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Essential oil nanoemulsions: Properties, development, and application in

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DOI: 10.1016/j.tifs.2022.01.026

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 Trends in Food Science & Technology 121 (2022) 1–13

Contents lists available at ScienceDirect

Trends in Food Science & Technology

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

Essential oil nanoemulsions: Properties, development, and application in

meat and meat products
Bruno Dutra da Silva a, b, c, Denes Kaic Alves do Rosário a, b, d, David A. Weitz e,
Carlos Adam Conte-Junior a, b, c, e, f, *
a
Center for Food Analysis (NAL), Technological Development Support Laboratory (LADETEC), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ,
21941-598, Brazil
b
Laboratory of Advanced Analysis in Biochemistry and Molecular Biology (LAABBM), Department of Biochemistry, Universidade Federal do Rio de Janeiro (UFRJ), Rio
de Janeiro, RJ, 21941-909, Brazil
c
Graduate Program in Food Science (PPGCAL), Institute of Chemistry (IQ), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, 21941-909, Brazil
d
Department of Food Engineering, Center for Agrarian Sciences and Engineering, Universidade Federal do Espírito Santo (UFES), Alegre, ES, 29500-000, Brazil
e
John A. Paulson School of Engineering and Applied Sciences (SEAS), Harvard University (HU), Cambridge, MA, 02138, USA
f
Nanotechnology Network, Carlos Chagas Filho Research Support Foundation of the State of Rio de Janeiro (FAPERJ), Rio de Janeiro, RJ, 20.020-000, Brazil

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

Keywords: Background: Essential oils have been investigated for their potential application as a natural ingredient in food
Nanoemulsion preservation. However, the low solubility in the aqueous phase, high volatility, and low long-term stability are
Essential oil limiting factors for its use as a natural preservative to replace traditionally used preservatives. With the
Meat products
advancement of nanotechnology, the development of essential oil nanoemulsions has become a promising
Antimicrobial
Antioxidant
alternative.
Safety aspects Scope and approach: Nanoemulsions have unique characteristics due to their nanometric size, which include
increased contact surface area and increased physicochemical stability. Meat matrices have a complex chemical
composition that can interact with the compounds present in EO and reduce their bioactivity. The essential oil
nanoemulsified is a conservation alternative to pure oil, with the ability to improve the interaction with specific
targets in meat and exert antimicrobial and antioxidant activity. This paper summarizes the essential charac­
teristics of nanoemulsions and presents the main advances in the application of essential oil nanoemulsified in
meat and meat products for preservation.
Key findings and conclusion: According to what is available so far from studies with essential oil nanoemulsified in
meat, the application can extend the shelf life of meat and meat products, with improved bioactivity compared to
pure oils. Bioactivity is directly associated with particle size, and both high and low-energy methods can be used
to prepare of nanoscale emulsions and achieve droplet sizes ≤100 nm. Therefore, nanoemulsions can be a
technological alternative to overcome the limitations of pure oils and be used in meat and meat products as a
natural preservative.

1. Introduction demand (Bhavaniramya, Vishnupriya, Al-Aboody, Vijayakumar, &

The profile of current consumers is increasingly demanding and
associated with easy access to information, has stimulated the search for
a healthier diet (Prakash et al., 2018). The use of traditional chemical
preservatives in meat and meat products negatively affects consumers
due to the possible toxicological effects of consumption. Therefore, the
food industry has been looking for natural alternatives to ensure the
conservation of meat and meat products and thus meet consumer
Baskaran, 2019; Pinelli et al., 2021). In this context, essential oils (EOs)
can be an interesting alternative due to their beneficial properties in
food matrices.
EOs consist of volatile bioactive molecules extracted from various
plant sources such as leaves, flowers, stems, roots, and seeds (Burt,
2004). The global demand for EO in 2020 was 247 kilotons, and the EO
market is expected to develop at an annual rate of 7.5% through 2027,
driven by the trend of natural product consumption (Grand View

* Corresponding author. 29 Oxford Street, Cambridge, MA, 02138, USA.

E-mail address: ccontejr@fas.harvard.edu (C.A. Conte-Junior).

https://doi.org/10.1016/j.tifs.2022.01.026
Received 6 November 2021; Received in revised form 3 January 2022; Accepted 16 January 2022
Available online 19 January 2022
0924-2244/© 2022 Elsevier Ltd. All rights reserved.
B.D. da Silva et al.
Research, 2020). The use of EO is compatible with meat and meat
products, and several EO has the safe status for consumption “GRAS”
(Generally Regarded as Safe”) according to the Food and Drug Admin­
istration (FDA, 2020). According to their chemical composition, EOs can
delay oxidative reactions, the formation of free radicals, and microbial
growth from in vitro and food preservation assays, which makes EOs
potential partial or total substitutes for traditional chemical pre­
servatives (Bhavaniramya et al., 2019; Gyawali & Ibrahim, 2014).
The main challenge for using EO in meat and meat products is linked
to the interaction of the constituents of the oils with a composition of the
meat matrix and the loss due to volatility of the compounds (Hyldgaard,
Mygind, & Meyer, 2012). The constituents of EO have a lipophilic
character and can become susceptible to oxidation and polymerization
with meat components during storage. Extrinsic factors (microbial
concentration, types of microorganisms, gaseous composition of the
environment, and temperature) and intrinsic factors of the meat matrix
(fat, proteins, water activity, salt concentration, and pH) can affect the
bioactivity of EO and reduce the conservation capacity (Hyldgaard et al.,
2012; Prakash et al., 2018). Due to the aromaticity of the EO, the
increased concentration used in the meat to overcome the limitation of
losses due to interaction with the meat and volatility during storage can
generate off-flavor that cause consumer rejection (Rosario, Rodrigues,
Bernardes, & Conte-Junior, 2020). The incorporation of pure oil in meat
matrices generally shows inferior results when compared to in vitro
preservation assays (Paparella et al., 2016).
Currently, nanotechnology is an emerging research field in food
science and technology. It is the target of study in several investigations,
as the size of the nanoparticles gives the nanomaterials distinct char­
acteristics that can be useful for application in food systems (Dammak,
Sobral, Aquino, Neves, & Conte-Junior, 2020). Due to the lipophilic
character of EO, the development of nanoemulsions stands out. The size
of droplets with a diameter between 20 and 200 nm expand the surface
area that generates numerous advantages such as increased solubility in
the aqueous phase, protection from oxidation of constituents of the EOs,
and reduction changes in sensory properties (Abdou, Galhoum, &
Fig. 1. Mechanisms of action of essential oils on microbial cells. (1) Cytoplasmic membrane rupture; (2) Leaking of intracellular constituents; (3) Loss of enzymatic
functionality; (4) DNA damage; (5) Impact on protein synthesis; (6) Interaction with quorum sensing mechanism; (7) Impact on ATP synthesis.
2
Trends in Food Science & Technology 121 (2022) 1–13
Mohamed, 2018; Barradas et al., 2015).
This paper has reviewed the detailed information on the potential
use of nanoemulsified EOs (NEO) as natural preservatives for meats and
meat products, focusing on applying nanotechnology to overcome the
limitations of using pure oil in current research with meat matrices.
2. Mechanism of action of essential oils
2.1. Antimicrobial activity
Meat presents favorable conditions for microbial growth, as a source
of nutrients, high water activity, low acidity, and even with meat pro­
cessing, conditions during storage such as temperature and oxygen
levels can influence the type of microorganism present and microbial
concentration (Doulgeraki, Ercolini, Villani, & Nychas, 2012). The
antimicrobial activity attributed to the EOs of each plant species varies
according to the structural configuration of the molecule. The main
metabolites synthesized by plants that make up EOs include terpenes,
phenylpropanoids, aldehydes, esters, alcohols, and ketones (Bhavanir­
amya et al., 2019). Due to the diversity of chemical constituents of EO,
there is no specific mechanism of action in microbial cells. However, one
of the most reported mechanisms involves increased cell membrane
permeability. Due to the lipophilic character of EOs, diffusion and
interaction with targets in the cytoplasmic membrane or with intracel­
lular constituents are facilitated. Increased membrane permeability can
also cause extravasation of cytoplasmic content and, consequently, cell
death (Fig. 1) (Burt, 2004).
According to Kedia, Prakash, Mishra, Dwivedy, and Dubey (2015),
an increase in electrolyte loss (Na+, K+, and Ca2+) was detected in
Aspergillus flavus after treatment with EO Trachyspermum ammi L.
concentration-dependent. According to Zhang, Liu, Wang, Jiang, and
Quek (2016), cinnamon EO (Cinnamomum zeylanycum) at inhibitory
concentrations increased extracellular nucleic acid levels in viable
Escherichia coli and Staphylococcus aureus cells. For Cui, Zhang, Li, and
Lin (2018), the EO from clove (Syzygium aromaticum) in bactericidal
B.D. da Silva et al.
concentrations increased extracellular ATP levels and caused a reduc­
tion in the enzymatic activity of ATPase. Another suggested mechanism
is the inhibitory action of EO during processes of strand DNA tran­
scription into mRNA and mRNA translation for protein synthesis, which
influences bacterial DNA replication (Xu, Liu, Hu, & Cao, 2016).
Besides the chemical composition of the EO, which is influenced by
plant growing conditions and climatic aspects, other factors during EO
application can influence the antimicrobial activity, such as the con­
centration of the oil used, microbial concentration, and cell structure
(Siddique, Parveen, Firdaus, & Mazhar, 2020). The outer membrane of
Gram-negative bacteria is composed of lipopolysaccharide, which can
act as a barrier against EO diffusion and protect the cytoplasmic mem­
brane (Nazzaro, Fratianni, De Martino, Coppola, & De Feo, 2013).
However, the resistance or sensitivity of a bacterium to EO action will be
primarily linked to the mechanisms of action and specific targets of the
oil constituents in the bacterial cell (Gyawali & Ibrahim, 2014). Several
researches report lower minimum inhibitory and bactericidal concen­
trations of EOs for some Gram-negative bacteria compared to
Gram-positive bacteria (Ferreira et al., 2019; Gómez-Sequeda, Cáceres,
Stashenko, Hidalgo, & Ortiz, 2020). According to Kachur and Suntres
(2020), the hydroxyl group of the molecule with delocalized electron
system in monoterpenoids such as thymol and carvacrol, present in some
EO, are responsible for damage to the outer membrane, which increases
the antimicrobial efficacy, even in Gram-negative bacteria.
2.2. Antioxidant activity
Oxidative deterioration is one of the main challenges related to the
conservation of meat and meat products, directly linked to the chemical
composition of the meat and forms of storage (Canto et al., 2016; Pateiro
et al., 2018). The presence of unsaturated lipids favors lipid and protein
oxidation in meat, heme pigments, metallic catalysts, and sulfur amino
acids in muscle tissue, which in exposure to reactive oxygen species and
free radicals can trigger the oxidative process (Cunha, Monteiro,
Costa-Lima, et al., 2018; Falowo, Fayemi, & Muchenje, 2014).
Oxidation control generally occurs through the presence of endoge­
nous or exogenous antioxidants that neutralize the oxidative process
through three mechanisms: hydrogen atom transfer, electron transfer,
and the ability to chelate transition metals (Granato et al., 2018). Many
EOs have antioxidant properties associated with the presence of
phenolic compounds with one or more free hydroxyls and other sec­
ondary metabolites, which can act through mechanisms of inhibition of
the oxidative process (Bhavaniramya et al., 2019). The EOs extracted
from various plant species are rich sources of oxygenated monoterpenes
(α-terpineol, menthol, linalool, and citronellal), phenolic terpenoids
(thymol and carvacrol), and terpenic hydrocarbons (α-pinene, myr­
cene), which are chemical compounds with an antioxidant activity
which can be used as a natural alternative for preserving meat products
(Pirbalouti, Izadi, Poor, & Hamedi, 2016; Valdivieso-Ugarte,
Gomez-Llorente, Plaza-Díaz, & Gil, 2019).
According to Snoussi et al. (2015), the EO from Mentha spicata rich in
carvone and limonene showed free radical scavenging activity equiva­
lent to butylhydroxyanisole (BHT), which is a traditional antioxidant
used in foods. Pirbalouti et al. (2016) reported similar antioxidant ca­
pacity compared to BHT in the EO of Ferulago angulata with a predom­
inance of α-pinene and cis-β-ocimene in its composition. Semeniuc et al.
(2018) evaluated different EO belonging to the Apiaceae and Lamiaceae
families and highlighted that the thyme EO presented greater antioxi­
dant capacity, emphasized by the chemical composition rich in phenolic
compounds. According to Jaradat et al. (2017), Ruta chalepensis EO
collected from three regions with different chemical profiles exhibited
free radical inhibiting activity. The variety of bioactive compounds with
antioxidant activity found in other plant species demonstrates the
relevance of using EO as a natural alternative to reduce the oxidative
deterioration of meat and meat products.
3
Trends in Food Science & Technology 121 (2022) 1–13
3. Limitations on the use of essential oils in meat and meat
products
In vitro studies with EO under controlled conditions exhibit broad
bioactivity. However, when applied in food systems, the efficacy of EOs
is reduced, which makes it a challenge for their application for food
preservation. The meat structure is heterogeneous and can give rise to
specific regions with different physicochemical properties that can in­
fluence the interaction of EOs with specific targets (Gutierrez,
Barry-Ryan, & Bourke, 2008). One of the main challenges with using
pure oils in meat and meat products is the diluting effect of EO con­
stituents in meat fat, which reduces its bioactivity in bacterial cells
(Hyldgaard et al., 2012). Raw meat has an average of 2.5% fat, which
can vary according to muscle location, animal species, gender, age,
genetics, and animal management (López-Bote, 2017). In meat products,
the amount of fat may be higher for some products due to the addition to
improve sensory and technological properties.
The constituents of EO can also interact with meat proteins through
electrostatic or hydrophobic interactions (van der Waals Forces) and
reduce their bioactivity (Weiss, Loeffler, & Terjung, 2015). The pH of the
medium can also influence the interactions of EO constituents. Food
systems with a lower pH (≤5) increase the hydrophobicity of EO and
may reduce the solubility in the aqueous phase of meat and meat
products. Generally, the pH of raw meat after rigor mortis is close to 5.5,
and in meat products, the pH is between 6.0 and 6.5 (Gutierrez et al.,
2008; Juven, Kanner, Schved, & Weisslowicz, 1994). In addition, the
meat nutrients can favor the recovery of microorganisms that have
suffered some type of stress due to the antimicrobial action of EO
(Doulgeraki et al., 2012). A study by Shekarforoush, Basiri, Ebra­
himnejad, and Hosseinzadeh (2015) with chicken meat reported that the
inhibitory effect of oregano EO against Listeria monocytogenes was
observed only in the first 24 h of storage at 8 ◦ C, despite the strong
antimicrobial activity in vitro reported. The same was reported by Porter,
Morey, and Monu (2020), in which the efficacy of white mustard EO in
nutrient broth was reduced when applied to chilled ground chicken
meat against Salmonella enterica. Alternatively, Corrêa et al. (2021), in a
study with cinnamaldehyde, the main compound of cinnamon EO, re­
ported that, despite the antibacterial activity shown in vitro, the appli­
cation in sliced ham was not effective in controlling S. Enteritidis and L.
monocytogenes.
Increasing EOs concentration in meat and meat products to over­
come activity loss can change sensory thresholds and negatively influ­
ence consumers’ acceptance (Hyldgaard et al., 2012). In a study by
Smaoui et al. (2016), higher concentrations of EO from Mentha piperita
decreased the overall acceptability of the minced meat due to the strong
odor from the volatilization of the EO. Alternatively, Silveira et al.
(2014) obtained good sensory acceptance for Tuscan sausage formulated
with EO from bay leaf (Laurus nobilis), but sausage without the addition
of EO was more accepted among consumers. According to Araújo,
Gumiela, Bordin, Luciano, and Macedo (2018), garlic EO (Allium sat­
ivum) at concentrations from 62.6 μL/kg applied to sausages reduced
acceptability by <70% for the attribute of odor when compared to the
sample without EO. The same was reported by Jaspal et al. (2021), in
which the smell and taste of chicken breast with lactic acid were within
the acceptable range during the entire storage period. However, EO
oregano negatively affected the sensory properties of chicken meat due
to its aromaticity. In a descriptive study of Al-Hijazeen (2018), trained
tasters could distinguish differences in oregano EO odor in concentra­
tion from 200 ppm compared to samples without added oil. A similar
result was also observed with consumers in a triangular sensory test in
the study of Kirkin and Gunes (2018), where the difference was signif­
icant (P < 0.05), with about 27% of consumers pointing to the sample
with Artemisia dracunlus EO as the different sample. Numerous factors
can reduce EO’s bioactivity, which hinders the direct application of EO
in meat matrices. Thus, it is necessary to apply other technologies
involving EO, which can overcome the limitations of use as a natural
B.D. da Silva et al.
preservative. With the advancement of science and technology, nano­
technology emerges as a possible solution to the limitations of using EO
for conservation in meat matrices, as they have different properties
compared to conventional emulsions (Salvia-Trujillo, Soliva-Fortuny,
Rojas-Grä, Julian, & Martín-Belloso, 2017).
4. General aspects of nanoemulsions
Emulsions widely used in commercial applications are oil-in-water
(O/W) or water-in-oil (W/O). In addition, there are bioactive com­
pound encapsulation technologies with multiple emulsion formation
such as W/O/W and O/W/O with improved chemical encapsulation
properties for components of different polarities and multiphase
modeling (Arriaga, Amstad, & Weitz, 2015; Michelon, Huang, de la
Torre, Weitz, & Cunha, 2019; Zhang et al., 2017; Zhao, Chen, Hui,
Weitz, & Middelberg, 2017). Another alternative is reducing droplet size
in emulsions to improve physicochemical properties and bioactivity.
Overall, nanoemulsions consist of oil droplets ranging from 20 to 200
nm with physicochemical and functional properties determined by their
size, chemical composition, electrical charge, and aggregation state
(Barradas & Silva, 2020; Kumar & Mandal, 2018).
As droplet size decreases, gradual changes in physicochemical
properties occur that distinguish nanoemulsions from macroemulsions:
(i) Nanoemulsions can be optically clear due to poor light scattering
through the droplets; (ii) are kinetically stable systems attributed to
Brownian motion droplet that prevails over gravitational forces; (iii) and
can have a higher chemical reactivity than conventional emulsions due
to the greater surface area (Pratap-Singh, Guo, Lara Ochoa, Fathor­
doobady, & Singh, 2021; Sheth, Seshadri, Prileszky, & Helgeson, 2020).
The formation of nanoemulsions with essential oils (NEO) has
numerous advantages such as protection against oxidation of volatile EO
compounds, increased solubility in the aqueous phase, controlled
release of bioactive compounds, and increased bioactivity due to
increased surface area of the emulsion. In addition, droplet sizes <40 nm
are smaller than visible wavelengths. With this, nanoemulsions form
transparent optical systems, making them useful for application in foods
with reduced sensory changes (Dammak et al., 2020; McClements, Das,
Dhar, Nanda, & Chatterjee, 2021). For drop sizes between 40 and 100
nm, nanoemulsions are turbid according to the chemical composition of
the EO. For drop sizes larger than 100 nm, the nanoemulsion becomes
white due to the multiple scattering of the light (Donsì & Ferrari, 2016).
By reducing droplet size, the antimicrobial action of NEO is
improved due to the facilitated transport of bioactive EO constituents
across the cell membranes of microorganisms and the increased surface
area of contact with specific targets in the cell (Moraes-Lovison et al.,
2017). In this context, Azizkhani, Jafari Kiasari, Tooryan, Shahavi, and
Partovi (2021) compared the bioactivity of pure oil and Artemisia dra­
cunculus L. NEO. The authors highlighted that the highest inhibitory
activity against Gram-negative and Gram-positive bacteria was achieved
by nanoemulsion. The same result is highlighted by Meneses et al.
(2019), in which the nanoemulsified clove EO (Eugenia aromatica) pre­
sented a lower minimum inhibitory concentration than the pure oil,
influenced by the increase in the surface area. Nirmala, Durai, Gopa­
kumar, and Nagarajan (2020) reported S. aureus cells in contact with
nanoemulsified celery seed EO (Apium graveolens) presented morpho­
logical change and the greater release of cytoplasmic content than pure
oil. These results reinforce the interaction mechanism with the cell
membrane and increase permeability.
The increase in the surface area generated by the high number of
droplets in the nanoemulsion also favors the interaction of the active
constituents of EO with free radicals and reactive oxygen species
(Balasubramani, Rajendhiran, Moola, & Diana, 2017). Seibert et al.
(2019) evaluated the antioxidant activity of Cymbopogon densiflorus EO
and reported that the nanoemulsion could reproduce the antioxidant
activity in vitro at a concentration four times lower than that of pure oil.
In a study by Soundararajan, Kandasamy, and Subramani (2021), the
4
Trends in Food Science & Technology 121 (2022) 1–13
antioxidant activity of Ocimum tenuiflorum NEO was superior to ascorbic
acid at the same concentrations (10–50 μg/μL) in DPPH radical scav­
enging assays. Similar results were also pointed out by Wang, Cen, Chen,
Zhou, and Fu (2021), in which the scavenging rate of DPPH radicals and
hydroxyl radicals were increased by the nanoemulsion of Litsea cubeba
EO. The authors emphasize that pure oil has a shorter reaction time due
to the high volatility of its compounds. However, the NEO provides
greater contact with free radicals due to the reduced droplet size.
5. Methods of preparing nanoemulsions
The input of energy into the system can be provided by applying
work to the fluid (High-energy methods) or by changes in the equilib­
rium state, through the chemical composition of the mixture or tem­
perature change (Low-energy methods) (Gupta, Eral, Hatton, & Doyle,
2016; Helgeson, 2016). The choice of preparation techniques influences
the particle size, the stability of the nanoemulsion, and the amount of
surfactant used, which can vary between 5 and 10% (Karthik, Ezhilarasi,
& Anandharamakrishnan, 2017). However, some researchers used more
than 10% of surfactant to form nanoemulsions of essential oils (Chue­
siang, Siripatrawan, Sanguandeekul, McLandsborough, & Julian
McClements, 2018; Tubtimsri et al., 2018). The main methods for pre­
paring nanoemulsions are shown in Fig. 2.
5.1. Low-energy methods
Spontaneous or “low-energy” emulsification requires little external
energy for droplet generation. The energy needed for the emulsification
process comes from the internal energy of the mixture out of chemical
equilibrium. The methods are based on the chemical energy released by
the phase transition phenomenon of the surfactant during the emulsion
process, induced by the change in temperature (Phase inversion tem­
perature or PIT) or composition (Phase inversion composition or PIC) of
the system (Solans & Solé, 2012). The formation of emulsions by
low-energy methods involves phase inversions of the surfactant, a pro­
cess in which a W/O system inverts into an O/W system and vice-versa
(Gupta et al., 2016; McClements & Jafari, 2018). There are two main
spontaneous emulsion methods: transition phase inversion and cata­
strophic phase inversion. The transition phase inversion phenomenon is
controlled by the interaction forces between the surfactant and the fluid
phases at different temperatures, also known as phase inversion tem­
perature (PIT). Alternatively, with catastrophic phase inversion (PIC)
emulsification, the system’s composition can be altered by the contin­
uous addition of dispersed phase at constant temperature (Bilbao-Sáinz,
Avena-Bustillos, Wood, Williams, & McHugh, 2010; Hien & Dao, 2021;
Shinoda & Saito, 1969). Phase inversion is preceded by an intermediate
bicontinuous phase or lamellar structure broken by changing the tem­
perature or adding a dispersed phase during the emulsification process,
giving rise to nanoscale droplets (Kumar, Li, Cheng, & Lee, 2015).
Low-energy methods for preparing nanoemulsions have a lower cost
for implantation. However, to achieve smaller drop sizes, low-energy
methods are dependent on controlling the composition system, the
concentration of surfactant used, surfactant properties, and conditions
of the system (Karthik et al., 2017). In a study by Tubtimsri et al. (2018)
with mint EO, the smallest droplet size (55.8 nm) was obtained from
using 20% surfactant in the emulsion system by the PIT method. Simi­
larly, Chuesiang et al. (2018), in the preparation of cinnamon NEO, used
15 and 20% of surfactant to achieve nanodroplets of 50.71 and 23.52
nm, respectively. According to research by Moraes-Lovison et al. (2017),
the 7% concentration of oregano EO used limited the phase inversion
phenomenon and made it difficult to form the interface with the sur­
factant in the bicontinuous phase.
5.2. High-energy methods
High energy methods are based on supplying mechanical energy to
B.D. da Silva et al.
Fig. 2. Main methods to form nanoemulsions. High-energy methods: (1) Ultrasound; (2) High-pressure homogenization; (3) Microfluidization; (4) High-speed
homogenization. Low-energy methods: (5) Phase inversion composition; (6) Phase inversion temperature.
generate disruptive forces capable of reducing the size of dispersed
phase droplets at the oil-water interface (Karthik et al., 2017). Obtaining
nanoemulsions by these methods generally involves two fundamental
steps. In the first step, conventional emulsions (500–1000 nm) are
formed by mixing the aqueous phase, oil, and surfactant in homoge­
nizers such as Ultraturrax® (Ghani, Barzegar, Noshad, & Hojjati, 2018;
Keykhosravy, Khanzadi, Hashemi, & Azizzadeh, 2020). Then, the
droplet diameter is reduced with the application of high-intensity me­
chanical energy at the droplet interface through equipment such as
high-pressure homogenizers, ultrasound, or microfluidizers (Barradas &
Silva, 2020). The curvature of the formed interface gives rise to a
pressure difference between the inside and outside of the drop, referred
to as Laplace’s stabilizing pressure. To achieve nanoscale droplet sizes,
the applied fragmentation stress must be sufficient to overcome the
Laplace stabilizing pressure (McClements & Jafari, 2018). The addition
of surfactants during the emulsification step reduces the interfacial
tension on the drop’s surface, facilitating the action of the disruptive
forces of high-energy drop size reduction methods (Gupta et al., 2016;
Karthik et al., 2017).
High-pressure homogenization (HPH) is a continuous process that
compresses a fluid through a micrometric channel at high pressures
(50–400 MPa). The passage of fluid through this chamber generates
intense mechanical stresses (shear, turbulence, and cavitation) that
reduce the size of the oil particles dispersed in the aqueous phase (Bai &
McClements, 2016). The fluid must pass through the high-pressure
chamber through several cycles to obtain particles at the nanoscale
and with reduced polydispersity indices (Dammak & Sobral, 2017).
Ultrasound (US) based preparation methods involve generating ul­
trasonic waves from a device with a rod or piston that oscillates at high
frequencies in the range of 20–40 kHz (Oca-Ávalos, Candal, & Herrera,
2017; Rosario et al., 2020). The cavitation phenomenon formed by ul­
trasound causes disturbance at the oil and water interface through rapid
changes in the formation and collapse of vapor bubbles. The implosions
caused by the collapse of the bubbles generate intense waves with local
pressures that can reach 100 MPa and travel through the entire solution
that causing rupture of the oil and water interface, thus reducing the size
of the droplets to nanometric scales (Barradas & Silva, 2020; Donsì &
Ferrari, 2016).
Microfluidizers (MF) are similar to the HPH mechanism for prepar­
ing nanoemulsions. Conventional force emulsions travel through narrow
bands within their chambers, where the homogenization pressure can
vary between 3.5 and 150 MPa (Dammak et al., 2020). The channels
5
Trends in Food Science & Technology 121 (2022) 1–13
inside a microfluidizer are designed to generate emulsion currents that
collide with each other, generating disruptive forces (cavitation, shear,
and turbulence), which cause the drop to rupture (Bai & McClements,
2016).
In addition to the commonly used methods, there is the possibility of
obtaining nanoemulsions from high-shear homogenization with a high-
speed rotor/stator (HSH) system. The rupture stresses are obtained by
high-frequency vibrations resulting from the rotation of the device
scissors (Artiga-Artigas, Montoliu-Boneu, Salvia-Trujillo, & Martín-Bel­
loso, 2019). The HSH system is more economical and industrially scal­
able, but it is limited due to the larger droplet size obtained in
nanoemulsions. This limitation is associated with the lower
fluid-mechanical intensity obtained from the system and the need for
high surfactant-oil ratio values to stabilize the nanoemulsion (Donsì &
Ferrari, 2016).
6. Phenomena of instability in emulsions
The stability of emulsions can be understood as the ability to resist
changes in physicochemical properties over time and under adverse
conditions. Droplet reduction to nanoscale provides improved kinetic
stability due to Brownian motion that favors random droplet distribu­
tion throughout the emulsion (Artiga-Artigas et al., 2019). Generally,
relatively small droplets (<100 nm) in an emulsion system are
controlled by Brownian motion, whereas larger droplets (>100 nm)
have a prevalence of gravitational forces. Some emulsion instability
phenomena involve aggregation, coalescence, gravitational separation,
and Ostwald ripening (Sheth et al., 2020).
Gravitational separation is one of the most common instability
phenomena in emulsions and is associated with differences in relative
density with a prevalence of gravitational forces between droplets
(>100 nm) of the dispersed and continuous phase, which can be
observed in the fluid as sedimentation or creaming. Essential oils have
lower densities than liquid water, and therefore there is a tendency for
the oil to move upwards, and the creaming phenomenon is more com­
mon in O/W emulsions (Helgeson, 2016; McClements et al., 2021).
According to Mcclements (2007), the occurrence of colloidal inter­
action between a pair of particles in emulsions can be described by the
sum of van der Waals interactions, electrostatic, steric, and hydrophobic
interactions. In nanoemulsions, the increase in surface area due to a
reduction in droplet size can promote or suppress contact between
droplet surfaces, depending on the magnitude and ranges of action of the
B.D. da Silva et al.
attractive and repulsive forces in the system. In the flocculation phe­
nomenon, a series of droplets come together and form an aggregate
where each droplet retains its integrity, while in coalescence, the
droplets fuse to form a single larger droplet. These phenomena are
susceptible to nanoemulsions with a high polydispersity index, indi­
cating heterogeneity in suspended droplet size (Karthik et al., 2017).
Ostwald ripening is an emulsion instability phenomenon character­
ized by the diffusion of smaller droplets to larger droplets through the
continuous phase. In O/W emulsions, the mechanism is associated with
the solubility of the oil phase in water, which in nanometric scales is
improved (Nakashima, van Haren, André, Robu, & Spruijt, 2021;
Zwicker, Hyman, & Jülicher, 2015). In nanoemulsions with non-zero
polydispersity, there is an energy mismatch between droplets of
Fig. 3. Main instability phenomena caused by interactions between droplets.
6
Trends in Food Science & Technology 121 (2022) 1–13
different sizes due to differences in interfacial energy, resulting in a
driving force that produces a dispersed phase liquid flow in larger
droplets from smaller droplets (Taylor, 1998). The main instability
phenomena are presented in Fig. 3.
Nanoemulsions must remain kinetically stable under adverse con­
ditions during storage, transport, and processing. The useful life of
nanoemulsions can be improved through stabilization strategies that
maintain stable droplet properties for extended periods (Dammak et al.,
2020). The polydispersity index (PDI) indicates the uniformity of droplet
size dispersed in the emulsion and can be evaluated using static light
scattering analyzers. PDI values between 0.10 and 0.25 indicate a uni­
form particle size distribution, and a value greater than 0.5 shows a
broad size distribution. The increase in PDI favors Ostwald ripening;
B.D. da Silva et al.
therefore, research on the preparation of nanoemulsions should evaluate
stability during storage and under different environmental conditions
(temperature and ionic strength) (Ferreira & Nunes, 2019; Pinelli et al.,
2021).
The zeta potential is another important physical characteristic for the
stability of emulsions, which corresponds to the electrical potential of
suspended particles. Thus, the surfactant interfacial layer in nano­
emulsions contributes considerably to the final droplet size. The zeta
potential is influenced by the electrical charge of the type of surfactant
used for adsorption and the solution conditions, such as pH and ionic
strength (Cardoso-Ugarte, López-Malo, & Jiménez-Munguía, 2016).
Ionic surfactants far from their isoelectric point, with values greater than
+30 mV/- 30 mV, provide stability to the aggregation of nanoemulsion
particles through electrostatic repulsion (Ferreira & Nunes, 2019; Liu &
Liu, 2020). The addition of non-ionic surfactants also provides stability
to emulsions through a bulky interfacial film formed around the drop­
lets, capable of providing steric repulsions (Helgeson, 2016).
7. Nanoemulsions applied in meat and meat products
Currently, around 3000 EO have already been produced by different
species of vegetables, and approximately 300 of these oils are
commercially used by the pharmaceutical, agronomic, cosmetic, per­
fumery, and food industries (Raut & Karuppayil, 2014). EO as natural
preservatives in the food industry is intended to replace traditionally
used preservatives in foods that present possible toxic effects associated
with excessive consumption (Cvetković, Živković, Lukić, & Nikolić,
2019; El-Nabarawy, Gouda, Khattab, & Rashed, 2020).
According to the World Health Organization (WHO), one in 10
people falls ill after consuming contaminated food, causing approxi­
mately 420,000 deaths annually (WHO, 2020). The increase in the
global market with more extensive and complex food chains favors
microbial contamination in foods (Doulgeraki et al., 2012). The main
contaminating pathogens in meat are Salmonella enterica, Campylobacter
jejuni, Staphylococcus aureus, and Escherichia coli. Pathogens such as
Listeria monocytogenes and Clostridium botulinum have a lower incidence
but have high mortality. The development of filamentous fungi can also
be a contamination problem with the production of mycotoxins, espe­
cially in meat products with high salt content, and the main contami­
nating fungi belong to the genera Aspergillus and Penicillium (Perrone,
Rodriguez, Magistà, & Magan, 2019; Pizzolato et al., 2018). The main
pathogens associated with foodborne diseases may be present in the
natural microbiota of meat or be contaminated along the processing
chain (Lianou, Panagou, & Nychas, 2017). The main studies involving
the use of NEO in meat and meat products in microbial development are
presented in Table 1.
Exposing meat to conditions that favor oxidative reactions shortens
its shelf life, and the process can reduce the quality of meat and its de­
rivatives through changes in sensory properties. Significant changes
include changes in color, reduction in nutritional value, off-flavor
development, reduction in tenderness, dripping, and formation of
toxic compounds (Coombs et al., 2018; Cunha, Monteiro, Lorenzo, et al.,
2018; Pateiro et al., 2018). Some of the authors highlighted in Table 1
also evaluated the antioxidant activity of NEO in their studies with meat
and meat products. The summary of some studies’ results on antioxidant
activity is presented in Table 2.
The size of nanoemulsified droplets that showed improved bioac­
tivity compared to pure oil ranged from 35 to 295.7 nm (Table 1). These
results demonstrate the effectiveness of EO at nanometric scales even at
different particle sizes and produced by high and low energy methods
(HPH, US, HSH, and PIT). The nanometer size of particles with a large
surface area makes NEO more effective than pure oil, as highlighted in
several investigations in Table 1. The difference in antimicrobial and
antioxidant action of NEO in vitro and meat matrices still occurs, but to a
lesser degree when compared to pure oil (Mendes et al., 2018; Pinelli
et al., 2021). Incorporating EO in nanoemulsion systems increases its
7
Trends in Food Science & Technology 121 (2022) 1–13
stability and solubility, facilitating the interaction of EOs bioactive
components with microorganisms in the aqueous phase, reactive oxygen
species, and free radicals (Prakash et al., 2018). In addition, the
controlled release of EOs from the encapsulation system guarantees a
long-term preservative action (Sun, Zhang, Bhandari, & Bai, 2021).
The chemical composition of EO is a crucial factor in determining its
bioactivity, and the constituents present may vary due to variations in
climate, growing regions, forms of EO extraction, and storage (Rahim­
malek, Heidari, Ehtemam, & Mohammadi, 2017). There is no single EO
mechanism of action, and these mechanisms are directly linked to the
presence and concentration of each compound present in the EO. In this
regard, the presence of a specific constituent can act synergistically or
antagonistically in the EO, even if the compound has excellent bioac­
tivity in its isolated form (Raut & Karuppayil, 2014). In research by
Amiri, Aminzare, Azar, and Mehrasbi (2019), the addition of cinna­
maldehyde in Zataria multiflora nanoemulsion decreased its antioxidant
activity. The blend of several nanoemulsified EO evaluated by Pinelli
et al. (2021) significantly improved the action against C. sporogenes. Sun
et al. (2021) reported that the EO from nanoemulsified Foeniculum vul­
gare showed excellent antimicrobial activity. However, no difference
was observed with the antioxidant activity with pure oil at the same
concentrations.
The most common way of applying NEO has been incorporation into
edible coatings. Coatings are a thin layer of edible materials applied to
the surface of food through immersion or spraying. The coatings provide
a semi-permeable barrier to reduce gas exchange, water loss, aromas,
and microbial contamination, in addition to incorporating several
compounds with bioactivity (Lopez-Polo, Monasterio, Cantero-López, &
Osorio, 2021). The mixture of EO directly into the meat mass can
compromise the availability of bioactive compounds depending on the
technological process that the meat is subjected (da Silva, Bernardes,
Pinheiro, Fantuzzi, & Roberto, 2021).
In macroemulsions, the thickness of the surfactant layer is generally
smaller than the radius of the hydrophobic core, the droplets mainly
being oil. However, in nanoemulsions, the thickness of the adsorbed
surfactant layer can have dimensions similar to those of the hydrophobic
core, directly influencing the general composition of the droplet and the
interaction properties (Tadros, Izquierdo, Esquena, & Solans, 2004). The
surfactants used during the preparation of nanoemulsions can form
electrically charged or non-electrically charged droplets. These proper­
ties can influence the interaction of the drops with food targets, whether
microorganisms or free radicals (Salvia-Trujillo, Martin-Belloso, &
Julian McClements, 2016). As coatings have been the main form of
application, interaction with food constituents is reduced, and the
impact on physicochemical properties is not significant, as demon­
strated in some studies (Amiri et al., 2019; Ghani et al., 2018; Mor­
aes-Lovison et al., 2017). With the increase in surface area and
surfactant properties, the solubility in the aqueous phase is improved
compared to the dilution in the predominant oil phase when applying
pure oils to meat and meat products (Gutierrez et al., 2008; Nakashima
et al., 2021). This allows the application of NEO in meat and meat
products regardless of the fat content of its composition.
Another critical point highlighted by the addition of NEO in meat
and meat products is the reduction in sensory impact caused by the
strong aromaticity of pure oil. Abdou et al. (2018) sensory evaluated the
coated chicken fillets and highlighted that the appearance, odor, and
flavor obtained better scores by adding coatings with nanoemulsified
cinnamon EO and curcumin. Similarly, Noori, Zeynali, and Almasi
(2018), ginger EO nanoemulsions with edible coating had higher scores
than conventional EO coatings. In research by Dini, Fallah, Bonyadian,
Abbasvali, and Soleimani (2020), the beef loins coated with nano­
emulsified cumin EO associated with the irradiation process reduced the
sensory impact. The color and odor aspects were more accepted than in
the samples that underwent only irradiation and control during the
storage period.
Based on the research presented, the high-energy and low-energy
B.D. da Silva et al. Trends in Food Science & Technology 121 (2022) 1–13

Table 1
Summary of papers evaluating the antimicrobial activity of NEO in meat and meat products (2017–2021).
Essential oil Food Preparation Droplet size Main results References
methods

Origanum vulgare Chicken pâté PIT 35 nm NEO was more effective in inhibiting E. coli than pure Moraes-Lovison et al.
oil over eight days of storage. However, the same result (2017)
was not observed for S. aureus.
Thymus vulgaris Beef Spontaneous 60 nm There was a reduction of 1.64 log CFU/g of E. coli in beef Stratakos & Grant
emulsification treated with NEO at the end of storage (7 days) (2018)
compared to the control. There was no comparison with
pure oil.
Eugenia brejoensis Ham slices HSH 143.1–356 nm NEO did not differ significantly from control samples in Mendes et al. (2018)
the growth of P. fluorescens over five days of storage at
10 ◦ C (P ≥ 0.05). There was no comparison with pure
oil.
Cinnamomum zeylanicum Beef US 141.2 nm NEO reduced 5.71 log CFU/g for TVC and 1.0 log CFU/g Ghani et al. (2018)
for yeast and molds in beef after eight days of storage at
4 ◦ C. There was no comparison with pure oil.
Allium sativum Chicken fillets US 9–40 nm After 12 days of storage at 4 ◦ C, there was a reduction in Abdou et al. (2018)
TVC of 97.8%; for psychrotrophic, the reduction was
95.7% for treatments with NEO coating. Molds and
yeasts were only detected after the 6th day of storage for
the NEO. In the control treatment, detection occurred
from the 3rd day.
Zingiber officinale Chicken US 57.4 nm The growth of psychrotrophic bacteria, molds, and Noori et al. (2018)
breast fillets yeasts were lower in the treatments coated with NEO
than the uncoated samples and those coated with
conventional emulsion after 12 days of storage at 4 ◦ C.

Thymus vulgaris Pork US 139.47 nm The NEO treatments showed a 6 days extended shelf life Liu and Liu (2020)
refrigerated compared to the control and did not exceed the
acceptable TVC limit during 12 days of storage at 4 ◦ C
(5.93 log CFU/g). There was no comparison with pure
oil.
Illicium verum Ready-to-eat US 97.1–116.3 nm Samples with NEO stored at 4 ◦ C for 20 days showed an Liu et al. (2020)
Yao meat extended shelf life of 12 days compared to the eight
days observed in control samples (TVC). For the growth
of E. coli, the results were similar to those of TVC, in
which NEO was more effective in antimicrobial activity.
There was no comparison with pure oil.
Zataria multiflora and Bunium Turkey meat US 130.2–154.26 The shelf life of turkey meat stored at 4 ◦ C was extended Keykhosravy et al.
persicum nm by 9 days after adding coating containing NEO. An (2020)
approximately 3 log CFU/g reduction in S. Enteritidis
counts and a 2 log CFU/g reduction in L. monocytogenes
were also observed after adding the NEO coatings.
There was no comparison with pure oil.
Origanum vulgare Pork loin HSH 48.49–53.09 Samples with NEO showed better results than samples Xiong, Warner, & Fang
nm with conventional emulsions of the same OE for TVC. (2020)
Cuminum cyminum Beef loins US 89.61 nm Compared to samples without edible coating, the shelf (Dini et al., 2020)
life of beef loins was extended to 8 days in samples with
NEO and chitosan. When combined with gamma
irradiation, the shelf life was increased to 14 days.
There was no comparison with pure oil.
Zataria multiflora Chicken meat HSH 176.6–184.7 Starch coatings containing NEO showed greater Abbasi, Aminzare,
nm antimicrobial activity than pure oil at the same Azar, & Rostamizadeh
concentration against several microorganisms (2020)
evaluated in chicken meat during 20 days of storage at
4 ◦ C.
Trachyspermum ammi Turkey fillets US 73.5 nm Samples coated with alginate and NEO showed the Kazemeini, Azizian, &
greatest reduction rate in L. monocytogenes counts Adib (2021)
compared to the control (1.99 log CFU/g). NEO also
showed more expressive antimicrobial effects than
conventional EO emulsions at the same concentrations,
with differences of up to 0.86 log CFU/g.
Origanum vulgare; Cinnamomum Mortadella US 45.5–47.3 nm The EO mixtures for the preparation of nanoemulsions Pinelli et al. (2021)
zeylanicum; Citrus aurantifolia; showed a greater reduction in the vegetative cell count
Elettaria cardamomum; Litsea of C. sporogenes compared to pure oil at the same
cubeba concentrations in mortadella samples with reduced
nitrite (75 ppm)
Foeniculum vulgare Pork meat HPH 295.7 nm Compared with pure oil at the same concentration, NEO Sun et al. (2021)
patties obtained greater reductions in TVC, molds, and yeasts.
There was an increase in the shelf life of patties from 6
to 10 days.
Mandarin (Citrus reticulata) Pork meat US 131.3–161.9 Control samples reached a count of 6 log CFU/g after six Song et al. (2021)
nm days of storage, characterized as unfit for consumption.
In treatments with NEO, all concentrations evaluated
maintained the microbial count below 6 log CFU/g
throughout the six days of storage. There was no
comparison with pure oil.

8
B.D. da Silva et al. Trends in Food Science & Technology 121 (2022) 1–13

PIT = Phase Inversion Temperature; HPH = High-Pressure Homogenization; US = Ultrasound; HSH = High-Speed Homogenization; TVC = Total viable counts; EO:
Essential oil; NEO: Essential oil nanoemulsified; CFU: colony-forming unit.

Table 2
Summary of papers evaluating the antioxidant activity of NEO in meat and meat products (2017–2021).
Essential oil Food Main results Reference

Allium sativum Chicken fillets All concentrations of NEO added to the coating significantly reduced the levels of thiobarbituric acid reactive Abdou et al. (2018)
substances (TBARS) compared to control samples over 12 days of storage at 4 ◦ C (P < 0.05).
Zingiber Chicken breast No significant antioxidant activity was observed during storage between NEO and pure oil treatments. Noori et al. (2018)
officinale fillets
Zataria Ground beef NEO added to cornstarch coatings had the lowest levels of TBARS in ground beef patties after 12 days of storage Amiri et al. (2019)
multiflora patties compared to control and pure oil. A similar result was also observed for the formation of free carbonyls resulting
from the degradation of the amino acid chain. Enrichment of NEO with cinnamaldehyde reduced its antioxidant
effect.
Citrus sinensis Emulsified meat All NEO concentrations evaluated showed a significant reduction compared to the control. The highest NEO Almaráz-Buendia et al.
system concentration evaluated showed a 2.7-fold decrease in malondialdehyde production, characterized as the (2019)
highest among the concentrations.
Origanum Pork loin Samples coated with NEO showed maintenance of meat color, reduced levels of TBARS, and lower loss of thiol Xiong, Warner, & Fang
vulgare groups from amino acid chains. Uncoated treatments suffered the effects of more severe lipid and protein (2020)
oxidation due to direct exposure to oxygen.
Cuminum Beef loins During the storage period, the TBARS level increased in all groups, while the rate of increase was greater in Dini et al. (2020)
cyminum groups without NEO addition. On day 21, the TBARS level was significantly lower (P < 0.05) in the groups with
NEO alone and combined with gamma irradiation.

NEO: Essential oil nanoemulsion; TBARS: Thiobarbituric acid reactive substances.

nanoemulsification methods can provide nanoemulsions with small
particle size, good stability, and the ability to preserve meat and meat
products for extended storage periods.
8. Current limitations of the use of NEO in food
Nanotechnology applied in EO for food formulations is an emerging
area in the food industry, showing promising results in conservation, as
highlighted in Table 1. The emergence and application of novel tech­
nologies always need to cross technological, social, and regulatory
barriers, mainly when applied to food (Chaudhry, Watkins, & Castle,
2017). However, the application of EO as a substitute for traditionally
used chemical preservatives is still at an early stage due to gaps that are
still under investigation (McClements et al., 2021). Some limitations,
such as effectiveness dependent on the composition and concentration of
the EO, cost-benefit ratios for the application of nanotechnology, and
mainly, the lack of regulations to standardize the use of nanotechnology
and EO in food, make its implementation difficult as a substitute for
traditionally used preservatives (Prakash et al., 2018).
Currently, the Food and Drug Administration (FDA) and the Euro­
pean Food Safety Authority (EFSA) are the main authorities with some
regulatory approaches to the use of EO in food. The FDA establishes a list
of EO, oleoresins, and natural extracts GRAS, including oregano, cloves,
basil, thyme, and cinnamon, for use in food (FDA, 2020). Alternatively,
the European Union defines individual compounds present in foods and
the restriction of use in various categories of foods (Regulation (CE) n.◦
1334/2008). The main limitation to regulatory issues is the diversifi­
cation of the chemical composition of the EO, which makes it difficult to
standardize acceptable daily intake levels (ADI) and no observed
adverse effect level (NOAEL) (Donsì & Ferrari, 2016).
EO can be toxic when ingested in high concentrations, especially
orally (Bhardwaj et al., 2020). Studies on the toxicity of EO and indi­
vidual compounds related to in vitro cell death induction were per­
formed in human liver and intestinal cell lines and reviewed by
Maisanaba et al. (2017). As nanoemulsions are usually made with ma­
terials of natural origin and allowed by legislation for use in food, the
main limitation is the presence of toxic compounds in the composition of
certain EO that can be easily absorbed by human cells due to their
nanometric size. Regarding possible EO toxicity, Sobczak, Kalemba,
Ferenc, and Zylinska (2014) compared the oxidative protection of
thymol and thyme EO at the same concentrations in neuronal tissue cells
modified to simulate brain aging. Brain aging is directly linked to the
reduction of endogenous antioxidant mechanisms. The authors high­
light that higher concentrations of thyme EO (400 μM) may have
increased cytotoxic effects with changes in neuronal cell morphology.
Some compounds in the EO of thyme can undergo oxidation in reactive
oxygen species and trigger oxidative processes in neuronal cells. The
authors also emphasize that thyme EO should be used with caution,
especially associated with consumption by older people. de Godoi et al.
(2017) evaluated the hemolytic action of nanoemulsified Eucalyptus
globulus EO and highlighted that at concentrations of 0.1%, there was
no toxicity in peripheral blood mononuclear cells in addition to having a
protective effect against possible cell damage when compared to the
pure oil. However, hemolysis was evidenced in the same study at higher
concentrations (≥0.2%). Alternatively, Bonferoni et al. (2017) evalu­
ated the toxicity of nanoemulsified Cymbopogon citratus EO and re­
ported that nanoemulsions in concentrations equivalent to antimicrobial
activity did not show cytotoxic effects in 4 cell lines (HEp-2, Caco-2,
WKD, and McCoy). In this context, there is a lack of research focused
on the cytotoxic effects of EO and the various compounds that constitute
them associated with consumption. As discussed in previous sections,
the nanometric droplet size and increased surface area improve the
interaction properties in the medium. Thus, a possible limitation would
be the behavior of nanodroplets in the human body upon the con­
sumption of foods with this conservation alternative. As highlighted,
some EO may show cytotoxicity when consumed in excess, and nano­
droplets may be able to lower the acceptable intake level. However, this
is a hypothesis since conclusive studies on the effects of NEO in human
cells are currently scarce due to the recent application of nanotech­
nology in food (Abd-Rabou & Edris, 2021; Marchese et al., 2020).
The possible adverse effects associated with the use of nanomaterials
in the biological system are still poorly understood based on the
behavior, interactions, destination, and toxicological effects of nano­
particles in the gastrointestinal tract (Chaudhry et al., 2017). Some
research projects are being undertaken to address the safety aspects of
the application of nanotechnology in food, including its toxicology, risk
assessment, risk exposure, and interaction mechanisms (Ferreira &
Nunes, 2019). An example of a project is NanoReTox, which seeks to
assess the implications of the use of free-engineered nanoparticles on
human health and the environment (CORDIS, 2017). Currently, the
EFSA and the FDA do not have any occurrences regarding the produc­
tion, application, and labeling of food-targeted nanoparticles.
Another possible limitation in the use of NEO in food is associated
with the cost of preparing nanoemulsions. Ultrasound is simpler and

9
B.D. da Silva et al.
cheaper equipment, easily scalable industrially. However, high-pressure
technologies such as HPH and microfluidizers have a high cost and are
currently more laboratory-scale. Low-energy methods reduce the pos­
sibility of application in food compared to high-energy methods, as they
require greater conditions for the formation of nanoemulsions. Mainly,
the amount of surfactant used can make the application in food unfea­
sible (Chemat et al., 2017). Although there is no specific standardization
for EO in food, some patents on nanoemulsions are being filed in several
countries. A South Korean patent application focused on cinnamon oil
nanoemulsions to inhibit microbial growth (Gi, Jung, Yeon, & Ji, 2016).
Alternatively, a patent application for a Litsea cubeba oil nanoemulsion
has been made in China for fresh canned salmon (Yanbo, Linglin, Jian, &
Feifei, 2020).
9. Conclusions and future perspectives
The global search for natural products with healthy appeal grows
every day. With this, replacing traditionally used preservatives becomes
a challenge for the meat industry, as the quality and safety of products
cannot be compromised. As reported in this review, several essential oils
are excellent natural sources of bioactive compounds with proven
antimicrobial and antioxidant activity. With the recent advance of
nanotechnology, the development of nanoemulsions with EO becomes
an alternative to overcome the limitation of low solubility in the
aqueous phase and high volatility that compounds present. Incorpo­
rating EO nanoemulsions in meat and meat products through edible
coatings has been a viable application alternative. It has shown prom­
ising results in extending shelf life and reducing undesirable sensory
changes, even pure oil.
However, as expected of any emerging technology, it is still neces­
sary to conduct several studies to elucidate gaps regarding standardi­
zation in particle size, synergistic blends between EO, stability of
nanoemulsions, and possible toxicological effects associated with the
consumption of nanometric particles. In addition, more research
directed towards the application of NEO in meat products must be car­
ried out to evaluate the different forms of interaction with the meat
matrix. From the development of more research on NEO in food
matrices, it will be possible to develop standardized legislation for food
application for conservation purposes. Thus, EO can become widely
commercially diffused as natural preservatives.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
The authors are thankful for the financial support provided by the
Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)
Brazil — grant number [E-26/203.049/2017], the Fundação de Amparo
à Pesquisa e Inovação do Espírito Santo (FAPES)/PDCTR grant number
[533/2020], the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) - grant number [300968/2021-3] and [140873/
2021-0].
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