Food Hydrocolloids 100 (2020) 105310

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

Preparation and digestibility of fish oil nanoemulsions stabilized by soybean T

protein isolate-phosphatidylcholine
Yang Lia,b,c,d,1, Mingda Lia,1, Yuman Qia, Li Zhenga, Changling Wua, Zhongjiang Wanga,∗,
Fei Tenga,∗∗
a
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang, 150030, China
b
Department of Food Science, Cornell University, Ithaca, NY, 14853-7201, USA
c
Harbin Institute of Food Industry, Harbin, Heilongjiang, 150030, China
d
Heilongjiang Academy of Green Food Science, Harbin, Heilongjiang, 150030, China

A R T I C LE I N FO A B S T R A C T

Keywords: Fish oil is used for its numerous health and nutritional benefits. Nevertheless, it is unstable during production,
Nanoemulsions storage, and application. Nanoemulsions can be used as an effective carrier for encapsulated nutraceuticals. The
soybean protein isolate effects of different components on the stability of fish oil nanoemulsions with soybean protein isolate-phos-
Phosphatidylcholine phatidylcholine (SPI-PC) were studied by characterizing particle size, polydispersity index, ζ-potential, turbidity,
Fish oil
and turbiscan stability index. The SPI-PC nanoemulsions with optimal stability were prepared with 2% SPI, 0.2%
In vitro digestion
PC, 1.5% fish oil, and 100 MPa homogenization pressure. Confocal laser scanning microscopy verified that the
oil droplet was encapsulated inside the nanoemulsions. Compared with a Tween 20 nanoemulsions control
group, the SPI-PC nanoemulsions have better storage and oxidative stability, and have better resistance to
certain concentrations (0.1–0.5 M) of Na+. However, we found that the SPI-PC nanoemulsions resistance to
acidic conditions was not as good as that of Tween 20. The SPI-PC nanoemulsions showed aggregation of
droplets during in vitro gastric digestion while the Tween 20 nanoemulsions did not. The release rate of free fatty
acids (FFA) reached 86.8% in the 2 h in vitro intestine digestion. SPI-PC nanoemulsions significantly increased
the digestibility of fish oil compared with Tween 20 nanoemulsions. Hence, SPI-PC nanoemulsions could be a
good way to stabilize fish oil and improve digestibility under simulated gastrointestinal conditions.

1. Introduction
Fish oil is an excellent source of polyunsaturated fatty acids
(PUFAs), particularly eicosapentanoic acid (EPA) and docosahexaenoic
acid (DHA) (García-Márquez, Higuera-Ciapar, & Espinosa-Andrews,
2017). Several studies have shown that fish oil plays a key role in the
prevention of cardiovascular disease, rheumatoid arthritis, and some
types of cancers (Kremer, 2000; Yaqoob, 2004; Garg, Wood, Singh, &
Moughan, 2006; Arab-Tehrany et al., 2012). However, fish oil usually
loses potential health benefits during processing, transportation, and
storage due to its low solubility in water, fishy smell and oxidative
instability. Thus, nanoencapsulated delivery systems such as polymeric
nanoparticles, nanoemulsions, and liposomes offer promising solutions
to improve the processability, water solubility, and bioavailability of
fish oil (Gupta, Eral, Hatton, & Doyle, 2016; Hörmann & Zimmer, 2016;
Kamaly, Yameen, Wu, & Farokhzad, 2016; Tan, Feng, Zhang, Xia, & Xia,
2016; Wackerlig & Schirhagl, 2015).
Nanoemulsions are emulsions with droplet sizes between 100 nm
and 500 nm (Xu, Mukherjee, & Chang, 2018). They are frequently used
as delivery systems due to their high optical clarity and resistance to
gravity separation and droplet aggregation (De Oca-Ávalos, Candal, &
Herrera, 2017). Some studies have reported increased bioavailability of
active compounds after encapsulation using nanoemulsions. For ex-
ample, the bioavailability of ramipil in nanoemulsion was increased to
539.49% compared with normal emulsion (Shafiq et al., 2007). The
bioavailability of vitamin E increased 1.6-fold when it was encapsulated
in nanoemulsions compared with marketed soft capsules (Gong et al.,
2012). Nanoemulsions are prepared mainly through high energy
methods, such as high-pressure homogenization, microfluidization, and
ultrasonication, which generate very uniform and small droplets by

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: wzjname@126.com (Z. Wang), tengfei@neau.edu.cn (F. Teng).
1
The first and second author contributed equally to this work.

https://doi.org/10.1016/j.foodhyd.2019.105310
Received 1 June 2019; Received in revised form 26 July 2019; Accepted 13 August 2019
Available online 16 August 2019
0268-005X/ © 2019 Published by Elsevier Ltd.
Y. Li, et al.
applying intense energy input. High energy emulsification methods
have wide applications in scale-up production in the food industry. At
present, high-pressure homogenization is one of the most common and
effective methods because it can effectively prepare uniform and highly
stable nanoemulsions (Li et al., 2018a).
Nanoemulsion stability is maintained through the interface formed
by a surfactant or emulsifier. Therefore, biological macromolecular
emulsifiers (particularly proteins) have been extensively used to further
enhance the stability of nanoemulsions (Cornacchia & Roos, 2011; Mao,
Yang, Xu, Yuan, & Gao, 2010; Qian, Decker, Xiao, & McClements,
2012). Soybean protein isolate (SPI) is an abundant plant protein that is
often used as an emulsifier. Some studies have reported that lecithin
can improve protein emulsification by the interaction between lecithin
and soybean proteins (Comas, Wagner, & Tomás, 2006; Fang &
Dalgleish, 1996). Phosphatidylcholine (PC) is an effective natural
emulsifier that can reduce the interfacial tension of emulsions. Hydro-
phobic fatty acid groups on the PC can bind with the oil surface, while
the hydrophilic group is oriented toward the surface of water. This
orientation helps to reduce the tension between oil and water and im-
prove the nanoemulsion stability (Sui et al., 2016). Some researchers
have proposed that the thermal stability of emulsions has been greatly
enhanced by composite protein and lecithin (Hirotsuka, Taniguchi,
Narita, & Kito, 1984; Kasinos et al., 2014; Van der Meeren, El-Bakry,
Neirynck, & Noppe, 2005). Comas et al. (2006) pointed out the inter-
action between SPI/denatured SPI and lecithin would reduce the
creaming rate by affecting the stability of composite emulsion.
Scuriatti, Tomás, and Wagner (2003) showed that the stability of the
composite system was stronger because of stronger interaction between
thermal denatured SPI and lecithin. Thus, SPI-PC is an excellent can-
didate for a composite emulsifier.
Presently, due to the low solubility and oxidative instability of fish
oil in most food systems, many researchers prepared fish oil nanoe-
mulsions, but with varying emulsifiers. Common protein emulsifiers
used include dairy-based proteins such as whey protein and casein.
Plant protein has also been used, however, it does not provide any
advantage in digestibility. For example, the release rate of free fatty
acids (FFA) of fish oil nanoemulsions stabilized by lentil protein was
only 59.6% (Primozic, Duchek, Nickerson, & Ghosh, 2018). The release
rate of FFA of soybean oil-in-water emulsions stabilized with soy or pea
protein isolates (PPI) was 22–25% during digestion (Fernandez-Avila,
Arranz, Guri, Trujillo, & Corredig, 2016). In our previous research, we
have successfully prepared β-carotene nanoemulsions stabilized by SPI-
PC using high-pressure homogenization (Li et al., 2018a), which proved
to be a potential delivery system. However, little information is avail-
able on encapsulated fish oil and its digestibility using the SPI-PC
composite emulsifier. The aim of this study was to examine the effect of
SPI-PC on the fish oil nanoemulsions formation by comparing the
characteristics (e.g., particle size, ζ-potential, polydispersity index
(PDI), and turbiscan stability index (TSI)) and evaluate stability and
digestibility of SPI-PC nanoemulsions compared with Tween 20. Our
research hope to establish a new green, low-cost, high-stability, and
high-digestibility nanoemulsions system and provide a new application
of fish oil in the food industry.
2. Materials and methods
2.1. Materials
SPI (protein content, 92%) was procured from the Yuwang Co.
(Yucheng, Shandong, China), it was made from low temperature (about
60 °C) defatted soybean meal using an alkali extraction and acid pre-
cipitation with a very low degree of denaturation according to the
manufacturer (Li et al., 2018b). Fish oil containing DHA (52.35 μg/mL)
and EPA (10.30 μg/mL) was purchased from the Yuwang Co. (Yucheng,
Shandong, China). PC and Tween 20 was obtained from the Sigma-
Aldrich (St. Louis, MO, USA). Other chemicals and reagents were
2
Food Hydrocolloids 100 (2020) 105310
procured from Tianjin Chemical Reagent Co. (Tianjin, China).
2.2. Preparation of nanoemulsions
SPI (1%, 2%, 3%, 4%, 5%, w/v) was mixed with PC (0, 0.2%, 0.4%,
0.6%, 1%, w/v) with varying ratios and they were dispersed in sodium
phosphate buffer solution (0.05 M, pH 7.4), stirring continuously at
room temperature (22oC–25 °C) for 12 h. Considering the fishy smell of
fish oil, the addition of fish oil (0.5%, 1%, 1.5%, 2%, 3%, w/v) was
selected for the experiment. Fish oil was added to above SPI-PC solution
followed by homogenization at room temperature by an Ultra-Turrax
T18 homogenizer (ANGNI Co. Ltd., Shanghai, China) at 20,000 r/min
for 3 min to form a coarse emulsion. Then, the coarse emulsion by high-
pressure homogenization using a D-6L ultra-high pressure homogenizer
(PhD Technology Co., Ltd., Saint Paul, MN, USA) at different pressures
was transformed into the nanoemulsions. The fish oil nanoemulsions
stabilized by Tween 20 contain 2% (w/v) Tween 20 and 1.5% (w/v)
fish oil.
2.3. Characterization of nanoemulsions
2.3.1. Measurement of particle size, PDI and ζ-potential
The particle size, PDI, and ζ-potential of the nanoemulsions were
measured using dynamic light scattering (Malvern Zetasizer Nano ZS90,
Malvern Instruments Ltd., Worcestershire, UK), according the method
of Sui et al. (2017). Considering the instrument sensitivity and reducing
multiple scattering effects, nanoemulsions were diluted 1000-fold and
100-fold in the sodium phosphate buffer solution for the determination
of particle size and ζ-potential, respectively. Particle size was expressed
as the volume weighted mean diameter D [4,3].
2.3.2. Measurement of turbidity
The turbidity was determined at 600 nm using a spectrophotometer
(UV2600, Shimadzu Co., Kyoto, Japan), according to the method de-
scribed by Li et al. (2018b). The nanoemulsions were diluted 40-fold
with the sodium phosphate buffer solution. The turbidity of nanoe-
mulsions (T) was:
T = 1.302AV / I (1)
Where A is the absorbance of the diluted emulsion at 600 nm, V is the
dilution factor (40), I is the optical path (0.01 m).
2.3.3. Measurement of TSI
The stability of the nanoemulsions was measured using the
Turbiscan Lab Expert Concentration System Stability Analyzer
(Formulaction Company, Toulouse, France). According to Luo et al.
(2017) with some modifications, 20 mL of nanoemulsions was taken in
turbiscan special glass cylindrical glass (27.5 × 70 mm). The sample in
the cell was scanned every 30 min for 6 h at 55 °C. TSI was calculated
with a Turbiscan lab expert.
2.3.4. Confocal laser scanning microscopy (CLSM)
The structure of nanoemulsions was recorded using Leica TCS SP2
confocal laser scanning microscope (Leica Microsystems, Heidelberg
GmbH, Heidelberg, Germany). Based on Bakry et al. (2016), Nile red
and Nile blue was dissolved in isopropanol with concentration of
0.001 g/mL Nile red and 0.01 g/mL Nile blue, respectively. 1 mL of fish
oil nanoemulsions was mixed with 20 μL Nile red and 25 μL Nile blue.
The sample dyed for 30 min after 30 s vortex mixing. Then, 5 μL of
nanoemulsions was transferred to the glass slide and covering the slide.
The samples were stored in the dark for 12 h and then observed by
CLSM.
Y. Li, et al.
2.4. Nanoemulsions stability
2.4.1. Storage stability
SPI-PC nanoemulsions and Tween 20 nanoemulsions were stored
under 4 °C, 25 °C and 50 °C for 30 days. The particle size and ζ-potential
of the nanoemulsions were measured every 5 day.
2.4.2. Oxidative stability
SPI-PC nanoemulsions and Tween 20 nanoemulsions were placed in
centrifugal tubes, respectively and were stored in a 50 °C incubator for
10 days. The contents of primary oxidation products (lipid hydroper-
oxides) and secondary oxidation products (2-thiobarbituric acid re-
active substances, TBARS) were determined by Yi et al. (2019) with
some modification.
Lipid hydroperoxides: 0.3 mL nanoemulsions was mixed with
1.5 mL solution of isooctane and isopropanol (3:1, v/v). Then, the or-
ganic solvent phase of the solution was collected by 1000×g cen-
trifugation for 2 min. An aliquot (approximately 200 μL) of the super-
natant was added in 2.8 mL mixture solution of methanol and butanol
(2:1, v/v). Then, 15 μL ammonium thiocyanate (3.94 M) and 15 μL
ferrous solution (0.132 M BaCl2 and 0.144 M FeSO4) were added into
above solution. After standing 20 min, the absorbance of the solution
was measured at 510 nm by UV-VIS Spectrophotometer (UV-1800,
Jinghua Co., Ltd. Shanghai, China). The hydroperoxides content was
determined by the standard curve measured by cumene hydroperoxide.
TBARS: The 1.0 mL nanoemulsions was mixed with 2.0 mL TBA
reagent (15% (w/v) trichloroacetic acid and 0.375% (w/v) thiobarbi-
turic acid in 0.25 M hydrochloric acid (HCl)). The solution was heated
in a boiling water bath for 15 min and filtered with 0.45 mm micro-
porous membrane. The absorbance of the filter liquor was determined
at 532 nm. The content of TBARS was calculated according to the
standard curve obtained from 1,1,3,3-tetraethoxypropane.
2.4.3. pH stability
Nanoemulsions were prepared in sodium phosphate buffer solution
with pH 3–8. Samples were stored at 25 ± 2 °C for 2 h and analyzed for
particle size and ζ-potential.
2.4.4. Ionic stability
The ionic strength of nanoemulsions was adjusted to different levels
(0–0.5 M) by adding appropriate amounts of NaCl. Samples were stored
at 25 ± 2 °C for 2 h and analyzed for particle size and ζ-potential.
2.5. In vitro digestion of nanoemulsions
A gastrointestinal model was constructed by referring to the method
of Wooster et al. (2014), including simulated gastric fluid (SGF), si-
mulated intestinal fluid (SIF). During the whole digestion experiments,
the temperature of the incubator was maintained at 37 °C.
2.5.1. Simulated gastric stage
Briefly, SGF was prepared by dissolving 1% (w/v) pepsin (3000 U/
g), 2 g of NaCl and 7 mL of HCl in 1 L deionized water, the pH was
adjusted to 2.0 with 1 M HCl. The 30 mL fish oil nanoemulsions and
30 mL SGF were mixed in the stopper beaker and incubated in a shaking
bed (200 r/min) at 37 °C for 2 h, maintaining pH at 2.0 by 0.1 M HCl.
After 2 h incubation, 30 mL of digestion emulsions was collected for the
subsequent intestinal digestion.
2.5.2. Simulated intestinal stage
SIF was prepared as described previously Liang et al. (2012a) with
slight modifications. The SIF contained 10 mg/mL pancreatin, 12 mg/
mL bile salt (0.012 g bile salt dissolved in 1 mL pH 7.5 sodium phos-
phate buffer solutions), the pH was adjusted to 7.5 using 0.1 M NaOH.
30 mL fish oil nanoemulsions after gastric digestion was transferred into
30 mL SIF in the stopper beaker and incubated in a shaking bed (200 r/
3
Food Hydrocolloids 100 (2020) 105310
min) at 37 °C for 2 h, maintaining pH at 7.5 by 0.1 M NaOH.
The volume of 0.1 M NaOH added to the samples was used to cal-
culate the concentration of FFA generated in the reaction beaker at 5,
10, 20, 30, 60, 90 and 120 min during intestinal digestion process. It is
assumed that each triacylglycerol molecule produces 2 FFAs under the
action of lipase. The percentage of released FFA was calculated through
eq (2) (Li & McClements, 2010):
VNaOH (t ) × CNaOH  × MWL    
FFA release% = × 100
2mL (2)
Where mL is the weight of total lipid in nanoemulsions, VNaOH(t) is the
volume of NaOH (mL) required for neutralizing FFA released during
lipid digestion, CNaOH is the molarity of the NaOH solution (in M), MWL
is the molecular weight of the fish oil (880 g/mol).
2.5.3. Optical microscope
The morphology of digestion nanoemulsions was performed by a
BH-2 type microscope (Olympus Corp., Tokyo, Japan). The original
nanoemulsions and digestion nanoemulsions were respectively trans-
ferred on the slide, then it covered with the cover glass. The sample was
observed by 40×, and photographed.
2.6. Statistical analysis
All the results were done at least triplicate. The results are expressed
as means ± standard deviations. The experiment data were evaluated
by ANOVA using SPSS 19 software (SPSS Inc., Chicago, IL, USA).
P < 0.05 was considered significant. The figures were made by Origin
pro 8.5.
3. Results and discussions
3.1. Effect of SPI addition on the stability of nanoemulsions
The effect of SPI addition on fish oil nanoemulsions stability is
shown in Table 1 and Fig. 1a. Particle size and PDI decreased sig-
nificantly (from 1% to 2%) with SPI, which may its low concentration
prevented SPI from fully covering the surface of the oil droplets. Thus,
the bare oil droplets that were not coated by emulsifier tended to ag-
gregate, forming large droplets. Similar results were also found by Li
et al. (2018a). When SPI was added at higher than 1%, the nanoe-
mulsions became relatively uniform and stable, indicating that the oil
droplet surface may be covered with enough protein to prevent ag-
gregation (Li, Ma, & Cui, 2014). However, when SPI concentration was
increased from 2% to 5%, the particle size and PDI value of the na-
noemulsions increased. This may be due to excessive protein aggregates
binding to form submicelles (Song, Zhou, Fu, Chen, & Wu, 2013), which
then form larger droplets. Unloaded protein molecules at the nanoe-
mulsion interface potentially flocculated due to osmotic aggregation
(Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012).
Similar results were obtained for turbidity, when SPI was above 2%,
the turbidity increased. It may be due to the fact that many unabsorbed
proteins could be removed from the surface of the emulsion, when the
oil droplet interface protein became saturated (Berton-Carabin, Ropers,
& Genot, 2014). The absolute value of ζ-potential decreased with in-
creasing SPI concentration. The stability of oil-in-water emulsion was
related to the surface charge of droplets. The ζ-potential was highest at
2.0% SPI with an absolute value of 32.3 mV. Previous studies suggest
that emulsion droplets can be effectively stabilized by electrostatic re-
pulsion when the ζ-potential value is higher than 31.20 mV (Sui et al.,
2017).
3.2. Effect of PC addition on the stability of nanoemulsions
The stability of the fish oil nanoemulsions with added PC is shown
in Table 1 and Fig. 1b. Particle size of the nanoemulsions without PC
Y. Li, et al. Food Hydrocolloids 100 (2020) 105310

Table 1
The Effect of components on stability of fish oil nanoemulsions stabilized by SPI-PC.
Component Parameter Particle Size (nm) PDI ζ-potential (mV) Turbidity TSI

SPI (%) 1 424.66 ± 1.93a

0.45 ± 0.02a
−26.03 ± 0.36d
3810.52 ± 38.34b
4.56 ± 0.05a
2 245.03 ± 2.54d 0.23 ± 0.01c −32.30 ± 0.40a 2244.65 ± 40.56e 3.04 ± 0.04e
3 251.30 ± 2.55cd 0.23 ± 0.02c −31.33 ± 0.35ab 2985.92 ± 111.88d 3.12 ± 0.03d
4 258.40 ± 4.57c 0.24 ± 0.01c −29.66 ± 0.25b 3577.89 ± 49.04c 3.17 ± 0.02c
5 316.20 ± 5.63b 0.37 ± 0.01b −28.50 ± 0.34c 4039.67 ± 20.09a 3.36 ± 0.04b

PC (%) 0 316.86 ± 4.07a 0.29 ± 0.03a −28.80 ± 0.35e 3695.94 ± 24.91a 4.24 ± 0.03a
0.2 245.03 ± 2.54e 0.23 ± 0.02a −32.30 ± 0.40d 2244.65 ± 40.56e 3.04 ± 0.04e
0.4 256.76 ± 3.54d 0.24 ± 0.01a −33.96 ± 0.53c 2697.74 ± 15.33d 3.12 ± 0.01d
0.6 271.96 ± 1.06c 0.25 ± 0.03a −34.66 ± 0.57b 2838.36 ± 29.46c 3.18 ± 0.02c
1 278.96 ± 2.57b 0.28 ± 0.01a −35.63 ± 0.18a 3025.84 ± 18.53b 3.34 ± 0.03b

Fish oil (%) 0.5 261.20 ± 1.59c 0.29 ± 0.01b −28.03 ± 1.52ab 2414.84 ± 31.91c 3.56 ± 0.01c
1 257.00 ± 5.32c 0.27 ± 0.01b −28.13 ± 0.25ab 2395.68 ± 19.48d 3.48 ± 0.02d
1.5 245.03 ± 2.54d 0.23 ± 0.02b −32.30 ± 0.40a 2244.65 ± 40.56e 3.04 ± 0.04e
2 306.63 ± 4.25b 0.25 ± 0.02b −29.43 ± 0.97ab 3859.13 ± 25.86b 4.65 ± 0.03b
3 422.46 ± 8.33a 0.42 ± 0.02a −29.06 ± 0.34ab 7383.20 ± 154.16a 5.43 ± 0.05a

Homogenization Pressure (MPa) 60 455.33 ± 10.11a 0.48 ± 0.02a −27.86 ± 0.46c 4336.52 ± 17.18a 5.83 ± 0.04a
80 403.53 ± 7.37b 0.45 ± 0.00b −28.86 ± 0.35b 2774.13 ± 41.30c 5.02 ± 0.03b
100 245.03 ± 2.54e 0.23 ± 0.02d −32.30 ± 0.40a 2244.65 ± 40.56d 3.04 ± 0.04e
120 313.71 ± 14.30d 0.35 ± 0.03c −31.58 ± 0.30a 2213.76 ± 20.09d 3.42 ± 0.01d
140 388.23 ± 6.02c 0.38 ± 0.01c −22.66 ± 0.26d 3185.62 ± 55.49b 4.88 ± 0.02c

Values is mean ± standard deviations (n = 3). The data with different letters are significantly different (p < 0.05).

Fig. 1. Particle size distribution of the fish oil nanoemulsions stabilized by SPI-PC on different components (a: SPI; b: PC; c: Fish oil; d: Homogenization pressure).

4
Y. Li, et al.
was 316.86 nm. The presence of PC (0–0.2%) significantly reduced the
particle size from 316.86 to 245.03 nm. The nanoemulsions have a
minimum particle size and PDI at 0.2% PC and gradually increased with
increasing PC from 0.2% to 1%. This may be because high concentra-
tions of surfactant could rapidly expand on the surface of oil droplets
and partially penetrate into the protein network (Wang, Li, Wang, &
Ozkan, 2010; Martínez, Ganesan, Pilosof, & Harte, 2011; Li, Li, & Guo,
2014). Excessive PC may replace soybean protein competitively at the
interface of the emulsion. The replaced soybean protein from the oil
surface aggregated with each other, leading to the increase in turbidity
(Table 1). The protein was replaced by competitive adsorption PC at the
O/W interface, resulting in the disintegration of the emulsified layer
and a decrease in emulsion stability (Matsumiya, Takahashi, Nakanishi,
Dotsu, & Matsumura, 2014; Wang et al., 2017).
As the added PC increased, the absolute value of ζ-potential for the
nanoemulsions also increased. This may be because the interaction of
PC with protein changed the charge distribution on the surface of the
protein. Previous studies found that PC can boost the negativity of the
SPI surface and increase repulsion between particles (Matsumiya et al.,
2014; Wang et al., 2017). García-Moreno, Horn, and Jacobsen (2014)
suggested that the ζ-potential of casein became more negative as le-
cithin increased. These results show that small particle size and high ζ-
potential could increase the stability of nanoemulsions.
3.3. Effect of fish oil on the stability of nanoemulsions
When the added fish oil increased from 0.5% to 1.5%, the particle
size and the PDI of nanoemulsions were reduced (Table 1 and Fig. 1c).
However, the particle size and PDI values significantly increased with
further addition of fish oil from 1.5% to 3%. It is possible that the
amount of protein was not sufficient to cover the surface of fish oil
droplets, accompanied by the fish oil adhering to the surface of the
droplets. The turbidity of the nanoemulsions increased significantly
with increasing fish oil. The value of ζ-potential was not significantly
different with the addition of fish oil. Previous studies also found no
correlation between ζ-potential and oil phase concentration in cur-
cumin nanoemulsions (Ma et al., 2017).
3.4. Effect of homogenization pressure on stability of nanoemulsions
When the homogenization pressure was increased from 60 MPa to
100 MPa, particle size, PDI, turbidity, and TSI all decreased, as shown
in Table 1 and Fig. 1d. The smallest nanoemulsions size was obtained at
100 MPa, similar to Tan and Nakajima (2004). Shear forces and tur-
bulence were the two main factors influencing particle size (Floury,
Desrumaux, & Lardieres, 2000; Floury, Desrumaux, & Legrand, 2002). A
faster shear rate could decrease particle size (Mason, Wilking, Meleson,
Chang, & Graves, 2006), however, particle size increased and exhibited
a bimodal peak distribution of size when the homogenization pressure
increased above 120 MPa. This may be due to the fact that high
homogenization pressure leads to a considerable increase in the specific
surface area of nanoemulsions droplets, composite emulsifier could not
cover all the droplet interfaces, leading to the instability of emulsion
and droplet aggregation. Similar results were also found by Fernandez-
Avila and Trujillo (2016).
With increasing homogenization pressure, the absolute value of ζ-
potential increased and the TSI value decreased. However, 140 MPa of
pressure led to instability and droplet cracking, leading the absolute
value of ζ-potential to decrease significantly. The increase of turbidity
at 140 MPa confirmed droplet cracking, consistent with Li et al.
(2018a).
Based on the above research, optimal nanoemulsion construction
parameters are 2% SPI, 0.2% PC, 1.5% fish oil, and 100 MPa homo-
genization pressure.
5
Food Hydrocolloids 100 (2020) 105310
3.5. CLSM analysis
CLSM imaging was used to observe the droplet distribution and
microstructure of the nanoemulsions. In Fig. 2a, red represents oil
droplets and green represents protein. The oil phase exhibits a spherical
shape, which indicates that oil droplets are completely encapsulated
inside the nanoemulsions. The spherical shape of the protein shows that
the SPI is adsorbed on the O/W interface of the nanoemulsions. How-
ever, the red label of the oil phase does not coincide perfectly with the
green label of the protein due to the fact that the droplets in nanoe-
mulsions are in constant Brownian motion. In Fig. 2b, the lipid droplets
were also relatively small and evenly dispersed in Tween 20 nanoe-
mulsions.
3.6. Storage stability
The shelf life of the product is essential for nanoemulsion-based
delivery systems in most practical applications (Chaari, Theochari,
Papadimitriou, Xenakis, & Ammar, 2018). The particle size and ζ-po-
tential of two fish oil nanoemulsions stored at 4 °C, 25 °C, and 50 °C for
30 days are shown in Fig. 3 and Fig. 4, respectively. After 30 days of
storage at 4 °C, 25 °C, and 50 °C, the particle size of the two nanoe-
mulsions increased with increasing storage temperature (Fig. 3a and b).
This suggested that emulsion droplets were more likely to collide due to
increased Brownian motion during long term high-temperature storage,
resulting in droplet flocculation (Liang et al., 2012b). Tween 20 na-
noemulsions particle size significantly increased when stored at 25 °C
and 50 °C. Due to the thermal dehydration of the hydrophilic surfactant
head groups at the oil-water interface, the polyoxyethylene moieties of
Tween 20 caused deformation and aggregation of the colloidal structure
(Dey, Banerjee, Chatterjee, & Dhar, 2018).
ζ-potential is another important indicator for evaluating the na-
noemulsions storage stability. Müller (1996) demonstrated that emul-
sion droplets can be stabilized by strong electrostatic repulsion alone,
when the absolute value of ζ-potential at 4 °C exceeded 30 mV. After
storage at 4 °C, 25 °C, and 50 °C for 30 days, the absolute ζ-potential
values of SPI-PC nanoemulsions gradually decreased to 27.7, 25.8, and
21.1, respectively (Fig. 4a). This instability may be due to the aging of
austenite and changes in protein conformation over time, resulting in
the formation of hydrogen bonds and hydrophobic bonds between ad-
jacent proteins at the interface (Tcholakova, Denkov, Ivanov, &
Campbell, 2006). The absolute ζ-potential value of the Tween 20 na-
noemulsions also decreased with increasing temperature during sto-
rage. The stability of the nanoemulsions at 4 °C is better than the sta-
bility at 25 °C and 50 °C. Some researchers also found that the stability
of carotene nanoemulsions and rapeseed oil nanoparticles (prepared
from whey protein isolate) was less at 25 °C than 4 °C (Adjonu, Doran,
Torley, & Agboola, 2014; Yi, Lam, Yokoyama, Cheng, & Zhong, 2014).
3.7. Oxidation stability
Oxidation of lipids is a major cause of nutrient loss in foods con-
taining unsaturated lipids. Therefore, we analyzed the contents of lipid
hydroperoxides and TBARS in SPI-PC and Tween 20 nanoemulsions. As
shown in Fig. 5a, the hydroperoxides content in Tween 20 nanoemul-
sions was higher than that of SPI-PC, which may be due to the fact that
Tween 20 contains some endogenous hydroperoxides (Yi et al., 2019).
In contrast, formation of hydroperoxides in the SPI-PC nanoemulsions is
much slower. This may be due to the antioxidant activities of soy
protein and phosphatidylcholine. Early studies have found that protein
emulsifiers inhibit lipid oxidation better than small-molecule surfac-
tants (Osborn & Akoh, 2004; Haahr & Jacobsen, 2008; Raikos, Duthie,
& Ranawana, 2016). In addition, the high degree of lipid oxidation in
Tween 20 nanoemulsions could also be related to its smaller particle
size compared to SPI-PC nanoemulsions, because the lipid oxidation
rate increases with decreasing droplet size (Imai, Maeda, Shima, &
Y. Li, et al.
Adachi, 2008; Sun & Gunasekaran, 2009). The TBARS content in Tween
20 nanoemulsions was higher than that of SPI-PC, and a certain amount
of TBARS was also present in freshly prepared nanoemulsions. The
TBARS formation rate was much faster for Tween 20 nanoemulsions.
The TBARS content increased with increasing storage time. These re-
sults were similar to that of hydroperoxides, as shown in Fig. 5a. This
may be because Tween 20 is a small molecular surfactant whose density
and stacking behavior at the interface could decrease antioxidant na-
noemulsion stability (Nejadmansouri, Hosseini, Niakosari, Yousefi, &
Golmakani, 2016). Thus, SPI-PC as a composite emulsifier can effec-
tively protect unsaturated fatty acids from oxidation.
3.8. pH stability
pH value affects the degree of ionization and emulsification of
soybean protein, and therefore changes the stability of nanoemulsions.
It is necessary to study the influence of pH value on the stability of fish
oil nanoemulsions. Table 2 shows that pH has little effect on the particle
size of fish oil nanoemulsions stabilized by Tween 20. However, abso-
lute value of ζ-potential in Tween 20 nanoemulsions increased with
increasing pH. Recent reports suggested Tween 20 is a non-ionic sur-
factant whose chemical structure contains no ionizable group (Taarji
et al., 2017). However, the addition of negative charges in Tween 20
nanoemulsions with increasing pH (Table 2) may be due to preferential
adsorption of hydroxyl ions (OH−) on the droplet's surface
(McClements, 2016). The change of particle size of SPI-PC nanoemul-
sions was larger in the pH 4.0–6.0 range but was not significant the pH
7.0–8.0 range. This may be due to the fact that the pH value was close
to the isoelectric point of soybean protein and the electrostatic repul-
sion among the droplets decreased, which promoted particle aggrega-
tion caused by the interaction of SPI-SPI. The ζ-potential of SPI-PC
nanoemulsions increased with decreasing pH value. When the pH value
6
Food Hydrocolloids 100 (2020) 105310
Fig. 2. Microstructure of fish oil nanoemulsions by
confocal laser scanning microscopy (a: fish oil na-
noemulsions stabilized by SPI-PC; b: fish oil nanoe-
mulsions stabilized by Tween 20). The red color re-
presents oil droplets and green color represents
protein in the nanoemulsions. Scale bar measures
10 μm. (For interpretation of the references to color
in this figure legend, the reader is referred to the Web
version of this article.)
was 3.0 and 4.0, the ζ-potential was positive (Table 2). However, the
absolute value of the ζ-potential was highest when the pH was 7.0 and
8.0. Rao and McClements (2011) also found that a relatively stable
nanoemulsion was formed at pH 6.0 and 7.0.
3.9. Ionic strength stability
Salt is a common food additive and also present in the human
gastrointestinal tract, which may affect the functional of the colloidal
delivery system in the food or digestion. Therefore, the effects of Na+
concentration (0.1–0.5 M) on the stability of fish oil nanoemulsions
were investigated. The addition of Na+ has no obvious effect on the
particle size of either Tween 20 or SPI-PC nanoemulsions, but the ab-
solute value of ζ-potential decreased gradually (Table 2), likely because
the salt reduced the electrostatic repulsion among molecules, reducing
the charge (Sari et al., 2015). Previous studies have shown that high salt
concentrations may lead to instability (e.g., aggregation) in nanoe-
mulsions, due to reduced electrostatic repulsion (McClements, 2016).
However, compared to Tween 20 nanoemulsions, the SPI-PC nanoe-
mulsions had a higher resistance to Na+, probably due to the higher
charge density of SPI/PC composite on the surface of the droplets. Na+
adsorbed on protein molecules by electrostatic attraction promoted an
increase in the number of protein molecules adsorbed on the surface of
oil droplets (Xu et al., 2018). It can be assumed that there is a large
electrostatic repulsion between the droplets in the nanoemulsions to
avoid charge neutralization of Na+ (Sharma et al., 2017).
3.10. In vitro digestion simulation
To evaluate the digestibility of fish oil nanoemulsions, we analyzed
structural changes of nanoemulsions in an in vitro simulated human
digestion system.
Y. Li, et al.
Fig. 3. Changes in particle size of nanoemulsions (a: fish oil nanoemulsions
stabilized by SPI-PC; b: fish oil nanoemulsions stabilized by Tween 20) at 4 °C,
25 °C and 50 °C for 30 days.
When the SPI-PC nanoemulsions were digested in a simulated sto-
mach (Table 3), the particle size increased significantly, and large ag-
gregated droplets could also be observed via optical microscopy
(Fig. 6), indicating that the nanoemulsion has been flocculated in si-
mulated gastric fluid. The pepsin in gastric fluid could hydrolyze the
SPI adsorbed on the surface of oil droplets, weakening the electrostatic
repulsion between lipid droplets and leading to the aggregation and
flocculation of droplets (Malaki, Wright, & Corredig, 2011a, b; Chang &
McClements, 2016). Sarkar, Goh, Singh, and Singh (2009) reported that
the particle size of β-lactoglobulin nanoemulsions increased due to
aggregation after stomach digestion. The addition of ions (such as
Ca2+, K+, and H+) may also affect the stability of the emulsion
(Verkempinck et al., 2018). The absolute value of ζ-potential of SPI-PC
nanoemulsions decreased significantly after gastric digestion. This may
be because low pH value of the simulated gastric fluid protonated some
anionic groups (e.g. -COO-→-COOH), which neutralized the negative
charge on the surface of the protein (Salvia-Trujillo & McClements,
2016). In addition, high ionic strength can decrease negative charge
density on the surface of the emulsion through an electrostatic shielding
effect (Chang & McClements, 2016; Chen et al., 2017). After gastric
digestion, the turbidity of SPI-PC nanoemulsions increased sig-
nificantly. That may be because the nanoemulsions droplets were de-
composed by pepsin hydrolysis and flocculation of the oil phase driven
by hydrophobic interactions. However, the particle size, PDI, TSI, tur-
bidity, and optical microscope images of Tween 20 nanoemulsions were
almost unchanged (Fig. 6) after exposure to simulated gastric fluid.
These results show that Tween 20 nanoemulsions are resistant to dro-
plet aggregation in simulated gastric fluid, while SPI-PC nanoemulsions
7
Food Hydrocolloids 100 (2020) 105310
Fig. 4. Changes in ζ-potential of nanoemulsions (a: fish oil nanoemulsions
stabilized by SPI-PC; b: fish oil nanoemulsions stabilized by Tween 20) at 4 °C,
25 °C and 50 °C for 30 days.
are not, which is consistent with the results of Van Aken, Bomhof, Zoet,
Verbeek, and Oosterveld (2011).
When SPI-PC nanoemulsions were digested in simulated intestine
(Table 3 and Fig. 6), the particle size of the nanoemulsions decreased to
226.66 nm. It is possible that the lipids were converted into free fatty
acids and monoacylglycerols by pancreatic lipase (Chang &
McClements, 2016). The emulsified layer was replaced by bile salt and
pancreatic lipase gradually adsorbed on the surface of emulsion, de-
creasing the particle size of the nanoemulsions (Qian et al., 2012).
Previous research demonstrated that the particle size of β-carotene
nanoemulsions increased in an in vitro intestinal fluid digestion ex-
periment (Kossena, Boyd, Porter, & Charman, 2003). However, oil
droplet aggregation did not appear in the fish oil nanoemulsions after
intestinal fluid digestion, which may be due to the fact that the hy-
drophobic interaction of SPI-PC enhanced the anti-polymerization ef-
fect of oil-water nanoemulsion interface. Optical microscopy showed
that the quantity of oil droplets decreased significantly after digestion
in simulated intestinal fluid (Fig. 6), which could be due to the diges-
tion of oil in mixed micelles. The particle size and microstructure of SPI-
PC and Tween 20 nanoemulsions were similar after the digestion of
intestinal fluid and they appear to be independent of the type of
emulsifier at the initial O/W interface. This may be due to the fact that
the original emulsifier was digested by enzymes or replaced by bile salt
and fatty acids.
When SPI-PC and Tween 20 nanoemulsions passed the intestinal
fluid, the absolute value of ζ-potential increased significantly. The
reasons may be the following: (1) The anionic bile salt in intestinal fluid
adsorbed on the surface of micelles, vesicles, and lipid droplets pro-
duced by lipid digestion, which led to the increase in negative charge;
or (2) FFAs with negative charge released by lipase-driven triglyceride
Y. Li, et al. Food Hydrocolloids 100 (2020) 105310

attributed in part to the effect of diluting the emulsion concentration.

3.11. Free fatty acid release

In Fig. 7, the effects of SPI-PC nanoemulsions and Tween 20 na-

Fig. 5. Hydroperoxides content and TBARS content of nanoemulsions used
different emulsifiers: (a) Hydroperoxides; (b) TBARS.
hydrolysis may accumulate on the surface of particles after lipid di-
gestion (Chang & McClements, 2016; McClements & Xiao, 2012; Singh,
Ye, & Horne, 2009). The turbidity of SPI-PC nanoemulsions decreased
significantly during intestinal digestion (Fig. 6) by biological enzymatic
hydrolysis and micellization. The decrease in turbidity can also be
noemulsions on the digestibility of fish oil were compared. After di-
gestion of simulated intestinal fluid for 120 min, FFA release of all
emulsions increased. The FFA release from SPI-PC nanoemulsions
reached 86.8%, however, release from Tween 20 nanoemulsions and
the (pure fish oil) control group were only 42.2% and 32.7%, respec-
tively. The digestibility of fish oil can be improved after encapsulation
by nanoemulsions because their smaller droplet size increased the di-
gestible interface area. In addition, the release of FFA of SPI-PC na-
noemulsions was 44.6% higher than that of Tween 20 fish oil nanoe-
mulsions. Previous research has proposed that Tween 20 as an
emulsifier can inhibit the activity of lipid hydrolysis. The surface ac-
tivity of Tween is stronger than that of the lipase, which prevents the
lipase from binding with the lipid droplet surface (Mun, Decker, &
McClements, 2007). Karthik and Anandharamakrishnan (2016) con-
cluded that the interaction between lipase and oil played a role in the
digestibility of lipids, as well as the composition of oil droplets and the
interface layer. These properties depend largely on the emulsifiers used
to stabilize the emulsion during the preparation process.
There are differences in the extent of digestion of lipids en-
capsulated by nanoemulsions in in vitro digestion in the literature.
Gumus, Decker, and McClements (2017) reported fish oil-in-water
emulsions stabilized with various pulse proteins from faba bean, lentil,
and pea in in vitro digestion showed 100% lipolysis, for instance.
However, a research reported FFA release in fish oil nanoemulsions was
59.6% (Primozic et al., 2018). That could be attributed to the type and
purity of emulsifiers used or the concentration of enzymes or bile salts,
and so on. Sarkar, Ye, and Singh (2016) showed that the FFA release of
sodium caseinate emulsions increased by almost 100% when the un-
adsorbed bile extract concentration increased from 1 to 5 mg/mL.
Karthik and Anandharamakrishnan (2016) reported the digestibility of
lipid depend largely on the emulsifiers used in the preparation process
(Karthik & Anandharamakrishnan, 2016). A recent study has found that
the non-digestible component and impurity in the protein may limit the
proteolysis extent in the simulated stomach, which may affect the re-
placement of proteins by bile salt from the surface of oil droplets and
thus reducing the degree of lipid digestion by lipase in the simulated
intestine (Primozic et al., 2018).
4. Conclusions
In this study, we prepared fish oil nanoemulsions with SPI-PC as

Table 2
The Effect of pH, ionic strength on particle size and ζ-potential of two fish oil nanoemulsions.
Effect of ionic strength Fish oil nanoemulsions stabilized by SPI-PC Fish oil nanoemulsions stabilized by Tween 20

Particle size (nm) ζ-potential (mV) Particle size (nm) ζ-potential (mV)

Control group 253.22 ± 1.41c −28.42 ± 0.45a 129.03 ± 0.22a −17.60 ± 0.42a
0.1 M NaCl 260.84 ± 2.80b −27.34 ± 0.13b 127.02 ± 1.21b −15.32 ± 0.10b
0.2 M NaCl 273.43 ± 1.51ab −27.22 ± 0.22b 129.40 ± 0.30a −14.40 ± 0.34c
0.3 M NaCl 276.52 ± 2.72a −26.80 ± 0.34bc 129.52 ± 0.48a −11.45 ± 0.25d
0.4 M NaCl 279.10 ± 2.83a −25.52 ± 0.22c 131.16 ± 0.65a −10.35 ± 0.46e
0.5 M NaCl 282.91 ± 4.02a −24.31 ± 0.22d 131.60 ± 0.32a −8.20 ± 0.29f

Effect of pH
pH = 3.0 520.61 ± 56.03c 22.80 ± 0.84e 137.70 ± 0.52a −6.30 ± 0.19e
pH = 4.0 664.42 ± 25.92b 12.04 ± 0.55d 134.41 ± 0.33b −13.51 ± 0.30d
pH = 5.0 812.20 ± 43.41a −3.59 ± 0.13c 131.62 ± 0.24c −14.80 ± 0.34c
pH = 6.0 638.53 ± 40.82b −15.74 ± 0.33b 128.03 ± 0.53e −18.53 ± 0.33bc
pH = 7.0 278.40 ± 4.63d −25.52 ± 0.21a 130.40 ± 0.25d −19.24 ± 0.24b
pH = 8.0 248.81 ± 4.02d −25.60 ± 0.25a 127.91 ± 0.27e −21.90 ± 0.55a

Values is mean ± standard deviations (n = 3). The data with different letters are significantly different (p < 0.05).

8
Y. Li, et al. Food Hydrocolloids 100 (2020) 105310

Table 3
Characterization of two fish oil nanomulsions during in vitro digestion.
Nanomulsion type Particle Size (nm) PDI ζ-potential (mV) Turbidity TSI

SPI-PC Initial 245.03 ± 2.54b

0.23 ± 0.02d
−32.30 ± 0.40 b
2244.65 ± 10.56 c
3.04 ± 0.04b
Stomach 641.96 ± 9.92a 0.64 ± 0.07a −23.3 ± 0.08c 4260.14 ± 0.21a 5.93 ± 0.02a
Intestine 226.66 ± 6.58c 0.33 ± 0.03b −40.6 ± 1.20a 2380.05 ± 0.10b 3.03 ± 0.03b
Tween 20 Initial 149.81 ± 1.90d 0.27 ± 0.00c −20.9 ± 0.14d 651.02 ± 0.01d 3.07 ± 0.01b
Stomach 144.76 ± 3.25d 0.33 ± 0.01b −15.7 ± 0.89e 442.30 ± 0.00e 3.02 ± 0.01b
Intestine 145.63 ± 4.30d 0.30 ± 0.06b −23.7 ± 1.34c 484.70 ± 0.01e 3.05 ± 0.01b

Values is mean ± standard deviations (n = 3). The data with different letters are significantly different (p < 0.05).

composite emulsifier and evaluated stability and digestibility. Our re-

sults showed that SPI-PC nanoemulsions have optimal stability at con-
ditions of 2% SPI, 0.2% PC, 1.5% fish oil, and 100 MPa homogenization
pressure. Compared with Tween 20 nanoemulsions, SPI-PC nanoemul-
sions have better storage stability, oil oxidation stability, and resistance
to Na+, due to the higher charge density of SPI/PC on the surface,
stronger electrostatic repulsion, and better inhibition of lipid oxidation.
However, the stability of SPI-PC nanoemulsions was not good in acidic
conditions due to the proximity of the isoelectric point of soybean
protein. The release of FFA in SPI-PC nanoemulsions reached 86.8%,
which was higher than that of Tween 20. Therefore, SPI-PC nanoe-
mulsions are a new type of nanoemulsion system for restraining oxi-
dation and improving digestibility of fish oil, and this research will take
us a step further in efficient utilization of fish oil in the future.

Conflicts of interest

None.
Fig. 7. Release rate of free fatty acids in pure fish oil, fish oil nanoemulsions
Acknowledgements stabilized by SPI-PC and Tween 20 during in vitro digestion.

We are very grateful to the support of the National Key Research Science Foundation of China (2018M641798) and Postdoctoral Project
and Development Program of China (research grant numbers in Heilongjiang Province (LBH-Z18030).
2017YFD0400202-04 and 2016YFD0401402),the National Natural
Science Foundation of China (research grant number: 31571876), Appendix A. Supplementary data
Major Project of Applied Technology Research and Development
Technology in Heilongjiang Province (GA17B002), the Natural Science Supplementary data to this article can be found online at https://
Foundation of Heilongjiang Province (C2018024), the Postdoctoral doi.org/10.1016/j.foodhyd.2019.105310.

Fig. 6. Microscopic observation images of fish oil nanoemulsions in vitro digestion. The digestion time of nanoemulsions in the stomach and intestine was 2 h,
respectively. The scale bar indicates 20 μm.

9
Y. Li, et al.
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