Ultrasound-assisted fabrication and stability evaluation of okra seed protein stabilized nanoemulsion

Abstract

The structure and functional properties of okra seed protein (OSP) were characterized, the ultrasonic homogenization process of OSP nano-emulsion was optimized by response surface methodology (RSM), and its stability was also evaluated in this study. The results suggested that OSP was a high-quality plant protein, rich in glutamic acid. The molecular weight of its main subunits distributed in the range of 10–55 kDa, and some subunits were connected by disulfide bonds. Although the water and oil holding capacities of OSP were inferior to those of soy protein isolate (SPI), its emulsifying ability was superior to that of SPI. And the OSP concentration, ultrasonic time and ultrasonic power had obvious effects on the droplet size of nanoemulsion. The optimum process of OSP emulsion was determined as follows: OSP concentration 2.4 %, ultrasonic power 600 W, ultrasonic time 340 s. Under these conditions, the median droplet size of the nanoemulsion was 192.03 ± 3.48 nm, close to the predicted value (191.195 nm). And the obtained nano-emulsion exhibited high stability to the changes of pH, temperature and ionic strength in the environment. Our results can provide reference for the application of OSP, and promote the development of plant protein-based nanoemulsions.

Full text

Ultrasonics Sonochemistry 104 (2024) 106807
Available online 15 February 2024
1350-4177/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Ultrasound-assisted fabrication and stability evaluation of okra seed protein
stabilized nanoemulsion
Lu Bai , Sheng Geng , Yingxuan Zhou , Hanjun Ma
*
, Benguo Liu
*
School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China
ARTICLE INFO
Keywords:
Okra seed protein
Nanoemulsion
Ultrasonic homogenization
Response surface methodology
Process optimization
ABSTRACT
The structure and functional properties of okra seed protein (OSP) were characterized, the ultrasonic homoge-
nization process of OSP nano-emulsion was optimized by response surface methodology (RSM), and its stability
was also evaluated in this study. The results suggested that OSP was a high-quality plant protein, rich in glutamic
acid. The molecular weight of its main subunits distributed in the range of 10–55 kDa, and some subunits were
connected by disulde bonds. Although the water and oil holding capacities of OSP were inferior to those of soy
protein isolate (SPI), its emulsifying ability was superior to that of SPI. And the OSP concentration, ultrasonic
time and ultrasonic power had obvious effects on the droplet size of nanoemulsion. The optimum process of OSP
emulsion was determined as follows: OSP concentration 2.4 %, ultrasonic power 600 W, ultrasonic time 340 s.
Under these conditions, the median droplet size of the nanoemulsion was 192.03 ±3.48 nm, close to the pre-
dicted value (191.195 nm). And the obtained nano-emulsion exhibited high stability to the changes of pH,
temperature and ionic strength in the environment. Our results can provide reference for the application of OSP,
and promote the development of plant protein-based nanoemulsions.
1. Introduction
Okra (Abelmoschus esculentus L.), native to Africa [1], is widely
cultivated in tropical, subtropical, and temperate regions [2]. It is an
annual herb of the genus Okra of Malvaceae. Okra is rich in dietary ber,
magnesium, potassium, folic acid,vitamin C, and bioactive components,
which has high nutritional value [3]. It is widely used in medicine to
prevent cholesterol [4], reduce gastritis, control hypertension and dia-
betes [5–7]. Although the edible part of okra is its tender pod, it is
generally believed that okra seed also has high nutritional value. Okra
seeds contain a signicant amount of protein and are rich in essential
amino acids, making them a potential protein supplement for cereal
foods [8]. Okra seed powder is commonly applied in the food industry as
a substitute for coffee or as a nutritional fortier in beverage production.
Castillo et al. found that okra seed protein not only met the individual’s
protein requirements but also alleviated symptoms of hypertension by
releasing bioactive peptides [9]. Yao et al. reported that OSP had
excellent antioxidant activity and a rich variety of amino acids, which
was suitable for the development of functional foods [10].
Nanoemulsion is a low viscosity, transparent or semi-transparent
dispersed system formed by the appropriate combination of water
phase, oil phase, and surfactants in suitable proportions, with the
droplet size range of 20–500 nm [11]. Because of its small droplet size
and large surface area, nanoemulsion possesses good stability for gravity
separation, occulation and coalescence. In addition to providing opti-
cal transparency for the product, the release and absorption of func-
tional nutraceuticals can also be controlled [12]. Small molecule
surfactants (such as Span and Tween) are often applied as emulsiers to
improve the stability of emulsions [13]. This is because emulsiers
adsorbed at the oil/water interface can form a stable physical barrier,
providing a certain protective effect [14], reducing interfacial tension
and increasing steric hindrance, thereby improving emulsion stability
[15]. However, recent studies have indicated that the safety of the
chemically synthesized surfactants is questionable [16,17]. Therefore,
natural surfactants have received widespread attention and become a
research hotspot in recent years [12,18,19]. Plant proteins are increas-
ingly being used in the preparation of nanoemulsions due to their ad-
vantages such as wide availability, low cost, and high nutritional value.
Xu et al. reported that soy protein isolate-stabilized nanoemulsions
exhibited good emulsifying and stability properties [20]. Akkam et al.
found that nanoemulsions developed by pea protein could effectively
protect vitamin D [21].
* Corresponding authors.
E-mail addresses: xxhjma@126.com (H. Ma), liubenguo@hist.edu.cn (B. Liu).
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson
https://doi.org/10.1016/j.ultsonch.2024.106807
Received 11 December 2023; Received in revised form 9 February 2024; Accepted 10 February 2024

Ultrasonics Sonochemistry 104 (2024) 106807
2
The preparation of nanoemulsions typically requires the application
of external energy to overcome the interfacial tension caused by the
reduction in droplet size of the dispersed phase. The preparation process
can involve both low-energy emulsication and high-energy emulsi-
cation. The high-energy method offers a wide range of options for sur-
factant selection and requires a relatively low dosage, thus nding
extensive applications in food industry. Proteins, polysaccharides, and
small molecule surfactants can all be used for the preparation of nano-
emulsions through the high-energy method. High-energy emulsication
is a method of producing nanoemulsions using mechanical devices that
generate powerful disruptive forces, including high-pressure homoge-
nization, high-speed shear, ultrasonic homogenization, and microuidic
emulsication [22]. Ultrasonic treatment is an emerging technology
that has been widely applied to enhance the performance and quality of
food materials. When used for the production of nanoemulsions, ultra-
sound generate strong mechanical vibration and cavitation effects that
break the oil–water interface, achieving molecular-level mixing. The
resulting nanoemulsion possesses small droplet size, narrow size distri-
bution, and high stability [23,24]. Abbas et al. prepared starch-
stabilized nanoemulsions using ultrasound at lower temperatures
(40–45 ◦C), resulting in reduced energy consumption [12]. Carpenter
et al. demonstrated that mustard oil-based nanoemulsions prepared
using ultrasound exhibited excellent storage stability, with a shelf life of
over 3 months at room temperature [25].
However, research on using OSP as an emulsier to construct
nanoemulsions is still lacking. In view of this, the structural and func-
tional properties of okra seed protein (OSP) were characterized, and
then the ultrasonic homogenization process of OSP nano-emulsion was
optimized by response surface methodology (RSM), and its stability was
evaluated.
2. Materials and methods
2.1. Materials and chemicals
Okra seeds were provided by Feifan Food Co., LTD (Hebi, China).
Soybean protein isolate (SPI) was from Mantianxue Food Manufacturing
Co., LTD (Anyang, China). Medium chain triglyceride (MCT) was pur-
chased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai,
China). All other chemicals were of analytical grade.
2.2. Preparation of OSP
The preparation of OSP was performed according to our previous
report [26]. The 100 g crushed okra seed was mixed with 1000 mL of n-
hexane and shaken for 4 h. The supernatant was discarded and the
process was repeated to obtain defatted okra seed powder. The powder
and ultra-pure water were well mixed at the ratio of 1:20 (w/v). The
mixture was adjusted to pH 9.0 with 1 mol/L NaOH solution, stirred at
25 ◦C for 2 h, centrifuged at 8000 ×g and 4 ◦C for 20 min, the super-
natant was collected, and the residue was re-extracted once. The com-
bined supernatant was adjusted to pH 4.5 with 1 mol/L HCl solution,
centrifuged at 8000 ×g and 4 ◦C for 15 min. The precipitate was
collected, redissolved in water and dialyzed at 4 ◦C for 24 h. After freeze-
drying, 9.6 g of OSP was obtained.
2.3. Amino acid composition measurement
The sample (20 mg) was placed into a hydrolysis tube, followed by
the addition of 10 mL 6.0 moL/L hydrochloric acid and 3 drops of
phenol. Nitrogen gas was then purged into the tube for 2 min, and the
tube was sealed. The hydrolysis tube was placed in a 110 ±1 ◦C oven
and left for 22 h. After cooling, the hydrolysate was ltered and diluted
to 100 mL. The obtained solution (1 mL) was transferred to a 50 mL
centrifuge tube and dried in a concentrator at 45 ◦C. After drying, 1 mL
of distilled water was added to dissolve the residue, followed by
ltration through a 0.22 µm lter membrane. The amino acid compo-
sition, excluding tryptophan, was measured using a Sykam S433D amino
acid analyzer (Munich, Germany).
2.4. SDS-PAGE electrophoresis
The SDS-PAGE measurement was performed according to the report
of Laemmli et al. [27]. The separation gel and concentration gel had
concentrations of 15 % and 5 % respectively. The OSP sample solution of
50
μ
L at a concentration of 5 mg/mL was mixed with 200
μ
L of SDS
buffer solution containing and not containing β-mercaptoethanol,
respectively. The mixture was boiled for 5 min and then cooled to room
temperature. The sample loading volume was 10
μ
L. The initial voltage
was 80 V, which was increased to 120 V after 20 min. After completion
of the test, the gel was soaked in Coomassie Brilliant Blue staining so-
lution for 30 min, followed by three rounds of destaining. The results
was recorded using a gel imaging system.
2.5. Determination of functional properties
2.5.1. Determination of emulsifying ability
According to the method of Vioque et al. [28], 0.2 % protein solution
was congured with 0.2 M NaH
2
PO
4
-Na
2
HPO
4
buffer solutions with
different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0). The salad oil (2 mL) and
protein solution (6 mL) were mixed and homogenized at 10000 rpm for
1 min. The 50
μ
L mixture was immediately taken from the bottom and
diluted into 5 mL with a buffer containing 0.1 % SDS. Its absorbance
value (A
0
) at 500 nm was determined by a spectrophotometer. After 10
min, the 50
μ
L mixture was taken out again and diluted to 5 mL with 0.1
% SDS buffer, and its absorbance (A
10
) at 500 nm was also read. The
emulsifying activity index (EAI) and emulsifying stability index (ESI) of
the sample could be calculated according to the following equations:
EAI (m2/g) = 2×T×A0×D
C×φ×10000 (1)
ESI (min) = A0
A0-A10
×10 (2)
where, T, 2.303; D, dilution ratio (100); C, protein concentration (0.2
%); φ, fraction of oil phase (0.25).
2.5.2. Determination of water holding capacity
According to the method of Lqari et al. [29], 0.2 g of protein was
mixed with 20 mL of ultrapure water and adjusted to different pH values
(3.0, 4.0, 5.0, 6.0, 7.0, 8.0). Then, 5 mL of the above sample solution was
transferred into a pre-weighed 10 mL centrifuge tube, and centrifuged
(5000 ×g, 30 min). After removing the supernatant, the mass of the
centrifuge tube was weighed. The water holding capacity (WHC) of the
sample could be determined using the following equation.
WHC =m2−m1
m×100 (3)
where, m is the sample mass; m
1
is the mass of the centrifuge tube; m
2
is
the mass of the centrifuge tube after removing the supernatant.
2.5.3. Determination of oil holding capacity
According to the method of Deeslie et al. [30], 0.5 g protein sample
and 5 mL salad oil were mixed in a pre-weighed centrifuge tube, and
centrifuged (5000 ×g, 30 min). The salad oil was removed, and the
weight of the centrifuge tube was measured. The oil holding capacity
(OHC) of the sample was calculated according to the following equation.
OHC =m2−m1
m×100 (4)
where, m is the sample mass; m
1
is the mass of the centrifuge tube; m
2
is
L. Bai et al.

Ultrasonics Sonochemistry 104 (2024) 106807
3
the mass of the centrifuge tube after removing the oil.
2.6. Measurement of interfacial tension
The effects of different concentrations of samples (0.5 %, 1 %, 2 %)
on the MCT/water interfacial tension were measured by a Theta Lite
optical contact angle meter (Biolin Scientic, Stockholm, Sweden), and
the ultrapure water was used as the blank control group. The tip of the
pipette of the high-precision syringe was immersed in MCT and dripped
a drop of sample solution. The shape of the droplet was recorded
continuously, and the interfacial tension of the sample was calculated by
the Young-Laplace equation.
2.7. Preparation of OSP nanoemulsion by ultrasonic homogenization
A certain amount of OSP was dissolved in ultra-pure water as water
phase and MCT as oil phase. The oil phase and the water phase were
mixed at the volume ratio of 5:95, and dispersed at 15000 rpm for 2 min
by a homogenizer to obtain the crude emulsion. The obtained crude
emulsion was placed in an ice bath, and treated under the designated
conditions by a Scientz JY99-IIDN ultrasonic homogenizer (Ningbo,
China). The pulse working time and the intermittent time were set to 2 s.
The droplet size and polydispersity index (PDI) of the obtained emulsion
were measured by a NANO ZS nanoparticle size analyser (Malvern In-
struments, Worcestershire, UK). Before measurement, the emulsion was
diluted 1000 times with water to reduce multiple light scattering effect.
2.8. Single factor experiment of ultrasonic homogenization
The effect of OSP concentration (0.5–3.5 %), ultrasonic power
(200–800 W) and ultrasonic time (60–480 s) on the median droplet size
and PDI of the nanoemulsion were evaluated by single factor
experiment.
2.9. Optimization of ultrasonic homogenization based on RSM
Based on the single factor experiment, the ultrasonic homogeniza-
tion process of the nanoemulsion was further optimized with OSP con-
centration (X
1
), ultrasonic power (X
2
) and ultrasonic time (X
3
) as
independent variables and median droplet size (Y) as response value.
2.10. Stability evaluation of OSP nanoemulsion
2.10.1. Effect of pH
The pH of the OSP nano-emulsion prepared under the optimum
conditions was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 using 1.0
mol/L HCl or NaOH solutions [31]. After 30 min, the changes in the
appearance and droplet size of the emulsion were recorded.
2.10.2. Effect of ionic strength
The Na
+
concentration in the nano-emulsion prepared under the
optimum conditions was adjusted to 0, 20, 40, 60, 80, and 100 mM by
using a 1.0 mol/L NaCl solution. After 30 min, the changes in the
appearance and median droplet size of the emulsion were recorded.
2.10.3. Effect of temperature
The nano-emulsion prepared under the optimum conditions was
placed in a water bath at different temperatures (40, 50, 60, 70, 80, 90,
100 ◦C) for 20 min, and rapidly cooled to room temperature [31]. The
changes in the appearance and median droplet size of the emulsion were
recorded.
2.11. Statistical analysis
Each test were conducted in triplicate, and expressed as mean ±SD.
SPSS 18 software was used for signicant analysis while Origin 8.0
software was employed for plotting. Design-Expert software was utilized
for RSM design and analysis.
3. Results and discussion
3.1. Amino acid composition analysis
The composition of essential amino acids required by the human
body is closer to that of animal proteins, but excessive intake of animal
proteins can lead to health problems such as obesity, hypertension, and
hyperlipidemia. This is because along with the intake of animal proteins,
a higher amount of saturated fat is also consumed. In comparison, the
fatty acid composition of oilseed crops that produce plant proteins is
more benecial for health [32]. The use of plant proteins as partial
substitutes for animal proteins has become a new trend in food industry.
As shown in Table 1, the amino acid composition of OSP is abundant.
The content of essential amino acids (excluding tryptophan) was 26.91
±1.39 %, accounting for 31.71 % of the total amino acids, which was
Table 1
Amino acid composition of OSP.
NEAA Content (%) EAA Content (%)
Asp 9.14 ±0.42 Thr 2.82 ±0.13
Ser 4.59 ±0.20 Val 4.39 ±0.21
Glu 17.01 ±0.82 Met 1.95 ±0.10
Gly 3.55 ±0.20 Ile 3.08 ±0.20
Ala 3.80 ±0.23 Leu 6.59 ±0.37
Cys 1.17 ±0.13 Phe 4.25 ±0.19
Tyr 3.19 ±0.22 Lys 3.83 ±0.19
His 2.81 ±0.09
Arg 9.95 ±0.52
Pro 2.73 ±0.22
Total NEAA 57.94 ±3.05 Total EAA 26.91 ±1.39
Fig. 1. SDS-PAGE analysis of OSP in the presence (reduced) and absence
(unreduced) of β-mercaptoethanol (MW: Molecular weight markers).
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Ultrasonics Sonochemistry 104 (2024) 106807
4
close to the FAO/WHO recommendation value (36 %) for evaluating
ideal protein resources. Similar to the results of the previous report [10],
glutamic acid was the most abundant amino acid in OSP (17.01 ±0.82
%), followed by arginine (9.95 ±0.52 %) and aspartic acid (9.14 ±0.42
%). Glutamic acid and its derivatives play an important role in cancer
prevention and have been widely used in bio-medicines [33]. Arginine
and histidine can meet the normal physiological needs of the elderly and
infants, and play an important role in metabolism and nutrition. The
content of sulfur-containing amino acid (cystine +methionine) in OSP
was the lowest, only 3.12 %. The decrease of sulfur-containing amino
acid content could be due to its interaction with other components in the
extraction process [34]. In terms of amino acid content and composition,
OSP is an excellent plant protein and can be used as a protein
supplement.
3.2. SDS-PAGE analysis
The SDS-PAGE results of OSP was shown in Fig. 1. In the reduced
state (Lane 2), the molecular weight of OSP was mainly distributed in
10–55 kDa, and there was an obvious subunit band between 10 and 17
kDa, a subunit band between 17 and 25 kDa and two subunit bands
between 43 and 55 kDa. It was worth noting that there was also an
obvious subunit band below the molecular weight of 10 kDa, indicating
that OSP was not a single pure protein, but a mixed protein. Disulde
bonds maintain the tertiary structure of proteins, and their changes
reect the degree of protein denaturation. Therefore, they have a crucial
inuence on the functional properties of proteins [35]. In order to study
the binding mode between protein subunits, SDS-PAGE measurement
was also performed in the non-reducing state (without adding β-mer-
captoethanol). At this time, the molecular weight distribution range of
OSP (Lane 1) was 10–180 kDa, and the distribution of protein subunits
changed signicantly. The bands below 10 kDa disappeared completely,
while new bands appeared between 55 and 180 kDa. The results indi-
cated that some subunits in OSP were connected by disulde bonds.
3.3. Functional properties
3.3.1. Emulsifying analysis
SPI occupies a leading position in the market because of its extensive
sources and excellent functional properties [36]. Emulsifying ability is
one of the most important functions of protein as food raw materials. It
affects the oil droplet size in food and determines the sensory and
texture properties of food. However, its emulsifying performance is
inuenced by environmental factors, such as pH. Good solubility is a
prerequisite for the emulsifying ability of proteins, and proteins exhibit
better emulsifying performance at pH values deviating from their iso-
electric point [37]. The EAI and ESI values of OSP and SPI at different pH
values were exhibited in Fig. 2A and B. With the increase of pH, both the
EAI and ESI values of OSP and SPI decreased at rst and then increased.
When pH =5.0, the emulsifying performance declined to the lowest.
This is because when the protein charge is zero near the isoelectric point,
its surface is not easily hydrated, leading to a strong tendency for ag-
gregation and the weakest emulsifying ability. When deviating from the
isoelectric point, the electrostatic repulsion improve the hydration de-
gree of proteins, leading to the diffusion of proteins to the water/oil
interface and an enhancement of emulsifying ability. Overall, the
emulsifying ability of OSP was superior to that of SPI, making it suitable
for widespread utilization in the food industry.
3.3.2. Water holding capacity analysis
The binding characteristic of protein and water is an index to reect
the degree of interaction between protein and water [38]. Its
Fig. 2. Functional properties of OSP (A, EAI; B, ESI; C, WHC; D, OHC).
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Ultrasonics Sonochemistry 104 (2024) 106807
5
hydrophilicity is the outcome of various factors such as protein molecule
size, shape, conformational characteristics, thermodynamic properties,
physicochemical environment, and solubility. Therefore, the water-
holding capacity varies signicantly among different proteins. As
shown in Fig. 2C, the water-holding capacities of OSP and SPI exhibited
signicant differences, with SPI demonstrating superior water-holding
capacity compared to OSP, which was closely associated with the hy-
drophilic structure of proteins. As the pH value changed, both proteins
demonstrated a trend of initially decreasing and then increasing, with
their hydrophobicity reaching the lowest point at pH =5.0. When the
pH was 6.0 to 8.0, their water holding capacities were gradually stable
and reached the maximum. This is because at the isoelectric point,
proteins exist in a molecular form and exhibit strong intermolecular
forces, while their interaction with water is weak. Conversely, proteins
have the highest water-holding capacity when they are far from the
isoelectric point.
3.3.3. Oil holding capacity analysis
The oil-holding capacity of proteins depends on internal factors such
as amino acid composition, protein conformation and surface hydro-
phobicity [39]. The oil-holding capacity of OSP and SPI was 262 g/100 g
and 446 g/100 g, respectively. The oil-holding capacity of SPI was
higher than that of OSP. The inuence of standing time on the oil-
holding capacity was shown in Fig. 2D. With the extension of standing
time, there was no signicant change in oil-holding capacity. The oil
bound by OSP and SPI could reach the saturated state in a short time, so
the change of oil holding capacity was not obvious by prolonging the
standing time. The mechanism of protein-lipid binding is not fully un-
derstood and may be inuenced by the presence of lipid/protein com-
plexes and protein content. The existence of hydrophobic groups may
also play a signicant role in facilitating the binding of lipids and pro-
teins [40].
3.4. Interfacial tension analysis
Interfacial tension is generated by the cohesive forces between par-
ticles, and emulsiers can reduce the oil/water interfacial tension,
thereby forming an emulsion [41]. The interfacial adsorption charac-
teristics of particles are reected by the magnitude of interfacial tension.
The lower the interfacial tension of particles, the stronger their
adsorption characteristics on the interface. The interfacial tension
analysis of OSP and SPI was exhibited in Fig. 3. Compared to the blank
(32.65 ±0.69 mN/m), both OSP and SPI could reduce interfacial ten-
sion, indicating that proteins could adsorb at the oil/water interface to
stabilize nanoemulsions. The interfacial tension decreased with
increasing protein concentration. At a protein concentration of 2 %, OSP
Fig. 3. Effect of OSP and SPI at different concentrations on the oil/water
interfacial tension.
Fig. 4. Effect of OSP concentration (A), ultrasonic power (B) and ultrasonic
time (C) on the median droplet size and PDI of OSP nanoemulsion.
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Ultrasonics Sonochemistry 104 (2024) 106807
6
reduced the interfacial tension to a minimum of 13.12 ±0.15 mN/m,
while SPI reduced the interfacial tension to 16.42 ±0.45 mN/m. At the
same concentration, the interfacial tension of OSP was lower than that of
SPI, which indicated that OSP had superior emulsifying performance.
This was consistent with the experimental results of emulsifying assay,
further conrming that OSP can be used as an emulsier to construct
nanoemulsions.
3.5. Analysis of factors inuencing ultrasonic homogenization
The droplet size of the nanoemulsion depends on the concentration
and type of the emulsier and the degree of destructive force or energy
applied to the liquid sample. Emulsiers can reduce the interfacial
tension of droplets and slow down the droplet aggregation rate, thus
providing stability for droplets [12]. Ultrasonic wave disrupts the oil/
water interface through intense cavitation effects and mechanical vi-
brations, which can achieve molecular mixing. A cavitation bubble
generated by the cavitation effect exerts signicant erosion on the
functional components by bursting, which further promotes the reduc-
tion of solid particle size and increase in surface area, facilitating the
preparation of nanoemulsions. To minimize energy loss and production
costs, it is crucial to determine the optimal ultrasonic homogenization
process for nanoemulsions.
3.5.1. OSP concentration
The physicochemical properties of nanoemulsions primarily depend
on their size and interfacial characteristics. The addition of emulsiers
signicantly inuences the physicochemical performance of nano-
emulsions, such as surface charge distribution and interfacial thickness,
ultimately affecting their colloidal stability, absorption rate, and
bioavailability [42]. Fig. 4A exhibits the effect of OSP concentration on
the median droplet size and PDI of the nanoemulsion. With the
increasing OSP concentration, the median droplet size of the nano-
emulsion declined. When the OSP concentration exceeded 2.5 %, the
median droplet size of the nanoemulsion was at the minimum (280.63
±9.28 nm) and tended to be stable. This may be due to the formation of
micelle systems through the aggregation of OSP molecules at low con-
centrations, which can enhance the stability of the emulsion. With an
increasing amount of protein added, the protein concentration at the
interface reaches the critical micelle concentration, and it no longer
increases with the increase in protein addition. As a result, the properties
of the emulsion tend to stabilize. The PDI values of nanoemulsions also
exhibited a trend of rst increasing and then decreasing. When the
protein concentration was 2.5 %, the PDI value of the nanoemulsion was
lower. This suggested that when the protein concentration was 2.5 %,
the smaller the droplet size distribution range in the emulsion dispersion
system, the better the dispersion [43].
3.5.2. Ultrasonic power
Fig. 4B illustrates the inuence of ultrasonic power on the droplet
size and PDI of the nanoemulsion. These indexes exhibited a decreasing
trend with increasing ultrasonic power. The number of cavitation bub-
bles generated by ultrasound increases with increasing ultrasound
power, leading to enhanced energy around the bubbles and easier
dispersion of droplets, resulting in a reduction in droplet size and
improved stability of the nanoemulsion [44,45]. When the ultrasonic
power exceeded 600 W, the nanoemulsion droplet size (239.50 ±7.25
nm) and PDI (0.37 ±0.01) reached smaller values and tended to be
stable. This showed that the dispersion of nano-emulsion droplets was
improved under high ultrasonic power. Although further increasing the
ultrasound power led to a slight reduction in the droplet size of the
nanoemulsion, this was unnecessary from an energy-saving perspective.
3.5.3. Ultrasonic time
The impact of ultrasonic time on the median droplet size and PDI of
the nanoemulsion is demonstrated in Fig. 4C. The droplet size of the
nanoemulsion is closely related to the ultrasonic time when the emul-
sier concentration and ultrasonic power are kept constant. The droplet
size exhibited an increasing trend followed by a decreasing trend with
the increase of ultrasonic time. After ultrasonic time exceeded 360 s, the
droplet size of the nanoemulsion reached its minimum value (270.57 ±
5.47 nm) and tended to stabilize. The cavitation effect of ultrasound
could induce the uniform dispersion of the water phase and the frag-
mentation of the oil phase, leading to an increase in the oil/water
interface area and contact frequency. Therefore, OSP rapidly resided at
the oil–water interface, exerting its emulsifying effect. Long-term ul-
trasonic treatment could result in protein aggregation and droplet coa-
lescence, thereby compromising the stability of nanoemulsion [46].
When the ultrasonic time was 360 s, the PDI value of the nano-emulsion
was also lower (0.62 ±0.04).
3.6. Process optimization of ultrasonic homogenization
According to the results of single factor test, the ultrasonic homog-
enization was optimized by RSM. The experimental design and results
were shown in Table 2. The regression term of the equation tted by
RSM was extremely signicant, and the “Lack of t” value was 0.1099 >
0.05. The linear relationship between the factor indexes of the tting
equation and the response value was signicant, and the “Adj R
2”
value
of the tting model equation was 0.9692. These indicators conrmed
Table 2
Experimental design and results of RSM.
RSM experiment ANOVA
Run X
1
(%) X
2
(W) X
3
(s) R (nm) Source Sum of squares df Mean square F-value P-value
1 1.5(−1) 500(0) 360(1) 310.17 ±1.60 Model 54001.30 9 6000.14 56.95 <0.0001
2 2(0) 500(0) 300(0) 293.77 ±4.45 X
1
6666.37 1 6666.37 63.27 <0.0001
3 1.5(−1) 400(−1) 300(0) 380.97 ±5.08 X
2
34069.11 1 34069.11 323.34 <0.0001
4 2.5(1) 600(1) 300(0) 192.03 ±3.48 X
3
4636.80 1 4636.80 44.01 0.0003
5 1.5(−1) 600(1) 300(0) 212.40 ±2.77 X
1
X
2
2443.67 1 2443.67 23.19 0.0019
6 2(0) 600(1) 360(1) 213.97 ±2.01 X
1
X
3
1597.36 1 1597.36 15.16 0.0059
7 2(0) 400(−1) 360(1) 327.83 ±7.54 X
2
X
3
785.88 1 785.88 7.46 0.0293
8 2(0) 400(−1) 240(−1) 404.23 ±11.80 X
1
2
1918.97 1 1918.97 18.21 0.0037
9 2.5(1) 500(0) 360(1) 224.53 ±2.160 X
2
2
1301.54 1 1301.54 12.35 0.0098
10 2(0) 500(0) 300(0) 294.23 ±6.38 X
3
2
601.43 1 601.43 5.71 0.0482
11 1.5(−1) 500(0) 240(−1) 318.13 ±9.92 Residual 737.56 7 105.37
12 2(0) 500(0) 300(0) 304.70 ±8.24 Lack of t 550.45 3 183.48 3.92 0.1099
13 2.5(1) 400(−1) 300(0) 261.73 ±2.40 Credibility analysis of the regression equations
14 2.5(1) 500(0) 240(−1) 312.43 ±5.09 Std. dev. 10.26 R
2
0.9865
15 2(0) 500(0) 300(0) 301.17 ±3.07 Mean 288.02 Adj. R
2
0.9692
16 2(0) 500(0) 300(0) 309.70 ±8.53 Second-order polynomial equation
17 2(0) 600(1) 240(−1) 234.30 ±1.74 R =300.71–28.87X
1
−65.26X
2
−24.07X
3
+24.72 X
1
X
2
−19.98X
1
X
3
+14.02X
2
X
3
−21.35 X
1
2
−17.58X
2
2
+11.95X
3
2
L. Bai et al.

Ultrasonics Sonochemistry 104 (2024) 106807
7
Fig. 5. Response surface plots for the ultrasonic homogenization of OSP
nanoemulsion.
Fig. 6. Effect of pH (A), Na
+
concentration (B) and temperature (C) on the
droplet size of OSP nanoemulsion.
L. Bai et al.

Ultrasonics Sonochemistry 104 (2024) 106807
8
the validity of the model equation. According to the F-value test, the
order of factors in terms of their inuence was as follows: ultrasonic
power >OSP concentration >ultrasonic time.
The effect of the interaction of factors was demonstrated in Fig. 5.
Increasing ultrasound power was benecial for reducing particle size,
with a greater impact than OSP concentration and ultrasonic time. With
the increase in ultrasonic power, strong ultrasonic treatment broke the
oil/water interface, allowing the preparation of nanoemulsion with
smaller droplet size. Tan et al. demonstrated that, the droplet size of
nanoemulsion depended on ultrasonic power in a short ultrasonic time
[47].
The regression model was analyzed by RSM method, and the optimal
process was determined as follows: OSP concentration 2.387 %; ultra-
sonic power 598.725 W; ultrasonic time 339.446 s. To adapt to the
actual operation, the process was modied as follows: OSP concentra-
tion 2.4 %; ultrasonic time 340 s, ultrasonic power 600 W. Under these
conditions, the measured median droplet size of the nanoemulsion was
192.03 ±3.48 nm, close to the predicted value (191.195 nm), which
proved that the regression model was reliable.
3.7. Stability of OSP nanoemulsion
3.7.1. pH
It is reported that the stability of protein nanoemulsion is higher than
that of small molecular surfactants, but they are more sensitive to the
change of pH [48,49]. When food emulsions are exposed to an acidic
environment, turbidity and precipitation occur. Therefore, the pH sta-
bility is crucial for the application of emulsions in food. As shown in
Fig. 6A, the droplet size of the nanoemulsion signicantly ascended as
the pH value increased from 3.0 to 5.0. It reached its maximum and
undergone phase separation at pH 5.0, indicating that the nanoemulsion
was highly unstable at this pH. Zhang et al. also reported that the droplet
size of the nanoemulsion increased sharply and occulation occurred at
pH 5.0 [50]. This may be due to the fact that at this pH, OSP is near its
isoelectric point, which makes the droplets prone to aggregation and
leads to a signicant increase in droplet size [51]. The pH value was
maintained within the range of 6.0 to 9.0, and the droplet size of OSP
nanoemulsion remained stable at around 210 nm, with no apparent
changes in the appearance of the emulsion, which attributed to the
electrostatic repulsion between droplets.
3.7.2. Ionic strength
The addition of ions can shield the electrostatic effects between
droplets and inuence the stability of nanoemulsion. In Fig. 6B, when
Na
+
was not added, the median droplet size of OSP nanoemulsion was
maintained at 202.56 ±3.75 nm. With the increase of Na
+
concentra-
tion from 0 mmol/L to 40 mmol/L, the droplet size also increased. In the
range of 40 ~ 100 mmol/L Na
+
content, the median droplet size of
nanoemulsion reached about 230 nm and tended to be stable. This was
due to the shielding effect of high ion concentration on the electrostatic
repulsion between oil droplets [52], which made van der Waals force
and hydrophobic gravity become the main forces, resulting in the trend
of droplet aggregation. Overall, the nanoemulsion maintained a small
droplet size (below 232 nm) even under high ionic strength, suggesting
that the OSP nanoemulsion possessed good ion stability. Throughout the
entire experimental process, there were no apparent changes in the
appearance of the nanoemulsion.
3.7.3. Temperature
The stability of temperature is an important parameter for assessing
the stability of food emulsions. For the partial denaturation of OSP, the
droplet size of the nanoemulsion gradually ascended when the tem-
perature rose to 60 ◦C (Fig. 6C). Although the droplet size demonstrated
an increasing trend, the growth amplitude was relatively small, which
was attributed to the strong electrostatic repulsion between droplets in
the nanoemulsion, preventing the interaction between protein
molecules on the droplet surface at high temperatures [12]. During the
temperature variation process, the nanoemulsion did not exhibit pre-
cipitation or aggregation, and its appearance remained unchanged.
Therefore, the OSP nanoemulsion possessed strong thermal stability,
making it a potential candidate for application in food emulsion systems.
4. Conclusion
OSP had reasonable amino acid composition and was rich in gluta-
mic acid. SDS-PAGE analysis showed that OSP had ve distinct subunit
bands, with molecular weights ranging from 10 to 55 kDa. Some sub-
units could be linked by disulde bonds. The emulsifying performance
and ability to reduce the interfacial tension of OSP were superior to
those of SPI, but its water-holding capacity and oil-holding capacity
were inferior to those of SPI. The ultrasonic homogenization method
could be used to prepare OSP nanoemulsion, and OSP concentration,
ultrasonic time and ultrasonic power had obvious inuence on the
droplet size of the emulsion. Through the RSM analysis, the optimum
process was as follows: OSP concentration 2.4 %, ultrasonic time 340 s,
ultrasonic power 600 W. Under these conditions, the median droplet size
of OSP nanoemulsion was 192.03 ±3.48 nm, close to the predicted
value. The obtained nanoemulsion exhibited high stability towards
variations in pH, temperature, and ionic strength in the environment.
The results can facilitate the utilization of okra seeds, and provide ref-
erences for the fabrication of novel nanoemulsion systems.
CRediT authorship contribution statement
Lu Bai: Writing – review & editing, Writing – original draft, Visu-
alization, Validation, Software, Methodology, Investigation, Formal
analysis, Data curation. Sheng Geng: Visualization, Validation, Meth-
odology, Investigation, Formal analysis, Data curation. Yingxuan Zhou:
Validation, Methodology, Investigation, Formal analysis. Hanjun Ma:
Writing – review & editing, Writing – original draft, Supervision, Re-
sources, Methodology, Funding acquisition. Benguo Liu: Writing – re-
view & editing, Writing – original draft, Supervision, Software,
Resources, Methodology, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
This work was supported by the Major Science and Technology
Project in Henan Province (No. 221100110500), the Natural Science
Foundation of Henan Province of China (No. 212300410005) and the
National Natural Science Foundation of China (No. 32072180).
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