Food Hydrocolloids xxx (2016) 1e8

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

Impact of ultrasonic treatment on an emulsion system stabilized with

soybean protein isolate and lecithin: Its emulsifying property and
emulsion stability
Xiaonan Sui a, b, c, 1, Shuang Bi b, 1, Baokun Qi b, c, 1, Zhongjiang Wang b, c, Min Zhang d,
Yang Li b, c, *, Lianzhou Jiang b, c, **
a
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University, Harbin, 150030, China
b
College of Food Science, Northeast Agricultural University, Harbin, 150030, China
c
National Research Center of Soybean Engineering and Technology, Harbin, 150030, China
d
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing, 102488, China

articleinfo abstract

Article history: The present study aims to investigate the impact of ultrasonic treatment on the emulsifying property and
Received 1 July 2016 emulsion stability of an emulsion system stabilized with soybean protein isolate (SPI) and lecithin. Ul -
Received in revised form trasonic parameters used were ultrasonic powers of 150, 300, and 450 W and ultrasonic durations of 12
9 October 2016 and 24 min. Emulsifying properties of emulsions were all improved with different extents after ultra -
Accepted 14 October 2016
sonic treatments. The emulsion treated at 150 W & 24 min showing the best emulsifying property and
Available online xxx
emulsion stability than the rest. However, the higher ultrasonic power of 450 W gave negative effects on
emulsion stability, with increased particle size and decreased absolute z-potential values due to protein
Keywords:
aggregation. Prolonged ultrasonic duration from 12 to 24 min resulted in a more stable emulsion under
Ultrasonic treatment
the ultrasonic power of 150 W. However, for ultrasonic powers of 300 and 450 W, the additional ul-
Emulsion system
Soybean protein isolate trasonic energy from prolonging ultrasonic duration from 12 to 24 min generated negative effects to
Lecithin emulsion stability.
Sunower oil 2016 Published by Elsevier Ltd.

1. Introduction approached around pH 4.8, which is the isoelectric point of soybean

Soybean protein is an attractive food ingredient due to its high
nutritional value, and its desirable avour in foods (Ma et al., 2015).
The specic surface properties of proteins allows them to be
adsorbed at oil-water interfaces, forming a thick protective layer
which reduces interfacial tension. Therefore, they act as effective
emulsiers by ensuring oil droplets remain in a stable condition in
an aqueous continuous phase (Imura et al., 2015). However,
emulsions prepared by soybean proteins are sensitive to changes in
ionic charge, which limits its application in food processing and
production (Nguyen et al., 2014). Cao et al. (2015) reported that the
stability of the emulsion was compromised once the system pH
* Corresponding author. College of Food Science, Northeast Agricultural Univer-
sity, Harbin, 150030, China.
** Corresponding author. College of Food Science, Northeast Agricultural Univer-
sity, Harbin, 150030, China.
E-mail addresses: liyanghuangyu@163.com (Y. Li), jlzname@163.com (L. Jiang).
1
The rst, second, and third author contributed equally to this work.
protein.
Lecithin, a type of a zwitterionic surfactant, is one of the most
effective natural emulsiers, and is extensively used in reducing the
interfacial tension of emulsions (Mottola, Vico, Villanueva, &
Fanani, 2015). Upon interacting with lecithin, soybean protein ex-
hibits different surface activity. Scuriatti, Tomas, and Wagner
(2003) and Mantovani, Cavallieri, Netto, and Cunha (2013) re-
ported that the addition of lecithin improved the stability of
emulsions consisting of soybean protein and whey protein,
respectively, even though the system was near the isoelectric point
of the proteins. Comas, Wagner, and Tomas (2006) also found that
the addition of lecithin enhanced the stability of emulsions pre-
pared by native or denatured soybean protein isolates, sunower
oil, and water. The hydrophobic interaction was found to play a key
role in maintaining the interactions between proteins and phos-
pholipids by incorporating the proteins into lecithin vesicles and
micelles (van Nieuwenhuyzen & Szuhaj, 1998). Recently, Kasinos
et al. (2013) and Li, Li, and Guo (2014) further concluded that
lecithin and soybean protein could interact through electrostatic

http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
0268-005X/ 2016 Published by Elsevier Ltd.

Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
2 X. Sui et al. / Food Hydrocolloids xxx (2016) 1e8

and hydrophobic interactions, which provide desirable changes to Table 1

the conformation of protein, and therefore result in improved The parameters of different ultrasonic treatments.

emulsifying ability. Sample number A B C D E F G

Ultrasound can be classied into low and high power ultrasound Ultrasonic power (W) 0 150 300 450 150 300 450
according to its frequency range (Awad, Moharram, Shaltout, Asker, Ultrasonic duration (min) 0 12 12 12 24 24 24
& Youssef, 2012). Low-power ultrasound is always used in ensuring
food quality and safety, while high power ultrasound is applied to
modify functional properties of different foods, including 2.3. Ultrasonic treatments
improving the stability of emulsions (Chemat & Khan, 2011). Kaltsa,
Gatsi, Yanniotis, and Mandala (2014) reported that in an emulsion Freshly-prepared emulsions were subjected to ultrasonic treat-
system containing a combination of whey protein isolate and ment using an ultrasonic processor (NingBo Scientz Biotechnology
xanthan, the droplet size of the oil phase was signicantly reduced Co. Ltd., Ningbo, China) with a 0.636 cm diameter titanium probe.
by either increasing ultrasonic power or the duration of ultrasonic An aliquot of 30 mL of the emulsion was poured into a 50 mL at
treatment. Similarly, an emulsion system using coconut milk pro- bottom breaker surrounded by a double-walled cooling water
tein was found to have a reduced mean droplet size and improved jacket, which helped to keep the temperature consistent. The
stability after ultrasonic treatment (Lad & Murthy, 2012). However, parameter of ultrasonic treatments was shown in Table 1. The ul-
few studies have investigated the change in the physicochemical trasonic treatments were performed at output intensity of 0, 150,
and functional properties of proteins after ultrasonic treatments 300, and 450 W, which cover the often used ultrasonic powers. The
(Chandrapala, Zisu, Palmer, Kentish, & Ashokkumar, 2011). More- ultrasonic treatment duration was set to 12 and 24 min, considering
over, to the best of our knowledge, no other study has investigated few study evaluated the two sonication durations.
the emulsifying property and emulsion stability of SPI and lecithin-
stabilized emulsions after ultrasonic treatments.
2.4. Determination of emulsifying properties
Thus, this work aims to evaluate the effects of ultrasonic power
and duration on the properties of emulsions prepared using SPI and
The emulsifying properties of control and ultrasound-treated
lecithin. Ultrasonic power of 150, 300, and 450 W were adopted for
emulsions were measured according to the method of Li, Huang,
two ultrasonic durations (12 and 24 min). The results of this work
Peng, Shan, and Xue (2014) with slight modications. After ultra-
would be useful in providing a novel and improved method of
sonic treatments, 50 mL of emulsions were immediately sampled
producing soybean protein based emulsions.
and diluted 100 times using 0.1% sodium dodecyl sulfate (SDS). The
absorbance of the emulsion at 500 nm was recorded immediately
(A0) and after 10 min (A10) using a spectrophotometer (SHJH Co.
2. Materials and methods
Ltd., Shanghai, China). The emulsifying activity index (EAI) and
emulsion stability index (ESI) were computed using Eqs. (1) and (2),
2.1. Materials
respectively.
Soybeans were purchased from Heilongjiang Agriculture Co. . . A0 N
EAI m2 g 2
T (1)
Ltd., Harbin, China. Lecithin, with a content of acetone-insoluble
10000 q L C
material (phosphatidylcholine, PC) > 95%, was obtained from
Sigma-Aldrich (St. Louis, MO, USA). Sunower oil (COFCO Co. Ltd., A0
Harbin, China) was purchased from a local shop. Nile Blue dye and
ESI min T10 T0 (2)
Nile Red dye were purchased from Sigma-Aldrich, Wicklow, A0 A10
Ireland. All other chemicals were of analytical reagent grade and where T equals to 2.303; A 0 is the absorbance at 0 min; N is the
deionized (DI) water was used throughout. dilution factor (100); q is the proportion of the oil phase (0.25); L is
the thickness of the cuvette (1 cm); C is the concentration of SPI (g/
mL); A10 is the absorbance at 10 min; T 0 represents 0 min; and T 10
2.2. Emulsion preparation represents 10 min.

SPI were prepared according to the method of Speroni, An ~ on,

2.5. Creaming stability measurement
and de Lamballerie (2010). Defatted soybean our was dispersed
in DI water (1:10, w/v), and the pH of the dispersion was adjusted to
Creaming index (%) represents the creaming stability of an
8.0 using NaOH (2 mol/L). The solution was subjected to alkaline
emulsion, whereby a lower creaming index indicates a lesser extent
extraction with continuous magnetic stirring for 2 h and then
of phase separation (Zisu, Schleyer, & Chandrapala, 2013). Control
centrifuged at 10,000g and 4 C for 30 min. The sediment was
and ultrasound-treated emulsions were placed inside hermetic
discarded and the supernatant was collected by adjusting the pH to
tubes and stored at room temperature for up to 7 days. The
4.5 using HCl (2 mol/L) and then centrifuging at 6000g and 4 C for
magnitude of creaming was measured once a day by measuring the
30 min. The obtained precipitate was dialyzed against DI water for
height of the clear liquid layer at the bottom (HC) and the height of
48 h at 4 C before neutralization using NaOH (2 mol/L), and then
total emulsion (HE). Creaming index can therefore be calculated
was freeze-dried for further use.
using Eq. (3).
SPI was mixed well with lecithin at a ratio of 10:1 (w/w) before
dispersing in 0.05 M phosphate buffer solution (1.1%, w/v), followed
Creaming index % HC=HE 100 (3)
by mixing at room temperature for 2 h using magnetic stirring.
Sunower oil was then added to the prepared slurry at a ratio of 1:3
(v/v), followed by homogenization at ambient temperature using
an Ultra-Turrax T18 homogenizer (ANGNI Co. Ltd., Shanghai, China) 2.6. Confocal laser scanning microscopy
at 20,000 rpm for 1 min. The freshly-prepared emulsion was then
subjected to the following studies. Proteins can be stained by Nile Blue dye, emitting green light. Oil

Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
 X. Sui et al. / Food Hydrocolloids xxx (2016) 1e8 3

droplets, however, are stained by Nile Red dye, which do not Table 2
uoresce in most polar solvents, but can uoresce intensely with The proximate composition of soybean protein isolates.

reddish colors upon placing in a lipid-rich environment. A Leica TCS Compositions Protein Crude fat Moisture Ash
SP2 confocal laser scanning microscope (Leica Microsystems, Hei-
Content (%) 90.11 0.15 1.43 0.20 3.95 0.10 4.51 0.20
delberg GmbH, Germany) was employed to examine the structure
of the ultrasonically-treated and control emulsions. An aliquot of
10 mL of stained emulsion was placed on a microscope slide, and
Statistical analysis was performed using Origin 9.1 software (Ori-
then gently covered with a coverslip. An Ar-Kr ion laser and a He-Ne
ginLab, Northampton, MA, USA).
laser operating at an excitation wavelength of 488 nm and 633 nm
respectively were used for imaging oil droplets and proteins,
3. Results and discussion
respectively. The observations were performed using an oil-
immersion objective at 40 magnication.
3.1. SPI composition analysis
2.7. Particle size distribution analysis
The composition of SPI is shown in Table 2. The content of
protein was 90.11%. The crude fat content was 1.43%. The quantity
The particle size distribution for emulsions was determined
of ash found in SPI was determined to be 4.51%, which was pro-
using a dynamic light scattering technique (Mastersizer 2000,
moted by salt formation during the process of protein extraction
Malvern Instrument Co., Ltd., Worcestershire, UK) according to the
(Sosulski & McCurdy, 1987). To check the quality of the prepared
method by Leong, Wooster, Kentish, and Ashokkumar (2009). Prior
SPI, we further did the poly acrylamide gel electrophoresis (SDS-
to analysis, the emulsions were diluted to a suitable concentration
PAGE) analysis according to the method of Laemmli (Laemmli,
in sodium phosphate buffer to prevent multiple scattering. The
1970). Result was shown in Fig. 1. The SPI consists two major
refractive index was taken as 1.46 for emulsion particle and 1.33 for
proteins, b-conglycinin (7S) and glycinin (11S). The composition of
dispersion medium. Results were represented as the volume
weighted mean diameter (de Brouckere mean diameter, D[4,3]) 7S was mapped by a0 , a, and b subunit, and their contents account
(Romero, Cordobes, & Guerrero, 2009). for 14.55, 6.26, and 6.54%, respectively (data not shown). In
comparison, the 11S consists acid and basic subunit with a
2.8. z-Potential measurement composition of 37.41 and 35.23%, respectively (data not shown).
The characteristic bands of SPI were consistent with previous
The z-potentials of oil droplets were evaluated using a Zeta research conclusion (Denavi et al., 2009) indicating the good
potential analyzer (Brookharen Instruments Corporation, New quality of SPI.
York, USA). The control and ultrasound-treated emulsions were
diluted to an appropriate concentration using DI water, and were 3.2. Emulsifying properties analysis
then injected into the instrument for measurements.
Emulsifying property is an indicator of the ability of a protein to
2.9. Solubility and surface hydrophobicity measurement adsorb to the oil/water interface. The emulsifying properties of an
emulsion can be evaluated by the emulsifying activity index (EAI)
To further investigate the effect of ultrasonic treatment on SPI in and emulsifying stability index (ESI) (Jamdar et al., 2010). The EAI
the emulsion system, the solubility and surface hydrophobicity of
SPI before and after ultrasonic treatment were therefore evaluated.
The emulsion was prepared following all the steps of Section 2.2
without adding sunower oil.

2.9.1. Solubility measurement

After ultrasonic treatments, the emulsion was stirred for 1 h and
then centrifuged at 12,000g at 25 C for 20 min. The protein content
of the supernatant were measured using the Lowrys method
(Classics Lowry, Rosebrough, Farr, & Randall, 1951) and bovine
serum albumin (BSA) served as the standard.

2.9.2. Surface hydrophobicity measurement

The surface hydrophobicity was determined according to the
method reported by (Kato & Nakai, 1980). An aliquots of 4 mL su-
pernatant (obtained from solubility measurement section) was
mixed with 40 mL of ANS (8.0 mmol/L in 0.05 mol/L neutral phos-
phate buffer). Fluorescence intensity (FI) was measured at 390 nm
(excitation) and 468 nm (emission) using an F-4500 spectrometer
(Hitachi, Ltd., Tokyo, Japan) with excitation and emission slit of
5 nm. The hydrophobicity was calculated from the linear regression
of initial slope of FI against protein concentration (mg/mL).

2.10. Statistical analysis

All the experiments were conducted in triplicate and results are

presented as mean standard deviation. Means were compared Fig. 1. SDS-PAGE analysis of prepared SPI. M denotes the molecular weight marker
using one-way ANOVA followed by Duncans test (p < 0.05). (Sigma-Aldrich Co., UK).

Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
4 X. Sui et al. / Food Hydrocolloids xxx (2016) 1e8
Fig. 2. Emulsifying properties of untreated (Sample A) and ultrasound-treated (Sample
B-G) SPI and lecithin stabilized emulsion. Experimental sample no. A) control; B)
150 W & 12 min; C) 300 W & 12 min; D) 450 W & 12 min; E) 150 W & 24 min; F)
300 W & 24 min; and G) 450 W & 24 min.
expresses the interfacial area stabilized per unit weight of a protein,
while the ESI measures emulsion turbidity (Zayas, 1997).
As shown in Fig. 2, the EAI and ESI of emulsions after ultrasonic
treatment were signicantly increased (p < 0.05). The emulsion
ultrasonically treated at 150 W for 24 min (Sample E) exhibited the
highest EAI (149.23 g/m 2) and ESI (221.03 min). Compared to the
control (Sample A), the ultrasonic treatment at 150 W for 24 min
effectively increased the EAI and ESI of the SPI-lecithin emulsion by
1.78 and 10.46 times, respectively. As for Sample G, although it
experienced the highest ultrasonic energy treatment, it exhibited
the lowest EAI (93.98 g/m2) and ESI (52.03 min) among all the ul-
trasonically treated emulsions. However, when ultrasonic power
was set greater than 300 W, the emulsifying properties were get-
ting lower. EAI and ESI for samples treated for 12 min with ultra-
sonic power of 450 W (Sample D) was lower than that of samples
treated with 300 W (Sample C). A similar trend was observed even
for treatment durations of 24 min, whereby EAI and ESI of Sample G
was lower than Sample F. A similar trend was reported by Zhang
et al. (2014), that no signicant difference in emulsifying activity
when the ultrasonic power exceeded 300 W.
3.3. Creaming stability analysis
The creaming of an emulsion is caused by the different densities
between the phases in an emulsion, usually oil and water phases
(Zayas, 1997). Thus, the creaming index (CI), as an indicator, pro-
vides information about the extent of the aggregation and sepa-
ration of oil droplets from the water phase in an emulsion. Fig. 3
shows the creaming index of emulsions. The emulsion without
ultrasonic treatment exhibited the worst stability with the highest
CI value (up to 72%). Upon visual inspection, phase separation had
already occurred in the control emulsion after standing at room
temperature for circa 2 h. This could be due to the inhomogeneous
distribution of oil droplets in the control emulsion, leading to ag-
gregation between oil droplets (Kuhn & Cunha, 2012). In compar-
ison, ultrasonically treated emulsions were more stable during
storage, reected by their lower CI values. Sample E showed the
lowest creaming index value of 40%, indicating that it possessed the
best stability. This was consistent with the aforementioned results
that Sample E had the highest EAI and ESI values. This can be
attributed to the emulsifying ability of ultrasound waves in dis-
rupting occulation and preventing creaming, thereby providing
Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
Fig. 3. Creaming index of untreated (Sample A) and ultrasound-treated (Sample B-G)
SPI and lecithin stabilized emulsion.
enough repulsive interaction between individual oil droplets
(Pongsawatmanit, Harnsilawat, & McClements, 2006). Other
possible reasons include the decrease in oil droplet size, and the
increase in repulsion between the electrical charges on the surfaces
of emulsion droplets (Chanamai & McClements, 2000). In addition,
the type of emulsier could be another factor that inuences the
stability of an emulsion. The experimental emulsion was composed
of DI water, SPI, lecithin, and sunower oil. In the emulsion, SPI
adhered to the surface of the sunower oil globules, and was
regarded as the dominant contributor to the stability of the emul-
sion against creaming. Moreover, the ultrasonic treatments were
also suspected to inuence the conformations of SPI and lecithin,
resulting in improvements in their interactions, thereby enhancing
the emulsion stability.
3.4. Confocal laser scanning microscopy analysis
As shown in Fig. 4, the red uorescence core and green uores-
cence periphery in confocal laser scanning microscopy (CLSM) mi-
crographs represent the oil and protein portion, respectively. As
expected, the biggest oil droplet size was observed in the control
emulsion (Sample A) indicating the lowest emulsion stability. For all
ultrasonic treatments investigated, with increasing the ultrasonic
power from 150 W to 450 W for both 12 min (B / D) and 24 min
(E / G) treatments, the oil droplet size was increased by 16.75% and
79.25%, respectively. Compared to Sample B, Sample C showed more
structured matrix (oil droplets surrounded by protein aggregations),
suggesting that the ultrasonic treatment of 300 W for 12 min offered
higher emulsion stability. The structured matrix was further rein-
forced by reducing the ultrasonic power to 150 W and increasing the
treatment time to 24 min (Sample E). During ultrasonic treatment,
the acoustic cavitation effects led to rapid molecular movement and
unfolding of protein chains, causing the increase in ow behaviours
such as viscosities, as well as more compact networks similar to what
has been reported for ultrasound-treated SPI dispersions (Hu et al.,
2013). However, micro-phase separation structures appeared to be
occurring in the emulsion (Sample G), which was possibly due to the
formation of insoluble protein aggregations under the ultrasonic
treatment of 450 W and 24 min. Our observation is in agreement
with Stathopulos et al. (2004) who also observed an acceleration of
sonication-induced protein aggregation with increasing sonication
time.
 X. Sui et al. / Food Hydrocolloids xxx (2016) 1e8 5

Fig. 4. Confocal laser scanning microscopy (CLSM) of SPI and lecithin stabilized emulsion. Fluorescence images of oil -in-water emulsions untreated (Sample A) and different ul-
trasonic treatments (Sample B-G). Images were obtained at 25 C. Magnication: 40. The bar represents 5 mm. Fat globules: red particles; protein network: green particle.
Experimental sample no. A) control; B) 150 W & 12 min; C) 300 W & 12 min; D) 450 W & 12 min; E) 150 W & 24 min; F) 300 W & 24 min; and G) 450 W & 24 min. (For
interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

3.5. Particle size distribution analysis
The stability of emulsion was further evaluated by particle size
distribution (PSD) analysis, which is known as one of the key pa-
rameters inuencing emulsion physicochemical and functional
properties, such as rheology, stability, and chemical reactivity
(McClements, 1996). The PSD of an emulsion formed during ho-
mogenization is attributed to the interaction between the particles
that are breaking up and coalescing. The particle breakup is
determined by the type and amount of shear force applied, and the
resistance of particle to deformation (Laplace pressure), while the
particle coalescence is dependent on the ability of the surfactant to
adsorb to the surface of formed particles (McClements, 2015;
Tadros, Izquierdo, Esquena, & Solans, 2004). When ultrasonic
waves pass through emulsions, re-arrangement of particle disrup-
tions and re-coalescence phenomena may occur simultaneously.
The PSD is shown in Fig. 5 (insert graph). Overall, Sample A, B, F,
and G show a bimodal PSD, while the rest samples exhibit a
unimodal PSD but with different peak values. The unimodal dis-
tribution always indicates a better emulsion stability since particles
in the emulsion have similar size and evenly distributed. After the
ultrasonic treatment, the bimodal PSD of control sample was
beginning to shift to unimodal PSD, however, the bimodal PSD was
again appeared of samples treated at high ultrasound power of 300
and 450 W for 24 min. Sample E shows the narrowest peak and
smallest particle size indicating its best stability. The PSD results are
in consistent with the CLSM observations.
The particle sizes were further expressed as D[4,3] mean di-
ameters (Fig. 5). Ultrasound-treated emulsions (Sample B-G) have
signicantly smaller D[4,3] values than the control sample (Sample
A). The particle sizes were decreased from 3.815 to 3.369 mm after
prolonging the ultrasonic treatment duration from 12 to 24 min at
the ultrasonic power of 150 W. The reduction in particle size could
be attributed to the extended turbulent ow induced by the
prolonged ultrasonic treatment, which consequently led to an
effective disruption of most particles (Kaltsa et al., 2014). As for the
higher ultrasonic power of 300 and 450 W, emulsions treated under
the longer ultrasonic duration of 24 min showed slight but signif-
icant increases in particle size, implying a tendency to re-
coalescence. The effect of long-duration ultrasonic treatment on
the particle size changes was termed as over-processing in the
literature (Desrumaux & Marcand, 2002; Kentish et al., 2008).
Kentish et al. (2008) reported a similar trend between emulsion
particle size and applied ultrasonic treatment, and they concluded
that after ultrasonic treatment, the particle size of an emulsion was
reduced to a minimum size at an intermediate power, but increased
in size at higher power values.
3.6. Surface charge density analysis
The z-potential, which is a measure of the surface charge density
of proteins, offers an indication of potential stability of an emulsion
system. The presence of proteins adhering to the surface of oil
droplets in emulsions may provide charges on the surface of oil
droplets (Shanmugam & Ashokkumar, 2014). The surface charge
density of proteins on the surface of emulsion droplets before and
after ultrasonic treatments measured is shown in Fig. 6A.
Negative z-potential values indicate the presence of anionic
molecules surrounding the surface of emulsion droplets
(Matsumiya, Takahashi, Inoue, & Matsumura, 2010). The unsoni-
cated emulsion stabilized by SPI and lecithin (Sample A) showed
the lowest absolute z-potential value of around 0.98 mV. In
comparison, all sonicated emulsions exhibited signicantly higher
(p < 0.05) absolute z-potential values, suggesting greater elec-
tronegativity. The increased absolute z-potential values of emul-
sions provided a high energy barrier between emulsion droplets,
thereby providing good electrostatic repulsion. When prolonging
the ultrasonic duration from 12 to 24 min at the ultrasonic power

Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
6 X. Sui et al. / Food Hydrocolloids xxx (2016) 1e8

Fig. 5. de Brouker mean diameter (D[4,3]) and particle size distribution (insert graph) of untreated (Sample A) and ultrasound-treated (Sample B-G) SPI and lecithin stabilized
emulsion.

Fig. 6. (A) z-potential values, (B) solubility, (C) surface hydrophobicity (H 0) of untreated (Sample A) and ultrasound-treated (Sample B-G) SPI and lecithin stabilized emulsion.
Experimental sample no. A) control; B) 150 W & 12 min; C) 300 W & 12 min; D) 450 W & 12 min; E) 150 W & 24 min; F) 300 W & 24 min; and G) 450 W & 24 min.

of 150 W, the absolute z-potential values were slightly more than
doubled. A greater z-potential values indicated a stronger inter-
particle electrostatic repulsion between SPI. The increased abso-
lute z-potential values indicated and disrupt protein aggregates
and prevent further aggregate formation (Shanmugam &
Ashokkumar, 2014). However, interestingly, the absolute z-po-
tential values were decreased after extending the ultrasonic
treatment duration from 12 to 24 min at an ultrasonic power of
300 W. In particular, the decrease of absolute z-potential values
was even greater for the ultrasonic treatment of 450 W & 24 min.
The reduced absolute z-potential values after prolonged ultra-
sonic treatment at the higher ultrasonic power of 300 and 450 W
was suspected to be caused by the formation of SPI aggregates,
that the exposed anionic molecules within SPI after
ultrasonication have emerged concomitantly with SPI aggregation
process.
3.7. Solubility analysis
Protein solubility is known as a crucial factor in determining
emulsifying ability (Zayas, 1997). The solubility of SPI after different
ultrasonic treatments is shown in Fig. 6B. After ultrasonic treat-
ments, the solubility of all samples was improved. Sample E
exhibited the highest solubility of 63.3% than the rest. Ultrasonic
treatment was reported to improve the solubility of SPI (Jambrak,
Lelas, Mason, Kresic, & Badanjak, 2009). The SPI molecule was
partially unfolded during ultrasonication, becoming more soluble
and more prone to interacting with lecithins (Jambrak et al., 2009).

Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
 X. Sui et al. / Food Hydrocolloids xxx (2016) 1e8
However, the increased ultrasonic power and duration resulted in a
reduction in solubility. This could be attributed to the formation of
SPI aggregates.
3.8. Surface hydrophobicity analysis
Surface hydrophobicity (H 0) represents the number of hydro-
phobic groups exist on the surface of a protein molecule
(Chandrapala et al., 2011). As shown in Fig. 6C, the H0 value of
control SPI was signicantly lower. After ultrasonic treatments, the
H0 values of SPI were increased. The increased H0 could be due to
the exposed hydrophobic residues of SPI after ultrasonic treatment.
Ultrasonic treatment was reported to cause a certain degree of
unfolding of SPI molecules, and therefore increased the amount of
hydrophobic groups as well as regions that were initially within the
SPI molecules to be exposed to the outside environment (Hu et al.,
2013). Similarly, the hydrophobicity of milk proteins was reported
to have increased after ultrasonication (Shanmugam &
Ashokkumar, 2014). Interestingly, the high ultrasonic power of
either 300 or 450 W didnt increase the surface hydrophobicity of
SPI, except for 300 W & 12 min that only slightly better in
enhancing the surface hydrophobicity of SPI than that of 150 W &
12 min. Our observation is in agreement with Chandrapala et al.
(2011), who observed that the surface hydrophobicity of recon-
stituted whey protein concentrate solutions increased at the rst
5 min of ultrasonic treatment, after which it decreased. They also
discovered that the reduced surface hydrophobicity causing protein
aggregation.
As aforementioned, SPI aggregation was concluded to be
responsible for the micro-phase separation structures (Section 3.4),
increased particle sizes (Section 3.5), decreased absolute z-poten-
tial values (Section 3.6), and reduced solubility (Section 3.7). Again,
we concluded that the reduced surface hydrophobicity was
attributed to the SPI aggregation. Although the formation of SPI
aggregates after ultrasonic treatment is still a matter of debate, the
widely accepted hypothesis are listed below.
(i) Chemical reactions: free radicals (H$ and OH$) produced by
water sonolysis further undergo various reactions resulting
in the formation of reactive oxygen species (H 2O2, O2, and
etc.) (Marchioni et al., 2009; Satheeshkumar & Jayakumar,
2002). The moieties on proteins react with the formed
reactive oxygen species yielding protein radicals, which
further be reacted, such as oxidation, chain reactions,
crosslinking, and cleavage reactions (Hawkins & Davies,
2001).
(ii) Physical interactions: the unfolding protein induced by
ultrasonication undergoes aggregation upon additional
ultrasonication. The ultrasonication-induced physical
shearing may break proteins native fold, but leave secondary
structure intact (Carrion-Vazquez et al., 2000; Jambrak et al.,
2009). The exposed secondary structure of protein could
form intermolecular interactions and aggregation.
4. Conclusion
Ultrasonic treatment of SPI and lecithin stabilized emulsion
resulted in an improved EAI and ESI. However, the continuous in-
creases of ultrasonic power and duration gave negative impacts on
the emulsion stability. It was suspected that the excess energy after
increasing ultrasonic power and prolonging ultrasonic duration
caused too many hydrophobic regions within the soybean protein
molecules to be exposed to the outside aqueous environment. This
in turn led to the aggregation of the proteins and nally reduced the
emulsion stability of SPI and lecithin stabilized emulsion.
Please cite this article in press as: Sui, X., et al., Impact of ultrasonic treatment on an emulsion system stabilized with soybean protein isolate and
lecithin: Its emulsifying property and emulsion stability, Food Hydrocolloids (2016), http://dx.doi.org/10.1016/j.foodhyd.2016.10.024
7
Acknowledgement
The authors would like to acknowledge the support for this
study by the National Natural Science Foundation of China
(research grant number: 31430067, 31601475, 31571876, and
31671807), the 13th Five-Year Plan (2016YFD0400402,
2016YFD0401402, and 2016YFD0400702), the National Key Tech-
nology Support Program (research grant number: 2014BAD22B01),
the Natural Science Foundation of Heilongjiang Province of China
(research grant number: ZD201302), the Fok Ying Tung Education
Foundation (research grant number: 151032), the Young Talents
Project of Northeast Agricultural University, and the Key Laboratory
of Soybean Biology in Chinese Education Ministry.
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