, Mass,
USA) as reported (C. Yu et al., 2018).
2.7. Protein solubility and turbidity measurements
YHP dispersion (10 mg/mL) centrifuged at 10,000g for 15 min at
20 ◦ C. The concentration of the supernatant was measured by the biuret
method. The absorbance of the supernatant was recorded at 540 nm. The
protein solubility was calculated as the percentage of protein content in
the supernatant relative to the protein content in the dispersion before
centrifugation.
Absorbance of YHP dispersion (1.0 mg/mL) was determined at 660
nm as an indicator of turbidity (Benjakul, Visessanguan, Ishizaki, &
Tanaka, 2001).
2.8. Rheological properties measurements
The rheological measurements were analyzed at room temperature
by a stress-controlled rotational rheometer with parallel plates (Dis­
covery HR-1, TA Instruments, New Castle, USA, with 60 mm diameter
and 1 mm gap). The viscosity properties of proteins were measured for
shear rate from 0.1 to 100 s− 1. Each sample was measured in triplicate.
The power law equation as follows was used to calculate the value of the
viscosity coefficients (k, Pa⋅s) and the flow behavior index (n).
τ=k×δn (1)
where τ is the shear stress (Pa); δ is the shear rate (s− 1).
2.9. Preparation of emulsion
Soybean oil and YHP dispersion (10 mg/mL) were mixed at 1:9 ratio
(v/v). The mixture was homogenized by a high-speed homogenizer
(model T25, IKA Labortechnik, Staufen, Germany) at 12,000 rpm for 1
min. Afterwards, the dispersion was passed through high-pressure ho­
mogenizer (Panda Plus 2000; GEA Niro Soavi, Parma, Italy) at 40 MPa
for 3 cycles.
2.10. Measurements of emulsifying activity index (EAI) and emulsifying
stability index (ESI)
A modified method was used to measure the EAI and ESI of YHP
C. Yu et al.
(Klompong, Benjakul, Kantachote, & Shahidi, 2007). Aliquots of 50 μL
freshly prepared emulsions were taken from the bottom of the beaker
and dispersed into 5 mL of a 0.1% sodium dodecyl sulfate solution. The
absorbance of the diluted emulsions was determined at 500 nm. The
calculation formulas of EAI and ESI are as follows:
/ 2 × 2.303 × A0 × DF
EAI ​ (m2 g) = (2)
c × ∅ × (1 − θ) × 10000
ESI ​ (min) =
​ A0 × Δt
(3)
A0 − A10
where A10 and A0 represent the absorbance at 500 nm after 10 min and
0 min, respectively; DF is the dilution factor; c is the concentration of
samples (g/mL), ∅ is optical path, and θ is the oil volume fraction; Δt is
the time interval.
2.11. Measurements of droplet size distribution in emulsions
The droplet size of YHP stabilized emulsions was determined by
acoustic/electroacoustic spectrometer DT-1202 (Dispersion Technol­
ogy, Inc., Bedford Hills, NY, USA) as previously reported (Cha et al.,
2019).
2.12. Optical microscopy analysis
An optical microscope (Leica DM2500, Leica Microsystems GmbH,
Wetzlar, Germany) was used to observe the microstructure of YHP sta­
bilized emulsion according to a previous report (Zhao, Wu, Xing, Xu, &
Zhou, 2019). Microimages were recorded at 200× magnification.
2.13. Measurements of adsorbed proteins (AP%)
AP% was determined using a previous method (Liang & Tang, 2013)
with a minor modification. The YHP stabilized emulsion was centrifuged
at 13,000g for 15 min. The aqueous phase at the bottom of the tube was
removed using a syringe and filtered through a 0.22 μm filter. After­
wards, the Lowry method was used to determine the protein concen­
tration of filtered aqueous phase (Cf). The initial YHP dispersion used to
stabilize the emulsion was subjected to centrifugation under the same
conditions. And then the protein concentration in supernatants (Cs) was
determined. The calculation formula of AP% was as follows:
Cs − Cf
AP ​ (%) = × 100% (4)
C0
where C0 is initial protein content.
2.14. Measurements of creaming index (CI)
The creaming index of YHP stabilized emulsion was determined ac­
cording to a previous report (Shen & Tang, 2012). Fresh emulsion was
added to clear glass bottles and stored vertically for 16 days at 25 ◦ C. Hs
stands for the height of cream layer and Ht represents the total heights of
emulsions. The value of Hs was determined every 4 days. CI was
calculated according to the following formula as:
Hs
CI (%) = × 100% (5)
Ht
2.15. Measurements of interfacial tension
Soybean oil was completely remove surface active impurities using
Florisil (Gaonkar, 1989). The mixture containing 100 mL of soybean oil
and 4 g of Florisil was stirred for 3 h and centrifuged at 8000g for 20 min.
The supernatant oil was obtained and mixed with 4 g Florisil, and then
proceeded the above steps including stirring and centrifugation. The
process was repeated 4 times and the purified oil was obtained. A
3
LWT 167 (2022) 113820
contact angle goniometer (DSA 25, Krüss GmbH, Germany) equipped
with a 1.056-mm-diameter needle was used. The pendant-drop method
was used to measure oil-water interfacial tension of YHP dispersion at
20 ◦ C for 12,000 s.
2.16. Statistical analysis
All measurements were carried out in triplicate. All data was re­
ported as mean value ± standard deviation (SD). Statistical analysis was
performed using ANOVA and Duncan’s test to analyze the significance of
the results (P < 0.05). Origin software was used to plot graphs.
3. Results and discussion
3.1. Effects of HIUS treatment on the conformational structure of YHP
3.1.1. Secondary structure analysis of HIUS-treated YHP
The CD spectroscopy of native and ultrasonicated protein dispersion
was determined. The ratios of α-helix, β-sheet, β-turn and random coil
were displayed in Table 1. With the increase of ultrasonic power
(150–600 W), α-helix content showed an increasing tendency, while the
content of β-sheet decreased gradually. In addition, the content of β-turn
increased first and then fluctuated within a narrow range. The content of
random coil barely changed. This change might be due to the intense
shear, turbulence, and cavitation force generated by HIUS treatment,
which facilitated interconversions of different secondary structures in
the proteins. It was reported that the secondary structure of protein not
only depends on the composition of amino acids but also on the inter­
action between different parts of the protein molecule (Hu et al., 2013).
Therefore, another possible explanation may be that HIUS treatment
unfolded the protein molecules and exposed the buried hydrophobic
regions, which lead to the reduction and fracture of intramolecular
hydrogen bonds (Li et al., 2021). Likewise, Hu et al. reported that
ultrasonication increased the α-helix content but decreased β-sheet
content of soy protein isolate (Hu et al., 2013). Nevertheless, contra­
dictory findings also be reported that HIUS treatment increased the
β-sheet content and decreased α-helix content of black bean proteins
(Jiang et al., 2014). Besides, Xiong et al. found that HIUS treatment had
almost no effect on the secondary structure of pea protein (Xiong et al.,
2018). This difference might be caused by different factors, such as
protein properties, structure type and ultrasonic conditions.
3.1.2. H0 of HIUS-treated YHP
H0 reflected the changes in the number of hydrophobic amino acid
residues on the surface of protein molecules (Wang et al., 2021), which
were closely related to the solubility and emulsifying ability. Fig. 1A
provides H0 of YHP dispersion as a function of HIUS power. Compared
with the control group, H0 of YHP significantly increased after HIUS
treatment from 150 to 600 W. Arzeni et al. showed that the hydrophobic
regions of native YHP were located inside the molecules, and ultra­
sonication induced the unfolding of the protein molecules, resulting in
more hydrophobic groups being exposed (Arzeni et al., 2012). This was
in agreement with published reports which indicated that
Table 1
Effect of different HIUS power on the secondary structure of YHP.
Ultrasonic power Secondary structure (%)
α-helix β-sheet β-turn Random coil
0W 31.7 ± 0.9a 42.3 ± 1.6a 3 ± 1.0a 22.9 ± 0.6a
150 W 36.1 ± 7.4ab 28 ± 4.6a 10.5 ± 0.4b 25.4 ± 2.7a
300 W 43.3 ± 4.4abc 8.8 ± 4.2a 20.2 ± 1.3c 27.9 ± 1.7a
450 W 48.8 ± 0.5bc 3.4 ± 1.8b 21.4 ± 1.1c 26.4 ± 1.2a
600 W 51.9 ± 1.7c Not detected 22.4 ± 0.6c 25.6 ± 1.2a
Note: Different lowercase letters within the table represent significant differ­
ences at P < 0.05.
C. Yu et al. LWT 167 (2022) 113820

Fig. 1. Effect of different HIUS power on the structure of YHP. Surface hydrophobicity (A), intrinsic emission fluorescence spectroscopy (B), particle size distribution
(C) and zeta potential (D).
Note: Different lowercase letters represent significant differences at P < 0.05.

ultrasonication increased the H0 of myofibrillar protein (Zhang,
Regenstein, Zhou, & Yang, 2017) and rapeseed protein (Li, Cheng, et al.,
2020).
3.1.3. Intrinsic fluorescence spectroscopy of HIUS-treated YHP
The intrinsic fluorescence spectroscopy reflects the changes in the
microenvironment of the tryptophan and tyrosine residues of proteins
(Liu et al., 2010). The intrinsic fluorescence emission spectra of YHP are
displayed in Fig. 1B. The wavelength of the fluorescence emission peak
of native and ultrasonicated YHP appeared at 406.5 nm. Compared with
native YHP, fluorescence intensity of the YHP decreased after HIUS
treatment. This phenomenon was due to that HIUS induced unfolding of
protein molecules, resulting in an increase in the amount of chromo­
phores exposed to the aqueous phase (Xiong et al., 2018). These results
are consistent with studies on peanut proteins (Zhang et al., 2014) and
pea proteins (Xiong et al., 2018).
3.1.4. Particle size of HIUS-treated YHP
The particle size reflects the degree of protein agglomeration,
disaggregation and flocculation (Arzeni et al., 2012). The effect of
ultrasonication on particle size distribution of YHP is displayed in
Fig. 1C. As the ultrasonic power increased from 0 W to 600 W, the
particle size of YHP decreased significantly. HIUS creates friction and
turbulence, leading to an increase in particle collisions, which effec­
tively break up the aggregates. As previously reported, ultrasonication
could reduce the particle size of myofibrillar protein (Zhang et al.,
2017). However, because of aggregates reintegration through non­
covalent and covalent interactions, the particle size of protein increased
after HIUS treatment (Jiang et al., 2014).
3.1.5. Zeta potential of HIUS-treated YHP
Proteins have non-polar hydrophobic residues and hydrophilic polar
groups, and their balance affects the final surface charge, which is the
main factor that determines their dispersion and aggregation (Li, Cheng,
et al., 2020). The higher absolute zeta potential of the protein
dispersion, the stronger repulsive force between molecules and the
better stability. As shown in Fig. 1D, zeta potential were all negative and
increased as the HIUS power increased, suggesting that negative amino
acids on protein surface gradually increased. The possible explanation
for this result is that HIUS treatment might disrupt aggregation of pro­
teins and exposed more surfaces, which contribute to increasing nega­
tive surface charge (Zhang et al., 2017). The electrostatic repulsions
between the particles were strengthened, which inhibited further ag­
gregation and improved the stability of protein dispersion. These results
are in accord with several studies such as ultrasonicated millet protein
concentrate (Nazari et al., 2018) and myofibrillar protein (Zhang et al.,
2017).
3.2. Effects of HIUS power on the functional characteristics of YHP
3.2.1. Solubility of HIUS-treated YHP
Solubility is the most important indicator to measure degree of
protein denaturation and aggregation, and it is also a prerequisite for a
protein to have good functional properties. The percentage solubility of
YHP as a function of pH (3.0–10.0) was measured and shown in Fig. S1.
In general, the samples exhibited a pH dependent U-shape curve, with a
lowest solubility at the range of pH 4.0–6.0, a higher solubility at pH 3.0,
and the highest solubility at the alkaline pHs (e.g., 9.0 and 10.0).
Considering that proteins are often used in neutral solutions, solubility
of HIUS-treated YHP at pH 7 was determined. As shown in Fig. 2A, the
solubility of YHP continuously increased as the ultrasonic power
increased from 0 to 600 W. The unfolding of the protein by ultra­
sonication treatment, which resulted in the hydrophilic amino acid
residues faced toward water, thereby increasing the solubility of the
protein (Jambrak, Mason, Lelas, Herceg, & Herceg, 2008). Ultra­
sonication can reduce the particle size of YHP, which enhances the
interaction between protein and water molecules, thereby increasing
solubility (Jiang et al., 2014). Besides, the physical force generated by
ultrasonication broken the hydrogen bonds and hydrophobic in­
teractions between protein molecules, thereby greatly improving

4
C. Yu et al. LWT 167 (2022) 113820

Fig. 2. Effect of different HIUS power on solubility (A), turbidity (B), viscosity (C) and shear stress (D) of YHP.
Note: Different lowercase letters represent significant differences at P < 0.05.

solubility (Zhang et al., 2017). In addition, the increased charged groups
after treatment also contribute to enhanced solubility (Jambrak et al.,
2008). Similar results were also obtained from myofibrillar proteins
(Zhang et al., 2017) and soy proteins (Hu et al., 2013). However, Jiang
et al. stated contrary tendency that the solubility of black bean proteins
decreased gradually after 450 W of ultrasonication. This may be due to
the protein molecules reassemble macromolecular aggregates through
non-covalent interactions (Jiang et al., 2014).
3.2.2. Turbidity of HIUS-treated YHP
Generally, turbidity is an index to measure protein aggregation,
higher turbidity corresponds to denser protein aggregation (Chen,
Zhang, Xue, & Xu, 2020). The effect of HIUS treatment on YHP turbidity
is shown in Fig. 2B. Turbidity of YHP continuously decreased with the
ultrasonic power increased from 0 to 600 W. This phenomenon may be
due to that the shear force, cavitation forces and turbulence of HIUS
destroyed the YHP original conformational structure and reduced the
particle size of YHP. Consequently, the specific surface area available for
light scattering increased, thus decreasing the protein turbidity (Cui
et al., 2021). In addition, turbidity may be related to protein-protein
interactions. After HIUS treatment, the tertiary and quaternary struc­
tures of the protein were changed, and the protein-protein interactions
were disrupted, thereby reducing the turbidity of the protein (Shi et al.,
2020). Other studies also found that HIUS treatment can reduce protein
turbidity of Cyperus esculentus seed proteins (Cui et al., 2021) and whey
protein isolate (Shi et al., 2020).
destroy internal hydrophobic interaction of protein (Wang et al., 2017).
The change of viscosity of YHP dispersion with the shear rate was
shown in Fig. 2D. The viscosity is a physical index reflecting the size of
the fluid friction. The lower the viscosity of the fluid, the better its
fluidity. The viscosity of YHP dispersion decreased gradually as the
shear rate increased, which suggested that proteins showed shear thin­
ning and behaved as pseudoplastic fluid. In addition, the shear stress of
YHP dispersion decreased with the increase of ultrasonic power. Table 2
showed that the n increased but k values decreased as the ultrasonic
power increased. The n value of all YHP dispersion is between 0 and 1,
indicating that the dispersion is a non-Newtonian fluid. The n value
decreased significantly as the ultrasonic power increased. This result
indicated that the shear thinning phenomenon of the YHP solution was
significantly increased. Several factors contributed to decreased vis­
cosity of YHP dispersion. Firstly, cavitation induced by HIUS treatment
reduced the particle size of YHP and contributed to decreased of vis­
cosity (Zisu, Bhaskaracharya, Kentish, & Ashokkumar, 2010). Secondly,
the increase in shear rate not only overcomed the Brownian motion, but
also broke the chemical bonds, so that the protein chains were aligned
parallel to the flow direction. This process greatly decreased the resis­
tance to flow and results in lower viscosity (Amiri, Sharifian, & Sol­
tanizadeh, 2018). In addition, HIUS treatment unfolded of the protein
molecules, causing more hydrophobic residues to be exposed to the
aqueous environment. This process improved the interaction between
the protein and water, resulting in decreased viscosity

Table 2
3.2.3. Rheology and viscosity analysis of HIUS-treated YHP
Effect of different HIUS power on viscosity coefficient and flow behavior index
The static rheological properties were used to reflect motion speed
of emulsions stabilized by HIUS-treated YHP.
and resistance of protein dispersion under external force (Wang, Yang,
Tang, Ni, & Zhou, 2017). Fig. 2C presents steady flow curves of YHP Ultrasonic Viscosity coefficient: k Flow Regression
power (Pa⋅s) behavior: n coefficients: R2
solutions at different shear rates. The shear stress of YHP dispersion
increased as the shear rate increased. In addition, the shear stress of YHP Control 0.124 0.445 0.98
150 W 0.110 0.450 0.96
dispersion decreased as ultrasonic power increased. This phenomenon
300 W 0.041 0.665 0.99
might be due to that acoustic cavitation induced by HIUS resulted in 450 W 0.031 0.718 0.99
stronger shear force and rapid molecular movement, which could 600 W 0.020 0.804 0.99

5
C. Yu et al.
(Arredondo-Parada et al., 2020).
3.3. Effects of HIUS power on emulsifying properties of YHP
3.3.1. EAI and ESI of emulsions stabilized by HIUS-treated YHP
EAI refers to the interfacial area stabilized by per unit weight of
protein. It represents the ability of protein to be adsorbed at the oil-
water interface (Sun et al., 2014). ESI represents the ability of protein
to stay at the oil-water interface after emulsion storage or heating (Amiri
et al., 2018). As shown in Fig. 3A, both EAI and ESI significantly
increased with the increasing ultrasonic power. The HIUS treatment
modified the structure of YHP, which changed the protein solubility,
hydrophobicity and particle size, thus contributing to improved emul­
sifying properties. surface hydrophobicity of ultrasonicated protein was
higher, which increased interaction between adjacent molecules at the
interface. This interaction decreased the adsorption barrier for oil-water,
resulting in a higher adsorption rate as well as increased EAI and ESI
(Resendiz-Vazquez et al., 2017). On the other hand, HIUS treatment
disrupted the conformational structure of protein, resulting in the
decrease of particle size, which further led to the increase of protein
adsorption at the oil-water interface (Nazari et al., 2018). In addition,
HIUS treatment reduces the interfacial tension between the oil and the
water, forming a more stable interfacial film to produce an emulsion
with higher stability (Cui et al., 2021). Our results were in agreement
with ultrasonicated millet protein (Nazari et al., 2018).
3.3.2. Droplet size distribution of emulsions stabilized by HIUS-treated YHP
The droplet size of emulsion is important because it determines the
Fig. 3. Effect of different HIUS power on the emulsifying properties of YHP. EAI and ESI (A), particle size distribution (B) and percentage of adsorbed proteins (C) of
emulsions stabilized by HIUS-treated YHP, dynamic interfacial tension at the oil-water interface of YHP (D), microstructure of emulsions (E).
Note: Different letters represent significant differences at P < 0.05.
6
LWT 167 (2022) 113820
stability and rheology of the emulsion (Boxall, Koh, Sloan, Sum, & Wu,
2012). Fig. 3B displays the particle size distribution of the emulsions
stabilized by YHP. As the ultrasonic power increased, the particle size
distribution of the emulsion shifted to the small particle size range. An
unimodal distribution was observed, which indicated that HIUS treat­
ment could make the emulsion more uniform. The decreased particle
size could be attributed to the intense turbulence induced by the
increased ultrasonic power, which consequently led to disruption of
most protein particles (Sui et al., 2017). The smaller protein particles
were more conducive to migration to the oil-water interface, thereby
preventing gravity separation, flocculation and coalescence. As result,
emulsion droplet size reduced (Li, Cheng, et al., 2020). Furthermore,
HIUS treatment induced the increase in the hydrophobicity of YHP,
thereby accelerating the adsorption rate of YHP to the interface, further
reducing the interfacial tension and resulted in the formation of smaller
emulsion droplet sizes (O’Sullivan, Arellano, Pichot, & Norton, 2014).
Similarly, studies on dairy proteins (O’Sullivan et al., 2014) and egg
white proteins (Arzeni et al., 2012) proved that HIUS treatment can
reduce the particle size of emulsion.
3.3.3. AP% of emulsions stabilized by HIUS-treated YHP
In general, the higher AP%, the better stability of emulsion (Taha
et al., 2019). As shown in Fig. 3C, the emulsions prepared by HIUS
treatment exhibited higher AP% values. As the ultrasonic power
increased from 0 W to 600W, AP% gradually increased. HIUS treatment
induced YHP to unfold and increased the hydrophobicity of the protein.
Hydrophobic groups tend to aggregate at the oil-water interface, which
increases the amount of YHP adsorption around the oil droplets (Taha
C. Yu et al.
et al., 2019). Furthermore, the particle size of ultrasonicated YHP
decreased, which accelerated the absorption rate of YHP around the oil
droplets (Yang et al., 2018). These results were consistent with previous
studies about soy protein (Yang et al., 2018).
3.3.4. Interfacial tension
Fig. 3D displays the interfacial tension for native and ultrasonicated
YHP at the oil-water interface. For all YHP dispersion, the interfacial
tension decreased rapidly at the beginning, indicating that protein
molecules rapidly absorbed and diffused at the oil-water interface
(Chen, Sheng, Gouda, & Ma, 2019). With the extension of measurement
time, the decline rate of surface tension was slower than initially.
Compared with native YHP, interfacial tensions of HIUS-treated YHP
were much lower. The higher ultrasonic power, the lower interfacial
tension. The reduced interfacial tension is conducive to rapid diffusion,
adsorbing, stretching and rearrangement of protein at the oil-water
interface (Sheng et al., 2018). HIUS treatment resulted in flexible and
loose structure of protein, which improved interface diffusion rate.
Therefore, protein was easier to adsorb on the oil-water interface, and
thereby reducing the interfacial tension (Sheng et al., 2018). In addition,
HIUS treatment significantly increased the surface hydrophobicity of
YHP, which contributed to the decrease of surface tension (Ai, Zhang,
Fan, Cao, & Jiang, 2021). A similar effect was obtained from ultra­
sonicated pea protein isolate (Xiong et al., 2018). The study on egg white
showed that interfacial tension first decreased and then increased with
the increase of ultrasonic power. They explained that excessive ultra­
sonication treatment possibly caused protein monomers to form dimers
or polymers through increasing free sulfhydryl groups (Sheng et al.,
2018).
3.3.5. Microstructure of emulsions stabilized by HIUS-treated YHP
The microstructure of the emulsion was shown in Fig. 3E. The
emulsion stabilized by native YHP distinctly showed large oil droplets,
oil flocculation and uneven droplet distribution. This phenomenon may
be due to the higher interfacial tension between oil and water (Fig. 3D).
The higher interfacial tension is the root cause of instability of the oil-
water interface (Kang et al., 2011), which forced oil droplets to aggre­
gate and form larger oil droplets (Chen et al., 2020). With the HIUS
power increased, the distribution of the emulsion stabilized by YHP was
more uniform, and the droplet size gradually decreased. This result
corresponded to the changes in droplet size distribution of the emulsions
described above (Fig. 3B). HIUS-treated protein had smaller size and
higher surface hydrophobicity, which was conducive to the adsorption
of protein on the surface of oil droplets. The higher the adsorption rate of
interfacial protein in the emulsion, the stronger the repulsive force
Fig. 4. Effect of different HIUS power on creaming index (A) and typical visual images (B) of emulsions stabilized by HIUS-treated YHP.
7
LWT 167 (2022) 113820
between the oil droplets, which inhibited the aggregation and phase
separation of the emulsion. Therefore, the uniformity of the emulsion
was improved (Ma et al., 2019). Similar results were also obtained from
ultrasonicated cod proteins (Ma et al., 2019) and mussel water-soluble
proteins (Zou et al., 2020).
3.3.6. Storage stability of emulsions stabilized by HIUS-treated YHP
The creaming index (CI) is related to the ability of emulsion to
maintain its own stability, and is used to evaluate the degree of emulsion
to resist flocculation, and aggregation (Huang, Zhang, Zhang, Fang, &
Zhou, 2021). A higher CI indicates poorer stability of the emulsion (Chen
et al., 2020). The changes in CI and visual observation of YHP-stabilized
emulsions stored at room temperature for 16 days are shown in Fig. 4A
and B. The results showed that the CI of emulsions decreased with the
increase of ultrasonic power, which was also correspond to visual
observation. On the fourth day, the bottom layer of native YHP and 150
W HIUS-induced emulsion showed transparency, indicating phase sep­
aration occurred. With the extension of storage time, phase separation
appeared in all samples. Phase separation of the emulsion stabilized by
YHP treated with higher ultrasonic power was not serious. These phe­
nomena indicated that HIUS treatment could effectively inhibit aggre­
gation and flocculation of emulsion droplets. HIUS treatment improved
surface hydrophobicity of protein and reduced the protein particle size,
which promoted the adsorption of YHP at the oil-water interface,
resulting in increased repulsive interactions between oil droplets.
Therefore, CI of the emulsion stabilized by ultrasonicated YHP is lower
(Sui et al., 2017). Similar results were obtained from emulsion stabilized
by ultrasonicated soy protein (Yang et al., 2018).
4. Conclusions
After HIUS treatment, the surface hydrophobicity of YHP increased
significantly, which indicated HIUS treatment caused certain unfolding
and dissociation of YHP. These changes also lead to an increase in ab­
solute zeta potential and solubility. The decrease in particle size and
turbidity of YHP indicates that HIUS treatment changed the interface
properties of YHP, thus improving EAI and ESI. In addition, from the
microstructure observation, the emulsion prepared by 600 W ultrasonic
treatment of YHP had the smallest particle size and the most uniform
distribution, thereby promoting the adsorption rate of YHP on the sur­
face of oil droplets. These results indicated HIUS treatment improved the
emulsifying properties of YHP as well as demonstrated the potential of
YHP as a natural plant-based functional ingredient in food.
C. Yu et al.
CRediT authorship contribution statement
Cuiping Yu: Supervision, Writing – review & editing. Sihui Li:
Writing – original draft, Investigation. Shuang Sun: Data curation,
Software, Preparation. Huijia Yan: Methodology, Investigation. Henan
Zou: Methodology, Data curation, Software, Investigation.
Declaration of competing interest
The authors declare that there are not any conflict interests in rela­
tion to the work described.
Data availability
Data will be made available on request.
Acknowledgements
This study is funded by the Initial Funding of Scientific Research for
the Introduction of Talents from Northeast Forestry University, the
National Natural Science Foundation of China (Grant no. 31801615)
and open project of Dalian Wenguanguo Engineering Technology
Research Center (Dalian Nationalities University).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2022.113820.
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