Food Packaging and Shelf Life 11 (2017) 4957

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Food Packaging and Shelf Life

journal homepage: http://www.elsevier.com/locate/fpsl

Antioxidant property of SiO2-eugenol liposome loaded nanobrous

membranes on beef
Haiying Cui, Lu Yuan, Wei Li, Lin Lin*
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

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

Article history:
Received 24 September 2016 In consideration of the strong volatility and chemical instability of eugenol, liposomes were introduced to
Received in revised form 13 December 2016 remain the activity of eugenol during processing and storage. Silica nanoparticle (SiO2) was used to
Accepted 2 January 2017 adsorb eugenol before liposome encapsulation for getting better physicochemical properties and
Available online xxx physical stability. The results of particle size, zeta potential and encapsulation efciency of liposomes
containing SiO2-eugenol were 315.7
0.7 nm, 47.7
0.3 mV, and 89.91
0.43%, respectively. The
Keywords: experiments testing the physical stability of liposomes containing SiO2-eugenol showed that SiO2 could
Antioxidant signicantly promote its stability. For the application in food antioxidant, electrospun nanobrous
SiO2
membranes were introduced to immobilize the SiO2-eugenol liposomes. In this study, the antioxidant
Eugenol
assay of SiO2-eugenol liposome loaded nanobrous membranes exhibited antioxidant activity on beef.
Liposome
Electrospinning 2017 Elsevier Ltd. All rights reserved.

1. Introduction physical and chemical stability of liposomes leads to troubles in the

Eugenol (4-allyl-2-methoxyphenol), the major compound of
clove oil, nutmeg oil and cinnamon oil, has been widely applied in
food preservation, cosmetics production and folk medicine. The
antibacterial activity, antioxidant activity, anti-inammatory and
local anesthetic of eugenol have been conrmed by researchers
(Phunpee et al., 2016). Unfortunately, the present application of
eugenol is limited in its volatility and chemical instability when
exposed in air, light, moisture, and high temperatures. The method
of liposomal encapsulation has been developed to reduce the
damage of eugenol during processing and storage.
Liposomes, articial lipid vesicles, are made up of one or
multiple concentric phospholipid bilayers embedding aqueous
cores. Liposomes can encapsulate hydrophilic and lipophilic drugs
(Pornpattananangkul et al., 2011). Liposome containing eugenol is
being examined as a natural and effective antioxidant but suffers
from drawbacks of liposomal instability. The success of the use of
liposome-encapsulated eugenol as an antioxidant depends on its
physical and chemical stability. Unfortunately, traditional lipo-
somes often encounter the low encapsulation efciency and
instability of vesicle aggregation, fusion, or rupture. The poor
* Corresponding author.
E-mail address: linl@ujs.edu.cn (L. Lin).
exploitation of eugenol liposomes as an antioxidant receivable
product. Therefore, it becomes a very important issue to obtain a
stable eugenol liposome for nally obtaining the safe and effective
solution.
Currently, some liposome-based formulations like polymer-
coated liposomes, hydrogel/liposome composites, nanoparticle-
stabilized liposomes, etc. have been exploited to ameliorate the
stability of liposomes (Kaminski, Sierakowski, Pontarolo, Santos, &
Freitas, 2015; Liu et al., 2016; Montenegro et al., 2013). Herein, we
show a simple strategy to form novel liposome-encapsulated SiO2-
eugenol as an antioxidant. This particular liposome-encapsulated
SiO2-eugenol has a supramolecular structure with colloidal
particles as a core encircled by a lipid shell (Liu et al., 2016).
SiO2 nanoparticles, a kind of food additives, served as core provide
a rigid bracing for the lipid bilayer thin lm and enhanced higher
physical stability of liposome (Cui, Liang, Yang, & Liu, 2016).
Besides, the porous properties and hydrophilic performance of
SiO2 nanoparticles make it easy to absorb more eugenol in case of
volatilization of eugenol (Okada, Tomita, & Kameshima, 1999;
Huang, Lan, & Liang, 2011). Hence, we think that the application of
SiO2 with an architectural function in liposomes offers the
promising prospect for the widespread application of eugenol-
based antioxidant.
Electrospinning has become a novel technique for forming
composite nanobers from various materials in recent years

http://dx.doi.org/10.1016/j.fpsl.2017.01.001
2214-2894/ 2017 Elsevier Ltd. All rights reserved.
50 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957
(Ghorani & Tucker, 2015; Moomand & Lim, 2015). Currently,
polyvinyl alcohol (PVA), chitosan (chitin), polycarbonate (PC), and
polyethylene oxide (PEO) have been directed at electrospinning on
account of their excellent properties in generating nanobers and
membranes (Bhushani & Anandharamakrishnan, 2014). Very few
researches immobilized liposome on electrospun nanobers for
food preservation (Li et al., 2014; Wickremasinghe, Kumar, &
Hartgerink, 2014). Furthermore, the SiO2-eugenolliposomes have
not been reported for electrospinning, while PEO was identied as
favorable food packaging material (FDA, 2009; FDA, 2012; Ge,
Zhao, Mo, Li, & Li, 2012; Peinado, Mason, Romano, Biasioli, &
Scampicchio, 2016). For these reasons, PEO/SiO2-eugenol lipo-
somes nanobrous membranes were electrospun for eugenol
immobilization.
Therefore, a novel nanobrous membrane of PEO containing
SiO2-eugenol liposomes was engineered. As a proof of concept, the
antioxidant activity of the nanober membrane on beef was
evaluated in this study.
2. Materials and methods
2.1. Materials
Tetraethyl orthosilicate (TEOS, 99%) was obtained from
Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Ammo-
nia solution (25%, AR), absolute ethanol (C2H5OH, AR), eugenol
(CP), and polyvinylpyrrolidone (PVP, GR) was purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lecithin
from soybean(BR) and cholesterol (AR) were purchased from
Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). PEO (99.99%) was
purchased from BASF SE (Ludwigshafen, Germany).
2.2. Experimental design
In order to reduce the volatilization and enhance the
encapsulated efciency of eugenol, the silica nanoparticle was
Fig. 1. Experimental design.
used to adsorb eugenol before liposomal encapsulation. Then, the
size, PDI, Zeta potential, encapsulation efciency, leakage rate and
storage stability of liposome containing SiO2-eugenol were
measured. Subsequently, the electrospun nanobrous membranes
containing SiO2-eugenol liposomes were prepared to prevent
oxidation of meat produce. As a proof of concept, the antioxidant
activity of the nanobrous membranes on beef was evaluated. The
entire experimental design was outlined in Fig. 1.
2.3. The synthesis and characterization of silica nanoparticle
5.0 mL of deionized (DI) water and 1.0 mL of ammonia solution
were added into 20 mL of absolute ethanol. The solution was then
stirred for 20 min at 60  C using a magnetic stirrer. After added
2.0 mL of TEOS drop wise, the solution was continuously stirred for
60 min at 60  C. Then, the solution was placed in an oven at 50  C
for 48 h to evaporate the solvent. Finally, powder was collected and
washed three times in DI water (Gangwar et al., 2013).
Morphologies and dimensions of the silica nanoparticles were
observed by a Scanning electron microscope (SEM) (JSM-7001F,
JEOL, Tokyo, Japan). The hydrodynamic size of the silica
nanoparticle (in PBS, pH 7.4) was measured using dynamic light
scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instru-
ments, Worcester, UK) setup. Measurements were performed in
triplicate at 25  C. The obtained silica sample was characterized by
X-ray diffractogram (XRD) performed on an X-ray diffractometer
(D8 ADVANCE, BRUKER, Germany) in the 2u range from 5 to 80
(Braun et al., 2016; Cui, Liang et al., 2016; Summerlin et al., 2016).
2.4. Eugenol adsorption
Adsorption was performed by adding 30 mg of eugenol into
60 mg of SiO2 (1:2 w/w) in the centrifuge tube. Then, the tube was
shaken for 24 h at 40  C. Using this approach, the nal silica loaded
with eugenol was prepared. The mixture of SiO2-eugenol was
stored in a refrigerator at 4  C (Janatova et al., 2015). Raman spectra
 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957 51

Fig. 2. Schematic formation of liposomes-encapsulated SiO2-eugenol.

were obtained by a laser Raman spectrometer (DXR, Thermo
Fisher, Massachusetts, USA). Raman measurements composed by
acquiring multiple spectral windows in the range 03500 cm1
(Stokes shifts) (Chowdhry, Ryall, Dines, & Mendham, 2015).
2.5. Evaporation test
The evaporation rate was measured at 22 and 37  C. In this test,
90 mg of SiO2-eugenol, which means 30 mg of pure eugenol, and
30 mg of unencapsulated eugenol in the pure form, were put into
centrifuge tubes in triplicate. The changes of weight were
determined after 24, 48, 72, 96, 120 and 144 h. The evaporation
rate was calculated as follow:
W0  Wt
ER  100%
30
Where ER was the evaporation rate (%), W0 was the weight of initial
tubes containing eugenol or SiO2-eugenol, and Wt was the weight
of tubes containing eugenol or SiO2-eugenol after evaporation at
different day (Janatova et al., 2015).
2.6. Fabrication of liposomes
0.2 g cholesterol and 1.0 g soybean lecithin (1:5 w/w) were
dissolved in trichloromethane in a round-bottom ask. The
solution was then evaporated in rotary evaporator until a thin
lm was formed. The lm was then dried at 30  C for 24 h using a
vacuum oven. Afterwards, the lm was suspended in 50 mL PBS
(pH 7.2) containing eugenol (250 mg) or SiO2-eugenol (750 mg)
and uniformized in cell ultrane grinding instrument (Ymnl-
1000Y, Nanjing Immanuel Instrument Equipment Co., Ltd.,
Nanjing, China) for 30 min at 350 W (Cui, Zhou, & Lin, 2016; Cui,
Zhao, & Lin, 2015). Finally, the liposomes-encapsulated eugenol or
liposomes-encapsulated SiO2-eugenol were obtained and stored in
a refrigerator at 4  C after centrifuged at 0.8952 kg for 10 min
(Fig. 2).
mass spectrometer analysis (GCMS, Agilent 6890N/5973N,
Agilent, USA). Subsequently, the liposome sample was centrifuged
at 10.1968875 kg for 3 h. To exam the content of unencapsulated
eugenol, the supernatant collection and analysis were performed
with GCMS. The amount of eugenol encapsulated in liposome was
expressed as encapsulation efciency calculated as follows:
C0  Ct
EE  100%
C0
Where EE is the encapsulation efciency (%), C0 is the total amount
of initial eugenol and Ct is the unencapsulated eugenol in the
supernatant (Lin et al., 2015).
2.7.3. In vitro release of eugenol from liposomes
The amounts of released eugenol during storage at 22  C and
37  C were tested by determining the total and free amount of
eugenol in the same way as the determination of EE. The release
rate of eugenol from liposomes in vitro was calculated by the
following equation:
Wrelease
RR  100%
Wtotal
Where RR was the release rate (%), Wrelease was the released
eugenol from liposomes, and Wtotal was the total amount of
eugenol encapsulated by liposomes (Cui, Li, Li, & Lin, 2016).
2.7.4. Storage stability of liposomes
To assess the physical stability, the relative particle size of
liposomes was determined periodically after storage at 22  C and

2.7. Characterization of liposomes

2.7.1. Size, polydispersity index (PDI) and zeta potential measurements

For size and zeta potential measurements, all specimens were
diluted for 10 times in ultrapure water (pH 7.4). Each sample was
measured in triplicate at 25  C with the dynamic light scattering
Zetasizer (Nano ZS90, Malvern Instruments, Worcester, UK). Data
was acquired and analyzed by the software Malvern v7.11 (Tan
et al., 2013).

2.7.2. Encapsulation efciency of eugenol in liposomes

Eugenol standard curve (0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL,
2.0 mg/mL and 4.0 mg/mL) was obtained via gas chromatography-
Fig. 3. Schematic diagram of the electrospinning setup.
52 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957

Fig. 4. The dynamic light scattering (DLS) measurements of SiO2 and scanning electron microscopy (SEM) image of SiO2 nanospheres (A), XRD patterns of SiO2 nanospheres
(B).

37  C for 60 d. Turbidity assay was a way to measure the relative
particle size of liposomes. The greater absorbance of liposomes
meant the larger relative particle size. The liposomes were
measured by a microplate reader (Innite 200 PRO, Tecan Austria
GmbH Untersbergstr, Grdig, Austria) at 500 nm.
Liposome is a thermodynamic instability dispersion system,
prone to occulation and precipitation. In addition, due to the
purity of liposome membrane material, soybean phospholipid will
hydrate insufciently and result in sedimentation. Therefore,
centrifugal sedimentation rate can indirectly reect the physical
stability of liposomes. When measuring, the primitive liposomes
samples (1.0 mL), without dilution in advance, were centrifuged at
0.50355 kg for 10 min. After centrifugation, the supernatant was
poured out and weighted. The centrifugal sedimentation rate was
determined periodically after storage at 22  C and 37  C and
calculated with the following formula:
W 1  W 0
CER  100%
WL
Where CER was the centrifugal sedimentation rate (%), WL was the
weight of 1.0 mL liposomes, W1 was the weight of centrifuge tube
containing sedimentary liposomes, W0 was the weight of empty
centrifuge tube (Duehrkop et al., 2016; Tan, Feng, Zhang, Xia, & Xia,
2016; Wang, Liu, & Song, 2014).
2.8. Antioxidation of the PEO/liposomes
2.8.1. H2O2 scavenging effect of liposomes
In the H2O2 scavenging activity assay, 1.0 mL sample (eugenol or
SiO2-eugenol liposomes) was mixed with 2.0 mL H2O2 solution
(20 mM in PBS solution). After incubation for 10 min, the
absorbance of the samples was measured at 230 nm using a
microplate reader (Innite 200 PRO, Tecan Austria GmbH
Untersbergstr, Grdig, Austria). The percentage of H2O2-scaveng-
ing activity was counted as follows:
A0  A1
H2 O2s  100%
A0

Fig. 5. Raman spectra of SiO2, eugenol and SiO2-eugenol (A), evaporation rate of SiO2-eugenol after different incubation time at 22  C and 37  C (B).
 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957 53

Table 1 Where DPPHs was the DPPH-scavenging activity (%), As was the
The characterization of liposomes.
absorbance of sample, Ac was the absorbance of control sample,
Eugenol liposome SiO2-eugenol liposome while Ab was the absorbance of sample blank (Azevedo et al., 2014;
Size (nm) 124.9
0.1 315.7
0.7 Tan et al., 2016; Zou et al., 2014).
PDI 0.277
0.004 0.196
0.001
Zeta potential (mV) 47.2
0.3 47.7
0.3 2.9. Electrospun nanobrous membranes preparation and
EE (%) 41.62
0.57 89.91
0.43
characterization
Values are expressed as mean
SE.
2.0 g PEO (10% w/v) and 4.0 mL SiO2-eugenol liposomes (40% v/
v) were added into16 mL deionized (DI) water to get the
electrospinning solution. The homogeneous solution was prepared
Where H2O2s was the H2O2-scavenging activity (%), A1 was the by stirring for 3 h at room temperature by magnetic stirrer.
absorbance of sample, A0 was the absorbance of control (H2O2 The homogeneous solution (PEO/liposomes) underwent elec-
solution without sample). trospinning (Fig. 3) (SNAN, MECC Co., Ltd., Fukuoka, Japan) from a
5.0 mL syringe with a injection needle with 0.9 mm inner diameter.
2.8.2. DPPH radical scavenging activity of liposomes The 0.6 mL/h feed rate was controlled by a syringe pump. Fibers
In the DPPH (1,1-diphenyl-2-trinitrobenzene hydrazine) radical were gathered on electrical connection of aluminum foil, which
scavenging activity test, 0.7 mL of SiO2-eugenol liposomes (or the was placed at a 12 cm vertical distance to the needle tip. The
direct mixture of eugenol-DMSO solution and blank liposomes) impressed voltage was set at 25 kV via a high voltage power supply.
was mixed with 0.2 mL of DPPH solution (0.4 mM in ethanol). The The morphology of PEO/liposomes nanobrous membranes were
eugenol in DMSO solution was adjusted to the same concentration characterized by SEM (JSM-7001F, JEOL, Tokyo, Japan) (Ge et al.,
as the SiO2-eugenol liposomes. Then, the solution was incubated at 2012).
room temperature in the dark for 40 min. DI water mixing with
DPPH solution instead of the sample was control. DI water mixed 2.10. Antioxidation of the PEO/liposomes nanobrous membranes on
with ethanol instead of DPPH was a sample blank. The absorbance beef
at 528 nm of the sample after incubation was measured by a
microplate reader. A low absorbance of the sample indicates a high Thiobarbituric acid reactive substances (TBARS) values of beef
free radical scavenging activity. The percentage of DPPH-scaveng- samples treated with liposomes nanobrous membranes were
ing activity was calculated as follows: measured to evaluate the oxidation level during 9 d storage at 4, 12
  and 22  C. The non-treated and BHA-treated beef samples were
As  Ac
DPPHs 1   100% negative and positive control groups respectively. TBARS values of
Ab
the samples were determined by Malondialdehyde Assay Kit (TBA
method) at 0, 3, 6 and 9 day. All the experiments were performed
triplicate.

2.11. Sensory evaluation

Same size and type of beef (control and PEO/liposomes

nanobrous membranes treated beef) were used for sensory
analysis by 50 panelists from Jiangsu University. Samples were
randomly allotted to a different order for each sensory evaluation.
Each sample was presented with a three-digit random code and
placed in a small clear plastic serving cup. The panelists evaluated
each sample by 9-point hedonic scale or intensity scale with a
score of 1 being either dislike extremely or extremely bland and a
score of 9 was like extremely or extremely avorful. The samples
were evaluated for overall like/dislike, like/dislike of avor, like/
dislike of color, like/dislike beefy avor, like/dislike of texture, and
like/dislike of juiciness (Kerth, Harbison, Smith, & Miller, 2015).

2.12. Statistical analysis

All experiments were performed in triplicate, and the results

were expressed in the form of means
standard error (SE) and
analyzed by the SPSS software (SPSS16.0 for Windows). The one-
way ANOVA was used to ascertain the level of signicance and
P < 0.05 was regarded as signicant.

3. Results and discussion

3.1. The characterization of SiO2 and SiO2-eugenol

In this study, we used TEOS to synthesize SiO2 nanoparticle.

 
Fig. 6. The release rate of liposomes stored at 22 C (A) and 37 C (B) for 60 days.
SiO2 was a near-perfect sphere with a smooth surface and a
Each symbol indicates the mean
SE of the mean for three independent diameter of 174.4 nm (Fig. 4A). As shown in Fig. 4B, the typical XRD
experiments. pattern of SiO2 nanoparticle was an amorphous peak with the
54 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957
Fig. 7. The absorbance at 500 nm of liposomes stored at 22  C (A) and 37  C (B) for different days. Each symbol indicates the mean
SE of the mean for three independent
experiments.
equivalent Bragg angle at 2u = 22 (Cui, Liang et al., 2016). Fig. 4
indicated that SiO2 nanoparticles were successfully synthesized in
this study.
As shown in Fig. 5A, for eugenol some bands were seen in the
Raman spectra at 1640 cm1 (CC, CH2), 2907 cm1 (CH3),
2941 cm1 (CH3), 2980 cm1 (CH2), and 3007 cm1 (CH3)
(Chowdhry et al., 2015; Ryall, Withnall, Dines, & Chowdhry,
2004). In case of SiO2, a weak band appeared at 500 cm1 (Lee &
Wachs, 2008; Sato, Funamori, & Yagi, 2011). For SiO2-eugenol-
composites, no additional bands appear in the Raman spectra. The
band at 500 cm1 was not observed in the Raman spectra of SiO2-
eugenol. Fig. 5A indicated that eugenol was successfully adsorbed
onto the surface of the SiO2nanospheres.
Desorption assays showed that SiO2-eugenol is vaporized
substantially slower than pure eugenol at 22  C and 37  C
(Fig. 5B). Besides, the eugenol and SiO2-eugenol appeared to be
more stable at 22  C than 37  C during the 144 h. The evaporation
rate assay conrmed that the adsorption of SiO2 could defer the
evaporation rate of eugenol.
3.2. Characterization of liposomes
Liposomes containing SiO2-eugenol was nally manufactured
with SiO2-eugenol as a core while liposome as a shell. After
prepared liposomes successfully, average size and zeta potential of
Fig. 8. The centrifugal sedimentation rate of liposomes stored at 22  C (A) and 37  C (B) for different days. Each symbol indicates the mean
SE of the mean for three
independent experiments.
liposomes were measured. The size distribution of liposomes
containing SiO2-eugenol was shown in Table 1. The average particle
diameter of SiO2-eugenol liposomes prepared at the optimum
conditions was 315.7
0.7 nm with a PDI of 0.196
0.001.
Comparing with the liposomes containing eugenol (average
particle size: 124.9
0.1 nm; PDI: 0.277
0.004), signicant
increase of liposomes containing SiO2-eugenol size was obtained
indicating the addition of SiO2-eugenol. And the PDI is an
important index of dispersion homogeneity and the values ranging
from 0 to 0.3 indicate a relatively homogeneous dispersion of
nanoparticles (Tan et al., 2013), so the SiO2-eugenol liposomes
prepared herein was homogeneous. Next as shown in Table 1, the
zeta potential of liposomes containing SiO2-eugenol was 47.7

0.3 mV. Compared to the liposomes containing eugenol (zeta
potential: 47.2
0.3 mV), the surface charges were increased
after loading SiO2-eugenol. While the relatively high charge could
prevent the particles from occulating to make the liposomes
become more stable (Tan et al., 2013). According to the above
results, the liposomes containing SiO2-eugenol possessed better
stability on account of their uniform particle size and high zeta
potential.
The linear regression equation between peak height of eugenol
and its concentration was y = 121,245,348.68x + 40,088,386.48,
R2 = 0.9923. When there was 5.0 mg/mL eugenol encapsulation,
the entrapment efciency of liposome containing SiO2-eugenol
 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957 55

Table 2
Antioxidant activity of eugenol and SiO2-eugenol liposomes after storage for 60 d at room temperature.

Storage time (d) H2O2 scavenging effect (%) DPPH radical scavenging activity (%)
a
Eugenol SiO2-eugenol liposome Eugenol SiO2-eugenol liposomeb
0 20.43
0.21 16.95
0.17 15.28
0.13 12.16
0.15
10 17.25
0.11 16.15
0.09 12.85
0.33 12.19
0.17
20 12.30
0.15 15.24
0.43 10.76
0.09 11.98
0.06
30 6.92
0.53 15.15
0.38 8.54
0.14 11.87
0.53
40 2.16
0.37 14.89
0.25 6.40
0.47 11.62
0.37
50 2.07
0.08 14.52
0.52 5.87
0.34 11.27
0.19
60 1.93
0.28 13.95
0.14 4.05
0.07 11.44
0.16
a,b
Values are expressed as mean
SE. The values were considered to be signicant (P < 0.05 versus the control group).

was 89.91
0.43% (Table 1). Compared to the liposomes containing
eugenol (entrapment efciency was 41.62
0.57%), the entrap-
ment efciency of liposomes containing SiO2-eugenol was
signicantly improved.
To assess the shelf life of liposomes, the release rates were
determined during 60 d storage at 22  C (Fig. 6A) and 37  C
(Fig. 6B). The encapsulated eugenol in liposomes underwent
different degrees of leakage. After 60 days of storage, the higher
release rate was observed at liposome containing eugenol with
27.7% and 46.9% values at 22  C and 37  C, respectively. It appeared
that the lipid bilayer can strongly retain the eugenol by the
adsorption of SiO2, while displaying a weak retaining capacity
when the liposome did not contain SiO2.
3.3. Liposomes stability
As shown in Fig. 7, absorbance value decreased as storage time
extended, obvious decreases of liposome containing eugenol or
SiO2-eugenol took place after storage at 22  C (Fig. 7A) and 37  C
(Fig. 7B). However, the change of liposome containing SiO2-
eugenol absorbance was not as obvious as liposome containing
eugenol. The results indicated that the liposome containing SiO2-
eugenol was more stability than liposome containing eugenol.
According to the centrifugal sedimentation rate of liposomes
after 60 day storage, the liposomes stability was evaluated at 22  C
(Fig. 8A) and 37  C (Fig. 8B). The centrifugal sedimentation rate of
liposomes increased with the increase of temperature and time, it
was mainly due to the occulent precipitate in liposomal
suspensions. When the thermodynamic motion was more intense,
the reveal of encapsulated eugenol was easier, hence liposomes
containing SiO2-eugenol obtained high stability.
3.4. Antioxidant activity of liposome
The H2O2 and DPPH methods, based on the reduction of H2O2
and DPPH in the presence of an H-donating antioxidant, are
frequently used for in vitro antioxidant activity determination (Tan
et al., 2016). The DPPH scavenging activity of eugenol and SiO2-

Fig. 9. SEM-micrographs of PEO nanober (A) and PEO/SiO2-eugenol liposome nanober (B), pictorial diagrams of PEO nanober (C) and PEO/SiO2-eugenol liposome
nanober (D).
56 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957

Table 3
Thiobarbituric acid reactive substances values (103 nmol/mg prot) of beef samples treated with liposomes nanobrous membranes and BHA at 4  C.

Different treatment 0d 3d 6d 9d
Control 0.2452
0.0006 0.3862
0.0005 0.5157
0.0008 0.6584
0.0006
Nanobera 0.2397
0.0012 0.2896
0.0006 0.3374
0.0006 0.4106
0.0008
BHAb 0.2383
0.0007 0.2651
0.0017 0.3092
0.0004 0.4187
0.0009
a,b
Values are expressed as mean
SE. The values were considered to be signicant (P < 0.05 versus the control group).

Table 4
Thiobarbituric acid reactive substances values (103 nmol/mg prot) of beef samples treated with liposomes nanobrous membranes and BHA at 12  C.

Different treatment 0d 3d 6d 9d
Control 0.2452
0.0006 0.4069
0.0004 0.5986
0.0006 0.7804
0.0001
Nanobera 0.2397
0.0012 0.3040
0.0001 0.3617
0.0008 0.4563
0.0004
BHAb 0.2383
0.0007 0.2962
0.0009 0.3271
0.0007 0.4272
0.0006
a,b
Values are expressed as mean
SE. The values were considered to be signicant (P < 0.05 versus the control group).

Table 5
Thiobarbituric acid reactive substances values (103 nmol/mg prot) of beef samples treated with liposomes nanobrous membranes and BHA at 22  C.

Different treatment 0d 3d 6d 9d
Control 0.2452
0.0006 0.4206
0.0009 0.6072
0.0002 0.8264
0.0002
Nanobera 0.2397
0.0012 0.3006
0.0007 0.3867
0.0009 0.4972
0.0006
BHAb 0.2383
0.0007 0.2996
0.0009 0.3526
0.0005 0.4377
0.0006
a,b
Values are expressed as mean
SE. The values were considered to be signicant (P < 0.05 versus the control group).

eugenol liposome was shown in Table 2. Owing to the directly
addition of eugenol in solution, eugenol has stronger H2O2 and
DPPH scavenging ability compared with SiO2-eugenol liposome.
However, H2O2 scavenging effect and DPPH radical scavenging
activity values decreased as storage time extended, obvious
decreases of eugenol took place after storage. The change of
SiO2-eugenol liposome was not as obvious as eugenol. One
probable reason was the enhanced stability by liposome encapsu-
lation. The results indicated that the liposome containing SiO2-
eugenol was more stability than eugenol.
3.5. Antioxidant activity of the PEO/liposomes nanobrous
membranes on beef
SEM images (Fig. 9A and B) and pictorial diagrams (Fig. 9C and
D) of PEO nanobers and PEO/SiO2-eugenol liposome nanobers
under the same condition were shown in Fig. 9. As shown in
Fig. 9A, the diameters of PEO nanobers were usually ranged from
400 nm to 500 nm. The SEM image of PEO/SiO2-eugenol liposome
nanobers was shown in Fig. 9B. It had been found that the
diameters of nanobers become larger when SiO2-eugenol
liposomes were added simultaneously (Fig. 9B). As shown in
Fig. 9B, the diameters of PEO/SiO2-eugenol liposome nanobers
were mostly 600700 nm.
To investigate the effect of liposome nanobrous membranes
on the lipid oxidations of beef samples, TBARS values of the system
were determined and the results were listed in Tables 35 . During
storage at 4  C (Table 3), TBARS values of all the samples displayed
increased with increasing storage time because of lipid oxidation.
The signicant differences between treatment groups and control
group (P < 0.05) were obtained. The TBARS values of nanober-
treated samples were lower than negative control groups, and
higher than positive groups. The results of 4  C were similar with
12  C (Table 4) and 22  C (Table 5). These indicated that nanober
exerted an inhibiting effect against oxidation and presented
moderate antioxidant activity in comparison with BHA.
Sensory evaluation data was shown in Table 6. The color values
altered signicantly after PEO/liposome nanobrous membranes

Table 6
The effect of PEO/liposome nanobrous membranes on sensory evaluation of beef treated for 10 d at 4  C, 12  C and 22  C.

Trait 4 C 12  C 22  C

Control Nanober Control Nanober Control Nanober

Overall like 5.87
0.31 5.89
0.04b 5.47
0.07 5.79
0.57a 5.29
0.35 5.74
0.21a
Flavor 5.82
0.83 5.85
0.44b 5.22
0.06 5.42
0.08a 5.10
0.13 5.35
0.19a
Color 5.53
0.09 5.76
0.31a 5.19
0.14 5.72
0.15a 5.17
0.07 5.38
0.06a
Beefy avor 5.82
0.08 5.85
0.62b 5.48
0.16 5.47
0.09b 5.42
0.28 5.37
0.09a
Texture 5.70
0.86 5.74
0.27b 5.27
0.36 5.29
0.37b 5.19
0.22 5.28
0.17a
Juiciness 6.35
0.69 6.39
0.68b 6.24
0.17 6.27
0.18b 6.12
0.12 6.19
0.16b

Values are expressed as mean
SE.

a
The values were considered to be signicant (P < 0.05 versus the control group).
b
The values were considered to be not signicant (P > 0.05 versus the control group).
 H. Cui et al. / Food Packaging and Shelf Life 11 (2017) 4957
treatment at 4, 12 and 22  C after storing for 10 d compared with
the control groups. Overall, the sensory evaluation values of
nanober treatment samples were better than control groups.
Therefore, PEO/liposome nanobrous membranes treatment could
not only minimize oxidation of beef, but also sustain the sensory
quality.
4. Conclusions
In conclusion, the physical stability of liposomes containing
SiO2-eugenol was signicantly improved by SiO2. The particle size,
zeta potential and encapsulation efciency of liposomes contain-
ing SiO2-eugenol were 315.7
0.7 nm, 47.7
0.3 mV, and
89.91
0.43%, respectively. Moreover, the antioxidant assay of
liposomes containing SiO2-eugenol exhibited stable antioxidant
activity during 60 d storage. Liposomes containing SiO2-eugenol
loaded electrospun nanobrous membranes exhibited excellent
antioxidant activity on beef. Hence, the novel antioxidant of SiO2-
eugenol liposomes loaded nanobrous membranes have promis-
ing prospect in the eld of food preservation.
Acknowledgments
The authors acknowledge the nancial support from National
Natural Science Foundation of China (grant nos. 31301573 and
31470594), Natural Science Foundation of Jiangsu Province (grant
no. BK20130493), China Postdoctoral Science Foundation (grant
nos. 2014M550419 and 2015T80873), Jiangsu Province Foundation
for talents of six key industries (grant no. NY-013), Innovation Fund
Designated for Graduate Students of Jiangsu Province (grant no.
SJLX16-0443), Jiangsu University Research Fund (grant no.
11JDG050), Priority Academic Program Development of Jiangsu
Higher Education.
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