This document summarizes a study investigating the use of thyme essential oil nanoemulsions (TEON) to inhibit Escherichia coli O157:H7. Three TEON were prepared using different emulsifiers (Tween 80, sodium dodecyl sulfate, cetylpyridinium chloride) and ultrasonication. Response surface methodology was used to optimize the nanoemulsion preparation conditions. The optimized nanoemulsions demonstrated good stability over storage and varying abilities to inhibit E. coli based on their measured minimum inhibitory concentrations and minimum bactericidal concentrations. Experiments showed the nanoemulsions interacted with and disrupted the E. coli cell membrane, causing release of intracellular contents. The study provides insights into using essential oil nano

1. 1

2. Introduction
• Essential oils (EOs) are derived from numerous bioactive chemical
components such as terpenes, terpenoids, and phenolics.
• Excellent antibacterial capacity of plant-derived essential oils against
food-borne pathogens and their contribution to food safety.
• Despite have drawbacks such as hydrophobicity, off-flavor, potential
toxicity at high concentrations, and oxidability limit their application
in food and pharmaceutical industries.
• Nano-encapsulation of essential oils into different delivery systems
is one possible strategy to solve these drawbacks.
• Nanoemulsions, homogeneous dispersion systems with mean
particle size diameters ranging from 1 to 1000 nm, are promising
and feasible approaches to encapsulate EOs.
2

3. Introduction
• Nanoemulsion are more stable as compared to bulk essential oils
and coarse emulsion, smaller droplet size and lower PDI besides
higher absolute zeta-potential values.
• Moreover, essential oil nanoemulsions exert excellent antimicrobial
activity against different bacterial pathogens which small droplet
size.
• Ultrasonication, broadly utilized to fabricate nanoemulsions with
excellent dispersion characteristics by high efficiency, simplicity,
and lower cost.
• Specific antibacterial mechanism of nanoemulsions with different
emulsifiers has not been thoroughly investigated yet.
3

4. Objectives
• Cell membrane’s unique functional and structural properties,
including outer membrane permeability and lipopolysaccharide layer,
were behind its resistance to bulk essential oils.
• Therefore, E. coli O157:H7 was selected as our target bacteria to
investigate the antibacterial ability of nanoemulsions.
• Response Surface Methodology (RSM) with Box-Behnken design
(BBD), the effect of emulsifier concentration, sonication time and
power on the dispersion characteristics (droplet size, PDI, zeta-
potential) and antibacterial efficacy [based on MIC and MBC] of
thyme essential oil nanoemulsions (TEON) were investigated.
• Furthermore, the storage stability and antibacterial mechanism of the
optimized nanoemulsions were also determined.
4

5. Material and Methods
• Thyme essential oil (TEO) (Sigma-Aldrich (St. Louis, MO, USA).
• Tween 80 (T80), sodium dodecyl sulfate (SDS) and
cetylpyridinium chloride (CPC) [Sinopharm Chemical Reagent
(Co., Ltd., Shanghai, China)].
• Escherichia coli O157:H7 ATCC 35150 [Guangdong Culture
Collection Center (Guangzhou, China)} and cultured on eosin
methylene blue (EMB) medium (Base BioTech Co., Hangzhou,
China).
5

6. Preparation of nanoemulsions
• Thyme essential oil and three emulsifiers (T80, SDS, CPC) were
chosen.
• Nanoemulsions formulation protocol followed a three-step process.
• Coarse oil-in-water (O/W) emulsion was prepared by adding thyme
essential oil and emulsifier into 50 mL deionized water.
• The mixture was then homogenized using a high-speed
homogenizer for 10 min at 10,000 rpm.
• Finally, 30 mL coarse emulsion was sonicated using a 20 kHz
ultrasonic processor with a Φ10 ultrasound probe at different power
(350, 450, 550 W) and time (5, 10, 15 min) combinations.
• After preparation, formed nanoemulsions were stored at 4 ◦C.
• The final concentration of TEO in TEON was obtained as 10
mg/mL.
6

7. Selection of nano-emulsification conditions
• Sonication time, ultrasound power, and emulsifier concentration
were the main preparation conditions affecting the nanoemulsions’
dispersion characteristics and antibacterial activity.
• Also, different nanoemulsions were prepared using the three
emulsifiers, and their comparative dispersion and antibacterial
properties were determined.
• For emulsifier concentration, considerations were given on each
emulsifier’s antibacterial activity so that the concentration selected
will have a minimal effect on the antibacterial activity of the
TEON.
7

8. Measurement of droplet size, PDI, and zeta-
potential
• Droplet size, PDI, and zeta-potential of nanoemulsions were
measured by Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK)
at 25 ◦C right after preparation.
• According to the equipment guidebook, corresponding emulsifier
solutions were selected to avoid the influence of pH and ionic
strength on zeta-potential measurement during dilution.
8

9. Preparation of E. coli suspension
• A single colony of E. coli was transferred from the EMB medium
into a 10 mL Nutrient Broth (NB) medium (Base BioTech Co.,
Hangzhou, China) and incubated for 17 h (37 ◦C, 180 rpm).
• After that, the culture was centrifuged at 5000 rpm for 10 min at 4
◦C.
• Then, the precipitate was resuspended twice with 0.85% normal
saline.
• Finally, the E. coli suspension was adjusted to a concentration of
approximately 5 log CFU/ mL through a serial dilution method
using 0.85% normal saline before inoculating in a 96-well
microtiter plate.
9

10. Determination of MIC and MBC
• Nanoemulsions were diluted to 4 mg/mL with the addition of NB.
• 100 μL adjusted E. coli suspension was inoculated into each well
and the plate was placed at 37 ◦C for 24 h.
• The lowest concentration in the well with OD600 nm less than 0.1
was regarded as MIC.
• Each group set positive (no nanoemulsion) and negative control
(normal saline ) group instead of E. coli suspension.
• A stock solution method was conducted for accurate MIC values.
• The MBC lowest conc. could kill 99.999% of treated cells of E.
coli. Approximately 100 μL, spread on Plate Count Agar (PCA) and
then incubated at 37 ◦C for 24 h.
• The lowest nanoemulsions concentration when no colony was
observed in the culture medium was regarded as MBC.
10

11. Antibacterial mechanism - Integrity of cell membrane
• Cell membrane integrity was determined by measuring the release of
proteins and nucleic acids.
• E. coli suspension cultured overnight was centrifuged, rinsed twice, and
adjusted to OD600 nm at 0.5 using PBS (0.01 M, pH = 7.0).
• Optimized nanoemulsions were set at MIC and MBC, whereas
nanoemulsions (containing emulsifier only) at MIC was set as the control.
• The concentrations of nucleic acids and protein released were measured at
260 nm and 280 nm, respectively, using a spectrophotometer.
• The concentration of nucleic acids was represented by the absorbance at
260 nm, whereas for the protein release concentration, a standard curve of
bovine serum albumin was used for the calculations.
• Results were presented as mg BSA equivalent per mL.
11

12. Analysis of the bacterial and nanoemulsion
interaction
• The interaction effect was characterized by detecting changes in the
particle size and zeta-potential of E. coli and nanoemulsions after
their contact.
• This was carried out using Zetasizer Nano ZS90. E. coli suspension
was diluted to 5 log CFU/mL, and the contact time was set at 10
min while the nanoemulsions concentration was maintained at the
MIC.
12

13. • The morphological changes of E. coli cells under different
treatments were observed by SEM.
• Briefly, cell suspension after treatment was fixed with 2.5%
glutaraldehyde, post-fixed with 1% OsO4 in phosphate buffer
and dehydrated in different ethanol concentrations (30, 50, 70,
80, 90, 95 and 100%, v/v).
• Samples were finally observed at 12.0 k magnification using
SEM (Hitachi Model SU-8010).
Scanning electron microscopy (SEM)
13

14. Results and Discussion
14

15. Table 1
Droplet size, PDI, Zeta-Potential, MIC and MBC
15

16. Storage Stability
Fig. 1. The dispersion characteristics of the optimized thyme essential oil nanoemulsions with different emulsifiers
(TEON-T80, TEON-SDS, TEON-CPC) stored at 4 ◦C for 14 days. (a) droplet size; (b) polydispersity index and (c)
zeta-potential.
16

17. Morphological changes of E. coli
Fig. 2. SEM images of E. coli treated with different nanoemulsions under different treatments. (a–c) ES, MIC,
and MBC treatment with TEON-T80. (d–f) ES, MIC, and MBC treatment with TEON-SDS. (g–i) ES, MIC, and
MBC treatment with TEON-CPC. 17

18. Fig 3. Interaction effect between E. coli and nanoemulsions measured by particle size (nm) and zeta-
potential (mV). A for particle size and B for zeta-potential.
Interaction analysis between E. coli and nanoemulsions
18

19. Fig. 4. Proposed interaction mechanism between E. coli and three nanoemulsions.
Mechanism between E. coli and three
nanoemulsions
19

20. • With optimal ultrasonication and three TEON enhanced dispersion
characteristics and antibacterial activity were obtained by response
surface methodology.
• Further storage stability and antibacterial mechanism were conducted
using optimized TEON.
• TEON-T80 possessed good storage stability but poor antibacterial
activity attributed to its controlled in vitro release, whereas TEON-
CPC exhibited excellent antibacterial activity due to its electrostatic
interaction effect of cationic emulsifier.
• In addition, although TEON-SDS had poor antibacterial activity, even
though it showed a good bactericidal effect based on antibacterial
mechanism experiments.
Conclusion
20

21. • TEON-CPC was suggested as the most optimal nanoemulsions
considering its superior antibacterial efficacy despite the need to
optimize further when expanding its application in the food industry.
• In sum, this study provides a reference for future efforts to optimize
essential oil nanoemulsions and study their antibacterial mechanism.
• Still, the antibacterial mechanism studies to further reveal the
specific bactericidal effect of the TEON and their antibacterial effect
against a wide range of microorganisms, particularly Gram-negative
bacteria, remain to be further explored.
Conclusion
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22. Thanks for listening me.!!
22

23. DI Water
Tween80
Citral oil
Span80 High-speed shear
13,000 rpm,
10 min
Citral oil
nanoemulsion
0
50
100
150
200
250
8 9 10 11 12 13 14 15
182.6
172.4 171.1
156.4 155.9 153.5
147.8
98.6
Particle
size
(nm)
HLB value
HLB value
(a)
HLB value
0
100
200
300
400
500
A B C D
Treatments
0 day
9 months
Particle
size
(nm)
0
100
200
300
400
500
A B C D
Treatments
0 day
9 months
(f)
Stability
1
100
10
4
10
6
10
8
0 200 400 600 800 1000 1200 1400
10
5
CFU/mL
10
6
CFU/mL
10
7
CFU/mL
10
8
CFU/mL
Time (min)
Amount
of
bacteria
(log)
y = 10^((-8.2*(1-exp(-0.0045...
Error
Value
NA
1.115e+16
Chisq
NA
0.708
R
2
y = 10^((-7.0*(1-exp(-0.0110...
Error
Value
NA
7.252e+8
Chisq
NA
0.9818
R
2
y = 10^((-6.1*(1-exp(-0.0136...
Error
Value
NA
1.042e+12
Chisq
NA
0.5266
R
2
y = 10^((-7.0*(1-exp(-0.0165...
Error
Value
NA
1.945e+13
Chisq
NA
0.8253
R
2
y = 10^((-8.2*(1-exp(-0.0045...
Error
Value
NA
1.115e+16
Chisq
NA
0.708
R
2
(e)
(E. coli)
1
100
10
4
10
6
10
8
0 200 400 600 800 1000 1200 1400
10
5
CFU/mL
10
6
CFU/mL
10
7
CFU/mL
10
8
CFU/mL
Time (min)
Amount
of
bacteria
(log)
y = 10^((-7.4*(1-exp(-0.0060...
Error
Value
NA
5.489e+15
Chisq
NA
0.8724
R
2
y = 10^((-5.4*(1-exp(-0.0170...
Error
Value
NA
1.141e+8
Chisq
NA
0.9969
R
2
y = 10^((-6.0*(1-exp(-0.0100...
Error
Value
NA
4.701e+11
Chisq
NA
0.8698
R
2
y = 10^((-7.2*(1-exp(-0.0090...
Error
Value
NA
8.136e+13
Chisq
NA
0.8043
R
2
y = 10^((-7.4*(1-exp(-0.0060...
Error
Value
NA
5.489e+15
Chisq
NA
0.8724
R
2
(f)
(S. aureus)
1
10
100
10
3
10
4
10
5
10
6
0 200 400 600 800 1000 1200 1400
E. coli
S. aureus
Time (min)
Amount
of
bacteria
(log)
(g)
Initial bacterial concentration Bacterial Strains
0
20
40
60
80
100
100 200 300 400
CK
1000 ppm
1500 ppm
2000 ppm
2500 ppm
Germination
rate
(%)
Particle size (nm)
a a
a
ba
b
a
b
cb
b
c
a
cd
cb
d
b
b
a
cbcb
c
(a)
0
20
40
60
80
100
1000 1500 2000 2500
ck
100 nm
200 nm
300 nm
400 nm
Germination
rate
(%)
Emulsion conc. (ppm)
a ba
bc
c c
a
c
a a
b
b
a
b
a
b
b
b
b
b
b
(b)
Germination rate of
Neoscytalidium dimidiatum
Characterization
Anti-bacterial
Anti-fungal
activity 23