International Journal of Biological Macromolecules 243 (2023) 125228

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Eco-friendly and effective antimicrobial Melaleuca alternifolia essential oil

Pickering emulsions stabilized with cellulose nanofibrils against bacteria
and SARS-CoV-2
Greiciele da S. Ferreira a, Daniel J. da Silva a, Alana G. Souza a, Eliana D.C. Yudice b,
Ivana B. de Campos b, Rute Dal Col b, Andre Mourão c, Herculano S. Martinho c, Derval S. Rosa a, *
a
Center for Engineering, Modeling, and Applied Social Sciences (CECS), Federal University of ABC (UFABC), Av. dos Estados, 5001, CEP 09210-210 Santo André, SP,
Brazil
b
Adolfo Lutz Institute, Santo André Regional Center, Av. Ramiro Colleoni, 240, CEP 09040-160 Santo André, SP, Brazil
c
Center for Natural and Human Sciences (CCNH), Federal University of ABC (UFABC), Av. dos Estados, 5001, CEP 09210-210 Santo André, SP, Brazil

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

Keywords: Melaleuca alternifolia essential oil (MaEO) is a green antimicrobial agent suitable for confection eco-friendly
Melaleuca alternifolia disinfectants to substitute conventional chemical disinfectants commonly formulated with toxic substances
Essential oil that cause dangerous environmental impacts. In this contribution, MaEO-in-water Pickering emulsions were
Nanocellulose
successfully stabilized with cellulose nanofibrils (CNFs) by a simple mixing procedure. MaEO and the emulsions
Oil-in-water Pickering emulsion
SARS-CoV-2
presented antimicrobial activities against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Moreover,
MaEO deactivated the SARS-CoV-2 virions immediately. FT-Raman and FTIR spectroscopies indicate that the
CNF stabilizes the MaEO droplets in water by the dipole-induced-dipole interactions and hydrogen bonds. The
factorial design of experiments (DoE) indicates that CNF content and mixing time have significant effects on
preventing the MaEO droplets' coalescence during 30-day shelf life. The bacteria inhibition zone assays show that
the most stable emulsions showed antimicrobial activity comparable to commercial disinfectant agents such as
hypochlorite. The MaEO/water stabilized-CNF emulsion is a promissory natural disinfectant with antibacterial
activity against these bacteria strains, including the capability to damage the spike proteins at the SARS-CoV-2
particle surface after 15 min of direct contact when the MaEO concentration is 30 % v/v.

1. Introduction
Currently, essential oils (EOs) have been considered an innovative
and technologically viable alternative to the manufacture of conven­
tional chemical disinfectants (sodium chloride, ethanol, quaternary
ammonium salts, among others) due to their biological activity against a
wide range of pathogenic bacteria, viruses, and fungi. The antimicrobial
capacity of EOs is directly associated with their chemical compositions
containing terpenes, terpenoids, and other substances capable of
directly destroying the biological structures of microorganisms (such as
proteins, lipids, and nucleic acids). Besides that, these substances can
lead to the generation of reactive oxygen species (ROS) that can cause
irreversible damage to microorganisms, compromising the cellular ma­
chinery and virion activities [1–4]. Despite the ability to act as dis­
infecting agents, EOs have high volatility, low solubility, high tendency
to degradation and oxidation that limit their direct use in the manu­
facture of antiseptic, pharmaceutical, cosmetic, and food products. In
this way, they are usually inserted into products in formulations con­
taining different components capable of stabilizing them, ensuring the
prolongation of the activity of their active principles and the effective­
ness of their antimicrobial properties.
Emulsification has been a technology widely used by the food,
cosmetic and pharmaceutical industries to stabilize or control the
release kinetics of an active ingredient or additive to protect the product.
In resume, emulsions are liquid mixtures based on an aqueous phase and
an oil phase, commonly stabilized by amphiphilic molecules or macro­
molecules that are well-established in the industry as surfactants and
emulsifiers. These auxiliary components guarantee outstanding emul­
sion stability over time to increase the shelf life of emulsion-based
products, which is a crucial factor for industries.

* Corresponding author.
E-mail addresses: dervalrosa@yahoo.com.br, derval.rosa@ufabc.edu.br (D.S. Rosa).

https://doi.org/10.1016/j.ijbiomac.2023.125228
Received 23 January 2023; Received in revised form 23 April 2023; Accepted 3 June 2023
Available online 7 June 2023
0141-8130/© 2023 Published by Elsevier B.V.
G. da S. Ferreira et al. International Journal of Biological Macromolecules 243 (2023) 125228

Fig. 1. Schematic illustration of the adopted methodology and characterizations of the MaEO/water Pickering emulsions stabilized with CNF.

2
G. da S. Ferreira et al.
Cellulose is the most abundant natural polysaccharide in the Earth's
crust, presenting several advantages of being applied instead of syn­
thetic emulsion stabilizers (surfactants and emulsifiers), such as biode­
gradability, renewable biomass, and non-toxicity that are relevant
characteristics to avoid environmental problems and toxicological issues
to biological systems. Among the several cellulose structures currently
and commercially available (cellulose nanocrystals, nanocrystalline
cellulose, and cellulose microfibrils, just to name a few), cellulose
nanofibrils (CNFs) are fibrous nanostructures with a nanoscale diameter
(lesser than 100 nm) that enable high surface area without high energy
and financial production costs [5–7]. In addition to being expensive, the
processes for nanocellulose isolation from the vegetable cell wall ma­
trixes generally involve the usage of toxic reagents and a high generation
of residues that cause several environmental impacts [5,8,9]. The
combination of high surface area and small size of these eco-friendly
solid cellulose nanostructures is important to their stabilization capac­
ity in EO-water Pickering emulsions, which involve a formation of an
interfacial CNF layer via intermolecular and steric hindrance respon­
sible for hampering the collapsing resistance of EO droplets due to
gravity and mechanical shear stress [10–12]. Moreover, the oil-in-water
(O/W) emulsion stabilization protects the antimicrobial compounds in
the EO droplets from volatilization and oxidation. For these peculiar
characteristics, CNFs have been applied as interfacial stabilizing agents
in several Pickering emulsions [6,12–16]. In addition to high stability,
Pickering emulsions have the advantage of using biodegradable, edible
or renewable solid particles as stabilizing agents instead of being less
harmful to the environment and safer for biota and fauna than synthetic
stabilizers [17,18].
Melaleuca alternifolia essential oil (MaEO) is extracted from the Tea
Tree (a native plant from Australia) by steam distillation of the leaves
and terminal branches. MaEO has both bactericidal and fungicidal ac­
tivity, including insecticidal capacity [19], standing out from other
essential oils for its strong bactericidal activity associated with shallow
minimum bactericidal concentration (MBC) values against several bac­
terial strains [20]. MaEO is mainly composed of volatile cyclic mono­
terpenes, which are summarily constituted by terpinen-4-ol, eucalyptol,
and α-terpineol (oxygenated monoterpenes) combined with α-terpinene,
γ-terpinene, and α-pinene (hydrocarbon monoterpenes) [21,22]. MaEO
has an antimicrobial activity due to these volatile compounds that lead
to the inhibition of the cellular respiration processes, microbial mem­
brane rupture, and leakage of potassium ions from cell membranes in
Gram-positive and Gram-negative bacteria [20].
Until now, few works have been published involving the stability of
Pickering emulsions of MaEO [23]. To the best of our knowledge, the
evaluation of the antiviral activity of Pickering emulsions of MaEO
against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
is still a gap in the scientific literature. In a previous work [24], we
evaluated the usage of Pickering emulsions of MaEO/water stabilized
with CNFs to confect wet disinfectant fabrics against different bacteria
and fungi. Therefore, it is necessary to evaluate the antiviral perfor­
mance of the MaEO/water Pickering emulsion against SARS-CoV-2
responsible for the global epidemy of coronavirus disease 2019
(COVID-19) since this emulsion type can be applied to design antimi­
crobial cosmetics and pharmaceutical products with sustainable and
renewable materials.
In this contribution, we evaluate several process parameters on the
stability of MaEO/water Pickering emulsions stabilized with CNFs using
design of experiments (DoE). For this purpose, cremation, sedimenta­
tion, stability index, and drop size of the emulsions were monitored over
time. Also, the antimicrobial performance of the MaEO and its emulsion
was evaluated against E. coli, S. aureus and SARS-CoV-2. Several exciting
points about the preparation and stability of the emulsions were dis­
closed here from a DoE perspective. The MaEO emulsion was able to
inactivate the coronavirus responsible for the biggest epidemic faced by
humanity in the last five years, showing great potential for use in rapid
sanitization products and food packaging formulations capable of
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International Journal of Biological Macromolecules 243 (2023) 125228
protecting food against SARS-CoV-2 and bacteria contamination.
2. Materials ad methods
2.1. Materials
Cellulose nanofibrils (CNF) (4.0 ± 1.0 μm of length and 66.3 ± 20.5
nm of diameter, length/diameter ratio of 60, ζ potential of − 33.2 ± 3.2
mV) were donated by Suzano Papel e Celulose (São Paulo, Brazil) [12].
Melaleuca alternifolia essential oil (MaEO) was purchased from Ferquima
Indústria e Comércio Ltda. (São Paulo, SP, Brazil). Rapid Antigen Test
kits were supplied by Panbio™ COVID-19 Ag rapid test (Abbott, USA).
Hexane and resazurin were purchased from Synth (São Paulo, Brazil).
Lysoform® and hypochlorite were purchased in a local Market (Santo
André, SP, Brazil).
2.2. Bacterial strains
Bacterial isolates were obtained from the Center for Interdisciplinary
Procedures for the Collection of Microorganisms of the Instituto Adolfo
Lutz (Brazil), affiliated with World Federation Culture Collections
(WFCC) # 282, Depository Collection - # 017/09-SECEX/CGEN/MMA:
Staphylococcus aureus (ATCC #6538) and Escherichia coli (ATCC
#11229).
2.3. Methods
2.3.1. Pickering emulsion preparation
CNF aqueous suspensions were homogenized in a high-intensity ul­
trasound (Sonics Vibra Cell, 400 W power and 24 kHz), for 10 min. The
MaEO was mixed with a previously dispersed CNF suspension, and this
mixture was homogenized in an Ultra-Turrax Blender, IKA T25 model
(IKA Werke, Staufen, Germany). The following parameters were varied:
CNF content in aqueous suspension (0.5 and 1 % wt/v), oil/water (O/W)
ratio (20 and 30 % v/v), mixing rotation (12,000 and 15,000 rpm), and
mixing time (5 and 7 min). Fig. 1 shows a schematic representation of
the adopted methodology. The emulsions were named XEO/YT/ZCNF/
WRPM, where X corresponds to the MaEO content (% v/v), Y is the
mixing time (min), Z is the CNF concentration (% wt/v), and W is the
mixing rotation (rpm).
2.4. Characterization
2.4.1. Gas chromatography coupled to mass spectrometry (GC–MS)
GC–MS analyses were carried out on a gas chromatograph coupled to
a mass spectrometer (3800 CG - 4000 EM system, Varian, USA) using a
DB-5MS chromatographic column (30 m × 0.25 mm × 0.25 μm) with a
stationary phase composed of 5 % diphenyl and 95 % poly(dimethyl
siloxane). The ion fragment analyzer was of the “Ion Trap” type, and the
detection range was 50–1000 m/z. The carrier gas used was Helium 6.0
(gas flow = 1 mL min− 1). The temperature program was as follows:
injector temperature = 250 ◦ C; initial column temperature = 60 ◦ C; final
column temperature = 270 ◦ C; heating rate = 10 ◦ C min− 1; column
pressure = 8.2 psi. For the analysis, 5 μL of essential oil was dissolved in
2 mL of hexane, and only 5 μL of the solution was injected into the
GC–MS oven. The essential oil compounds were identified with the NIST
MS Search 2.0 and MS Data Review Software (Varian), using the NIST
MSMS and NIST 02 MS databases.
2.4.2. Optical microscopy
The oil droplets in the emulsions were analyzed using a Leica DM KM
optical microscope (Leica Microsystems GmbH, Wetzlar, Germany). An
emulsion droplet was added to a glass slide, and representative images
were obtained on days 0 and 30 (statical storage). The droplet diameters
were measured using the ImageJ software as an average number of 120
droplets.
G. da S. Ferreira et al.
2.4.3. Emulsion storage stability
The samples were added to glass tubes and stored at room temper­
ature. The stability of the emulsions was evaluated through the crema­
tion phenomenon, evaluated in duplicates on 0–30 days. The cremation
index (CI) was calculated using Eq. (1), where Htotal and Hcream are the
total heights of the emulsion and the cream layer, respectively.
Hcream
CI = × 100 (1)
Htotal
The sedimentation ratio (Sr) was measured according to Eq. (2),
where h1 is the sedimentation height (observed at the bottom of the test
tubes), and H is the height of the total suspension.
h1
Sr = × 100 (2)
H
The emulsion stability index (ESI) was measured in duplicates on
0–30 days and calculated according to Eq. (3) [25,26].
Hs
ESI = × 100 (3)
Ht
where, Hs is the height of the aqueous layer and Ht is the total height of
the emulsion layer.
2.4.4. Fourier-transform Raman spectroscopy (FT-Raman)
The emulsions were analyzed in the FT-Raman model MultiRaman
(Bruker Optics, USA) spectrometer using the laser at the wavelength of
1064 nm as an excitation source (laser power of 150 mW). The data
acquisition was carried out in the spectral window of 600–4000 cm− 1
using 64 scans and 2 cm− 1 spectral resolution.
2.4.5. Fourier-transform infrared spectroscopy (FTIR)
Fourier-transform infrared spectroscopy (FTIR) with attenuated total
reflectance (ATR) diamond accessory was performed on Frontier 94,942
equipment (PerkinElmer Inc., Massachusetts, USA). The spectra were
collected with 4 cm− 1 spectral resolution, 64 scans, from 4000 to 400
cm− 1. The emulsions were dripped onto the ATR crystal until covered,
and then the spectra were collected.
2.4.6. Essential oil releasing
The MaEO releasing from the emulsions was monitored in the
equipment STA 6000 (PerkinElmer®, Inc., Massachusetts, USA) with
alumina pans, using 20–25 mg of the emulsion. The release was
measured under isothermal conditions at 37 ◦ C under an N2 atmosphere
(gas flow = 20 mL.min− 1). Since the MaEO was released with the water,
the weight fraction (wt%) of oil released was obtained from the TGA
measurements correcting the weight data by considering the specific
mass (ρ) and volume fraction of water (ρ = 1 g.cm− 3) and MaEO (ρ = 0.9
g.cm− 3) in the emulsions.
2.4.7. Minimal inhibition concentration (MIC) and minimal bactericidal
concentration (MBC)
The MIC and MBC assays were determined by the resazurin protocol
involving successive microdilution in a culture broth [27]. Twelve serial
dilutions (40, 16, 6.4, 2.6 % v/v, until 0.002 % v/v) were performed in a
96-well polystyrene plate (Kasvi). The first plate was made up of 40 % v/
v of the antimicrobial sample (MaEO or emulsion), diluted sequentially
in 150 μL of Mueller-Hinton nutrient broth until the plates reached a
total volume of 250 μL. Then, all diluted mixtures were inoculated with
100 μL of a previous bacteria inoculum (1.5 × 108 CFU mL− 1) and
incubated for 24 h at 36 ± 1 ◦ C. Then, 50 μL of the serial inoculates were
plated in Petri dishes filled with Mueller-Hinton nutrient agar (Oxoid)
and incubated at 36 ± 1 ◦ C for 24 h. The MBC corresponds to the
minimum concentration at which there is no formation of bacteria
colony-forming units (CFUs), using resazurin as an indicator of cell
growth [27,28].
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International Journal of Biological Macromolecules 243 (2023) 125228
2.4.8. Bacteria inhibition zone
Stock bacterial cultures were grown in Mueller-Hinton broth with 15
% glycerol and stored at − 70 ◦ C. Stock cultures were grown on Mueller-
Hinton agar (Oxoid) and inoculated at 37 ◦ C for 24 h. The inoculating
culture (for 24 h with turbidity equivalent to 0.5 Mc Farland, a con­
centration equivalent to 1.5 × 108 CFU mL− 1) prepared the isolated and
standard bacterial strain suspensions. Then, agar (30 mL) was melted
and cooled to 48 ± 1 ◦ C. Subsequently, 500 μL of the bacterial inoculum
at 1.5 × 108 CFU mL− 1 was mixed with the molten agar. Then, this
mixture was transferred to a Petri dish. After solidification, a diameter
hole (0.9 cm) was made in the middle of the dishes, and the emulsion
(0.2–0.5 mL) was allocated into this hole. Finally, the inoculated dishes
were incubated for 24 h at 36 ± 1 ◦ C. The appearance of inhibition halos
around the orifice with the product was evaluated. These inhibition
halos correspond to the diameter of an inhibition zone merged around
the antimicrobial samples. We used common commercial sanitizers
(hypochlorite and Lysoform®) as antimicrobial controls.
2.4.9. Bacteria colony-forming counting assays
For the bacteria counting, 100 μL of a previously adjusted stock
bacteria inoculum (105 CFU.mL− 1 in Nutrient Broth, HIMEDIA, India)
was mixed with 9.9 mL of nutrient broth (HIMEDIA, India) and 100 μL of
the essential oil or emulsion. The samples were incubated at 36 ± 1 ◦ C
for 24 h with 85 % humidity. Then, 100 μL of the incubated samples
were mixed with 9,9 mL of agar nutrient medium (HIMEDIA, India),
pour-plated into Petri dishes (with 90 mm diameter) and incubated at 36
± 1 ◦ C for 24 h with 85 % humidity. The inoculum viability was
confirmed via a pour-plate method on agar nutrient as the positive
control. The number of counting forming units was measured with
ImageJ software. The tests were carried out in triplicate. The total
bacterial count (in CFU.mL− 1) was determined by multiplying the
number of bacterial colonies counted in the dishes by the dilution factor
(10− 4) of the plated bacterial inoculum.
2.4.10. SARS-CoV-2 virucidal assays
Nasopharyngeal samples collected from patients and resuspended in
saline were tested for SARS-CoV-2 in the laboratory routine diagnostic
assay by the RT-qPCR method (real-time reverse transcription-
polymerase chain reaction), and the spare sample was applied in this
study. Then, 100 μL of a positive sample were diluted in 100 μL of saline
using a swab from the kit to mix. This swab was dipped into 300 μL of the
buffer from the kit, and five drops of the diluted positive SARS-CoV-2
inoculum were added to the Rapid Antigen Test device to confirm the
integrity of the viral particles, according to the manufacturer's in­
structions. The run-time of chromatography was 15 min. After obtaining
a positive control, a mixture of 100 μL of the same SARS-CoV-2 positive
sample and 100 μL of the essential oil or the MaEO/water emulsion was
prepared. The samples were tested at different contact times (0, 5, 15,
and 20 min). The tests were performed in duplicate.
2.4.11. Statistical analysis and design of experiments (DoE)
One-way analysis of variance (ANOVA) was performed in GraphPad
Prism 7 (Dotmatics, Massachusetts, USA) to evaluate the significant
difference between data, using Tukey's test with a confidence level of 95
%. The effects of the parameters used to prepare the emulsions were
evaluated by a factorial DoE, using ANOVA and factor analysis with t-
test and Bonferroni test. The effects of these factors and their in­
teractions on the emulsions' stability properties were presented in Pareto
charts. DoE analyses were carried out on Design Expert 11 (Stat-Ease,
Inc., Minnesota, USA). In the Pareto charts, positive effect means that
the factor increases the property, and negative effect means that the
factor reduces the property, according to the results obtained by factor
analysis.
G. da S. Ferreira et al. International Journal of Biological Macromolecules 243 (2023) 125228

Table 1
Chemical composition of Melaleuca alternifolia essential oil (MaEO) from GC–MS data.
Chemical component Retention time (min) Area Peak area percentage (%)

Terpinen-4-ol 7.671 14,800,000 40.40

γ-Terpinene 5.871 8,134,000 22.20
α-Terpinene 5.309 4,539,000 12.39
Cimene 5.407 2,047,000 5.59
α-Terpineol 7.847 1,877,000 5.12
Terpinolene 6.27 1,659,000 4.53
1R-α-pinene 4.243 1,218,000 3.32
Eucalyptol (cineole) 5.541 973,252 2.66
Limonene 5.477 580,725 1.59
α-Tujene 4.124 198,543 0.54
α-Phellandrene 5.169 130,438 0.36
1S-β-pinene 4.87 122,567 0.33
1R-β-pinene 4.82 103,764 0.28
3-Carene 7.095 68,112 0.19
1S-α-pinene 4.717 57,517 0.16
(Z,Z,Z)-8,11,14-eicosatrienoic acid, methyl ester 4.022 60,373 0.16
4-Carene 6.846 50,472 0.14
Thujone 7.192 8,920 0.02
Camphene 6.493 7,130 0.02

3. Results and discussion ketones, and phenols [30,31].

3.1. GC–MS
3.2. Design of experiments and optical microscopy and emulsion stability

The MaEO chemical composition was evaluated by GC–MS, and the

The CNFs (in 20 and 30 wt/v %) effectively stabilized the MaEO/
nineteen constituent substances identified are detailed in Table 1. The
water Pickering emulsions against creaming and sedimentation within
GC–MS chromatogram and spectra are presented in Material Supple­
the 30-day shelf life under static conditions, as shown in optical mi­
mentary. The main chemical components in the MaEO are ternenen-4-ol
croscopy images and droplet size distributions in Figs. S2 and S3 (Sup­
(40.4 %), γ-terpinene (22.2 %), α-terpinene (12.39 %), cymene (5.59 %),
porting Information). The Pareto charts are shown in Figs. S4 and S5,
and α-terpineol (5.12 %) that are a chemical composition very similar to
indicating the effects of EO content, CNF content, mixing rotation, and
other results reported in the literature [20,22,29], with differences in
mixing time adopted in preparing the emulsions. As can be identified in
the percentage composition that is expected since it depends on the part
Pareto charts, the MaEO average droplet diameter in the emulsions is
of the plant used in extraction, extraction method used, characterization
enhanced by the increase of oil content, mainly at the 30-day storage
method, crop region and climate [22].
time. The increase in CNF content and mixing time prevents the oil
γ-Terpinene, α-terpinene, and cymene are terpenes (also known as
droplets' coalescence due to the Ostwald ripening mechanism [12],
isoprenoids) that are hydrocarbons based on isoprene (2-methylbuta-
principally at the 30-day storage according to the t-value and Bonferroni
1,3-diene) units rearranged into cyclic or aliphatic chemical structures.
limit tests. Also, the mixing rotation and CNF content have significant
Ternenen-4-ol and α-terpineol are terpenoids that are terpene modified
synergetic effects on the droplet stability right after preparing the
with different functional groups with oxygen atoms (i.e., oxygenated
emulsion.
terpene derivatives). Terpenoids are usually found in essential oils of
The emulsion stability index (ESI) tends to be increased by the CNF
different classes, such as alcohols, aldehydes, esters, ether, epoxides,
content that can be connected with the high and preferential

Fig. 2. Optical microscopy images of the main MaEO/water Pickering emulsions: (a) 30EO/5T/0.5CNF/15RPM and (b) 20EO/5T/0.5CNF/12RPM samples.

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G. da S. Ferreira et al. International Journal of Biological Macromolecules 243 (2023) 125228

Fig. 3. (a) FTIR spectra of MaEO and MaEO/water emulsions stabilized with CNF. Zoom in the regions: (b) 500–1600 cm− 1; (c) 1500–2500 cm− 1; and (d)
2500–4000 cm− 1. Spectral data from MaEO (black line), 20EO/5T/0.5CNF/12RPM (blue line) and 30EO/5T/0.5CNF/15RPM (red line) samples. Each color of the
sets is associated with signals from different substances: cellulose (light green), water (cyan), terpinen-4-ol (orange), γ-terpinene (pink), α-terpinene (navy blue),
terpineol (yellow) and terpinolene (purple).

interactions between the CNFs, leading to CNF output of the oil/water

Table 2 interface and the cellulose aggregation over storage time. The ESI values
Signals observed in the FTIR spectra from the MaEO and MaEO/W emulsions
are decreased by the MaEO content within the concentration range used
stabilized with CNF.
to prepare the emulsions within the 30-days of emulsion storage. Mixing
Chemical FTIR signals (cm− 1) rotation and mixing time improves the emulsion stability along the
compound
storage time. However, no emulsion preparation factors affect the ESI
Cellulose 1155 (C–O–C asymmetrical stretching) data predominantly, but they act simultaneously and synergetically with
1160 (C–O–C asymmetrical stretching)
very similar intensity over the ESI results in a complex way, according to
1315 (OH vibration in the cellulose rings)
1430 (symmetric CH2 bending) the Pareto charts from DoE data.
2019 and 2129 (C––C stretch vibration) The 30EO/5T/0.5CNF/15RPM and 20EO/5T/0.5CNF/12RPM
2850 (CH2 group vibration) emulsions are the main stable emulsions concerning the smallest droplet
2910 (CH group vibration) sizes and ESI values, including the lowest variations of these parameters
Water 1640 (H2O scissor vibration mode)
2100 (H–O–H bending vibration)
along the 30 days of storage as shown in Fig. 2. Both emulsions were
3330 (OH stretching vibrations) stabilized with 0.5 % wt/v CNF and using 1 % wt/v visually tends to
Terpinen-4-ol 799, 865, 889, 985, 1160 and 1310 (C–O and C–C aromatic increase ESI, as indicated by Pareto diagrams. Besides the lowest
ring vibrations) manufacturing cost, CNFs have a high aspect ratio and flexibility to
3450 (OH vibrations)
stabilize over time by adsorption and fiber entanglement to be more
γ-Terpinene 780, 815, 830,1160 and 1375 (C–C aromatic ring vibrations)
2820 and 2872 (CH3 and CH2 vibrations) effective via steric hindrance much lower than cellulose nanostructures
α-Terpinene 950 and 1470 (C–C aromatic ring vibrations) as CNCs with a lower aspect ratio that cannot involve all the surface area
1640 (C––C vibrations) of the oil droplets by adsorption [26,32].
2967 (CH3 and CH2 vibrations)
3330 (O–H vibrations)
Terpineol 1365 and 1440 (C–O and C–C aromatic ring vibrations)
3450 (O–H vibrations) 3.3. FTIR
Terpinolene 1020, 1125 and 1514 (C–O and C–C aromatic ring vibrations)
1640 and 1750 (C––C vibrations) The FTIR spectra of MaEO and more stable emulsions (30EO/5T/
2910 (CH3 and CH2 vibrations) 0.5CNF/15RPM and 20EO/5T/0.5CNF/12RPM samples) are shown in
3390 (O–H vibrations)
Fig. 3. The FTIR spectra from MaEO reflect the chemical structures of the
main chemical groups identified by GC–MS. The infrared bands between

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G. da S. Ferreira et al. International Journal of Biological Macromolecules 243 (2023) 125228

Fig. 4. (a) FT-Raman spectra of MaEO and MaEO/water emulsions stabilized with CNF. Zoom in the spectral regions: (b) 500–1600 cm− 1; (c) 1500–2500 cm− 1; and
(d) 2500–4000 cm− 1. Each color of the sets is associated with signals from different substances: cellulose (light green), water (cyan), terpinen-4-ol (orange),
γ-terpinene (pink), α-terpinene (navy blue), terpineol (yellow) and terpinolene (purple).

1600 and 1800 cm− 1 correspond to alkene chemical bonds (C– – C), while
the bands in the 1370–1470 cm− 1 region are associated with –CH2–
and –CH3 scissoring vibrations [33]. The FTIR signals around 2900
cm− 1 are from the CH3 and CH2 vibrations on the hydrocarbon struc­
tures of the terpenes and terpenoids [34]. Other notable bands from
C–O aromatic ring vibrations and C–C vibrational modes in hydro­
carbon cyclic skeletons found below 1500 cm− 1 are influenced by the
position and chemical nature of the aromatic ring's substituent. Table 2
summarizes the FTIR signals detected by infrared spectroscopy.
The band intensity at 3600–3000 cm− 1 is due to the OH vibrations
from terpenoids in MaEO, such as terpinen-4-ol, terpineol, α-terpinene,
and terpinolene [35]. However, α-terpinene, terpinolene and terpenes
strongly absorb infrared radiation with wavenumber from 3200 to 3600
cm− 1 due to C–H asymmetric and symmetric stretching modes. It is
highlighted in Fig. 3b, c and d the FTIR characteristics signals from
terpinen-4-ol (799, 865, 889, 985, 1160, 1310, and 3450 cm− 1), γ-ter­
pinene (780, 815, 830,1160, 1375, 2820, and 2872 cm− 1), α-terpinene
(950, 1470, 1640, 2967 and 3330 cm− 1), terpineol (1365, 1440 and
3450 cm− 1), and terpinolene (1020, 1125, 1514, 1640, 1750, 2910 and
3390 cm− 1) [35,36].
The presence of CNF and water in O/W emulsions improves the in­
tensity of the absorption band from 3200 to 3600 cm− 1 due to the
overlap among the water, MaEO, and cellulose absorption bands since
there is an increase in the OH moiety from the cellulose and water
(Fig. 3). The FTIR band from 2000 to 2300 cm− 1 is associated with the
H–O–H bending vibration forming a hydrogen bonding network of
liquid water in the Pickering emulsions that can be influenced by tem­
perature, including the presence and concentration of anions and cations
in the water media [37]. Moreover, the high water content in Pickering
MaEO/water emulsion causes an overlap of the FTIR characteristic ab­
sorption signals from the MaEO compounds located at 500–1500 cm− 1
and 3200–3600 cm− 1. Also, due to the outstanding water concentration,
the infrared signals from cellulose nanostructures are not observed in
the FTIR spectra of the emulsions. According to the literature [24,38],
FTIR characteristic signals for the cellulose are C–O–C asymmetrical
stretching at 1155 cm− 1, C–O–C asymmetrical stretching at 1160
cm− 1, OH vibration in the cellulose rings at 1315 cm− 1, symmetric CH2
bending at 1430 cm− 1, C– – C stretch vibration at 2019 and 2129 cm− 1,
CH2 group vibration at 2850 cm− 1, and CH groups at 2910 cm− 1.
The slight shift of the FTIR signals at 2500–3000 cm− 1 spectral re­
gion (CH2 and CH3 vibrational modes) to higher wavenumbers in Fig. 3d
can be connected with modification in molecular vibrations of the MaEO
components (γ-terpinene, terpinolene, and α-terpinene) due to their
physical and intermolecular interactions with the surface of the CNFs in

7
G. da S. Ferreira et al.
Table 3
Raman shift signals observed in the FT-Raman spectra from the MaEO and
MaEO/W emulsions stabilized with CNF.
Chemical Raman shifts (cm− 1)
compound
Cellulose 1090 (glycosidic C–O–C, ring vibration)
1155 (asymmetric C–C and C–O ring elongation)
1375 (CH2, HCC, HCO and COH vibrations)
1460 (amorphous phase)
2895 (CH and CH2 elongation)
3250 (O–H elongation)
Water 1600 (H2O scissor vibration mode)
3200–3500 (water network of hydrogen bonds)
Terpinen-4-ol 728 (ring's stretching and deformation)
1375, 1430 and 1445 (CH3 and CH2 flexion and elongation
modes)
1680 (C––C stretching)
2880, 2920 and 2960 (C–H symmetrical and asymmetrical
stretching)
755 (ring's stretching and deformation)
γ-Terpinene
1420 and 1450 (CH3 and CH2 flexion and elongation modes)
1700 (C––C stretching)
2880, 2920 and 2960 (C–H symmetrical and asymmetrical
stretching)
α-Terpinene 1380 and 1465 (CH3 and CH2 flexion and elongation modes)
1660 (C––C stretching)
2880, 2920 and 2960 (C–H symmetrical and asymmetrical
stretching)
Terpineol 1310 and 1435 (CH3 and CH2 flexion and elongation modes)
2880, 2920 and 2960 (C–H symmetrical and asymmetrical
stretching)
Terpinolene 723 (ring's stretching and deformation)
1445 (CH3 and CH2 flexion and elongation modes)
1660 (C––C stretching)
2880, 2920 and 2960 (C–H symmetrical and asymmetrical
stretching)
the emulsions. These interactions with cellulose cause changes in the
vibration modes and vibrational energy levels available to the MaEO
compounds in the multiphasic system stabilized by the CNF.
Souza et al. [12] observed that cellulose in the form of CNF prefer­
entially interacts with OH groups of the components of different
essential oils (cinnamon, cardamon, and Ho wood). In contrast, our FTIR
results here indicate changes in the vibration modes of CH2 and CH3 due
to interactions between terpene groups with non-polar groups in cellu­
lose, suggesting that the OH groups in the terpinen-4-ol molecules (the
major component of MaEO) probably cannot interact effectively with
the polar groups of cellulose due to steric hindrance caused by the iso­
propyl substituent group on terpinen-4-ol.
3.4. FT-Raman
The FT-Raman spectrum of the MaEO is shown in Fig. 4. All terpe­
noids also showed bands around 650–800 cm− 1, typically associated
with ring stretching and deformations. The band up to 1160 cm− 1 refers
to C–O elongation in terpenoids. Ring vibrations from the cyclohexane
exhibit bands at 1020 and 950 cm− 1. Symmetrical and asymmetrical
C–H stretching modes of terpenes and terpenoids are associated with
bands from 2750 to 3050 cm− 1 [39]. The band observed at 1445 cm− 1 is
linked to the flexion and elongation modes of the CH3 and CH2 groups.
The bands around 1600 cm− 1 in the MaEO spectra are associated with
C–– C stretching [40,41], as listed in Table 3.
Cellulose Raman spectra bands are seen in Fig. 4a–d at 1090
(glycosidic COC, ring vibration), 1155 (asymmetric CC and CO ring
elongation), 1375 (CH2, HCC, HCO and COH vibration modes), 1460
(amorphous cellulose), 2895 (CH and CH2 elongation) and 3250 cm− 1
(OH elongation) [42,43]. The pure water band is reported at 1600 and
3400 cm− 1 [44], but O/W emulsions show Raman signals at 1600, 3200
and 3350 cm− 1. This reduction in the water band in the region
3200–3350 cm− 1 indicates changes in the network of H–O–H bonds in
8
International Journal of Biological Macromolecules 243 (2023) 125228
the aqueous medium, as highlighted in the inset shown in Fig. 4d. This
phenomenon can be attributed to the formation of hydrogen bonds be­
tween water and cellulose nanofibrils in the emulsion with less energy
than the strong hydrogen bonds between water molecules [44].
The characteristic bands in the 700–800 cm− 1 region from cellulose
due to OH bond vibrations can be identified by FTIR spectroscopy.
However, such vibrational modes commonly are not detected by Raman
spectroscopies because there is a great dipole moment and low polar­
izability associated with the OH bond among cellulose macromolecules.
However, the FT-Raman spectra of the emulsions display Raman pattern
shifts at 725 (CCC, COC, OCO, CCO, and OH out-of-plane bending) and
800 cm− 1 (OH bending), suggesting strong interactions between the
CNFs with water and the oxygenated terpenes in the MaEO by hydrogen
bonds [45]. Fig. 5 illustrates the possible intermolecular interactions
between water and MaEO compounds with the hydrophobic and hy­
drophilic cellulose sites [46], as the FTIR and FT-Raman data suggested.
3.5. Essential oil releasing
We propose the usage of thermal analysis to evaluate the controlled
release of MaEO from emulsions at a temperature similar to that used in
bactericidal assays (37 ◦ C). The method is an accelerated test for the oil
release since the carrier gas helps release the essential oil by convection,
making it the fastest release test that allows a comparative investigation
of the MaEO release rate and stability between emulsions.
According to the isothermal curves (Fig. S6), MaEO in the emulsions
starts to be liberated at the beginning of the experiment. It takes 64 and
58 min for the MaEO to be released entirely from the 20EO/5T/0.5CNF/
12RPM and 30EO/5T/0.5CNF/15RPM emulsions, respectively.
The oil-releasing kinetics follows a zero-order model, i.e., MaEO is
released from the emulsion by a linear relationship with the time ac­
cording to Eq. (4) [47–49].
Mt/M = k0 t + C0 (4)
∞
where Mt/ corresponds to the fraction of MaEO released at time t, and
M∞
k0 is a constant connected with the oil release rate.
A zero-order model suggests that the diffusion rate is practically
independent of the emulsion stability [24]. The coefficient parameters
for the kinetic mechanism involved in the MaEO liberation are listed in
Table 4. The k0 values are − 0.6 and − 0.3 (% v/v).min− 1 for the 30EO/
5T/0.5CNF/15RPM and 20EO/5T/0.5CNF/12RPM samples, indicating
that the MaEO releasing is faster for the emulsion with the highest ratio
between the oil and CNF content what is expected since there is a higher
amount of oil to be stabilized by the same CNF content.
3.6. MIC and MBC
MIC and MBC assays were performed to evaluate the bactericidal
performance of the MaEO against E. coli (Gram-negative bacteria strain,
G− ) and S. aureus (Gram-positive bacteria strain, G+). The MIC and
MBC values are detailed in Table 5. The MIC value of MaEO is 0.2 ± 0.1
% v/v against E. coli, but 2.3 ± 0.9 % v/v against S. aureus. The MIC/
MBC ratio for the MaEO against both G+ and G− strains evaluated are
≤4, indicating that MaEO can be considered a bactericidal agent in
which it is possible to kill 99.9 % of the bacteria cells under adequate
concentrations [50]. A MIC/MBC ratio higher than 4 is related to a
bacteriostatic drug.
The O/W emulsions present higher MIC and MBC values than the
MaEO due to the lowest amount of antimicrobial compounds in the
emulsified system. The MIC and MBC doses of the emulsions are 6.4 ±
0.7 % v/v against E. coli and do not vary with the amount of MaEO
content. The MIC and MBC data are identical for 20EO/5T/0.5CNF/
12RPM and 30EO/5T/0.5CNF/15RPM emulsion against S. aureus,
although they are different conceptual concepts. Garzoli et al. [51]
observed a similar phenomenon for MIC and MBC results for essential
G. da S. Ferreira et al. International Journal of Biological Macromolecules 243 (2023) 125228

Fig. 5. Schematic representation of the

chemical interactions at the cellulose/
water and cellulose/MaEO interfaces in
the Pickering emulsions. Hydrogen bonds
between cellulose and the terpenoids and
CH-CH dipole-induced-dipole interactions
(van der Waals forces) between cellulose
terpenes in the MaEO at the cellulose/oil
interface. At the cellulose/water interface,
there are hydrogen bonds between cellu­
lose and water molecules. The hydrogen
bonds between the CH-OH cellulose
groups are the main interactions that
contribute to the cohesion of CNFs sur­
rounding the oil droplets [46].

oils from the Lavandula genus against G− bacteria strains.

Table 4 For these outstanding bactericidal features and broad-spectrum and
Zero-order kinetic model parameters for the MaEO releasing from the emulsion antimicrobial activity, MaEO is commonly applied as an eco-friendly
at 37 ◦ C. topical antiseptic and antibacterial component in cosmetics with po­
Sample Parameters tential use in the food industry to prolong the shelf life of food products
k0 [(% v/v).min− 1] C0 (% v/v)
[52]. S. aureus is an opportunistic pathogen that, when installed in the
R2
host, leads to superficial (suppurative infection in soft tissues) and deep
20EO/5T/0.5CNF/12RPM − 0.3 ± 0.1 20.2 ± 0.1 0.99
(systemic spread by bacteremia) infections, even toxigenic infections
30EO/5T/0.5CNF/15RPM − 0.6 ± 0.1 30.1 ± 0.1 0.99
characterized by the production of toxins by systemic action [53]. E. coli
are facultative anaerobic bacteria and a member of the human intestinal
microbiota, which can cause intestinal and urinary tract infections by
Table 5 different mechanisms [53].
Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentra­ The antimicrobial performance of the MaEO has been attributed to
tion (MBC) of Melaleuca alternifolia essential oil (MaEO) against S. aureus (ATCC its components, mainly terpinen-4-ol and α-terpineol, which cause
#6538) and E. coli (ATCC #11229). several injuries to the bacteria cells via membrane-damaging mecha­
Sample S. aureus E. coli nisms [52,54]. It is reported that MaEO inhibits cellular respiration and
MIC (% v/ MBC (% v/ MIC (% v/ MBC (% v/ disbalances the potassium ions at the cytoplasmic membrane that pro­
v) v) v) v) voke cellular morphological damages, accompanied by disruption of the
MaEO 2.3 ± 0.9 4.0 ± 1.5 0.2 ± 0.1 0.3 ± 0.1
cell permeability barrier for the S. aureus and E. coli strains [54]. Other
20EO/5T/0.5CNF/ 16.0 ± 1.6 16.0 ± 1.6 6.4 ± 0.7 6.4 ± 0.7 authors indicate the coagulation of cytoplasmic constituents in E. coli
12RPM cells after direct contact with MaEO due to monoterpene penetration
30EO/5T/0.5CNF/ 2.6 ± 2.5 2.6 ± 2.5 6.4 ± 0.7 6.4 ± 0.7 into the cell associated with a previous cell permeability barrier
15RPM
compromising that was occasioned by irreversible membrane injuries by
MaEO constituents [21]. Moreover, monoterpenes in MaEO can damage
the lipid constituents of the microbial membranes [54]. Then,

9
G. da S. Ferreira et al.
Fig. 6. Inhibition halos for hypochlorite, Lysoform, MaEO, and MaEO/water emulsions against E. coli (a, c) and S. aureus (b, d). The values are presented as mean ±
standard deviation from triplicate measurements. Only the sample data without statistically significant differences are marked on the graphs (ns, where p > 0.05),
according to Tukey's test with a 95 % confidence level. *The scale bars of graphs (a, b) are in different scales.
alternative effectiveness and synergistic interactions between antimi­
crobial MaEO compounds may result in simultaneous and different
bactericidal mechanisms that lead to whole-cell lysis or compromising of
the cell machinery, eventually leading to the bacteria's death.
MBC corresponds to the concentration at which the antimicrobial
sample can inactivate >99.99 % of the bacteria in the medium [55]. The
MBC results in Table 5, the G+ strain seems more resistant to the
bactericidal components in the MaEO, evidencing different susceptibil­
ity to tea tree essential oil as reported by other authors [21,56]. This
differential susceptibility effect is still not well understood. Even more, it
is an effect contrary to what was expected since G+ bacteria have only
an outer layer, which is ineffective in hampering the interactions be­
tween external agents with the cytoplasmic membrane, generally mak­
ing the G+ bacteria more susceptible and fragile to antimicrobial agents
[56]. Antagonastly, G− bacteria cells have an additional outer mem­
brane responsible for protecting the inner cytoplasmic membrane,
which generally makes them more resistant than G+ strains to external
antimicrobial agents [56]. Carson et al. reported that the primary anti­
microbial mechanism against S. aureus is a complex delayed rupture of
the cytoplasmic membrane marked by inducing the leakage of cyto­
plasmic material [57]. Terpinen-4-ol cannot lyse the S. aureus cells
completely, while α-pinene, β-pinene, and limonene inhibit the respi­
ratory activity of the mitochondria.
Surface charges and chemical composition of the cell and outer-cell
membranes of the G− and G+ bacterial strains must be connected with
their susceptibility to MaEO. G+ strains present a cytoplasmatic
10
International Journal of Biological Macromolecules 243 (2023) 125228
membrane surface (covered by a peptidoglycan cell wall) predominantly
charged with negative charges from phosphate groups in teichoic acids,
which are essential components of the lipopolysaccharide cell wall to
stabilize the G+ cell wall layer through electrostatic interactions with
cations, such as potassium [58]. Besides G− strains having an additional
outer lipidic membrane responsible for protecting the peptidoglycan cell
wall and the inner cytoplasmic membrane, they are also predominantly
negatively charged since their outer lipidic membranes have a strong
negative charge conferred by the lipopolysaccharide that, with lipo­
proteins and phospholipids, cover the peptidoglycan cell layer [58].
Moreover, the plasma membrane of G+ bacteria is surrounded by a
thinner cell wall with several peptidoglycan layers, forming a thickness
of 20–100 nm. In contrast, G− strains display a thin cell wall, showing a
few nanometric thick (1–8 nm) due to the presence of a single pepti­
doglycan layer or a reduced number of peptidoglycan layers [59–61].
These differences between the cell wall thickness of the G+ and G− cell
walls must play a crucial role in the bactericidal resistance of these
bacteria strains against MaEO since monoterpenes are potent destroyers
of lipid structures [54].
3.7. Bacteria inhibition zones
The inhibition zone technique using the agar diffusion test was
performed to evaluate the bacteriostatic capacity of the MaEO and
MaEO/emulsions. The formation of inhibition halos indicates that the
product under analysis has bacteriostatic properties, evidencing the
G. da S. Ferreira et al.
Fig. 7. Bacterial counting and relative bacterial reduction for the MaEO and MaEO/water emulsions against E. coli (a, b) and S. aureus (c, d). The values are presented
as mean ± standard deviation from triplicate measurements. *Sample data are significantly different (*p > 0.05), according to Tukey's test with a 95 % confi­
dence level.
inhibiting of bacterial growth and reproduction without causing the
bacteria death. The inhibition zone diameter depends on the diffusion of
the agar preparation. As a comparative control test, we also evaluated
the formation of inhibition halos on agar diffusion plate assays with
hypochlorite and Lysoform®, which are effective disinfectants
commercially available and commonly used by the Brazilian population.
As shown in the graphs in Fig. 6a, the inhibition zone diameter
observed for the MaEO is equal to 2.95 ± 0.35 cm against E. coli, which
is a value 50 % higher than that detected for Lysoform® (1.65 ± 0.05
cm) but statically equal to that obtained with hypochlorite (3.15 ± 0.52
cm). Fig. 6c and d presents the images of the agar diffusion dishes with
the inhibition zones. The MaEO/water emulsions (30EO/5T/0.5CNF/
15RPM and 20EO/5T/0.5CNF/12RPM samples) also display significant
inhibition zone diameters than that observed for the hypochlorite
against E. coli and S. aureus.
According to Fig. 6b, the 30EO/5T/0.5CNF/15RPM and 20EO/5T/
0.5CNF/12RPM samples lead to the formation of inhibition zone di­
ameters similar to the obtained for the Lysoform® against S. aureus (2.95
± 0.10 cm), but they are less than that identified by the MaEO (4.15 ±
0.37 cm) that can be justified by the high content of volatile antimi­
crobial agents in the tea tree essential oil. These highlightable antimi­
crobial results indicate that the Pickering emulsions are suitable to
11
International Journal of Biological Macromolecules 243 (2023) 125228
release the volatile MaEO bactericidal agents gradually within the MIC
values, which is essential to ensure the prevention of bacteria prolifer­
ation of both G− and G+ strains in a long-standing, avoiding a quick loss
of antimicrobial activities of the MaEO [12]. Then, the protection of the
CNFs of these agents against the decomposition occasioned by external
agents (e.g., sunlight) does not compromise the bactericidal perfor­
mance of the MaEO.
3.8. Bacteria colony-forming counting assays
MaEO presents bacterial colony-forming counting of 4.9 ± 0.1 log
(CFU.mL− 1) and 5.1 ± 0.3 log(CFU.mL− 1) against S. aureus and E. coli,
corresponding to a bacterial reduction of around 40–42 % (in regarding
the bacterial counting from positive controls) with significantly different
(*p > 0.05), according to Tukey's test with a 95 % confidence level, as
shown in Fig. 7. It is important to mention that the bacteria counting
tests were performed with MaEO content below the MIC value deter­
mined against the S. aureus. 2-log(CFU/mL− 1), i.e., 102 CFU.mL− 1 that
corresponds to a bacteriostatic action and indicates a 99 % reduction in
bacteria that involves inhibition of the bacteria growth and reproduc­
tion without killing the bacteria cells. Bactericidal activity is associated
with an antibacterial agent that completely prevents bacterial growth
G. da S. Ferreira et al.
Fig. 8. SARS-CoV-2 antiviral tests for the MaEO (a) and MaEO/water emul­
sions (b, c) after 0, 5, 15, and 20 min of direct contact. Negative results are
highlighted in red squares.
and reproduction, characterized by a 3-log10 reduction of the bacterial
counting (in CFU.mL− 1) in the initial inoculum (i.e., a reduction like ⩾
99.9 % in bacterial counting) [62].
The bacterial counting for 30EO/5T/0.5CNF/15RPM and 20EO/5T/
0.5CNF/12RPM emulsions are 7.2 ± 0.3 and 6.9 ± 0.1 log(CFU.mL− 1)
against S. aureus, respectively. In contrast, the S. aureus positive control
presents a bacterial counting equal to 8.5 ± 0.2 log(CFU.mL− 1). How­
ever, the relative bacterial reduction from 30EO/5T/0.5CNF/15RPM
and 20EO/5T/0.5CNF/12RPM emulsions are statistically similar
(15–20 %) against this G+ bacteria strain. Concerning the bacterial
12
International Journal of Biological Macromolecules 243 (2023) 125228
counting tests against E. coli, the 20EO/5T/0.5CNF/12RPM and 30EO/
5T/0.5CNF/15RPM emulsion also showed statistically identical relative
bacterial reduction results (5–10 %), which is the antimicrobial per­
formance less than that for the MaEO (42.1 ± 3.1 %).
Similar to the inhibition zone results, bacterial count and ANOVA
data indicate that MaEO and MaEO/water CNF-stabilized emulsions
have significantly greater antimicrobial activity against S. aureus than
against E. coli. In contrast, the MIC and MBC results suggested the lowest
bacteriostatic and bactericidal doses of MaEO against E. coli than those
against S. aureus. D'Arrigo et al. [54] observed similar contradictory
behavior for the antimicrobial performance of MaEO, and they reported
that the E. coli strain is more susceptible than S. aureus to the antimi­
crobial compounds in MaEO over time from using time-killing assays,
while the MIC and MBC indicate that S. aureus is more susceptible to this
essential oil. However, these authors did not explain these conflicting
results for the MaEO antimicrobial sensibility from the MIC, MBC, and
time-kill assay data. In another work, Cox et al. [21] also observed a
lower MIC and MBC of MaEO against E. coli than that against S. aureus,
but identified that E. coli undergoes greater respiratory inhibition and
are more susceptible to K+ efflux due to membrane-damaging effects
than S. aureus. These findings are connected with differences in the
susceptibility of the different bacterial strains to MaEO, which seem not
to be only influenced by the bacterial cell structure and physical/
chemical factors (warmth, moisture, pH and oxygen levels). The anti­
microbial test method also influences the findings on the bactericidal
properties of an antimicrobial agent due to its experimental variables
that affect the results and lead to experimental errors. Just to exemplify,
the final colony-forming units obtained by bacterial colony counting
assays are influenced by the growth and proliferation kinetics of the
bacteria strains, which depends on the bacterial generation time.
3.9. SARS-CoV-2 virucidal assays
The SARS-CoV-2 antigen-detecting rapid diagnostic test kits were
applied to evaluate the integrity of the spike proteins (S proteins) at the
surface of the SARS-CoV-2 virions, identifying their viability to infect a
new host [63]. The rapid test used here is a chromatographic immu­
noassay for qualitatively detecting the S-specific proteins of the SARS-
CoV-2 particles in the nasopharynx inoculum. The denaturation/
disruption of these viral proteins on the surface of the virus promoted by
our samples is indicated by the absence of the red color in the “test line”
on the rapid diagnostic test kits, presented as negative result, because
their antibodies cannot bind to these antigens once damaged, as shown
in Fig. 8.
MaEO deactivated all SARS-CoV-2 virions immediately upon con­
tacting it, as indicated in Fig. 8a. The 30EO/5T/0.5CNF/15RPM emul­
sion could inactivate all virions in 15 min of direct contact since all the S
viral proteins were damaged in the inoculum (Fig. 8b). In contrast, the
20EO/5T/0.5CNF/12RPM emulsion did not inactivate all viral particles
within 20 min (Fig. 8c), which can be explained by its lowest MaOE
content. However, the reduction in the intensity of the red coloration in
the “test line” indicates partial inactivation of the virus; thus, this 20EO/
5T/0.5CNF/12RPM emulsion can probably lead to complete inactiva­
tion of all viable virus particles at longer direct contact times.
Several studies demonstrated the antiviral activity of MaEO against a
wide range of viruses, even at doses below the cytotoxic doses, such as
Herpes labialis, Tobacco mosaic, Herpes simplex (HSV), and Influenza A
viruses [64–68]. The viral inactivation mechanism depends on virus
morphology, including viral replication and viral infection molecular
mechanisms. So far, no studies show the antiviral mechanism of MaEO
against SARS-CoV-2. However, our antiviral assay results indicate that
the inactivation mechanism of SARS-CoV-2 virions involves the
destruction/denaturation of Spike protein trimers that play a critical
role in the host cellular infection mechanism. Consequently, the infec­
tion ability of the SARS-CoV-2 particles is abrogated [63,69].
G. da S. Ferreira et al.
4. Conclusions
In conclusion, we have shown that MaEO and MaEO/water CNF
stabilized emulsion present antibacterial performance against E. coli and
S. aureus, including virucidal performance against SARS-CoV-2. Mela­
leuca alternifolia essential oil (MaEO) deactivates the SARS-CoV-2 par­
ticles immediately by damaging their S proteins, but its emulsions take
much longer to deactivate. FT-Raman and FTIR data indicate different
interactions between water and CNF with different essential oil com­
ponents in emulsions, depending on their structure and chemical
composition. The MaEO/water emulsion stabilized with a 0.5 % wt/v
CNF suspension, 30 % v/v MaEO content, mixed by 5 min using a mixing
rotation of 15 rpm, presented the highest statical stability and was
capable of deactivating the SARS-CoV-2 virions in 15 min of direct
contact. Factorial DoE indicates that the improvement of the mixing
time is essential to stabilizing the emulsions within a 30-days storage
time.
Here, we evidence that the bactericidal performance of the MaEO
and the Pickering emulsions against E. coli and S. aureus is influenced by
the antimicrobial assay type, which can lead to conflicting conclusions
about the greatest antimicrobial susceptibility among these bacteria
strains to the “green” bactericidal agents. Then, the antimicrobial sus­
ceptibility of one bacteria strain to the essential oil must be evaluated by
different microbiological assays to identify such inconsistencies,
enabling a better decision on which antimicrobial agent is most effective
against a given bacterial strain.
Ethical statement
The antiviral tests against SARS-CoV-2 were performed following the
standard regulations approved by the Research Ethics Committee of the
Federal University of ABC and Instituto Adolfo Lutz CAAE:
49573421.2.3001.0059, and by the Technical-Scientific Council of
Instituto Adolfo Lutz, CTC 18-N/2021. All procedures followed the
biosafety standards of the Instituto Adolfo Lutz of the Regional Center of
Laboratories of Santo André.
Funding
This research was funded by Fundação de Amparo à Pesquisa do
Estado de São Paulo (2020/12208-9), Conselho Nacional de Desenvol­
vimento Científico e Tecnológico (305819/2017-8), CAPES-Pandemias
(88881.504639/2020-01).
CRediT authorship contribution statement
Greiciele da S. Ferreira: Conceptualization, Methodology, Valida­
tion, Formal analysis, Investigation, Writing – original draft, Writing –
review & editing, Visualization. Daniel J. da Silva: Conceptualization,
Methodology, Validation, Formal analysis, Investigation, Writing –
original draft, Writing – review & editing, Visualization. Alana G.
Souza: Conceptualization, Methodology, Validation. Eliana D.C.
Yudice: Methodology, Validation, Formal analysis. Ivana B. de Cam­
pos: Methodology, Validation, Formal analysis. Rute Dal Col: Meth­
odology, Validation, Formal analysis. Andre Mourão: Methodology,
Validation, Formal analysis, Writing – review & editing. Herculano S.
Martinho: Formal analysis, Supervision, Writing – review & editing.
Derval S. Rosa: Resources, Writing – review & editing, Supervision,
Project administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
13
International Journal of Biological Macromolecules 243 (2023) 125228
Data availability
Data will be made available on request.
Acknowledgment
The authors thank the CAPES (Code 001), CAPES-Pandemias
(8881.504639/2020-01), MULTIUSER CENTRAL FACILITIES (UFABC),
UFABC, and REVALORES Strategic Unit.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2023.125228.
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