LWT - Food Science and Technology 117 (2020) 108656

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LWT - Food Science and Technology

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Inhibition of Escherichia coli O157:H7 biofilm on vegetable surface by solid T

liposomes of clove oil
Haiying Cuia, Chenghui Zhanga, Changzhu Lib, Lin Lina,∗
a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China
b
Department of Bioresource, Hunan Academy of Forestry, Changsha, 410007, China

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

Keywords: Escherichia coli O157:H7 (E. coli O157:H7) easily forms biofilms on pores and veins of plant, which poses a
Escherichia coli O157:H7 serious hazard to safety of ready-to-eat vegetables. Therefore, this study was designed to evaluate inhibition
Biofilm mechanism of clove oil on E. coli O157:H7 biofilm and apply it to vegetable for reduce risk of contamination. The
Clove oil results of crystal violet staining showed that clove oil could effectively remove biofilm. After treatment of biofilm
Solid liposome
with clove oil (2 MBIC), the results of resazurin method showed that bacterial metabolic activity decreased by
Extracellular polymer
61.48%, and results of phenol-sulfuric acid method and Coomassie brilliant blue method showed that extra-
cellular protein and polysaccharide content decreased by 62.76% and 47.5%, respectively. This indicates that
clove oil can affect biofilm through above aspects. Finally, solid liposomes (SLPs) were prepared and applied to
vegetables, which was first time that SLPs were used to remove biofilms. The results showed that clove oil SLPs
exhibited excellent anti-biofilm activity at 4 °C and 25 °C without affecting senses and quality of vegetables.
Therefore, the clove oil SLPs can exert a long-lasting antibacterial effect, effectively removing E. coli O157:H7
biofilm on vegetable surface, and prolonging the shelf life.

1. Introduction
E. coli O157:H7 is one of the most important foodborne pathogens.
The study found that outbreak of E. coli O157:H7 is associated with
contaminated agricultural products (cucumber, lettuce, spinach). E. coli
O157:H7 readily forms biofilms on the pores, epidermis, stratum cor-
neum and vein surfaces of plants (Lee et al., 2014). Biofilm is one of the
important reasons for spread of bacterial contamination, and biofilm is
resistant to antibacterial agents, which poses a great hazard to the
safety of ready-to-eat vegetables. Therefore, an effective antibacterial
agent is needed to reduce risk of biofilm contamination of E. coli
O157:H7.
Recently, natural plant sources, animal-derived antibacterial sub-
stances have become a research hotspot (Al-Sokari & El Sheikha, 2015;
El Sheikha AF, 2017). Plant essential oils are a general term for oily
liquids extracted from plants and have good antibacterial activity and
broad-spectrum antibacterial properties (Shukla, Singh, Prakash, &
Dubey, 2013). Echeverría, LópezCaballero, Mauri, and Montero (2018)
prepared an active nanocomposite membrane of soy protein - mon-
tmorillonite - clove oil to preserve frozen bluefin tuna (Thunnus
thynnus) fillets. Girardi, García, Passone, Nesci, and Etcheverry (2017)
encapsulated essential oil of Lippia turbinata by complex coacervation
and used it to control fungal pathogens of peanut seeds. Meral, Ceylan,
and Kose (2019) used ultrasonic technology to prepare thyme essential
oil nanoemulsion and used it to inhibit growth of microorganisms in
fish fillets. As one of them, clove oil has made great progress in research
of its antibacterial activity. Therefore, this study investigated the in-
hibitory effect of clove oil on E. coli O157:H7 biofilm. However, clove
oil is volatile and unstable under light and heat conditions (Kringel
et al., 2017). Therefore, liposome encapsulation was chosen to increase
stability and utilization of clove oils (Cui, Li, & Lin, 2017).
Liposomes are lipid bilayer vesicle structures composed of phos-
pholipids and cholesterol. It has advantages of biocompatibility, sus-
tained release, and increased stability of the encapsulated substance.
Currently, liposomes have been widely used in fields of biomedicine
and food. Liposome particles are unstable during storage, which can
undergo aggregation, fusion, flocculation and precipitation (Xia & Xu,
2005). This drawback leads to limited application of liposomes.
Therefore, clove oil SLPs were prepared by freeze-drying to improve
liposomes stability (Lin, Zhu, Thangaraj, Abdel-Samie, & Cui, 2018).
In this study, clove oil was encapsulated into liposomes, and then
clove oil SLPs were prepared. Firstly, the scavenging effects of clove oil
SLPs on E. coli O157:H7 biofilm were detected by tube experiments and
scanning electron microscopy (SEM) and Laser scanning confocal

∗
Corresponding author.
E-mail address: linl@ujs.edu.cn (L. Lin).

https://doi.org/10.1016/j.lwt.2019.108656
Received 5 June 2019; Received in revised form 16 September 2019; Accepted 17 September 2019
Available online 18 September 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
H. Cui, et al.
microscope (LSCM). At the same time, the partial inhibition mechanism
of clove oil SLPs on E. coli O157:H7 biofilm was explored. Finally, it was
apply to vegetable surface. This is first study to inhibit biofilms by
encapsulating antibacterial agents with solid liposomes.
2. Materials and methods
2.1. Materials
Clove oil was purchased from J.E International (Caussols, France).
Cholesterol and soy lecithin were purchased from Sigma Aldrich
Chemical Company (St. Louis, Missouri). The MTT kit was purchased
from Biyuntian Biotechnology Research Institute. Resazurin was bought
from Shanghai Sinopharm Chemical Reagent Co., Ltd. E. coli O157:H7
was bought from China Industrial Culture Collection Center (Beijing).
Strain was cultured in a nutrient broth medium at 37 °C for 24 h with
shaking.
2.2. Antimicrobial activity of clove oil against E. coli O157:H7 biofilms
2.2.1. Determination of minimum biofilm inhibition concentration (MBIC)
and minimum biofilm eradication concentration (MBEC)
The clove oil was diluted in tryptone soy broth (TSB) (4 mg/mL to
0.25 mg/mL) in 48-well plate. E. coli O157:H7 was inoculated in plate
to give a suspension of 105–106 CFU/mL. The plate was statically cul-
tured at 37 °C for 3 d. After washing free bacteria, the number of biofilm
was quantified by MTT method. MBIC is the lowest concentration
without blue-violet.
Biofilm was established in a 48-well plate. Then, it was treated with
clove oil (4 mg/mL - 0.25 mg/mL) for 24 h. The number of viable
bacteria was quantified by MTT method (Sabaeifard, Abdi-Ali, Soudi, &
Dinarvand, 2014). MBEC is defined as the lowest concentration at
which no bacteria are detected.
2.2.2. Time-kill analysis of clove oil on E. coli O157:H7 biofilm
Sterile stainless steel pieces were added to TSB medium supple-
mented with E. coli O157:H7 (105−6 CFU/mL), and cultured at 37 °C for
3 d. After biofilm formation, plate was washed with PBS and then
placed in clove oil (MBEC and 2 MBEC). A sample without clove oil was
used as control group. After 5, 10, 20, 40 and 80 min of incubation,
stainless steel pieces were removed into a centrifuge tube containing
10 mL of PBS. The biofilm is dispersed by sonication to form a single
free bacterium. Finally, the number of surviving bacteria was measured
by plate coating.
2.2.3. Determination of scavenging effect of clove oil on E. coli O157:H7
biofilm by crystal violet staining
Crystal violet staining is most commonly used for quantification of
biofilm biomass (Ommen, Zobek, & Meyer, 2017). 200 μL of bacterial
suspension (105−6 CFU/mL) was inoculated into a 96-well plate, and
clove oil was added to suspension to get 1/4 MBIC-2MBIC, respectively,
and cultured at 37 °C for 3 d. A sample without clove oil was used as
control. The planktonic bacteria were washed away with PBS and fixed
with methanol for 1 h, then washed again with PBS. 1% crystal violet
was added for 30 min and finally washed with PBS. After membrane
was dried, the cells were suspended in 200 μL of ethanol:
acetone = 80:20 (v/v) and detected at 570 nm.
2.2.4. LSCM analysis
Sterile slides (2 × 2 cm2) were placed in a 6-well plate, and biofilm
was cultured according to method of 2.3.2 and treated with clove oil.
The clove oil was not added as control. The slides were washed with
PBS, and then DAPI (4′, 6-diamidino-2-phenylindole) stain was added,
and allowed to stand for 15 min in the dark. Next, it was washed with
PBS, and the excess liquid on slide was blotted, fixed with 40% glycerol,
and observed with LSCM.
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LWT - Food Science and Technology 117 (2020) 108656
2.3. Inhibition mechanism of clove oil on E. coli O157:H7 biofilm
2.3.1. SEM analysis
The biofilm was cultured on sterilized nylon sheets (1 × 1 cm2).
Samples were then prepared for SEM observation according to method
of Zhang, Ma, and Ye (2018).
2.3.2. Effect of clove oil on metabolism of E. coli O157:H7 biofilm
The biofilm was cultured onto sterilized steel sheets (2 × 2 cm2).
After biofilm matured, they were treated with clove oil (1/4MIC-2MIC)
for 4 h. The biofilm was suspended into PBS by ultrasound. Next, re-
sazurin solution was added to bacterial suspension (100 μg/mL). The
test tube was shaken for 2 h, and after centrifugation, the supernatant
was taken for detection. No essential oil treatment group was used as a
control.
2.3.3. Effects of clove oil on extracellular protein and extracellular
polysaccharides of E. coli O157:H7 biofilm
Sterile stainless steel sheets were added to TSB medium supple-
mented with bacterial (105−6 CFU/mL), and then clove oil was added
(1/4 MBIC-2 MBIC). No essential oil treatment group was used as
control. Biofilm extracellular polysaccharides and proteins were mea-
sured after standing culture for 3 d at 37 °C.
The biofilm was peeled off into sterile water by ultrasound and then
pH was adjusted to neutral. The extracellular polysaccharide was
measured by phenol-sulfuric acid method (Xi, Wei, Wang, Chu, & Xiao,
2010). The extracellular protein was determined by Coomassie Brilliant
Blue method.
2.4. Preparation and characterization of clove oil liposomes
The clove oil liposomes were prepared by a film-ultrasonic disper-
sion method (Cui, Li, et al., 2017). Particle size distribution, poly-
dispersity coefficient (PDI) and Zeta potential of clove oil liposome
were measured by a dynamic light scattering with a Zetasizer (Nano
ZS90, Malvern Instruments, Worcester, UK)(Guan et al., 2015). The
turbidity (in NTU) of clove oil liposomes was determined using a tur-
bidimeter; the pH of clove oil liposomes was measured with a pH meter.
A standard curve was first obtained by GC-MS (Agilent 6890 GC/5973
NMSD, NYSE: A, USA) analysis of clove oil. Some clove oil liposome
was added to a centrifuge tube, and after centrifugation at high speed
for 10 min, the upper layer of free essential oil was removed, and an
equal amount of absolute ethanol was added for ultrasonic treatment
for 3 h. After centrifugation (6000 rpm/min, 15 min), the filtrate was
obtained by a 0.22 μm filter and used for GC-MS analysis. The package
ratio is calculated as follows:
EE = C1/ C0 × 100%
where C1 is the concentration (mg/mL) of clove oil in liposome, and C0
is the initial concentration of clove oil (mg/mL).
2.5. Preparation and characterization of SLPs
2.5.1. Preparation of SLPs
SLPs were prepared by freeze-drying. A 10% (w/v) β-cyclodextrin
(β-CD) solution was added as a protective agent to clove oil liposomes
(β-CD: liposome (w:w) was 6:1) (Lin et al., 2018). It was frozen to
−80 °C, dried for 24 h, and finally stored at 4 °C for further study.
2.5.2. SEM analysis
The clove oil SLPs powder was adhered to a copper table in a high
vacuum evaporator through a gold sputtering module under an argon
atmosphere. The morphology of blank liposome and clove oil SLPs
powder was then observed by SEM (JSM-7001 F, JEOL, Tokyo, Japan).
H. Cui, et al.
Fig. 1. (A)Time-kill curve of clove oil against E.coli O157:H7 biofilm; (B) The inhibitory effect of clove oil on the formation of E.coli O157:H7 biofilm by crystal violet
staining.(C) LSCM image of E. coli O157:H7 biofilm before and after treatment with clove oil. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
2.5.3. FTIR analysis
Infrared spectroscopy analysis of clove oil SLPs and different com-
ponents thereof was carried out using a Nicolet 6700 Fourier transform
infrared spectrometer. The scan range was 600–4000 cm−1 and 32
scans were collected for each sample.
2.5.4. Storage stability
The clove oil SLPs were stored at 4 °C and 25 °C, respectively.
Samples were taken every 3 d and rehydrated, and then the particle
size, PDI and Zeta potential of liposome solution were determined ac-
cording to method in 2.5.2.
2.6. Application of clove oil SLPs on different vegetables
2.6.1. In vitro release rate of clove oil from SLPs
0.1 g of clove oil SLPs was added to 5 mL of absolute ethanol and
then sonicated for 3 h. The SLPs solution was centrifuged and analyzed
by GC-MS to calculate total concentration of clove oil. Then, the same
amount of clove oil SLPs was added to 5 mL of PBS, and incubated at
4 °C and 25 °C. Samples were taken every 3 d and centrifuged to de-
termine the concentration of clove oil in reaction solution (Lin, Zhang,
Zhao, & Cui, 2016). Release rate is calculated as follows:
RR = C1/ C0 × 100%
where, RR was release rate (%), C1 was the concentration of clove oil
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LWT - Food Science and Technology 117 (2020) 108656
released after a period of culture and C0 was the concentration of clove
oil in 0.1 g clove oil SLPs.
2.6.2. Determination of anti-biofilm of clove oil SLPs in vitro
After biofilm formation on a sterilized stainless steel plate
(1 × 1 cm2), it was immersed in a 5 mg/mL and 10 mg/mL clove oil
SLPs solution, respectively. No clove oil SLPs treatment group was used
as a control. A piece of steel was transferred to a tube containing 10 mL
of PBS at 0, 1, 2 and 3 d, respectively, and the tube was then sonicated
for 10 min. Finally, the number of residual bacteria was counted (Cui,
Bai, Rashed, & Lin, 2017).
2.6.3. The elimination effect of clove oil SLPs on different vegetables
The vegetables were cut into 1 × 1 cm 2 samples. To reduce natural
microbial community, samples were treated with UV light for 30 min.
Next, the sample was inoculated into E. coli O157:H7 suspension
(103−4 CFU/mL) for 1 h to adhere bacteria. The sample was dried and
incubated at 25 °C for 48 h, and then was immersed in a solution of
5 mg/mL and 10 mg/mL clove oil SLPs solution, respectively. No clove
oil SLPs treatment group was used as a control. The samples were in-
cubated at 4 °C or 25 °C. After 0, 1, 2, and 3 d, respectively, each set of
samples was transferred to a sterile homogenized bag supplemented
with 10 mL of PBS, homogenizer (Ymn1-9D, Rimaneili Instruments Co.,
Ltd, Nanjing, China) homogenization for 5 min. Subsequently, the
number of bacteria was counted.
H. Cui, et al. LWT - Food Science and Technology 117 (2020) 108656

Fig. 2. (A)SEM images of E. coli O157:H7 biofilm before and after treatment with clove oil; (B)The effect of clove oil on the extracellular protein and extracellular
polysaccharides of O157 biofilm; (C) Effect of clove oil on the metabolism of E.coli O157:H7 biofilm bacteria.

Table 1
The characterization of clove oil liposomes.
Control 2 mg/mL 3 mg/mL 4 mg/mL 5 mg/mL 6 mg/mL

PDI 0.187 ± 0.0021a 0.255 ± 0.0018e 0.234 ± 0.0019c 0.233 ± 0.0015c 0.194 ± 0.0013b 0.246 ± 0.0014d
particle size(nm) 65.49 ± 3.54a 75.88 ± 4.23b 84.35 ± 3.75c 114.53 ± 5.22d 139.56 ± 4.87e 156.21 ± 5.12f
Zeta potential −29.6 ± 1.23a −25.4 ± 1.03b −23.1 ± 1.35b −25.9 ± 1.19b −29.9 ± 1.24a −25.9 ± 1.15b
Turbidity/NTU 234 ± 23.5a 298 ± 21.1b 437 ± 19.7c 563 ± 20.53d 684 ± 22.9e 815 ± 25.61f
PH 6.99 ± 0.003b 7.04 ± 0.002b 7.12 ± 0.002c 7.03 ± 0.001b 6.92 ± 0.003a 7.11 ± 0.004c
Encapsulation efficiency (%) 0a 25.1 ± 0.21b 27.9 ± 0.28b 30.6 ± 0.36c 31.2 ± 0.44c 25.1 ± 0.49b

Values are expressed as mean ± SD.

a-f
different superscripts within the same column indicates significant differences (p < 0.05).

2.6.4. Chroma and sensory evaluation 3. Result and discussion

To investigate whether treatment of clove oil SLPs has an effect on
the sensory and quality of vegetables, the color and texture of samples
were evaluated. The samples were color-measured using a colorimeter
(Color Quest XE, HunterLab, USA) and expressed as brightness (L*), red
(a*), and yellow (b*). Sensory evaluation of vegetables was performed
on a randomly selected group of 50 people. Evaluation parameters in-
clude appearance, color, odor, and overall acceptability. Sample quality
assessments were scored between 1- (very dislike) and 9 (very like)
(Cui, Wu, Li, & Lin, 2017).
2.7. Statistical analysis
All experiments were performed in triplicates. All data were pre-
sented as mean ± standard deviation (SD). The results were analyzed
by one-way ANOVA using SPSS software (SPSS 16.0 for windows).
P < 0.05 was regarded as significant.
3.1. Antimicrobial activity of clove oil against biofilms
3.1.1. Scavenging effect of clove oil on E. coli O157:H7 biofilm
The MBIC value of clove oil against E. coli O157:H7 biofilm was
0.5 mg/mL and the MBEC value was 1 mg/mL. Compared with control
group, the number of bacteria in clove oil group was significantly re-
duced, and the higher the concentration of clove oil, the longer the
action time, and the more significant the effect (Fig. 1A). After 80 min
of treatment with 2MBIC, the number of bacteria decreased by 95.27%.
Crystal violet staining is commonly used to quantify biofilms
(Sabaeifard et al., 2014), and it can be seen from Fig. 1B that the results
are consistent with plate count results. Compared with control group,
the MBIC and 2MBIC concentration treatment groups were reduced by
65.74% and 84.78%, respectively. From the above, clove oil can ef-
fectively remove E. coli O157:H7 biofilm.

4
H. Cui, et al.
Fig. 3. SEM images of (A)blank SLPs and (B)clove oil SLPs; (C)FTIR spectra of Clove oil, β-CD, Blank SLPs and Clove oil SLPs.
3.1.2. LSCM analysis
The LSCM was used to observe the scavenging effect of clove oil on
E. coli O157:H7 biofilm. In control group, bacterial adhered to each
other and accumulated in layers to form a dense membrane structure.
In clove oil treatment group, the fluorescence area was significantly
reduced, and some bacteria still adhered to pieces, cloud-like, and
biofilm structure was sparse (Fig. 1C). The results indicate that clove oil
has a good scavenging effect on E. coli O157:H7 biofilm.
3.2. Inhibition mechanism of clove oil on E. coli O157:H7 biofilm
3.2.1. SEM analysis
The number and morphology of biofilm after treatment with clove
oil SLPs are shown in Fig. 2A. In control group, a tight biofilm structure
was formed on the nylon sheet, and the cells were rod-shaped. In clove
oil SLPs treatment group, the structure of biofilm was from the whole to
the dispersion, and some of the surface of the bacteria collapsed, which
may be caused by the exudation of the contents. It is speculated that
clove oil can affect the extracellular polymer structure of the biofilm, so
that the barrier effect of the extracellular polymer on the cells dis-
appears.
3.2.2. Effect of clove oil on metabolism of E. coli O157:H7 biofilm
Resazurin is a redox indicator that penetrates into bacteria and
degrades into high fluorescence intensity intermediates by the action of
different oxidoreductases in bacteria. This conversion is directly pro-
portional to the number of metabolically active irreversible bacteria
(Macia, Rojo-Molinero, & Oliver, 2014). As concentration of clove oil
increased, the percentage of bacteria with metabolic activity gradually
decreased. After treatment of biofilm with 2MBIC clove oil, 61.48% of
the bacteria lost their metabolic activity (Fig. 2B).
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LWT - Food Science and Technology 117 (2020) 108656
3.2.3. Effects of clove oil on extracellular protein and extracellular
polysaccharides of E. coli O157:H7 biofilm
The biofilm extracellular polymer is a complex mixture. They play
an important role in formation of biofilms, including strengthening the
adhesion of bacteria to solid surfaces, acting as the main structure of
biofilms and protecting bacteria (Angelo, Pierluigi, Emanuela, Sergio, &
Adriana, 2016). Therefore, the effect of clove oil on the secretion of
polysaccharides and proteins, the main components of extracellular
polymers, was investigated. Compared with control group, the extra-
cellular polysaccharide and protein formation of clove oil treatment
group decreased (Fig. 2C). When concentration of clove oil reached
2MBIC, extracellular polysaccharide and protein content decreased by
62.76% and 47.5%, respectively. So, it is presumed that clove oil affects
the formation of biofilm by inhibiting the secretion of extracellular
polysaccharides and proteins.
3.3. Preparation and characterization of clove oil liposomes
The characterization results are shown in Table 1. The average
particle size of clove oil liposomes ranged from 65.49 nm to 156.21 nm,
indicating that liposomes were well distributed. The PDI was between 0
and 0.3, indicating that liposomes were very stable and the particle size
distribution was uniform. The Zeta potential fluctuated between −30
mV and −20 mV, which is relatively stable because the surface of li-
posome is negatively charged, maintaining the stability of liposome due
to the mutual repulsion of surface charges (Cui, Li, et al., 2017). The
turbidity of liposome was related to the particle size, which was risen
with the increased concentration of essential oil. PH is also an indicator
of stability of liposomes, which can be seen to remain at around 7. The
maximum encapsulation efficiency of liposomes was 31.2% with 5 mg/
mL essential oil. Based on the comprehensive encapsulation efficiency
and relevant stability index, the optimal essential oil concentration was
5 mg/mL, which was selected for subsequent experiments.
H. Cui, et al.
Fig. 4. Storage stability of clove oil SLPs at (A) 4 °C and (B)25 °C; (C)In vitro release rate of clove oil from SLPs; (D)Anti-biofilm of SLPs of clove oil in vitro.
3.4. Preparation and characterization of clove oil SLPs
3.4.1. SEM analysis
The morphology and structure of the SLPs were observed by SEM,
and it was seen that blank SLPs and clove oil SLPs showed similar
particle distribution and morphology, (Fig. 3A and B). The results in-
dicate that SLPs were successfully prepared. The control group and the
clove oil group are similar in shape because the ratio of β-CD as a
protective agent is high in the preparation of SLPs.
3.4.2. FTIR analysis
The spectra of different samples are shown in Fig. 3C. In spectrum of
clove oil, there are two peaks representing the C]C vibration of aro-
matic compound at 1602 cm−1 and 1514 cm−1, the absorption peak at
1742 cm−1 represents the C–H overtone peak on the benzene ring, and
the absorption peak at 1240 cm−1 represents phenol –OH in-plane
stretching vibration. In the spectrum of β-CD, 3367 cm−1 represents the
–OH stretching vibration absorption peak, and 2925 cm−1 represents
the C–H stretching vibration absorption peak, which appears at
1645 cm−1, 1157 cm−1 and 1082 cm−1. C–O stretching vibration ab-
sorption peak (Hu et al., 2012). The spectra of SLPs and clove oil SLPs
retained characteristic peaks of β-CD at 3367 cm−1, 2925 cm−1 and
1157 cm−1, since β-CD was added as a protective agent to liposomes in
a high amount. In addition, clove oil SLPs retained two peaks of clove
oil at 1742 cm−1 and 1240 cm−1, but both peaks were weakened, while
other characteristic peaks disappeared, indicating that clove oil had
been encapsulated. At the same time, the encapsulation amount of clove
oil in SLPs is not high, and the content of β-CD is high, which may be
the reason for the weakening of characteristic peak. It can be seen from
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LWT - Food Science and Technology 117 (2020) 108656
above that clove oil SLPs have been successfully prepared.
3.4.3. Storage stability
The clove oil SLPs were stored at 4 °C and 25 °C for 15 d, respec-
tively, and their physical properties are shown in Fig. 4A and B. At 0 d,
the particle size of SLPs was slightly increased compared to that before
lyophilization and increased from 162.3 nm to 203.5 nm after 15 d of
storage. In addition, the change in the PDI value and the zeta potential
of SLPs stored at 4 °C is also in a relatively stable range. When SLPs
were stored at 25 °C, the particle size, PDI and zeta potential varied
greatly. Some measurement points exceed stability range, that is par-
ticle size < 300 nm, PDI < 0.3, zeta potential < -30 mV or > 30 mV
(Lu et al., 2014). According to above results, the temperature is an
important factor affecting storage stability, and the low temperature
storage is more favorable for maintaining the stability of SLPs to utmost
extent.
3.5. Application of clove oil solid liposome on different vegetables
3.5.1. In vitro release rate of clove oil from SLPs
The release rate of clove oil from SLPs was determined at 4 °C and
25 °C to evaluate antibacterial activity of clove oil SLPs (Lin et al.,
2018). As shown in Fig. 4C, the clove oil is released rapidly during
initial time, and then the release slows down and gradually stabilizes.
The release rate of clove oil at 25 °C was significantly higher than 4 °C.
After 7 d of storage, the cumulative release rate of clove oil in SLPs was
32.24% at 4 °C and 64.87% at 25 °C. Therefore, it is concluded that the
SLPs is more stable under low temperature conditions and is more
conducive to storage.
H. Cui, et al. LWT - Food Science and Technology 117 (2020) 108656

Fig. 5. The scavenging effect of clove oil SLPs on the surface of E.coli O157:H7 biofilm on cucumber skin at (A) 4 °C, (B)25 °C. The scavenging effect of clove oil SLPs
on the surface E.coli O157:H7 biofilm of lettuce at (C) 4 °C, (D)25 °C.

Table 2
Effects of clove oil SLPs on the quality of cucumber and lettuce at 4 °C.
Sensory parameter Cucumber Lettuce

0d 3d 0d 3d

Control Control Clove oil SLPs Control Control Clove oil SLPs

L* 25.46 ± 2.57a 27.35 ± 8.27b 26.13 ± 6.23b 57.44 ± 8.1a 61.50 ± 4.4b 62.82 ± 5.08b
a* −3.35 ± 0.82b −5.98 ± 0.82a −5.33 ± 0.98a −9.47 ± 0.46b −10.96 ± 0.46a −10.44 ± 0.93a
b* 3.45 ± 1.33a 10.24 ± 1.33b 9.55 ± 2.76b 28.04 ± 2.32a 37.7 ± 2.32c 35.57 ± 2.2b
Appearance 8.31 ± 0.34b 7.62 ± 0.39a 7.79 ± 0.36a 8.11 ± 0.38b 7.35 ± 0.38a 7.42 ± 0.37a
Color 8.29 ± 0.29b 7.59 ± 0.42a 7.83 ± 0.46a 8.26 ± 0.24b 7.34 ± 0.24a 7.65 ± 0.39a
Odor 8.22 ± 0.27b 7.44 ± 0.31a 7.62 ± 0.23a 8.13 ± 0.31b 7.61 ± 0.31a 7.82 ± 0.43a
Overall acceptability 8.31 ± 0.32b 7.42 ± 0.33a 7.65 ± 0.32a 8.24 ± 29b 7.25 ± 29a 7.58 ± 0.41a

Values are expressed as mean ± SD.

a-d
different superscripts within the same column indicates significant differences (p < 0.05).

3.5.2. Determination of anti-biofilm of clove oil SLPs in vitro
The scavenging effect of clove oil SLPs on E. coli O157:H7 biofilm
was evaluated in vitro (Fig. 4D). The number of bacteria in control
group remained substantially unchanged after 3 d. The number of
bacteria in clove oil SLPs treated group was reduced compared to
control group. The reduction effect of 5 mg/mL clove oil SLPs group
was not significant, while the number of bacteria in 10 mg/mL clove oil
SLPs group decreased from 6.377 Log CFU/cm2 to 2.431 Log CFU/cm2,
and the clearance rate reached 99.99%. This result indicated that clove
oil was slowly released from SLPs and acts to scavenge biofilm.

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H. Cui, et al. LWT - Food Science and Technology 117 (2020) 108656

Table 3
Effects of clove oil SLPs on the quality of cucumber and lettuce at 25 °C.
Sensory parameter Cucumber Lettuce

0d 3d 0d 3d

Control Control Clove oil SLPs Control Control Clove oil SLPs

L* 25.46 ± 2.57a 30.15 ± 2.89b 29.64 ± 5.02b 57.44 ± 8.1a 65.84 ± 2.91c 59.63 ± 2.65b
a* −3.35 ± 0.82a −3.67 ± 0.37a −3.53 ± 0.39a −9.47 ± 0.46b −7.95 ± 2.14c −10.15 ± 0.63a
b* 3.45 ± 1.33a 5.09 ± 0.74b 5.04 ± 1.22b 28.04 ± 2.3a 37.99 ± 1.17b 37.92 ± 1.59b
Appearance 8.24 ± 0.34b 7.57 ± 0.34a 7. 61 ± 0.46a 8.19 ± 0.38c 6.13 ± 0.32a 6.96 ± 0.43b
Color 8.35 ± 0.29b 7.33 ± 0.29a 7.59 ± 0.37a 8.23 ± 0.24b 7.09 ± 0.31a 7.17 ± 0.38a
Odor 8.18 ± 0.27b 7.38 ± 0.27a 7.46 ± 0.38a 8.21 ± 0.31b 7.14 ± 0.29a 7.38 ± 0.31a
Overall acceptability 8.27 ± 0.32b 7.21 ± 0.32a 7.49 ± 0.47a 8.18 ± 29b 6.58 ± 0.33a 6.89 ± 0.44a

Values are expressed as mean ± SD.

a-d
different superscripts within the same column indicates significant differences (p < 0.05).

3.5.3. The elimination effect of clove oil SLPs on different vegetables
In the application, clove oil SLPs has a lower ability to clear biofilms
than in vitro biofilm resistance, so the concentrations of 10 mg/mL and
15 mg/mL were chosen for testing (Fig. 5). For cucumber skin, at 25 °C,
the number of bacteria in control group increased rapidly after 3 d,
while the 10 mg/mL group and the 15 mg/mL group decreased by
96.67% and 99.98%, respectively. At 4 °C, the number of bacteria in
control group increased slowly compared with 25 °C. After 3 d, 10 m/
mL group and 15 mg/mL group decreased by 99.81% and 99.99%, re-
spectively, compared with control group. The application of clove oil
SLPs in lettuce shows similar effects. The results indicate that clove oil
SLPs can effectively remove E. coli O157:H7 biofilm on surface of cu-
cumber and lettuce.
3.5.4. Chroma and sensory evaluation
The chroma analysis and sensory evaluation results are shown in
Tables 2 and 3. Compared with 0 d, the sensory parameters of cucumber
and lettuce in control group decreased after storage for 3 d, and the
chromaticity parameters changed significantly, indicating that the
freshness of vegetables decreased. Besides, there were no significant
difference in sensory parameters and chromaticity parameters between
treatment group and control group. From the above results, it was
confirmed that the treatment of clove oil SLPs is advantageous for ve-
getable preservation and does not adversely affect sensory of vege-
tables.
4. Conclusion
In summary, the prepared clove oil SLPs has good storage stability
and the ability to remove E. coli O157:H7 biofilm on plant surface. The
results of mechanism study indicated that clove oil SLPs affects biofilm
by inhibiting bacterial metabolic activity in membrane and inhibiting
secretion of extracellular polymer. The results of FTIR indicated that
clove oil SLPs was successfully prepared. In addition, the release effi-
ciency indicates that clove oil achieves sustained release from SLPs,
which is beneficial for long-lasting antibacterial. Finally, the applica-
tion results show that clove oil SLPs can effectively remove E. coli
O157:H7 biofilm on vegetable surface, thus extending shelf life.
Therefore, it can be proposed that the application of clove oil SLPs to
vegetable preservation has broad prospects in field of food safety.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
This research project was financially supported by Hunan Science
and Technology Major Project (Grant no. 2016NK1001-3), Natural
Science Foundation of Jiangsu Province (Grant no. BK20170070),
Jiangsu Province Research Fund (Grant no.NY-013 and JNHB-131) and
Jiangsu University Research Fund (Grant no. 11JDG050).
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