LWT - Food Science and Technology 107 (2019) 280–290

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LWT - Food Science and Technology

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Carvacrol and astaxanthin co-entrapment in beeswax solid lipid T

nanoparticles as an efficient nano-system with dual antioxidant and anti-
biofilm activities
Mohammad Shakeria, Seyed Hadi Razavia,∗∗, Shahryar Shakerib,∗
a
Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran, Karaj, Iran
b
Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran

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

Keywords: Carvacrol (Car) and astaxanthin (Asta) are widely used in the food industry as antimicrobial and antioxidant
Simultaneous entrapment agents. This study investigated simultaneous entrapment of them in beeswax solid lipid nanoparticles (BW.SLNs)
Carvacrol in order to increase their biological activities. Constituents of optimized formulation (lecithin (7.31 mg), tween
Astaxanthin 80 (0.81% v/v), beeswax (25.04 mg), Asta (500 μg) and Car (5 mg)) were determined by response surface
Beeswax solid lipid nanoparticles
methodology. Car/Asta loaded BW.SLNs (Car/Asta-BW.SLNs) were prepared with size and zeta potential of
Dual activities
58.8 nm and −36 mV, respectively. High entrapment efficacy (EE) for Asta (94.6 ± 0.72%) and Car
(88.5 ± 1.9%) was achieved. In-vitro release study showed that Car was released into the buffer solution two
times more than Asta. Entrapped Car and Asta were more stable under acidic and alkaline conditions. Also, 98.2
and 91.37% of entrapped molecules were intact in BW.SLNs under high oxidative condition. Car/Asta-BW.SLNs
(1.5 mg/mL) showed efficiency to remove 92.87 and 84.25% and kill 94.81 and 83.26% of total Pseudomonas
aeruginosa and Staphylococcus aureus biofilms cells. In conclusion, simultaneous entrapment of Car and Asta by
BW.SLNs is reported for the first time and Car/Asta-BW.SLNs can be applied as an efficient nano-system with
dual antioxidant and anti-biofilm functions.

1. Introduction
Antimicrobial and antioxidant compounds originated from natural
sources have been applied in the food industry (Aziz & Karboune,
2018). Among them, carvacrol and astaxanthin are predominant in
oregano and microalgae Haematococcus pluvialis, respectively (Ambati,
Phang, Ravi, & Aswathanarayana, 2014; Marinelli, Di Stefano, &
Cacciatore, 2018). The existence of phenolic hydroxyl groups in car-
vacrol and extended polyene chain of conjugated double bonds in as-
taxanthin are responsible for their antimicrobial and antioxidant ac-
tivities (Higuera-Ciapara, Felix-Valenzuela, & Goycoolea, 2006; Xu,
Zhou, Ji, Pei, & Xu, 2008). Antimicrobial activity of carvacrol against
pathogenic bacteria is well known in food packaging material
(Rodriguez-Garcia et al., 2016). Also, its anti-biofilm activity against
biofilm forming pathogenic bacteria has been studied (Nostro et al.,
2009). Reactive oxygen species (ROS) and oxidative stresses (OS) are
considered as main concerns to cause human diseases (Pizzino et al.,
2017). Astaxanthin, the most powerful antioxidant among carotenoids,
presents free radical scavenging and anti-inflammatory activity and can
protect the human body against adverse effects of ROS and OS.
Like many other essential oils and carotenoids, carvacrol and as-
taxanthin are hydrophobic and lipid-soluble. They have poor solubility
in water, which has limited their applications as food additives.
Furthermore, some physicochemical properties of foods may change the
stability and antioxidant activity of these bioactive compounds
(Anarjan, Tan, Nehdi, & Ling, 2012; Marinelli et al., 2018). Also, car-
vacrol is a volatile compound and could be oxidate or decompose under
the air or light conditions (Nostro et al., 2009). Hence, increasing their
half-life, oxidation stability, and water solubility is necessary for the
food and beverage industries. To overcome these limitations, various
research works were carried out to entrap or encapsulate these bioac-
tives in nanocarriers.
Several nano-formulations have been investigated for entrapment of
carvacrol such as chitosan (Keawchaoon & Yoksan, 2011), poly-
hydroxybutyrate (PHB) (Shakeri, Shakeri, & Hojjatoleslami, 2014), poly
(lactic-co-glycolic acid (PLGA) (Iannitelli et al., 2011) and zein

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: mohammad.shakeri@ut.ac.ir (M. Shakeri), srazavi@ut.ac.ir (S.H. Razavi), sh.shakeri@kgut.ac.ir (S. Shakeri).

https://doi.org/10.1016/j.lwt.2019.03.031
Received 31 December 2018; Received in revised form 7 March 2019; Accepted 9 March 2019
Available online 13 March 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
M. Shakeri, et al.
nanoparticles (Wu, Luo, & Wang, 2012). Recently, a nanoemulsion was
used for delivery of carvacrol. This nanoemulsion was effective to in-
activate microflora on fresh-cut vegetables (Sow, Tirtawinata, Yang,
Shao, & Wang, 2017).
Also, nanocarriers made from stearic based solid lipid nanoparticles
(SLNs) (Li, Zahi, Yuan, Tian, & Liang, 2016), polysaccharides (Anarjan
& Tan, 2013c) and liposomes (Pan, Zhang, Gu, & Zhang, 2018) have
been applied for astaxanthin nano-delivery with the aims of increasing
their stability and water solubility for the medical and food industry
applications (Weiss et al., 2008). More stable formulations, controlled
release kinetics of bioactives and their protection against physico-
chemical factors can be achieved by using SLNs (Kumar & Randhawa,
2013). SLNs made from beeswax (BW) and carnauba wax were em-
ployed for entrapment of ketoprofen (Kheradmandnia, Vasheghani-
Farahani, Nosrati, & Atyabi, 2010). Beeswax is usually used in the food
industry and packaging as edible film mixed with chitosan (Velickova,
Winkelhausen, Kuzmanova, Moldao-Martins, & Alves, 2015) or poly-
lactic acid (PLA) (Lim, Kim, Ko, & Park, 2015). It is non-toxic and edible
biomaterial that can be used for the preparation of SLNs due to its low
cost. Furthermore, its hydrophobic property makes it a suitable matrix
for lipid-soluble active compounds.
Interestingly, co-entrapment or co-encapsulation of carvacrol and
astaxanthin simultaneously in beeswax solid lipid nanoparticles
(BW.SLNs) is not investigated up to now. Therefore, the current re-
search work focused on the preparation and optimization of carvacrol/
astaxanthin loaded BW.SLNs (Car/Asta-BW.SLNs). Response surface
methodology (RSM) was used to optimize the preparation conditions of
nano-formulations. RSM consists of a group of mathematical techniques
based on the fit of empirical models to the experimental data obtained
in relation to experimental design (Bezerra, Santelli, Oliveira, Villar, &
Escaleira, 2008). In a previous study, RSM was applied to investigate
the optimal levels of some processing parameters to optimize the mi-
crowave-assisted extraction of astaxanthin from Phaffia rhodozyma
(Zhu, Han, Chen, & Han, 2010, pp. 2104–2109). Also, the detailed
characterization, pH, storage and oxidative stability, radical scavenging
and anti-biofilm activities of Car/Asta-BW.SLNs were investigated. A
schematic illustration representing the Car/Asta-BW.SLNs and their
dual activity is shown in Fig. 1.
2. Materials and methods
2.1. Materials
Refined beeswax (BW) with purity of 99% and melting point of
61–65 °C, astaxanthin (Asta), carvacrol (Car), egg yolk lecithin (L-α-
phosphatidylcholine) and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) were
obtained from Sigma Aldrich (St Louis, Mo., USA). Tween-80, nutrient
broth (NB), tryptic soy broth (TSB), triphenyltetrazolium chloride
Fig. 1. Schematic illustration of Car/Asta-BW.SLNs preparation by nano-emulsion method and their dual antioxidant and anti-biofilm activities (beeswax (BW),
astaxanthin (Asta), carvacrol (Car), lecithin (Lec).
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LWT - Food Science and Technology 107 (2019) 280–290
(TTC) and crystal violet (CV) were purchased from Merck (Darmstadt,
Germany). Acetonitrile and all other solvents were used in this study
were of the highest grade commercially available. Microtiter plates
were purchased from Dynatech, Immulon. The reference strains used
for the testing of biofilm removal and killing were Pseudomonas aeru-
ginosa (ATCC 15442) and Staphylococcus aureus (ATCC 6538). Bacterial
strains were stored in glycerol stock (15%v/v) at −80 °C.
2.2. Statistical experimental design, optimization of data and validation of
response surface methodology (RSM)
Central Composite statistical design was employed with 5 factors, 3
levels and 26 runs for the optimization study using Design-Expert
software (Design-Expert 10.0.7.0). Lecithin (X1), tween 80 (X2),
beeswax (X3), astaxanthin (X4) and carvacrol (X5) were selected as
independent variables and they were set at low, medium and high le-
vels on the basis of the results of initial trials (Supplementary material
S1 and Table S1). Then, different batches were prepared with different
independent variables at different levels and response like entrapment
efficiency was obtained. The data was substituted to the design expert
software and then, polynomial equations were determined (Anarjan &
Tan, 2013b).
2.3. Preparation of Car/Asta-BW.SLNs, Car-BW.SLNs, Asta-BW.SLNs and
unloaded BW.SLNs (U-BW.SLNs)
A nano-emulsion technique was used for the preparation of Car/
Asta-BW.SLNs (Kheradmandnia et al., 2010). Briefly, specified amounts
of BW (25.04 mg), lecithin (7.31 mg), Car (5 mg) and Asta (500 μg)
were melted in a screw cap glass tube in the water bath at 90 C°. In the
same way, 5 mL of aqueous solution (deionized water) of tween 80
(0.81% v/v) was heated at 90 °C for 10 min and was added to the
molten beeswax-lecithin mixture, leading to the formation of an
emulsion. The resulting primary emulsion was gently stirred for 1 min
and the resulting emulsion was dispersed at 24 kHz within 2 min using
an ultrasonic homogenizer. Finally, this emulsion was gradually poured
by means of a syringe into 50 mL cold water (4 °C) (volume ratio 1:10)
with magnetic stirring (1100 rpm) for 2 min to allow the formation of
Car/Asta-BW.SLNs. Also, this method was used for the preparation of U-
BW.SLNs, Car-BW.SLNs and Asta-BW.SLNs. The compositions used for
the preparation of unloaded and loaded BW.SLNs are shown in Table 1
and supplementary material S2.
2.4. Characterization of unloaded and loaded BW.SLNs
2.4.1. Yield determination of BW.SLNs
To measure the yield of prepared BW.SLNs, 10 mL of prepared
BW.SLNs was freeze dried. Then the resulting nanoparticles were
M. Shakeri, et al.
Table 1
The compositions used for the preparation of Car/Asta-BW.SLNs, Car-BW.SLNs, Asta-BW.SLNs and U-BW.SLNs.
Formulation Beeswax (mg) Lecithin (mg)
U-BW.SLNs 25.04 7.31
Car-BW.SLNs 25.04 7.31
Asta-BW.SLNs 25.04 7.31
Car/Asta-BW.SLNs (optimized) 25.04 7.31
weighed and the mass of the sample was obtained.
2.4.2. Scanning electron microscopy (SEM)
Morphology of nanoparticles was investigated by using a JEOL
LSM5600LV scanning electron microscopy. Samples of unloaded and
loaded BW.SLNs were purified by centrifugation. The resulting nano-
particles were frozen at −20 °C and freeze-dried by a freeze dryer.
These samples were used for analysis by SEM.
2.4.3. Determination of size and zeta potential of unloaded and loaded
BW.SLNs
Determination of size and dimension was performed by means of a
Zetasizer Nano DLS (Silas, France). Deionized water was used as a
background. Zeta potential of the nanoparticles was determined by
Beckman coulter delsa™ nano zeta potential analyzer. BW.SLNs were
dispersed in deionized water and were subjected to zeta potential
analysis at room temperature.
2.5. Determination of entrapment efficacy (EE) of Car and Asta
One milliliter of each nanoparticle suspension (Car/Asta-BW.SLNs,
Car-BW.SLNs, and Asta-BW.SLNs) was centrifuged at 4500×g for
10 min and resulting nanoparticles phase (upper phase) was removed
and added to 5 mL of acetonitrile. This solution was stirred at 1000 rpm
for 30 min and then was centrifuged at 4500×g for 10 min. Then, ab-
sorption at 270 and 470 nm was measured to calculate Car and Asta by
UV spectrophotometry, respectively. The entrapment efficacy for Car
and Asta by BW.SLNs was calculated based on the standard curve and
the following Eq. (1) (Keshavarzi, Shakeri, & Kiani, 2015):
Entrapment efficacy (EE%) = (loaded carvacrol or astaxanthin (mg)) /
(Total added carvacrol or astaxanthin (mg)) × 100 (1)
2.6. In-vitro release profile of Car and Asta from BW.SLNs
The release profile of Car and Asta from BW.SLNs was studied by
dialysis method. Car/Asta-BW.SLNs suspension was poured inside dia-
lysis membranes with molecular weight cut-off 12000 Da. Then, the
dialysis tube was placed into the external aqueous phase of the PBS
solution (pH: 7.4) with magnetic stirring (500 rpm) at 30 °C. Finally,
1 mL of samples inside the dialysis membrane was collected at defined
time intervals and the amount of released Car and Asta was determined
by UV spectrophotometry as the above method for determination of
entrapment efficacy. Cumulative release of Car and Asta was calculated
by Eq. (2).
Cumulative release (%) = (Released amount of carvacrol or astax-
anthin (mg)) / (Total loaded carvacrol or astaxanthin (mg)) × 100
(2)
2.7. Stability of loaded Car and Asta in BW.SLNs under acidic and alkaline
pH
The structural properties of Car and Asta can be easily changed in
acidic and alkaline fluids. The chemical stability of free carvacrol (F-
Car) (100 μg/mL aqueous solution), free astaxanthin (F-Asta) (10 μg/
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LWT - Food Science and Technology 107 (2019) 280–290
Carvacrol (mg) Astaxanthin (μg) Tween 80 (% v/v)
– – 0.81
5 – 0.81
– 500 0.81
5 500 0.81
mL acetonitrile solution) and Car/Asta loaded in BW.SLNs (1 mg/mL
aqueous suspension) were tested with the pH values of 2 and 9. The pH
of formulations was adjusted to 2 or 9 and formulations were incubated
for 7 days, at room temperature, in the dark. After defined incubation
times, 1 mL of each formulation was collected and Car and Asta were
extracted and measured by HPLC. Finally, pH stability was calculated
by the following Eq. (3) (Li et al., 2016):
pH stability (%) = (Residual amount of carvacrol or astaxanthin (mg))
/ (Original amount of carvacrol or astaxanthin (mg)) × 100 (3)
2.8. Stability of loaded Car and Asta in BW.SLNs under oxidative condition
(H2O2)
The oxidative stability of F-Car, F-Asta, and Car/Asta loaded in
BW.SLNs was studied in hydrogen peroxide (H2O2) solution (Li et al.,
2016). All samples including F-Car (100 μg/mL aqueous solution), F-
Asta (10 μg/mL acetonitrile solution) and Car/Asta-BW.SLNs (1 mg/mL
aqueous suspension)) were prepared with various amounts of H2O2
(0.1, 0.5, 1%). The samples were incubated at room temperature for 2 h
in the dark. Then, 1 mL of samples was collected and then, Car and Asta
were extracted and their oxidative stability was measured by HPLC and
calculated by the following Eq. (4):
Oxidative stability (%) = (Residual amount of carvacrol or astaxanthin
(mg) / (Original amount of carvacrol or astaxanthin (mg)) × 100 (4)
2.9. Stability of size and zeta potential of Car/Asta-BW.SLNs after 30 days
Size and zeta potential of Car/Asta-BW.SLNs can be changed during
storage conditions. Storage stability of BW.SLNs loaded with Car and
Asta was measured in 4 °C for 30 days. Nanoparticles suspensions were
placed into the sealed chamber for 30 days at 4 °C. Then, particle size
and zeta potential of Car/Asta-BW.SLNs were analyzed by a dynamic
light-scattering instrument. Each test was done in triplicates.
2.10. Determination of DPPH radical scavenging activity of F-Car, F-Asta,
U-BW.SLNs and Car/Asta-BW.SLNs
Radical scavenging ability of F-Car (100 μg/mL), F-Asta (10 μg/mL),
U-BW.SLNs (1 mg/mL) and Car/Asta-BWSLNs (1 mg/mL) was assessed
using DPPH method with some modification (Pan et al., 2018). These
amounts of F-Car and F-Asta which were used for stability test or DPPH
assay were almost equal to their amounts which were entrapped in
BW.SLNs (1 mg/mL aqueous solution). 1 mL of samples was added to
2 mL of DPPH solution (0.1 mM ethanol) and mixed well. The solutions
were incubated in the dark at room temperature for 30 min. Then, the
solutions were centrifuged at 4500×g for 10 min and the absorbance
value of supernatant was determined at 517 nm by the spectro-
photometer. Ethanol was used as control experiment. Finally, radical
scavenging activity (%) was calculated by Eq. (5):
Radical scavenging activity (%) = (Abs control-Abs sample) / (Abs
control) × 100 (5)
M. Shakeri, et al. LWT - Food Science and Technology 107 (2019) 280–290

Table 2
Obtained responses of 26 runs of experiments according to central composite design. Abbreviation: (encapsulation efficacy: EE).
Run Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Response 1 Response 2 Response 3

X1 Lecithin X2 Tween 80 X3 Beeswax X4 Astaxanthin X5 Carvacrol Y EE for Astaxanthin – EE for Carvacrol – Size

mg % mg μg mg % % nm

1 6.25 1.5 25 500 5 97.46 86.91 62.5

2 12.5 1 50 300 2.5 83.74 81.82 70.7
3 18.75 0.5 75 100 5 98.53 87.9 66.2
4 12.5 1 50 300 5 94.83 89.5 68.1
5 6.25 1.5 75 100 5 96.54 91.43 73.1
6 18.75 1.5 25 100 5 98.6 92.7 57.4
7 12.5 1 50 500 2.5 86.83 79.45 73.7
8 12.5 1 50 300 2.5 81.25 83.51 69.4
9 6.25 0.5 75 500 5 95.64 87.72 74.9
10 12.5 1 50 300 0 39.34 0 72.5
11 12.5 1 50 300 2.5 84.87 80.95 68.3
12 6.25 0.5 25 100 0 25.91 0 69.8
13 18.75 0.5 75 500 0 58.7 0 75.7
14 12.5 1 50 100 2.5 74.19 83.65 70.2
15 12.5 1.5 50 300 2.5 84.66 77.03 73.9
16 12.5 1 50 300 2.5 81.17 81.6 72.6
17 18.75 1.5 25 500 0 41.47 0 77.5
18 18.75 0.5 25 500 5 96.78 87.51 60.4
19 12.5 1 25 300 2.5 83.8 80.3 70.6
20 6.25 1 50 300 2.5 85.71 78.5 69.4
21 12.5 1 75 300 2.5 85.27 78.27 72.9
22 6.25 1.5 75 500 0 56.48 0 77.1
23 18.75 1.5 75 100 0 33.4 0 76.9
24 12.5 1 50 300 2.5 82.7 82.19 71.6
25 18.75 1 50 300 2.5 83.56 75.64 71.3
26 12.5 0.5 50 300 2.5 76.56 85.12 70.1

2.11. HPLC analysis of Car and Asta
Car and Asta were analyzed by HPLC (Shimadzu system) equipped
with an analytical reversed phase C18 column (4.6 × 250 mm, 5 μm,
Diamodsil™). For carvacrol detection, the mobile phase was acetoni-
trile-water (40:60). The flow rate was 1.5 mL/min and the temperature
of the column was kept at 30 °C. The detection wavelength was set at
276 nm (Hajimehdipoor, Shekarchi, Khanavi, Adib, & Amri, 2010). For
astaxanthin detection, the mobile phase was methanol, acetonitrile, and
water (20%, 75.5% and 5% v/v, respectively). The flow rate and tem-
perature of the column were 1 mL/min and 30 °C, respectively. The
wavelength was set at 476 nm and the injection volume was 10 μL (Lu,
Zhang, Zhao, Zhou, & Yu, 2010).
2.12. Assessment of the potential of F-Car, F-Asta, U-BW.SLNs and Car/
Asta-BW.SLNs against bacterial biofilms
The antibiofilm activity of different formulations including: F-Car,
F-Asta, U-BW.SLNs and Car/Asta-BW.SLNs was assessed at various
concentrations (0, 0.3, 0.5, 0.8, 1.1 and 1.5 mg/mL) either to remove or
to kill biofilm of Pseudomonas aeruginosa (ATCC 15442) and
Staphylococcus aureus (ATCC 6538).
2.12.1. Bacterial growth
Pseudomonas aeruginosa and Staphylococcus aureus were used to form
biofilm because these strains have a good biofilm formation capacity at
37 °C in the microtiter plate. An overnight culture of P. aeruginosa and
S. aureus were prepared in tryptic soy broth (TSB) medium and then
used for determination of minimum inhibitory concentration (MIC) and
biofilm formation in 96 wells microtiter plates.
2.12.2. Biofilm formation in 96-well microtiter plates
One milliliter of each overnight culture was transferred to 10 mL of
sterile TSB medium. Then, 250 μL of an inoculated TSB medium was
transferred into the wells of the microtiter plate. Blank wells contained
TSB medium, only. Plates were made in triplicate, covered, and in-
cubated for 48 h at 37 °C. After the incubation period, the planktonic
suspension and nutrient solutions were aspirated and biofilm washed
with water. The plates were vigorously shaken in order to remove all
non-adherent planktonic cells. Then, different nano-formulations were
applied to bacterial biofilm.
2.12.3. Assessment of the potential of formulations either to remove or to
kill biofilm cells
These formulations were separately used in a disinfection regimen
to evaluate their effect on biofilm removal and killing. Formulations
were applied to wells immediately after washing the biofilm. All wells
in a column received the same treatment. In addition to the formula-
tion-treated columns, each plate had one control (untreated) biofilm
column and one blank (sterile) column. After 1 h, suspensions were
removed and wells were stained with 2% CV (2% w/v) for 5 min or 2, 3,
5- triphenyl tetrazolium chloride (TTC, 2% w/v) for 1.5 h in the dark-
ness. In this study, TTC was used to measure active metabolism and
respiration by bacterial cells that survived during treatment with for-
mulations. After staining, the wells were rinsed and then filled with
96% ethanol. After 15 min incubation, the plates were vigorously
shaken prior to reading absorbance at 570 nm (CV-stained wells) and
450 nm (TTC- stained wells) (Shakeri, Kermanshahi, Moghaddam, &
Emtiazi, 2007). A measure of formulation efficacy (i.e. the percentage
reduction in stain) was calculated from the blank, control and treated
absorbance values (Eq. (6)) (Pitts, Hamilton, Zelver, & Stewart, 2003):
Killing or removal efficacy (%) = [(CeB)-(T-B)] / (CeB) × 100 (6)
Where C denotes the average absorbance for control wells, B denotes
the average absorbance for blank wells, and T denotes the average
absorbance for treated wells.
2.13. Statistical analysis
Statistical comparisons with statistically significant differences (p-

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M. Shakeri, et al.
Fig. 2. 3-D response surface plots: the effects of Car and Asta (A) and the effect of Car and BW (B) on EE of Asta by BW.SLNs. Contour plots: the effects of Car and Asta
(C) and the effect of Car and BW (D) on EE of Asta by BW.SLNs.
value < 0.05) were prepared using statistical software (SAS).
3. Results and discussion
3.1. Optimization results
In this study, our aim was the preparation of BW.SLNs with si-
multaneous high efficiency for loading of carvacrol and astaxanthin.
BW.SLNs were applied as a nano-system with dual antioxidant and anti-
biofilm activities. Different batches were prepared with different in-
dependent variables at different levels and response such as entrapment
efficiency (EE%) was obtained (Table 2, supplementary material S3 and
Table S2). Three dimensional and contour plots (for response Y) were
prepared and are shown in Fig. 2A–D. The 3D-response graphs and
contour plots showed that EE of Asta was enhanced after increasing of
the content of BW in nanoparticles formulations. Also, maximum EE of
Asta in BW.SLNs was achieved when Car was incorporated in for-
mulations. The composition of optimized formulation was included BW
(25.04 mg), lecithin (7.31 mg), tween 80 (0.81%), Car (5 mg), and Asta
(500 μg) to fulfill requisites of an optimized Car/Asta-BW.SLNs for-
mulation.
3.2. Preparation of Car/Asta-BW.SLNs, Car-BW.SLNs, Asta-BW.SLNs and
U-BW.SLNs
Car/Asta-BW.SLNs, Car-BW.SLNs, Asta-BW.SLNs and U-BW.SLNs
were prepared and used for size analysis, stability tests, antioxidant
activity and assessment of their anti-biofilm potential against Gram-
negative and positive bacteria.
3.3. Morphology, size and zeta potential analysis of nanoparticles
3.3.1. Car/Asta-BW.SLNs
The composition of optimized formulation was included: BW
(25.04 mg), lecithin (7.31 mg), tween 80 (0.81%), Car (5 mg), and Asta
(500 μg). Car and Asta were entrapped simultaneously into the
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LWT - Food Science and Technology 107 (2019) 280–290
BW.SLNs. Prepared nanoparticles were spherical in shape and their size
was 58.8 ± 6 nm (Fig. 3A1, B1). Zeta potential of nanoparticles was
−36 mV (Fig. 3C1). The stable aqueous suspension of Car/Asta-
BW.SLNs was prepared and no aggregate or agglomerate of nano-
particles was observed.
3.3.2. Car-BW.SLNs
For the preparation of Car-BW.SLNs, Asta was removed from opti-
mized formulation and then, nanoparticles were prepared and char-
acterized. Spherical Car-BW.SLNs were prepared by the same method
and the same compositions of the optimized formulation. But, the size
of the nanoparticles was increased to 61.8 ± 8.6 nm (Fig. 3A2, B2).
Zeta potential of these nanoparticles was increased to −43 mV
(Fig. 3C2). Suspension of nanoparticles was very stable and no ag-
gregate and agglomerate of nanoparticles was observed in aqueous
suspension. Fan, Xu, Xia, and Zhang (2008) prepared salidroside nano-
liposomes with particle size of less than 100 nm. Their findings in-
dicated that unloaded liposomes had zero electric charge on their sur-
face. But, the zeta potential of nano-liposomes significantly increased in
the range of −10 and −20 mV after loading with salidroside. They
concluded that interaction between the hydroxyl group of salidroside
and choline generates the dipole tropism and consequently enhance
surface electric charge of nanoparticles. In our study, it could be con-
cluded that the hydroxyl group of carvacrol could interact with the
polar region of phosphatidylcholine to increase the zeta potential of
Car-BW-SLNs.
3.3.3. Asta-BW.SLNs
Also, Asta-BW.SLNs were prepared spherically and their size was
determined 75.5 ± 9.7 nm (Fig. 3A3, B3). Nanoparticles were mono-
modal, but a significant difference of zeta potential was observed be-
tween Asta-BW.SLNs and Car-BW.SLNs or Car/Asta-BW.SLNs. Zeta po-
tential of Asta-BW.SLNs was decreased to −28.8 mV after entrapment
of Asta into the nanoparticles (Fig. 3C3).
M. Shakeri, et al.
3.3.4. U-BW.SLNs
U-BW.SLNs were prepared with 71.9 ± 4.8 nm in diameter and
mono-modal distribution in aqueous suspension (Fig. 3A4, B4). Nano-
particles were very stable in suspension and zeta potential analysis
showed that U-BW.SLNs had a negative surface charge (−36.9 mV)
(Fig. 3C4). It was shown that zeta potential or surface charge of
BW.SLNs was the main affected factor by loading or unloading of Car
and Asta into the nanoparticles. Loading of Car into the BW.SLNs in-
creased zeta potential of nanoparticles from −36.9 (unloaded nano-
particles) to −43 mV (Car-BW.SLNs). In the other side, entrapment of
Asta into the BW.SLNS decreased zeta potential of unloaded nano-
particles from −36.9 to −28.8 mV in Asta-BW.SLNs. Interestingly, si-
multaneous entrapment of Asta and Car into the BW.SLNs enhanced
zeta potential of nanoparticles from −28.8 to −36 mV in Car/Asta-
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LWT - Food Science and Technology 107 (2019) 280–290
Fig. 3. SEM image (A1), size distribution (B1) and
zeta potential (C1) of Car/Asta-BW.SLNs. Spherical
and uniformly distributed Car/Asta-BW.SLNs were
prepared. Size and zeta potential of nanoparticles
were assessed as 58.8 ± 6 nm and −36 mV, re-
spectively. SEM image (A2), size distribution (B2)
and zeta potential (C2) of Car-BW.SLNs. Spherical
Car-BW.SLNs were observed and the size of nano-
particles was assessed as 61.8 ± 8.6 nm. SEM image
(A3), size distribution (B3) and zeta potential (C3) of
Asta-BW.SLNs. Size and zeta potential of Asta-
BW.SLNs were determined as 75.5 ± 9.7 nm and
−28.8 mV, respectively. SEM image (A4), size dis-
tribution (B4) and zeta potential (C4) of U-BW.SLNs.
BW.SLNs. The formulations containing only Asta showed increased
nanoparticles size. With incorporating Car into the formulations, the
size of nanoparticles was decreased. Also, Car increased zeta potential
of Asta-BW.SLNs from −28.8 to −36 mV in Car/Asta-BW.SLNs.
The possible reason behind the reduction of the size of the nano-
particles might be the property of Car for increasing the surface charge
of BW.SLNs. The finding of Keawchaoon and Yoksan (2011) showed
that the diameter of nanoparticles was increased as a function of initial
carvacrol content. Also, similar to our work, the surface positive charge
of chitosan nanoparticles decreased from +42.1 (unloaded chitosan
nanoparticles) to +29.4 mV in carvacrol loaded chitosan nanoparticles
(chitosan: carvacrol ratio, 1:1.25 w/w). These results showed that the
surface positive and negative charges can be changed by loading and
entrapment of carvacrol into the nanoparticles.
M. Shakeri, et al.
Table 3
Characterizations of unloaded and loaded BW-SLNs with Car and/or Asta. The data are shown as mean ± standard deviation (SD) (n = 3). Different letters (a-c) are
significantly different (p < 0.05). Abbreviation: Entrapment efficacy (EE).
Formulation Size (nm) Zeta potential (mV)
U-BW.SLNs 71.9 ± 10.5a −36.9 ± 1.2b
Car-BW.SLNs 61.8 ± 8.6a −43.0 ± 1.9a
Asta-BW.SLNs 75.5 ± 9.7a −28.8 ± 0.6c
Car/Asta-BW.SLNs (optimized) 58.8 ± 7.4a −36.0 ± 1.8b
Fig. 4. Release profiles of Car (-●-) and Asta (-■-) from BW.SLNs in PBS (pH
7.4) and 500 rpm at 30 °C. Three release phases (the first initial burst release
(IBR) from 1 to 3 h, second release phase from 3 to 24 h and final third release
phase during 24–168 h) were observed. The data are shown as mean ±
standard deviation (SD) (n = 3).
Also, the results of da Silva et al. (2015) research work showed that
chitosan nanoparticles loaded with Salvia officinalis (sage) extract have
less positive surface charge (+20.8 to +27.8 mV) than unloaded
chitosan nanoparticles (+30 mV). It was shown that the loading of
eugenol by chitosan nanoparticles decreased zeta potential from
+37.7 mV to +16.23–33.5 mV (Woranuch & Yoksan, 2013). Also,
increasing of eugenol content in nanoparticles decreased their surface
positive charge. In our study, more negative surface charge around the
BW.SLNs makes them more stable in aqueous solution.
3.4. Determination of yield and EE
The yield of prepared unloaded and loaded nanoparticles was ob-
tained between 94 and 96%. EE of Car in Car-BW.SLNs and Car/Asta-
BW.SLNs was 89.4 ± 1.85 and 88.5 ± 1.9%, respectively (Table 3).
Shakeri et al. (2014) used polyhydroxybutyrate (PHB) nanoparticles for
entrapment of carvacrol. EE was obtained 11 and 21%, by dialysis and
nanoprecipitation methods, respectively. In another research work,
Poly (DL-lactide-co-glycolide) (PLGA) nanoparticles were used for en-
capsulation of carvacrol and the results showed that only 26% of car-
vacrol was encapsulated by PLGA nanoparticles (Iannitelli et al., 2011).
In comparison to PHB and PLGA nanoparticles, human serum albumin
nanoparticles (HSA) were able to encapsulate 32–48% of carvacrol by
desolvation or emulsion/desolvation methods, respectively (Keshavarzi
et al., 2015).
Encapsulation efficacy of 60–80% was achieved when Wu et al.
(2012) used zein nanoparticles as carvacrol delivery system. In our
research, results showed that BW based SLNs have the potential to
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LWT - Food Science and Technology 107 (2019) 280–290
Dispersity Yield (%) Carvacrol EE (%) Astaxanthin EE (%)
Monomodal 96 ± 1.3a – –
Monomodal 94.2 ± 0.9a 89.4 ± 1.8a –
Monomodal 95.5 ± 1.4a – 43.1 ± 2.7b
Monomodal 96 ± 1.6a 88.5 ± 1.9a 94.6 ± 0.7a
entrap more than 88% of Car inside of nanoparticles. This high en-
trapment efficacy of carvacrol by nanoparticle system is reported by
this research for the first time. Also, EE of Asta in Asta-BW.SLNs and
Car/Asta-BW.SLNs was 43.1 ± 2.7 and 94.6 ± 0.72%, respectively.
However, the EE of Asta in Asta-BW.SLNs was much lower than Car/
Asta-BW.SLNs. Interestingly, maximum EE for Asta (94.6%) was
achieved, when this compound was loaded simultaneously with Car
into the nanoparticles (optimized formulation). Various colloidal and
nano-dispersion systems have been used for encapsulation and delivery
of astaxanthin (Anarjan & Tan, 2013a; Anarjan et al., 2012; Li et al.,
2016). But, liposomes that were prepared by Pan et al. (2018), showed
the highest encapsulation efficacy of 97.68% for astaxanthin. They used
soy phosphatidylcholine based liposomes for encapsulation of astax-
anthin and optimized nano-liposomes were prepared with the highest
astaxanthin content. They concluded that the possible interaction be-
tween astaxanthin and lipid bilayer containing cholesterol and lecithin,
could maintain astaxanthin efficacy.
In our study, BW based SLNs were prepared and showed EE of
94.6%. In our study, carvacrol, as a liquid oily compound, helped as-
taxanthin to dissolve more homogeneously in beeswax-lecithin phase.
Also, the results showed that with increasing of Car amount in for-
mulations, the EE of Asta was increased. The high EE of Asta was
achieved in the entire Car containing formulations. Changing of poly-
morphism and crystallinity miscibility of solid lipid-based nanoparticles
has an increasing or decreasing effect on encapsulation efficacy of drugs
(Jores et al., 2004; Severino, Pinho, Souto, & Santana, 2011). Adding of
carvacrol to beeswax based nanoparticles formulations, might de-
creases melting point of SLNs (Kumar & Randhawa, 2013) or affects
crystallinity of solid lipid nanoparticles (Souto, Wissing, Barbosa, &
Muller, 2004), so increased solubility of astaxanthin and consequently
it's more interaction with beeswax-lecithin lipid phase could enhance
its entrapment efficacy in BW.SLNs.
3.5. In-vitro release study of Car and Asta from BW.SLNs
Cumulative release of Car and Asta was evaluated by dialysis
method in PBS (pH 7.4), 500 rpm at 30 °C (Fig. 4A and B). Three release
phases (the first initial burst release phase (IBR) from 1 to 3 h, second
release phase from 3 to 24 h and final third release phase during
24–168 h) were observed and monitored. IBR of Car and Asta was
measured during 3 h of incubation. The IBR was 16.2 and 6.89% for Car
and Asta, respectively (Fig. 4B). The IBR could be related to weakly
adsorbed molecules of Car and Asta on the surface of nanoparticles
(Campos et al., 2018).
During the second phase (3–24 h), the cumulative release of Car and
Asta was increased to 25.3 and 11.02%, respectively. These results of
the second phase showed that Car was released into the buffer two
times more than Asta. Higher aqueous solubility of carvacrol (slightly
soluble: about 0.11 mg/mL) (Ben Arfa, Combes, Preziosi-Belloy,
Gontard, & Chalier, 2006) than astaxanthin (very poor soluble in water)
(Lockwood, O'Malley, & Mosher, 2003) can be contributed in its faster
release into the buffer solution. The release of Car and Asta was con-
tinued and reached 45.8 and 33.62% of total entrapped Car and Asta,
after 168 h (Fig. 4A). It means that more than 54 and 66% of Car and
Asta were still entrapped into the BW.SLNs after 168 h, respectively.
M. Shakeri, et al.
These initial and then slowly releases also were observed by Hosseini,
Zandi, Rezaei, and Farahmandghavi (2013) for oregano essential oil.
3.6. Stability of Car/Asta-BW.SLNs under acidic and alkaline pH
The chemical stability of F-Car, F-Asta and entrapped Car and Asta
were studied in aqueous solutions with pH values of 2 and 9. Stability of
F-Car under acidic pH of 2 was obtained as 83.5, 71.9 and 57.4% during
1, 72 and 168 h, respectively (Fig. 5A). Entrapped Car was more stable
under acidic pH and more than 82% of loaded molecules were intact in
nanoparticles in acidic suspension, after 168 h of incubation. Car was
susceptible to alkaline pH (pH 9) and only 53.3% of free molecules of
Car were remained intact after 168 h of incubation (Fig. 5B). BW.SLNs
enhanced the stability of entrapped Car to 77.74% during 168 h of in-
cubation in alkaline pH. It has been shown that some food-related
parameters such as lipids, proteins, and pH can change stability and the
inhibitory effect of free carvacrol against food pathogens (Carvalho, de
Jesus Medeiros, Chaves, de Souza, & Magnani, 2018). Campos,
Madureira, Sarmento, Gomes, and Pintado (2015) showed that the type
of wax or lipid which are used for the preparation of nanoparticles
matrix can increases stability of entrapped herbal extract and phenolic
compounds (Campos et al., 2015).
Free and entrapped molecules of Asta were more sensitive to the
acidic and alkaline pH than free and entrapped molecules of Car. Only,
52.27% of free molecules of Asta maintained their stability under pH of
2 at 1 h and this retention rate was decreased to 39.4% after 168 h of
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LWT - Food Science and Technology 107 (2019) 280–290
Fig. 5. Stability of F-Car and entrapped Car
in BW.SLNs under acidic pH (A) and alka-
line pH (B). Stability of F-Asta and en-
trapped Asta in BW.SLNs under acidic pH
(C) and alkaline pH (D). The experiments
were performed in triplicate. The data are
shown as mean ± standard deviation (SD).
Different letters in the figure denote that
difference is significant at p < 0.05.
Fig. 6. The oxidation stability of F-Car and
entrapped Car (A) and F-Asta and entrapped
Asta (B) in BW.SLNs in aqueous solutions
and suspensions with various amount of
H2O2. The experiments were performed in
triplicate. The data are shown as mean ±
standard deviation (SD). Different letters in
the figure denote that difference is sig-
nificant at p < 0.05.
incubation (Fig. 5C). Stability of entrapped Asta under acidic pH was
obtained 78.47, 76.1 and 71.52% during 1, 72 and 168 h, respectively.
Alkaline pH showed adverse effects on free molecules of Asta same as
the acidic pH and more than 56% of free molecules of Asta were af-
fected by pH 9, after 168 h (Fig. 5D). Our results are in accordance with
the results of Qian, Decker, Xiao, and McClements (2012). Their results
showed that the acidic pH of 3 promotes degradation of beta-carotene.
Also, it could be concluded that the molecules of Car and Asta which are
entrapped into the matrix of BW.SLNs, are protected from the adverse
effects of acidic and alkaline pH.
3.7. Stability of Car/Asta-BW.SLNs under oxidative condition (H2O2)
Oxidation stability of F-Car, F-Asta and entrapped Car and Asta in
BW.SLNs was investigated in various amounts of H2O2 solutions (0.1,
0.5 and 1% v/v). Results showed that F-Car and F-Asta were more
sensitive to oxidation by H2O2 (Fig. 6A and B). However, Car/Asta-
BW.SLNs showed better protection of Car and Asta toward oxidation by
H2O2 and about 98.2 and 91.37% of entrapped Car and Asta were intact
in nanoparticles under high oxidative condition (H2O2: 1% v/v). The
inhibition of carvacrol and astaxanthin oxidation, might be due to the
incorporation of their molecules in lipid nucleation which limits their
exposure to the oxidative factors (Li et al., 2016).
M. Shakeri, et al.
Fig. 7. The storage stability of Car/Asta-BW.SLNs at 30 °C for 30 days (A). DPPH radical scavenging activity of F-Car, F-Asta, U-BW.SLNs and Car/Asta-BW.SLNs (B).
The data are shown as mean ± standard deviation (SD) (n = 3). Different letters in the figure denote that difference is significant at p < 0.05.
3.8. Stability of size and zeta potential of Car/Asta-BW.SLNs during the
time and their radical scavenging activity
Size and zeta potential were studied after 1-month incubation of
Car/Asta-BW.SLNs suspensions at 4 °C. The results showed that the size
and zeta potential were changed from 58.8 nm and −36 mV to
77.46 nm and −32.8 mV, respectively (Fig. 7A). Nevertheless, the ag-
gregation of nanoparticles did not observe in suspensions after 1-
month. It was reported that mannitol at 10% (w/v) is a proper cryo-
protectant for phenolic compounds loaded nanoparticles and SLNs
suspensions during 90 days of storage (Campos, Madureira, Sarmento,
Pintado, & Gomes, 2017). In our study, the storage stability of nano-
particles was evaluated in suspension without using any cryoprotec-
tants. Our results showed nanoparticles were stable in suspension until
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LWT - Food Science and Technology 107 (2019) 280–290
Fig. 8. Removal (A, B) and killing (C, D) of
Pseudomonas aeruginosa and Staphylococcus aureus
biofilms by F-Car (-■-), U-BW.SLNs (-▲-) and Car/
Asta-BW.SLNs (-●-). Removal and killing of biofilms
by each suspension were assessed by crystal violet
(CV) and triphenyltetrazolium chloride (TTC)
staining using the microtiter plate test. The data are
shown as mean ± standard deviation (SD) (n = 3).
30 days of storage. Also, the results of radical scavenging activity in-
dicated that F-Car and F-Asta scavenged 13.7 and 18.4% of free radi-
cals, respectively. Ue W.SLNs were able to scavenge free radicals less
than 4% (Fig. 7B). Interestingly, more than 47% of free radicals were
scavenged by Car/Asta-BW.SLNs. Wu et al. (2012) reported that
0.67 mg/mL carvacrol loaded zein nanoparticles showed 50% inhibi-
tion of DPPH. In our study, 1 mg/mL of BW.SLNs that was containing
119 and 12.8 μg of Car and Asta, respectively, inhibited 47.1% of DPPH.
In our study, entrapped Car and Asta showed more scavenging activity
than their free forms. Entrapment of F-Asta in BW.SLNs can enhance its
water solubility and dispersibility which consequently improve its ra-
dical scavenging activity (Pan et al., 2018).
M. Shakeri, et al.
3.9. Biofilm removal and killing efficacy of F-Car, F-Asta, unloaded and
loaded BW.SLNs
The MIC, removal and killing efficacy of F-Car, F-Asta, U-BW.SLNs
and Car/Asta-BW.SLNs were evaluated against planktonic and biofilm
forms of Gram-negative bacterium (Pseudomonas aeruginosa) and Gram-
positive bacterium (Staphylococcus aureus). MIC of F-Car, F-Asta, U-
BW.SLNs and Car/Asta-BW.SLNs were determined by the broth dilution
method. Results of observation of the test tubes containing F-Asta and
U-BW.SLNs showed a turbid broth culture, indicating growth of both
bacterial strains. These results suggested that F-Asta and U-BW.SLNs
didn't possess inhibitory effect against P.aeruginosa and S. aureus at the
concentration below 1.5 mg/mL. The MIC of F-Car and Car/Asta-
BW.SLNs against P.aeruginosa and S. aureus was 0.35 and 0.2 mg/mL
and 0.5 and 0.3 mg/mL, respectively. These results of MIC suggested
that F-Car and Car/Asta-BW.SLNs had antibacterial effect against tested
bacterial strains. In the next step, assessment of removal and killing
efficacy of formulations was carried out for 1 h at 37 °C, without
shaking in 96 wells microtiter plate. Asta was not effective to remove or
kill Pseudomonas or Staphylococcus biofilms (data not shown). But, F-
Car, U-BW.SLNs and Car/Asta-BW.SLNs were effective to remove 20.43,
74.85 and 92.87% of total Pseudomonas biofilm at concentration of
1.5 mg/mL, respectively (Fig. 8A).
Enhanced removal activity of BW.SLNs suspension was observed
from 74.85 to 92.87% against P. aeruginosa biofilm when Car and Asta
were entrapped into the nanoparticles. The results showed that the
removal activity of F-Car against Staphylococcus biofilm was ranged
from 24.8 to 56.37% (Fig. 8B), which is more than its removal activity
on Pseudomonas biofilm (8.47–20.43%). But, profiles of removal ac-
tivity of U-BW.SLNs and Car/Asta-BW.SLNs were almost the same and
no significant difference was observed in concentration between 0.8
and 1.5 mg/mL. It has been reported that the negative surface charge of
Pectobacterium carotovorum was decreased after treatment with carva-
crol. Therefore, stronger cell surface negative charge and electrostatic
repulsion between biofilm cells can promote biofilm detachment and
removal from the surfaces (Gutierrez-Pacheco et al., 2018).
The results showed that the killing activity of 0.3–1.5 mg/mL of F-
Car ranged from 36.5 to 81.6% of the total Pseudomonas biofilm cells
(Fig. 8C). This killing activity for U-BW.SLNs was obtained between 5.2
and 27%. Also, Car/Asta-BW.SLNs killed 68–94.81% of Pseudomonas
biofilm cells at the concentration of 0.3–1.5 mg/mL. In the case of the
killing of Staphylococcus biofilm cells, F-Car and U-BW.SLNs were not
effective to kill more than 18.2 or 28.6% of biofilm cells (Fig. 8D). But,
killing activity of BW.SLNs was enhanced up to 83%, when Car and Asta
were entrapped into the BW.SLNs.
Nostro et al. (2009) showed the potential antimicrobial activity of
carvacrol against staphylococcal biofilm in liquid and vapor phases.
Their results determined that staphylococcal biofilms can be inactivated
by liquid and vapor contact of this important component of essential
oils. Also, the antimicrobial activity of carvacrol loaded chitosan na-
noparticles was assessed against the planktonic form of S. aureus by
Keawchaoon and Yoksan (2011). Their results showed the inhibitory
activity of carvacrol loaded nanoparticles with MIC of 0.257 mg/mL.
The inhibitory effect of carvacrol on quorum sensing (QS) in P. aeru-
ginosa and Chromobacterium violaceum biofilms has been evaluated
(Tapia-Rodriguez et al., 2017). MIC of carvacrol was obtained 7.9 mM,
but in a lower concentration of carvacrol, pyocyanin and violacein
production that were related to QS, were decreased up to 60%. Simi-
larly, reduced bacterial biofilm formation and inhibition of QS in
Chromobacterium violaceum were observed by carvacrol (Burt, Ojo-
Fakunle, Woertman, & Veldhuizen, 2014).
4. Conclusion
Optimized Car/Asta-BW.SLNs were very effective for simultaneous
entrapment of high amount of Car and Asta. It was found that
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LWT - Food Science and Technology 107 (2019) 280–290
entrapment efficacy of Asta was correlated with the incorporation of
Car into the nanoparticles formulation. Initial burst release showed that
about 16.2 and 6.89% of Car and Asta might be absorbed on the surface
of nanoparticles and can be released very fast into the buffer solution.
Also, BW.SLNs were able to protect entrapped molecules of Car and
Asta against acidic and alkaline pH and oxidative conditions.
Furthermore, no aggregate or agglomerate was observed after one
month of storage of Car/Asta-BW.SLNs at 4 °C. Simultaneous entrap-
ment of Car and Asta in BW.SLNs increased their radical scavenging
activity because of improving their water solubility and dispersibility.
Finally enhanced removal and killing activities of Car/Asta-BW.SLNs
were observed against Pseudomonas and Staphylococcus biofilms. In
conclusion, this BW based nano-system can be applied with efficient
dual antioxidant and anti-biofilm activities against bacterial biofilms.
Conflict of interest statement
The authors declare that they have no competing interests.
Authors contributions
S. H. Razavi and S. Shakeri designed the study and interpreted the
results. M. Shakeri carried out the experimental work and interpreted
the results. Also, the manuscript was prepared by S. H. Razavi and S.
Shakeri.
Acknowledgment
This work was supported by the University of Tehran, Faculty of
Agricultural Engineering and Technology (Grant No. 95.621) and
Graduate University of Advanced Technology, Department of
Biotechnology. Also, the authors are grateful to Abdul-Hamid Rezaei for
electron microscopy.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lwt.2019.03.031.
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