Food Bioscience 28 (2019) 57–65

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Antibacterial and antibiofilm activities of linalool nanoemulsions against T

Salmonella Typhimurium
Anand Prakasha, Vellingiri Vadivela,∗, Durairajan Rubinib, Paramasivam Nithyanandb
a
Chemical Biology Lab (ASK-II), School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur-613401, Tamilnadu, India
b
Biofilm Biology Lab, School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur-613401, Tamilnadu, India

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

Keywords: Linalool is the active constituent of essential oils of several plant species like coriander, rosewood and cinnamon,
Linalool which is known to have strong antibacterial activity. However, its application in food systems is limited because
Nanoemulsions of low water solubility, high volatility and low stability. The present study was undertaken to overcome such
Salmonella Typhimurium problems by encapsulating the linalool into nanoemulsions using Tween 80 and water using ultrasonic assisted
Antibiofilm
emulsification. The stable nanoemulsions with a mean droplet diameter of 10.9 ± 0.1 nm was obtained at a 1:3
(v/v) ratio of linalool and Tween 80. The antibacterial and antibiofilm activities of the stable nanoemulsions
were further investigated. The linalool nanoemulsions showed twofold enhanced antibacterial activity against
Salmonella Typhimurium through their increased ability to disrupt cell membrane integrity. Moreover, compared
to the pure linalool, the linalool nanoemulsions showed an 11.5% higher antibiofilm activity. Nanoemulsions
also effectively reduced the S. Typhimurium biofilm on the surface of cut pineapple. Sensory analysis showed
acceptance of fresh cut pineapple with linalool nanoemulsions treatment. Therefore, results of the present study
indicated the potential use of linalool nanoemulsions as a natural antibacterial and antibiofilm agent against S.
Typhimurium, which could find applications in the food industry.

1. Introduction
An estimated 600 million people in the world fall ill after consuming
contaminated food that results in over 420000 deaths every year (WHO,
2017). Food contamination by Salmonella leads to 94 million cases of
gastroenteritis and 155000 deaths globally each year (Ros-Chumillas,
Garre, Mate, Palop, & Periago, 2017). The intake of contaminated food
products from poultry, pigs and cattle have been reported to be the
primary cause of Salmonella outbreaks among consumers. But of late, a
considerable number of outbreaks have also been linked to consump-
tion of fresh fruits and vegetables contaminated with Salmonella spp.
(Simm, Ahmad, Rhen, Le Guyon, & Romling, 2014). The formation of
Salmonella biofilms is another serious concern in the food processing
industry as bacteria encased in the biofilms are generally well protected
against disinfectants (Milan, Agostinetto, Conceicao, Gonzalez, &
Timm, 2015; Steenackers, Hermans, Vanderleyden, & De Keersmaecker,
2012). Even in fresh fruits and vegetables, conventionally used che-
mical sanitation techniques do not completely eliminate the threat of
Salmonella contamination and pose a serious risk factor for humans
(Amrutha, Sundar, & Shetty, 2017). Furthermore, the changing con-
sumer's preference for food products with safe preservatives suggests
the need for the development of natural antimicrobial agents. The
broad spectrum antimicrobial activity of the essential oils (EOS) and
their constituents has been studied as promising food preservatives
(Burt, 2004).
The direct incorporation of EOS and their constituents as food
preservative has several technological challenges owing to their low
solubility, poor chemical stability and high volatility (Moghimi,
Ghaderi, Rafati, Aliahmadi, & McClements, 2016). Such constraints can
be overcome by encapsulating the EOS so that they are compatible with
the food system (Donsì & Ferrari, 2016). Moreover, encapsulation can
reduce the adverse interaction of EOS with food constituents and en-
hance their antimicrobial efficacy by promoting contact with the pa-
thogens (Majeed et al., 2016). Among the encapsulation systems, na-
noemulsions have been reported to be highly appropriate for
application in food systems (McClements & Rao, 2011). Nanoemulsions
are mixtures of oils with surfactants and co-surfactants that form fine
oil-in-water (O/W) or water-in-oil emulsions (W/O) with the droplet
size of 10–100 nm (McClements & Rao, 2011). Nanoemulsions show
several distinct characteristics such as optical clarity, reduced rate of
gravitational separation and flocculation (Donsì & Ferrari, 2016). Na-
noemulsions can be formed using various low and high energy

∗
Corresponding author.
E-mail address: vadivel@carism.sastra.edu (V. Vadivel).

https://doi.org/10.1016/j.fbio.2019.01.018
Received 10 May 2018; Received in revised form 7 January 2019; Accepted 17 January 2019
Available online 28 January 2019
2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
A. Prakash et al. Food Bioscience 28 (2019) 57–65

Abbreviations MIC Minimum inhibitory concentration

MTT Methyl thiazolyldiphenyl tetrazolium bromide
BIC Biofilm inhibitory concentration O/W Oil-in-water
DMSO Dimethyl sulfoxide PDI Polydispersity index
EOS Essential oils SCD Soyabean casein digest
HLB Hydrophilic lipophilic balance SEM Scanning electron microscope
LN Linalool nanoemulsions TEM Transmission electron microscope
MBC Minimum bactericidal concentration WI Whiteness index

fabrication methods. In high energy techniques, introduction of large
amount of mechanical energy using a microfludizer, high-pressure
homogenizer or ultrasonicator gives nanoemulsion droplets (Prakash,
Baskaran, Paramasivam, & Vadivel, 2018). Ultrasound is an energy
efficient technique for the generation of nanoemulsions using bubble
formation, growth and implosive collapse in a liquid medium
(Sivakumar, Tang, & Tan, 2014).
Another problem with EOS is their variability, as they are obtained
from plant species, whose chemical profile may vary according to their
geographical origin and harvesting period, which might result in var-
iation of their antimicrobial efficacy (Burt, 2004). Hence, the major
individual chemical constituents of EOS could be explored as natural
antimicrobial agents. Linalool (3,7-dimethyl-1,6-octadien-3-ol) is one
such major compound found in the EOS of several plant species like
coriander, rosewood and cinnamon (Aprotosoaie, Hancianu, Costache,
& Miron, 2014; Farisa Banu et al., 2017). It is an acyclic monoterpene
alcoholic fragrant constituent used in hygiene products and is classified
as generally recognized as safe by the US Food and Drug Administration
(Hsu, Lai, Chuang, Lee, & Tsai, 2013). An acceptable daily intake of
0–0.5 mg linalool/kg body weight/day has been established by the
Joint FAO/WHO Expert Committee on Food Additives (Aprotosoaie
et al., 2014). Further, linalool has been documented as a broad spec-
trum antibacterial agent against several foodborne pathogens like Sal-
monella sp., Staphylococcus aureus and Listeria monocytogenes (Sokovic,
Glamoclija, Marin, Brkic, & van Griensven, 2010; Zengin & Baysal,
2014).
Formulation of linalool nanoemulsions (LN) could give a viable
solution for its use as a natural antibacterial agent in food systems. The
objectives of the present study were (1) to optimize conditions to for-
mulate ultrasonic-assisted LN with small droplet size and high stability
(2) to assess the antibacterial activity of LN against Salmonella
Typhimurium and elucidate its mechanism of action (3) to study the
biofilm inhibitory efficacy of LN using in vitro and fresh cut pineapple
models and (4) to investigate the sensory acceptability of linalool and
LN treated fresh cut pineapple.
2. Materials and methods
2.1. Preparation of LN
Nanoemulsions were prepared based on high energy ultrasonic
homogenization using linalool (97% purity, Sigma-Aldrich, Saint Louis,
MO, USA), Tween 80 (Merck, Mumbai, Maharashtra, India) and dis-
tilled water using the method described by Ghosh, Mukherjee, and
Chandrasekaran (2013a) with slight modifications. A linalool con-
centration of 5% (v/v) was kept constant for all the formulations.
Initially, coarse emulsions were prepared by mixing linalool and Tween
80, followed by the addition of distilled water. Three different nanoe-
mulsions with varying ratio of linalool and Tween 80, i.e., LN1 (1:1),
LN2 (1:2) and LN3 (1:3) were prepared (Table 1). To reduce the droplet
size, coarse emulsions were sonicated using a probe sonicator (PRO-
250, Labman, Chennai, Tamilnadu, India) which had a power output of
250 W and frequency of 50 kHz for 10 min at 4°C with 30 s pulse on and
30 s off. Heat generated during ultrasound was decreased by keeping
the emulsion beaker in an ice bath.
2.2. Characterization of LN
Particle size, polydispersity index (PDI) and zeta potential of the
formulated nanoemulsions were determined using a particle size ana-
lyzer (Zeta Sizer Nano-ZS, ZEN3600, Malvern Instruments, Malvern,
UK). All the formulations were 100-folds diluted with distilled water to
reduce the multiple scattering effects and to eliminate the effect of
viscosity. The samples were analyzed in a disposable capillary cell
(DTS1070, Malvern Instruments) at 25°C with an equilibration time of
60 s. All the results were obtained using the instrument's software (Zeta
sizer software version v7.11).
The viscosity of the LN was measured with 10 ml samples using a
viscometer (Brookfield DV-II + Pro, Middleboro, MA, USA) with a
spindle (S18, Brookfield, MA, USA) at 25°C. The pH value of the LN was
measured with a pH meter (LMPH-12, Labman) at 25°C. The turbidity
of the LN was measured at 600 nm using an UV–visible spectro-
photometer (Model Evolution 201, Thermo Scientific, Waltham, MA,
USA). Color values (L*, a*, b*) of LN were measured using a Hunter lab
color measuring system (Labsan-XE, Hunter Associates Laboratory,
Reston, VA, USA). The instrument was standardized against a black and
white tile (Hunter Associates Laboratory). The results were expressed as
the whiteness index (WI) according to the equation from Salvia-Trujillo,
Rojas-Grau, Soliva-Fortuny, and Martin-Belloso (2015), i.e., WI = 100 –
[(100 – L) 2 + (a2 + b2)] 0.5.
The stability of LN was evaluated by subjecting the samples to dif-
ferent thermomechanical stress conditions. To measure the physical
stability, samples were centrifuged at 800×g for 30 min at room tem-
perature (Remi CM-12 Plus, Mumbai, Maharashtra, India). Samples
were also subjected to consecutive cycles of heating-cooling (storage at
45 and 4°C) and freeze-thawing (storage at −21 and 25°C) for 12 h each
to evaluate the droplet stability (Azeem et al., 2009). Each cycle was
repeated three times and visually observed for any form of instability
(separation, creaming or flocculation). Stability of the LN during one
month of storage at room temperature was studied using the particle
size analyzer. Among the three formulations, the most stable nanoe-
mulsion (LN3) was selected for further studies.

Table 1
Physicochemical characteristics of linalool nanoemulsions.
Sample Oil: Surfactant ratio Appearance Zeta size (nm) Zeta potential Polydispersity Whiteness index pH Absorbance Viscosity (cP)
(v/v) (mV) index (600 nm)

LN1 1:1 Milky white 85 ± 1 - 20 ± 2 0.44 ± 0.04 21.4 ± 0.04 6.90 ± 0.01 2.54 ± 0.01 1.49 ± 0.01
LN2 1:2 Slightly creamy 35 ± 1 - 10.3 ± 0.1 0.15 ± 0.01 43.4 ± 0.02 6.89 ± 0.01 2.56 ± 0.02 1.99 ± 0.01
LN3 1:3 Transparent 10.9 ± 0.1 - 18.1 ± 0.5 0.21 ± 0.02 86.8 ± 0.3 6.95 ± 0.01 0.14 ± 0.01 4.23 ± 0.01

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A. Prakash et al.
Fig. 1. Appearance of different formulations of linalool nanoemulsions (a); Zeta
size image of LN3 formulation (b); TEM image of LN3 formulation showing
spherical shaped droplet diameter range in the range of 13–16 nm (c).
The morphology and size of LN3 were characterized using trans-
mission electron microscope (TEM). The sample was prepared by pla-
cing a drop of LN3 on a 200-mesh carbon-coated copper grid (Electron
Microscopy Sciences, Hatfield, PA, USA) and dried under vacuum. The
sample was then negatively stained with 2% (w/v) ammonium mo-
lybdate, dried and visualized using a TEM (JEOL-JEM 1011, Akishima,
Tokyo, Japan) at a magnification of 40,000x and an acceleration vol-
tage of 200 kV.
2.3. Antibacterial assays
Minimal inhibitory concentration (MIC) of linalool which was dis-
solved in 5% dimethyl sulfoxide (DMSO; Himedia, Mumbai,
Maharashtra, India) and LN3 against Salmonella enterica subsp. enterica
serovar Typhimurium (ATCC 13311, Manassas, VA, USA) was de-
termined using a broth microdilution method (Farisa Banu et al., 2017).
Briefly, 50 μl of serially diluted linalool and LN3 (5–0.009%) were
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Food Bioscience 28 (2019) 57–65
Table 2
Minimal inhibitory concentration (MIC) and minimal bactericidal concentra-
tion (MBC) of linalool and LN3.
S. No. Sample MIC (v/v, %) MBC (v/v, %)
1 Linalool 1.25 1.25
2 Linalool nanoemulsion (LN3) 0.625 0.625
Fig. 2. Time kill kinetics of linalool and LN3 against Salmonella Typhimurium.
added to 50 μl test culture and 100 μl of nutrient broth in a 96 well
plates (Tarson, Kolkata, West Bengal, India). DMSO (5%, v/v) served as
a negative control. Plates were then incubated at 37°C for 24 h and the
MIC was determined as the lowest concentration of the samples that
inhibited the bacterial growth. Twenty μl of 5 mg/ml of methyl thia-
zolyldiphenyl tetrazolium bromide (MTT; Sigma-Aldrich) was added to
each well and incubated at 37°C for 30 min. To determine the minimum
bactericidal concentration (MBC), 100 μl of the aliquots from wells
where no visible growth was observed were plated on nutrient agar
(Himedia). The plates were incubated at 37°C for 24 h and observed for
any bacterial growth. The lowest concentration that showed the ab-
sence of a viable colony was the MBC.
The time kill assay was done to determine the exact time point when
linalool and LN3 had their bactericidal effect on S. Typhimurium.
Linalool and LN3 at their respective MIC were added to S. Typhimurium
cells (107 CFU/ml). Then, an aliquot (0.1 ml) was taken from each
sample after 0, 2, 4, 8, 10, 16 and 24 h and serially diluted and spread
plated on nutrient agar (Himedia). After incubation for 24 h at 37°C, the
colonies were counted and the time kill curve was obtained.
2.4. Integrity of cell membrane
The membrane integrity of S. Typhimurium treated with linalool
and LN3 was studied by determining cell contents release as described
by Sugumar et al. (2013) with slight modifications. Briefly, an over-
night bacterial culture grown at 37°C was harvested, washed, re-sus-
pended and adjusted to 1 × 107 CFU/ml with sterile saline. Bacterial
suspension (0.5 ml) was added to 4.5 ml of the MIC of linalool and LN3
and then incubated at 37°C with agitation for 1 h. The samples were
then centrifuged at 3600×g for 10 min. The cytoplasmic contents re-
leased in the supernatant were measured at 260 and 280 nm.
The membrane damage caused by linalool and LN3 was visualized
using scanning electron microscope (SEM) according to the method
described by Sugumar, Ghosh, Nirmala, Mukherjee, and
Chandrasekaran (2014) with some modifications. In brief, S. Typhi-
murium (107 CFU/ml) treated with linalool and LN3 at their MIC were
A. Prakash et al.
Fig. 3. The effect of linalool and LN3 on cell constituents release in Salmonella Typhimurium at 260 nm (a) and 280 nm (b). Bars with asterisk indicate significant
difference (P < 0.05) of treatment with respect to control. SEM image of Salmonella untreated (c); Linalool treated (d) and LN3 treated (e). Arrow indicates damage
seen in the cell membrane.
fixed with 20% glutaraldehyde (Sigma-Aldrich) for 1 h and thereafter
successively washed with a gradient concentrations (30, 50, 70, 90 and
100%) of ethanol (Changshu Hongsheng Fine Chemicals, Suzhou,
Jiangsu, China). Finally, the samples were fixed on a support, gold
sputtered using an Auto Fine Coater (JFC-1600, Jeol, Tokyo, Japan) and
visualized using a SEM (Tescan Vega 3, Brno, Czech Republic) at
35000x magnification.
2.5. Biofilm inhibition studies
The ability of linalool and LN3 to disrupt S. Typhimurium biofilms
was determined using a crystal violet assay as described by Farisa Banu
et al. (2017). For formation of biofilms, 1 ml of soyabean casein digest
(SCD) broth (Himedia) with 0.6% yeast extract was added to 100 μl of
cell suspension (108 cells/ml) in a 24 well plate (NEST Biotechnology,
Wuxi, Jiangsu, China) and incubated for 24 h. Then, 50 μl of linalool
and LN3 at their respective sub-inhibitory concentrations (½ and ¼
MIC) were added to the wells and further incubated for 24 h at 37°C. S.
Typhimurium cells without treatment were kept as the control while
the cells treated with 5% DMSO served as the vehicle control. The spent
media was discarded and biofilms were stained with 0.4% crystal violet
(SRL, Mumbai, Maharashtra, India). Finally, the biofilms were dissolved
in 95% ethanol and the absorbance was read at 595 nm and the percent
inhibition was calculated using the formula [(Control OD – Test OD/
Control OD) X 100].
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Food Bioscience 28 (2019) 57–65
The inhibition of biofilms by linalool and LN3 was visualized using
light microscopy and SEM. Glass slides (1 × 1 cm) were placed in 24
well plates and biofilms were allowed to form as described above. Later,
biofilm inhibitory concentration (BIC), i.e., ½ MIC of the samples were
added to the wells and further incubated for 24 h at 37°C. The spent
media was then removed and the biofilms formed on the glass slides
were stained with crystal violet dye, air dried and viewed using a light
microscope (Nikon Eclipse, Ti 100, Tokyo, Japan) at a magnification of
40x. Similarly, for SEM visualization, biofilms were allowed to form on
glass slides, which were then fixed and visualized using SEM according
to the methodology described by Thenmozhi, Nithyanand, Rathna, and
Karutha Pandian (2009). Briefly, the S. Typhimurium biofilms were
formed on the glass slides in the presence or absence of linalool and
LN3 and fixed with 20% glutaraldehyde for 1 h. Later, the glass slides
were dehydrated through a gradient series of ethanol as previously
described, gold sputtered and examined using the SEM at 3000, 8000
and 15000x magnifications.
2.6. Biofilm reduction on a pineapple surface
Fresh pineapples purchased from a local market were aseptically cut
using a sterile knife into thin section of 2 × 2 cm slices and placed in 24
well plates. The biofilm formation, treatment, processing and SEM vi-
sualization of pineapple slices were done as described above. For the
treatment, the BIC of linalool and LN3 were used.
A. Prakash et al. Food Bioscience 28 (2019) 57–65

Fig. 4. Biofilm inhibitory effect of linalool and LN3 (a). Bars with asterisk indicate significant difference (P < 0.05). Light microscopic visualization of antibiofilm
activity of linalool and LN3 (b).

2.7. Sensory evaluation strong”. For pineapple odor and linalool odor, 2.5 was set as set as low
or detection threshold and 7.5 was set as high or saturation threshold.
The sensory effect of linalool and LN3 on the cut pineapple was
evaluated as described by Chapman, Lawless, and Boor (2001) with
2.8. Statistical analysis
slight modifications. Uniformly cut pineapples slices (3 × 3 cm) were
dipped in a solution containing the MIC of linalool or LN3 for 5 min.
All the experiments were carried out in triplicates. Significant dif-
Control samples were dipped in water. The samples were then air dried
ference among means for each group was determined using ANOVA
and presented in random order to a semi-trained panel of 10 members
followed by Tukey post hoc test (P < 0.05) using GraphPad prism
from the School of Chemical and Biotechnology, SASTRA Deemed
version 5.0 (GraphPad Software Inc., San Diego, CA, USA).
University. Panels were acquainted with the linalool odor before the
sensory evaluation. Appearance, pineapple odor, linalool odor, texture
and overall acceptability attributes were assessed on an intensity rating 3. Results and discussion
scale which ranged from 0 to 9, where less than 3 was considered
“poor”, 4 to 6 was “fair” and 7 to 9 was “good”. In the intensity scale 0 3.1. Characterization of nanoemulsions
signified “attribute not detected” and 9 signified “attribute extremely
Three different LN, i.e., LN1, LN2 and LN3 were formulated as

61
A. Prakash et al.
Fig. 5. Scanning electron microscopic image on antibiofilm activity of linalool and LN3.
described in Table 1. The droplet size of the nanoemulsions was af-
fected by the amount of surfactant present in it. The highest droplet
diameter of 85 nm was observed in LN1 which had a 1:1 (v/v) ratio of
linalool and surfactant. The mean droplet diameter of the nanoemul-
sions was found to decrease with an increase in surfactant concentra-
tion, i.e., 35 and 10.9 nm in LN2 and LN3, respectively. Similarly,
Ghosh et al. (2013a) also achieved minimum droplet size in basil EOS at
low oil to surfactant ratio. Surfactant is known to stabilize the emulsion
by reducing interfacial tension at the O/W interface (Ghosh, Mukherjee,
& Chandrasekaran, 2014). With LN1 and LN2, the nanoemulsions could
have been stabilized by surfactant due to a low surfactant to oil ratio,
while the nanoemulsions in LN3 could have been stabilized due to high
surfactant to oil ratio and subsequent micelle formation around linalool
(McClements, 2012). LN2 showed a relatively low zeta potential
(−10.3 mV) as compared to other formulations, which indicated its low
stability. LN3 showed good stability among all formulations and
showed a zeta potential of −18.1 mV, which was found to be stable for
one month with a marginal increase in the droplet size (Table 1 and
Supplementary Fig. S1). On the other hand, phase separation was ob-
served in LN1 and LN2 formulations after 20 and 10 days of storage,
respectively.
Visual appearance of the nanoemulsions is shown in Fig. 1a and
Table 1, which indicated LN1 and LN2 were turbid while LN3 was clear.
LN1 and LN2 showed a higher absorbance value of 2.54 and 2.56 when
compared to LN3 (0.14). This could be because of the larger particles in
LN1 and LN2 which scattered light more intensely than the smaller
ones. Likewise, the WI of the nanoemulsions was found to be 21.4, 43.4
and 86.8 in LN1, LN2 and LN3 formulations, respectively. The pH of all
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Food Bioscience 28 (2019) 57–65
nanoemulsions was found between 6.89 and 6.95. The concentration of
surfactant showed a positive correlation with viscosity, which is in
accordance with the previous report of Ghosh et al. (2013a).
The thermodynamic stability of the nanoemulsions is shown in
Supplementary Fig. S2. Phase separation was observed in LN1 and LN2
after centrifugation, whereas LN3 was found to be stable. Likewise, LN1
and LN2 also could not withstand temperature variation and showed
phase separation in both heating-cooling and freeze-thaw cycles. The
improved stability of LN3 could be attributed to its smallest droplet size
of 10.9 nm. On reduction of droplet size, the strength of the repulsive
force increases more rapidly than the attractive force (Ghosh et al.,
2014) which resulted in enhanced stability of LN3 and therefore all the
further studies were carried out only with LN3.
The morphology and size of the LN3 was observed using TEM
(Fig. 1c). Spherical droplets of LN3 from 13 to 16 nm were observed,
which almost matched the results obtained using the particle size
analyzer (10.9 nm). Similarly, Ghosh, Saranya, Mukherjee, and
Chandrasekaran (2013b) also showed spherical shaped nanoemulsions
with nanometric diameters from EOS of Cinnamomum zeylanicum.
3.2. Antibacterial activity
LN3 showed a lower MIC value (0.625%) than linalool (1.25%)
(Table 2). Similar to the observations in this study, Moghimi et al.
(2016) also observed a 10-fold higher antibacterial activity of Thymus
daenensis EOS nanoemulsions. The same authors suggested that the
small particle size of nanoemulsions could have better interaction with
the cell membrane surface as compared to EOS.
A. Prakash et al.
Fig. 6. Biofilm inhibitory effect of linalool and LN3 on the surface of fresh cut pineapple observed using SEM: Cut surface of pineapple (a); Control untreated (b);
Linalool treated (c); LN3 treated (d). Arrow marks indicate Salmonella biofilms.
The change in the viability of S. Typhimurium over time on inter-
action with linalool or LN3 was established by determining the killing
kinetics (Fig. 2). Within 2 h of interaction with LN3 no viable cells of S.
Typhimurium were observed, whereas linalool treatment showed 1.2
log CFU/ml. These results showed that LN3 was more effective than
linalool in killing bacterial cells within a short period of time. A pre-
vious report also showed rapid inactivation of Staphylococcus aureus
cells with eugenol nanoemulsions treatment (Ghosh et al., 2014).
3.3. Integrity of the cell membrane
Nanoemulsions are known to fuse with the lipid bilayer of bacterial
cell membrane, and destabilize its integrity which eventually leads to
cell lysis (Ghosh et al., 2013b). The cytoplasmic contents released
during cell lysis can be quantified by measuring the absorbance at 260
and 280 nm which corresponds to the nucleic acids and proteins, re-
spectively, released from the cells. A significant increase in absorbance
at both 260 and 280 nm, compared to control was observed when S.
Typhimurium cells were treated with linalool and LN3 (Fig. 3a and b).
Compared to linalool, LN3 at its MIC showed a 4 and 4.5-fold increase
in optical density of the supernatant at 260 and 280 nm, respectively. A
slight increase in the efficacy of LN3 was observed at 2 x MIC. There-
fore, these results suggested that linalool and LN3 caused damage to the
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Food Bioscience 28 (2019) 57–65
S. Typhimurium membranes, which led to the release of internal con-
stituents.
SEM images also showed alteration of the surface morphology of the
bacterial cells upon treatment with linalool and LN3 (Fig. 3c–e). The
control (untreated) cells of S. Typhimurium showed rod shaped struc-
tures with an intact cell membrane. A distortion of the membrane was
seen in bacterial cells treated with LN3 than linalool. As discussed
earlier, this enhanced efficacy of LN3 could be due to its easy access to
the cell membrane as compared to linalool.
3.4. Biofilms inhibition
Biofilms inhibition studies of the linalool and LN3 were carried out
at their respective sub-MIC concentrations (½ and ¼ MIC). At ½ MIC of
LN3 and linalool, 64.4 and 56.9%, respectively, biofilms inhibition was
observed (Fig. 4a). The results indicated a ∼11.5% enhanced biofilms
inhibition of the LN3 as compared to linalool. In another study, Lou
et al. (2017) also showed an increased antibiofilm activity of Citrus
medica EOS nanoemulsions against Staphylococcus aureus as compared
to EOS. The same authors proposed that antibiofilm ability of the EOS
nanoemulsions could be due to their ability to inhibit the attachment of
bacteria on the surfaces. The antibiofilm efficacy of the linalool and
LN3 were also confirmed using light microscopy, wherein it was found
A. Prakash et al.
that sub-MIC of LN3 significantly reduced the biofilm formation as
compared to linalool (Fig. 4b).
To further confirm the results of light microscopy, samples were also
observed using SEM and the results are shown in Fig. 5. Control showed
dense aggregates of bacterial cells, which is a typical characteristic of
biofilm formation. On the other hand, the dense aggregates of cells
were reduced upon treatment with the LN3 in comparison with linalool.
The formation of Salmonella biofilms in food processing is a major
problem (Silva et al., 2018). Moreover, the mechanical removal of
biofilm forming bacteria is challenging as they show more resistance to
disinfectants than their planktonic counterparts. As LN3 showed anti-
biofilm potential in the present study, it could possibly be used as an
effective disinfectant in food industry.
3.5. Biofilms reduction on pineapple surface
Development of biofilms on the surface of cut fruits can be a threat
to food safety (Tarver, 2009). The linalool and LN3 showed a clear
reduction of S. Typhimurium biofilms on the surface of pineapple
(Fig. 6). Control samples showed a dense aggregation of biofilms, which
was reduced on linalool treatment. In accordance with the in vitro
studies, LN3 was more effective than linalool in reducing the biofilms
on the surface of pineapple as well. Studies in the past have also re-
ported a higher microbial reduction in different fruit models like ba-
nana, papaya and dragon fruit upon nanoemulsions treatment (Zahid,
Ali, Manickam, Siddiqui, & Maqbool, 2012). Recently, Li, Chang,
Saenger, and Deering (2017) showed the reduction of E. coli O157:H7
and S. Typhimurium biofilms in lettuce and blueberries treated with
thymol nanoemulsions. Hence, LN3 holds promise as a potential anti-
biofilm agent for its application in the food industry.
3.6. Sensory evaluation
Sensory evaluation results showed acceptance of linalool and LN3
treatment in fresh cut pineapple (Supplementary Fig. S3). Stronger li-
nalool odor was perceived in linalool treated pineapple samples than in
LN3 treated samples. The overall acceptance of the pineapple was in the
following order: Control > LN3 treated > Linalool treated. Linalool
masked the characteristic pineapple odor more than LN3, which might
have been the reason for higher acceptability of LN3 treated samples.
The sensory acceptance of food samples incorporated with EOS is
highly influenced by compatibility between specific EOS compounds
and the food matrix (Prakash et al., 2018). Therefore, future studies on
a suitable blend of linalool with other EOS or its components might
further enhance the consumer acceptability of linalool.
4. Conclusions
The nanoemulsions of linalool were formulated using an ultrasonic-
assisted emulsification method and their characteristics were studied.
The stable LN3 showed increased antibacterial and antibiofilm activ-
ities against S. Typhimurium in comparison with linalool. Mechanistic
studies showed that the higher antibacterial activity of the LN3 could be
due to its increased efficacy to disrupt the bacterial membrane. Further,
the LN3 also showed high antibiofilm activity in both in vitro and fresh
cut pineapple models. Moreover, sensory analysis showed acceptance of
LN3 treated fresh cut pineapple. Thus, the LN3 formulation could be
explored as a prospective antibacterial and antibiofilm agent to prevent
Salmonella contamination in the food industry.
Conflicts of interest
The authors declare that they have no conflict of interest.
64
Food Bioscience 28 (2019) 57–65
Acknowledgements
AP acknowledges a Junior Research Fellowship provided by CSIR,
India (09/1095/(0020)/2017-EMR-I), DR acknowledges SASTRA
Deemed University for a Teaching Assistantship, VV (YSS/2014/
000332) and PN (SB/YS/LS-284/2013) acknowledges DST-SERB.
Authors are grateful to the management of SASTRA Deemed University
for their support and provision to use analytical facilities from other
labs and the DST-FIST facility (SR/FST/ETI-331/2013).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fbio.2019.01.018.
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