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Nanoemulsification of Satureja khuzestanica essential oil and pure carvacrol; comparison of physicochemical properties and antimicrobial activity against food pathogens
Accepted Manuscript Nanoemulsification of Satureja khuzestanica essential oil and pure carvacrol; comparison of physicochemical properties and antimicrobial activity against food pathogens Zeinab Mazarei, Hasan Rafati PII:
S0023-6438(18)30947-2
DOI:
https://doi.org/10.1016/j.lwt.2018.10.094
Reference:
YFSTL 7560
To appear in:
LWT - Food Science and Technology
Received Date: 18 September 2018 Revised Date:
29 October 2018
Accepted Date: 30 October 2018
Please cite this article as: Mazarei, Z., Rafati, H., Nanoemulsification of Satureja khuzestanica essential oil and pure carvacrol; comparison of physicochemical properties and antimicrobial activity against food pathogens, LWT - Food Science and Technology (2018), doi: https://doi.org/10.1016/j.lwt.2018.10.094. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Nanoemulsification of Satureja khuzestanica essential oil and pure carvacrol; Comparison
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of physicochemical properties and antimicrobial activity against food pathogens
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Zeinab Mazarei a, Hasan Rafati a,*
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a
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Institute, Shahid Beheshti University, Tehran, Iran
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Department of Phytochemistry & Chemical Engineering, Medicinal Plants and Drugs Research
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Address for correspondence: Department of Chemical Engineering, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983963113, Tehran, Iran
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Phone: +982129904042
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Email: [email protected]
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Abstract
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The aim of the present study was to fabricate a stable carvacrol-rich Satureja khuzestanica
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essential oil (SKEO) nanoemulsion and evaluate its antibacterial activity against food borne
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pathogens. The effect of determining factors including preparation method, surfactant type,
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surfactant to oil ratio and hydrophilic lipophilic balance (HLB) were evaluated on the mean
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particle size and stability of nanoemulsions. The optimized formulation prepared by high speed
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homogenization method containing 3%w/w EO and 9%w/w surfactant mixture (Tween 80+Span
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80) with HLB value of 10 that produced stable nanoemulsion with mean particle diameter of 95
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nm. The defined conditions were applied for fabrication of pure carvacrol nanoemulsion. Both
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formulations showed appreciable long-term stability. The antibacterial activity of pure SKEO,
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carvacrol and their nanoemulsions were examined against three food-borne bacteria. The results
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showed an improvement in antibacterial activity for carvacrol and SKEO nanoemulsions against
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Salmonella enterica and Staphylococcus aureus.
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Key Words: Nanoemulsions; Essential oil; Antibacterial activity; Ostwald ripening; Carvacrol
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1. Introduction Satureja khuzestanica Jamzad which is called Marzeh Khuzestani in Persian and is an aromatic
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endemic plant wildly grown and distributed in the south-western part of Iran. This species is
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described as an analgesic, antiseptic and sedative agent in the folk medicin (Siavash Saei-
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Dehkordi, Fallah, Heidari-Nasirabadi, & Moradi, 2012). Due to the high amounts of natural
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monoterpenoid, carvacrol, the essential oil (EO) of Satureja Khuzestanica has been taken into
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consideration, with the carvacrol levels reported in some cases of this species up to 94% of the
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total oil (Farsam, Amanlou, Radpour, Salehinia, & Shafiee, 2004; Hashemi, Niakousari,
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Saharkhiz, & Eskandari, 2012). Carvacrol is well known for many diverse biological activities
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including antimicrobial, antitumor, analgesic, antiinflammatory, antiparasitic, antihepatotoxic
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and hepatoprotective activities (Can Baser, 2008).
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Since significant antimicrobial properties have been reported for this monoterpenoid, the
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carvacrol-rich EO of Satureja khuzestanica has the potential to be used as an antimicrobial agent
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in the food industry. However, the low water-solubility and high volatility are main drawbacks
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that limit the utilization of EO in commercial products (Ma, Davidson, & Zhong, 2016).
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Encapsulation of EO in suitable colloidal delivery systems such as nanoemulsions may be used
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to overcome many of these limitations (Moghimi, Aliahmadi, McClements, & Rafati, 2016).
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Nanoemulsion is a colloidal dispersion containing small oil droplets (d = 20 – 200 nm)
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suspended in an aqueous phase (Moghimi, Aliahmadi, McClements, & Rafati, 2017).
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Different mechanisms have been reported for instability of nanoemulsions, including
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flocculation, Ostwald ripening, creaming, phase separation, coalescence and sedimentation
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(Karthik, Ezhilarasi, & Anandharamakrishnan, 2017). Ostwald ripening –known as important
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challenge of nanoemulsion´s instability- is described by the growth of larger oil droplets at the
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expense of smaller oil droplets due to diffusion of oil molecules through the intervening aqueous
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phase (Chang, McLandsborough, & McClements, 2012). Two main strategies are suggested to
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prevent Ostwald ripening; adding a water-insoluble component into the oil phase and second,
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formation of a strong interfacial layer (Chebil, Desbrières, Nouvel, Six, & Durand, 2013).The
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second approach, also known as utilization of surface ripening inhibitor, is preferable since the
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concentration of bioactive component in the system can be increased (Chang et al., 2012;
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Ghaderi, Moghimi, Aliahmadi, McClements, & Rafati, 2017).
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Nanoemulsions can be fabricated by a number of different processing methods, which are
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usually categorized as either high- or low-energy methods (McClements & Rao, 2011). High
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energy approaches utilize different devices to generate intense disruptive forces leading to the
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formation of the tiny oil droplets e.g., high pressure homogenizers, high speed homogenizers,
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microfluidizers and ultrasonication methods (Ryu, McClements, Corradini, & McLandsborough,
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2018). High speed homogenizers apply shear force for producing tiny droplets in emulsion
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systems (Karthik
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nanoemulsion by cavitation phenomenon where formation and collapse of vapor cavities in the
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liquid medium occurred due to high intensity ultrasound (Mahdi Jafari, He, & Bhandari, 2006).
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A literature survey revealed that no study has been conducted on the fabrication of Satureja
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khuzestanica EO (SKEO) nanoemulsion. However, in recent years several reports have been
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published concerning nanoemulsification of carvacrol as an antimicrobial agent (Chang,
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McLandsborough, & McClements, 2013; Donsi, Annunziata, Vincensi, & Ferrari, 2012; Landry,
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Chang, McClements, & McLandsborough, 2014; Landry et al., 2016; Nash & Erk, 2017; Tastan,
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Ferrari, Baysal, & Donsì, 2016). Ostwald ripening is known as the most important mechanism
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affecting the stability of carvacrol nanoemulsions (Chang et al., 2013; McClements & Rao,
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2011). To the best of our knowledge, all methods reported for fabricating of carvacrol
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2016). Ultrasonication
process
produces
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nanoemulsions, utilize carrier oils e.g. MCT oil or sunflower oil in their formulations that
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simultaneously decrease the rate of droplet growth due to Ostwald ripening (Chang et al., 2013;
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Donsi et al., 2012; Landry et al., 2014; Landry et al., 2016; Nash & Erk, 2017). However, in
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most cases, when the percentage of carvacrol in the nanoemulsions exceeds 2.5%, a significant
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increase in particle size is observed after one month. Therefore, Ostwald ripening is still
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recognized as the most important challenge for carvacrol containing nanoemulsions.
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In the present study, we investigated the possibility of fabricating a stable nanoemulsion from
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carvacrol-rich SKEO without the incorporation of carrier oil. A series of surfactants mixture
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were utilized and the efficiency of two high energy methods was evaluated to determine the best
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preparation method that led to the production of stable nanoemulsions with fine droplets. The
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optimized nanoemulsion properties were applied for fabrication of carvacrol nanoemulsion that
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showed appreciable stability over long time storage. Finally, the antimicrobial effects of pure
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carvacrol and SKEO as well as their nanoemulsion formulations were evaluated against some
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selected food-borne bacteria including Escherichia coli, Staphylococcus aureus and Salmonella
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enterica.
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1. Materials and Methods
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2.1. Chemicals
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Tween 80, Span 80, Tween 20 and Span 20 were purchased from Merck Millipore (Darmstadt,
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Germany). Carvacrol (98%) was purchased from Sigma-Aldrich (Germany). Satureja
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khuzestanica essential oil was kindly provided by Dr. J. Hadian from Khorraman Pharmaceutical
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Co.
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2.2. GC chromatography/Mass spectrometry (GC-FID, GC-MS)
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To analyze the EO components, GC analysis was carried out using a TRACE mass spectrometer
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(Thermoquest, Manchester, UK), equipped with a flame ionization detector (FID) and a DB-5
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column (30 m × 0.25 mm × 0.25 µm film thickness). Nitrogen was used as a carrier gas with a
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flow rate of 1.1 mL/min. The oven temperature was gradually increased from 60 to 250°C at the
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rate of 5℃/min, and then maintained at 250℃ for 10 min. The temperatures of the injection
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chamber and detector were maintained at 250 and 300℃, respectively.
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The constituents of the EO were identified using a GC coupled to a mass spectrometer
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(Thermoquest-Finnigan, Manchester, UK) equipped with a DB-5 column (60 m × 0.25 mm ×
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0.25µm film thickness). Helium gas with a flow rate of 1.1 mL/min was used as the mobile phase
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and ionization energy was set to 70 eV. The temperature settings were similar to those given
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above for the GC analysis.
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2.3. Nanoemulsion Preparation
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Essential oil nanoemulsions were prepared from a mixture of SKEO (2 or 3% w/w), surfactants
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(including Tween 20 or 80 and Span 20 or 80) with surfactant to oil ratio (SOR) of 2, 3 (4, 6 or
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6, 9% w/w) and deionized water. The aqueous phase (solution of Tween in deionized water) was
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poured into the oil phase consists of SKEO and Span. Nanoemulsions were then fabricated either
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by utilizing a high speed homogenizer (SilentCrusher M, Heidolph, Germany) or a probe
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sonicator (MPI, Dattwil, Switzerland). Homogenization process was performed 5 min at 15000
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rpm applying a 12F dispersion tool which immersed in 5 mL sample. For fabricating of
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nanoemulsions by ultrasonication method, a probe sonicator was used with 20.5 KHz frequency
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and 30% maximum of power output (120 W). The energy input was provided using an ultrasonic
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probe containing a piezoelectric crystal with a maximum probe diameter of 8 mm. The sonicator
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was dipped into 3 mL of the mixture placed in ice bath and ultrasonic waves were applied for 10
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min. The hydrophilic lipophilic balance (HLB) value of SKEO was optimized according to the
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surfactant HLB values ranging from 8 to 15 using different combinations of Span 80 (HLB 4.3)
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and Tween 80 (HLB 15.0) (Nirmal, Mereddy, Li, & Sultanbawa, 2018).
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2.4. Particle Size Measurements
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Measurement of droplet size and particle size distribution of the nanoemulsions was performed
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using a Dynamic Light Scattering (DLS) instrument (Nanophox Sympatec GmbH, Claushtal,
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Germany). Nanoemulsions were diluted with deionized water to have a specified particle count
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range, between 200 and 1000 kCPS.
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2.5. Nanoemulsion Stability
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Long-term stability of nanoemulsions was evaluated by determination of mean particle diameter
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for 60-90 days storage at room temperature and refrigerator respectively.
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2.6. Transmission Electron Microscopy
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Morphology and structure of optimum nanoemulsion were evaluated using transmission electron
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microscopy (TEM). A 20 L drop of the sample was placed on a Carbon film coated on 300 mesh
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copper grid (Agar) for 2 min. Excess liquid was absorbed with filter paper then negatively
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stained with a 20 L drop of 2% w/v uranyl acetate for 1-2 min and the grid was allowed to air
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dry. Grid was examined on a Zeiss EM900 transmission electron microscope operating at an
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accelerating voltage of 80 kV.
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2.7. Antimicrobial Activity
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Determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations
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The antimicrobial activity of pure SKEO, pure carvacrol and their related nanoemulsions were
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evaluated against Escherichia coli PTCC1339, Staphylococcus aureus ATCC25923, or
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Salmonella enterica PTCC1639 using a serial dilution method. Aliquots of samples were serially
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diluted in Mueller Hinton Broth (MHB) medium in 96 well plates, to produce a concentration
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range of 0.03 to 64 mg/mL. To prevent possible solubility problem in MIC determination of pure
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SKEO or carvacrol, a 0.5% v/v Tween 80 solution was added to the medium. The final
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concentration of microorganisms was adjusted to 5 × 106 CFU/mL. Plates were incubated at
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37ºC for 24 h and the MIC values were determined as the lowest concentration of samples that
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demonstrated no visible growth of microorganisms. For MBC determination, 100 µL of the
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samples from wells without visible growth were transferred onto Mueller Hinton Agar (MHA)
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and were incubated for 24 h at 37ºC. The lowest concentration of the sample that could kill all of
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the initial bacterial population is reported as the MBCs.
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2.8. Statistical analysis:
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Each experiment was performed in triplicates and all values are reported as mean ± standard
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deviation (SD) by Microsoft Excel. The data was statistically analyzed by one-way analysis of
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variance (ANOVA) and p values less than 0.05 were considered statistically significant.
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3. Results and Discussions
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3.1. Essential Oil Analysis
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The SKEO was analyzed by GC-FID and GC-MS methods. Twenty-one components were
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identified representing 99.12% of the total oil. The qualitative and quantitative EO compositions
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are presented in Table 1, where compounds are listed according to the elution time on the DB-5
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column. The major constituents of the EO were carvacrol (87.16%) and p-cymene (6.39%). High
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level of carvacrol content (between 87.7 and 93.9%) in the SKEO were reported in previous
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studies which proposes this endemic medicinal plant as a natural source of carvacrol for
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application in food industries (Farsam et al., 2004; Hashemi et al., 2012).
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3.2. Effect of Surfactant Type on Particle Size
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Initially, a SKEO nanoemulsion was prepared by homogenization of 3% w/w EO with 6%w/w
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surfactants (Tween 80 and Span 80) with a required HLB 10 (determined based on the
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preliminary studies), and 91%w/w water. To assess whether the defined HLB value could be
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suitable for fabrication of stable SKEO nanoemulsions, different combinations of surfactants
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including Tween 20, Tween 80, Span 20 and Span 80 were utilized for determination of the best
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surfactant mixture. The mean particle diameter of the resulting nanoemulsions, just after
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preparation and a week are represented in Figure 1A. The results implied that the initial diameter
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of nanoemulsion droplets was strongly depended on the type of the oil soluble surfactant. Using
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Span 80 as the oil soluble surfactant in the presence of either Tween 20 or Tween 80 resulted in
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the production of nanodroplets with almost the same particle size (p>0.05). The use of Span 20
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resulted in the production of much smaller nanoemulsion droplets at the preparation time
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compared to Span 80. Since usually emulsifiers having different HLB value are being used in
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nanoemulsion preparation, hydrophobic emulsifiers having low HLB value are located inside
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the oil droplets and the hydrophilic emulsifiers are outside, which makes hydrocarbon chains in
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tail in close contact (Cho, Kim, Bae, Mok, & Park, 2008). Span 20 contains a saturated 12
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carbon atom chain of lauric acid with a fairly linear structure compared to the more kinked
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structure of unsaturated oleic acid moiety presents in Span 80. This spatial difference affects the
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arrangement of the surfactant molecules in the oil-water interface that can cause more compact
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structure and facilitate the production of smaller droplets when Span 20 is used as the oil soluble
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surfactant. In the case of Span 20, the utilization of Tween 80 as the water soluble surfactant
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results to production of smaller droplets compared to Tween 20 (p<0.05). Tween 20 contains a
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fairly linear structure of saturated lauric acid compared to the more kinked structure of
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unsaturated oleic acid moiety presents in Tween 80 similar to those of Spans. It has been
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reported that the presence of double bond in the hydrophobic tail of nonionic surfactants is
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preferable for producing of smaller nanoemulsion droplets (Wang, Dong, Chen, Eastoe, & Li,
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2009).
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It is noteworthy that there is not a strong correlation between the primary particle size of
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nanoemulsions and stability over a period of time. Nanoemulsions that contain Span 20 as the oil
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soluble surfactant produced smaller droplets at the preparation time, however, the significant
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enhancement of droplet size was observed after a week. Using Span 80 as the oil soluble
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surfactant resulted in the production of nanoemulsions which were more resistant against droplet
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size enhancement due to Ostwald ripening. It has been reported that packing form of the
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surfactant molecules at the oil-water interface that conducted from their molecular geometry, has
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a significant impact on the formation of nanoemulsions (Israelachvili, 2011). The packing
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parameter (p) of a surfactant defined as the cross-sectional area of the tail group relative to that
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of the head group (p=aT/aH) (Komaiko & McClements, 2016). Span 20 has a saturated linear tail
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group whereas Span 80 has an unsaturated kinked one. Thus, Span 80 would be expected to have
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a higher packing parameter (due to a larger aT) that correlates to production of more stable
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droplets compared to those produced by Span 20. Therefore, it can be concluded that, in addition
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of the hydrophilic-lipophilic balance (HLB) value of surfactants mixture, the geometry of
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surfactants plays a crucial role in the stability of nanoemulsions droplets (Schmidts, Dobler,
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Nissing, & Runkel, 2009).
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3.3. Effect of Nanoemulsion Preparation Method on Particle Size
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To evaluate the impact of preparation method on the primary droplet size and stability of
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nanoemulsions, the above mentioned nanoemulsions were fabricated using a probe sonicator.
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The mean particle diameter of the resulting nanoemulsions that measured at preparation time and
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after one week represented in Figure 1B. Comparison of the mean particle diameter of prepared
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nanoemulsions with those fabricated by high speed homogenizer, revealed that the
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homogenization method is more efficient in the producing of smaller droplets. Although higher
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energy level is used in the ultrasonication method and consequently, smaller droplets are
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expected to be produced, but the cavitation phenomenon does not seem to provide the sufficient
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time required for the proper arrangement of surfactant molecules around the oil. This could be
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correlated to the high surfactant concentration used to prepare nanoemulsions that led to the
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aggregation of surfactant molecules around the oil droplets. Both homogenization and
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ultrasoniaction method showed similar pattern in the primary particle size and stability of
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nanoemulsions over a week. Some probable consideration was implied in the previous section.
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3.4. Effect of Surfactant and EO Concentration on Particle Size and Long-Term Stability
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To evaluate the effect of surfactant and EO concentration on the primary droplet size and long
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term stability, fabrication of SKEO nanoemulsion was performed by homogenization of 2 or 3
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wt% EO and surfactant to oil ratio (SOR) of 2 and 3 (Tween 80 and Span 80 in HLB 10).
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Preliminary experiments indicated that the equal concentration of surfactant and EO (SOR=1)
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could not result in the formation of nanoemulsion. Figure 2 represents the changes in mean
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particle diameter of nanoemulsions over a period of two months. As expected, increasing the
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percentage of EO led to the formation of larger droplets when the percentage of surfactants
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remained constant. It is also clear that when the percentage of the EO is constant, the particle
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size decreases with increasing surfactant percentage. Increasing the surfactant concentration
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leads to the accumulation of more surfactant molecules at the interface and from the organic
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phase into the aqueous phase that facilitates the formation of smaller droplets at the oil-water
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interface. In addition, concentrated surfactant solutions provide different structural arrangements
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of the surfactant, oil and water molecules in the system, and some of these arrangements are
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favorable for nanoemulsion formation (Lamaallam, Bataller, Dicharry, & Lachaise, 2005). Long-
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term stability study of nanoemulsions based on the mean particle diameter measurement revealed
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that some fluctuations were observed for nanoemulsion contain 2 % EO with SOR=3. Three
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other nanoemulsions showed acceptable stability over two months storage at room temperature.
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3.5. Effect of Different HLB Values of SKEO on Particle Size and Long-Term Stability
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For determination of the best HLB system, SKEO nanoemulsions were prepared by
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homogenization (5 min, 15000 rpm) of 3 wt% EO and 9 wt% surfactant mixture (Tween 80 and
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Span 80) with HLB values ranging from 8 to 15. The first nanoemulsion with HLB value of 8,
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resulted in an apparent creaming process and was eliminated from further investigations. A
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portion of each of the nanoemulsions was stored in the refrigerator and the other part was kept at
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ambient temperature. Figure 3 represents the changes in mean particle diameter of
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nanoemulsions over a period of two and three month storage at room and refrigerator
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temperatures. The primary particle diameter of prepared nanoemulsion with HLB=9 was 148 nm
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and nanoemulsions with HLB values of 10 to 15 formed smaller droplets ranging from 70 to 95
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nm. The long-term stability studies indicated that nanoemulsions with HLB values more than 10
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showed significant enhancement in the mean particle diameter so that phase separation was
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occurred for the nanoemulsion with HLB=15 after six weeks. This remarkable difference in the
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long-term stability of nanoemulsions indicates that the HLB value of surfactants mixture could
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be considered as an important factor that controls the rate of droplets growth due to Ostwald
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ripening mechanism. In addition, as shown in Figure 3B, the storage of the nanoemulsions at a
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refrigerated temperature showed that the rate of droplet growth decreases significantly. This
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observation is in accordance with the theory proposed by Lifshitz-Slezov and Wagner that
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implied the rate of droplet growth due to Ostwald ripening is inversely correlated to the absolute
260
temperature (Delmas et al., 2011; Ee, Duan, Liew, & Nguyen, 2008).
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Since the optimum nanoemulsion with HLB value of 10 represented appreciable stability over
262
long time storage, the pure carvacrol nanoemulsion was fabricated based on the defined
263
properties with the same method. No significant differences were observed between SKEO
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nanoemulsion and pure carvacrol nanoemulsion based on the mean particle diameter and long-
265
term stability (data not shown). Previous reports concerning nanoemulsification of carvacrol
266
utilized carrier oils in their formulation that couldn’t inhibit the Ostwald ripening completely
267
when carvacrol was more than 2.5% in formulation (Chang et al., 2013; Donsi et al., 2012;
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Landry et al., 2014; Landry et al., 2016). Therefore, the present study is the only report that has
269
been able to form a nanoemulsion containing 3% carvacrol without the incorporation of carrier
270
oil, and showed considerable long term stability. In addition, as shown in Figure 4, the small
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polydispersity index (PDI) value of optimized formulation (<0.1) indicated that the particles
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were of a narrow size range.
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3.6. Antimicrobial Efficacies of Carvacrol and SKEO Nanoemulsions
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The antimicrobial effects of SKEO, carvacrol and their optimized nanoemulsions against two
275
strains of foodborne Gram negative (Escherichia coli PTCC1339 and Salmonella enterica
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PTCC1639) and Gram positive (Staphylococcus aureus ATCC25923) pathogenic microorganism
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are shown in Table 2. The results indicated that the optimized nanoemulsions showed better
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antimicrobial activity than the bulk oil against S. enterica and S. aureus for carvacrol and SKEO,
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respectively. An improved antimicrobial activity of the nanoemulsions compared to the bulk oil
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was also observed in our previous study (Moghimi, Ghaderi, Rafati, Aliahmadi, & McClements,
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2016). In other cases, the antimicrobial activity of optimized nanoemulsions was similar to that
282
of the bulk oils for all three bacterial strains. These results are in agreement with some previous
283
reports that indicated nanoemulsions have similar antimicrobial activity to bulk oils (Ghaderi et
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al., 2017). In addition, no significant difference was observed in the antimicrobial activity of
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carvacrol, SKEO and their optimized nanoemulsions against both gram positive and gram
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negative bacteria.
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It has been reported that hydrophobicity of natural EO components such as carvacrol could be
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an advantage for inducing antibacterial properties. It is well known that lipophilic compounds
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possess a high affinity for cell membranes and their insertions induce changes in membrane
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physicochemical properties. The interactions of antimicrobial compounds and cell membranes
291
are considered to affect both the lipid ordering and the bilayer stability, resulting in a membrane
292
integrity decrease and potential depolarization (Ben Arfa, Combes, Preziosi-Belloy, Gontard, &
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Chalier, 2006; Xu, Zhou, Ji, Pei, & Xu, 2008). The bacterial membrane perturbations caused by
294
carvacrol, lead to the leakage of intracellular ATP, proton and potassium ions and ultimately cell
295
death (Liolios, Gortzi, Lalas, Tsaknis, & Chinou, 2009). Microencapsulation prevents reactivity
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of bioactive component with the environment (water, oxygen, light) and decreases the
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evaporation or the transfer rate to the outside region (Liolios et al., 2009).
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nanoemulsions in order to deliver EOs bioactive components to the biological membranes, can
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also provide the necessary protection against their oxidation and evaporation (Flores et al.,
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2011), while the incorporation of food antimicrobials could aid in the protection of food products
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against growth of spoilage and pathogenic microorganisms.
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4. Conclusion
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In the present work, carvacrol and SKEO nanoemulsions were fabricated without the
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incorporation of any other carrier oil. Hydrophilic Lipophilic Balance (HLB) value and
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molecular geometry of surfactants showed significant impact on initial droplet size and
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nanoemulsions stability. Comparison of two high energy preparation methods implied that high
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speed homogenization was more efficacious than ultrasonication in the production of smaller
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droplets. In addition, the impact of surfactant to oil ratio and HLB value of surfactants mixture
310
were evaluated on the nanoemulsions stability during long-term storage. Optimized
311
nanoemulsions containing 3 % EO and 9 % surfactant mixture (Tween 80+Span 80) with HLB
312
value of 10 showed appreciable stability against droplet growth due to Ostwald ripening. The
313
antimicrobial activity of optimized carvacrol and SKEO nanoemulsions improved compared to
314
that of the bulk oils. Therefore, the optimized formulation could be considered as a stable
315
carvacrol delivering system that preserved the antibacterial activity for utilization in food
316
industries.
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Acknowledgment
319
This work has been supported by Shahid Beheshti University Research Council and the authors
320
gratefully acknowledge the support provided by MPDRI. The kind assistance of Ms. Lida
321
Ghaderi in determination of antibacterial effects of nanoemulsions is gratefully appreciated. The
322
authors appreciate help provided by Khorraman Pharmaceutical Co.
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Chemical composition, antioxidative capacity and interactive antimicrobial potency of
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Satureja khuzestanica Jamzad essential oil and antimicrobial agents against selected food-
421
related microorganisms. International Journal of Food Science & Technology, 47(8),
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1579-1585.
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424
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Hydrocolloids, 61, 756-771.
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Wang, L., Dong, J., Chen, J., Eastoe, J., & Li, X. (2009). Design and optimization of a new self-
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Xu, J., Zhou, F., Ji, B. P., Pei, R. S., & Xu, N. (2008). The antibacterial mechanism of carvacrol
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and thymol against Escherichia coli. Letters in Applied Microbiology, 47(3), 174-179.
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Figure captions:
433
Figure 1. Effect of surfactant type on mean particle diameter of SKEO nanoemulsions produced
434
by (A) homogenization method and (B) ultrasonication method.
435
T80: Tween 80, T20: Tween 20, S80: Span 80, S20: Span 20. (HLB value of surfactants mixture
436
=10). (
437
Figure 2. Effect of surfactant to oil ratio (SOR) on mean particle diameter and stability of SKEO
438
nanoemulsions. (
439
SOR 3) SKEO: Satureja khuzistanica essential oil
440
Figure 3. Effect of HLB value of surfactant mixture (Tween 80 and Span 80) on mean particle
441
diameter of SKEO nanoemulsions and storage stability at different temperatures (A) 25°C and
442
(B) 4°C. (
443
Satureja khuzistanica essential oil
444
Figure 4. (A) Particle size distribution diagram of optimized SKEO nanoemulsion measured by
445
DLS (B) Transmission electron microscopy (TEM) image of optimized SKEO nanoemulsion.
446
SKEO: Satureja khuzistanica essential oil
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After one week) SKEO: Satureja khuzistanica essential oil
10,
11,
12,
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2% EO, SOR 3.
3% EO, SOR 2.
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Compounds
RI
%
1
α-pinene
935
0.63
2
camphene
951
0.05
3
β-myrcene
992
4
α-phellandrene
1007
5
3-carene
1013
6
α-terpinene
1018
7
p-cymene
8
γ-terpinene
9
p-cymenene
1091
0.17
10
linalool
1100
0.51
11
borneol
1167
0.7
12
terpinen-4-ol
1178
0.08
13
L-α-terpineol
1191
0.23
14
carvacrol methyl ether
1242
0.11
15
thymol
1294
0.25
carvacrol
1325
87.16
carvacrol acetate
1372
0.53
trans-β-caryophyllene
1418
0.09
trans-α-bergamotene
1432
0.09
β-bisabolene
1506
0.67
α-bisabolene
1538
0.05
17 18 19
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0.89
0.06
0.06
0.34
1027
6.39
1059
0.06
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Table 1. Chemical composition of the essential oil of Satureja khuzistanica Jamzad
RI: retention indices relative to C9-C22 n-alkanes on the DB-5 column
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Table 2. Antibacterial activity of carvacrol, SKEO and their optimized nanoemulsions against selected bacteria
SKEOa SKEO nanoemulsion
ATCC25923
PTCC1639
MIC
MBC
MIC
MBC
MIC
MBC
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
0.25
0.25
0.125
2
0.5
0.5
0.25
0.25
0.125
1
0.125
0.25
0.25
0.25
0.25
0.25
0.25
0.125
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SKEO: Satureja khuzestanica essential oil
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PTCC1339
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nanoemulsion
Salmonella enterica
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Carvacrol
Staphylococcus aureus
2
0.25
0.25
1
0.25
0.25
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Carvacrol
Escherichia coli
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•
SKEO : Satureja khuzestanica essential oil, HLB : hydrophilic lipophilic balance
2
•
Stable nanoemulsions were fabricated from carvacrol and SKEO
3
•
The best surfactants mixture defined as Tween 80: Span 80 with HLB value of 10
4
•
Ostwald ripening was inhibited by optimization of HLB value of surfactants
5
•
The MIC value of nanoemulsions improved 2-4 times compared to the bulk oils in two
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S0023-6438(18)30947-2
DOI:
https://doi.org/10.1016/j.lwt.2018.10.094
Reference:
YFSTL 7560
To appear in:
LWT - Food Science and Technology
Received Date: 18 September 2018 Revised Date:
29 October 2018
Accepted Date: 30 October 2018
Please cite this article as: Mazarei, Z., Rafati, H., Nanoemulsification of Satureja khuzestanica essential oil and pure carvacrol; comparison of physicochemical properties and antimicrobial activity against food pathogens, LWT - Food Science and Technology (2018), doi: https://doi.org/10.1016/j.lwt.2018.10.094. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Nanoemulsification of Satureja khuzestanica essential oil and pure carvacrol; Comparison
2
of physicochemical properties and antimicrobial activity against food pathogens
3
Zeinab Mazarei a, Hasan Rafati a,*
4
5
a
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Institute, Shahid Beheshti University, Tehran, Iran
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Department of Phytochemistry & Chemical Engineering, Medicinal Plants and Drugs Research
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Address for correspondence: Department of Chemical Engineering, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983963113, Tehran, Iran
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Phone: +982129904042
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Email: [email protected]
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Abstract
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The aim of the present study was to fabricate a stable carvacrol-rich Satureja khuzestanica
15
essential oil (SKEO) nanoemulsion and evaluate its antibacterial activity against food borne
16
pathogens. The effect of determining factors including preparation method, surfactant type,
17
surfactant to oil ratio and hydrophilic lipophilic balance (HLB) were evaluated on the mean
18
particle size and stability of nanoemulsions. The optimized formulation prepared by high speed
19
homogenization method containing 3%w/w EO and 9%w/w surfactant mixture (Tween 80+Span
20
80) with HLB value of 10 that produced stable nanoemulsion with mean particle diameter of 95
21
nm. The defined conditions were applied for fabrication of pure carvacrol nanoemulsion. Both
22
formulations showed appreciable long-term stability. The antibacterial activity of pure SKEO,
23
carvacrol and their nanoemulsions were examined against three food-borne bacteria. The results
24
showed an improvement in antibacterial activity for carvacrol and SKEO nanoemulsions against
25
Salmonella enterica and Staphylococcus aureus.
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Key Words: Nanoemulsions; Essential oil; Antibacterial activity; Ostwald ripening; Carvacrol
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1. Introduction Satureja khuzestanica Jamzad which is called Marzeh Khuzestani in Persian and is an aromatic
29
endemic plant wildly grown and distributed in the south-western part of Iran. This species is
30
described as an analgesic, antiseptic and sedative agent in the folk medicin (Siavash Saei-
31
Dehkordi, Fallah, Heidari-Nasirabadi, & Moradi, 2012). Due to the high amounts of natural
32
monoterpenoid, carvacrol, the essential oil (EO) of Satureja Khuzestanica has been taken into
33
consideration, with the carvacrol levels reported in some cases of this species up to 94% of the
34
total oil (Farsam, Amanlou, Radpour, Salehinia, & Shafiee, 2004; Hashemi, Niakousari,
35
Saharkhiz, & Eskandari, 2012). Carvacrol is well known for many diverse biological activities
36
including antimicrobial, antitumor, analgesic, antiinflammatory, antiparasitic, antihepatotoxic
37
and hepatoprotective activities (Can Baser, 2008).
38
Since significant antimicrobial properties have been reported for this monoterpenoid, the
39
carvacrol-rich EO of Satureja khuzestanica has the potential to be used as an antimicrobial agent
40
in the food industry. However, the low water-solubility and high volatility are main drawbacks
41
that limit the utilization of EO in commercial products (Ma, Davidson, & Zhong, 2016).
42
Encapsulation of EO in suitable colloidal delivery systems such as nanoemulsions may be used
43
to overcome many of these limitations (Moghimi, Aliahmadi, McClements, & Rafati, 2016).
44
Nanoemulsion is a colloidal dispersion containing small oil droplets (d = 20 – 200 nm)
45
suspended in an aqueous phase (Moghimi, Aliahmadi, McClements, & Rafati, 2017).
46
Different mechanisms have been reported for instability of nanoemulsions, including
47
flocculation, Ostwald ripening, creaming, phase separation, coalescence and sedimentation
48
(Karthik, Ezhilarasi, & Anandharamakrishnan, 2017). Ostwald ripening –known as important
49
challenge of nanoemulsion´s instability- is described by the growth of larger oil droplets at the
50
expense of smaller oil droplets due to diffusion of oil molecules through the intervening aqueous
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phase (Chang, McLandsborough, & McClements, 2012). Two main strategies are suggested to
52
prevent Ostwald ripening; adding a water-insoluble component into the oil phase and second,
53
formation of a strong interfacial layer (Chebil, Desbrières, Nouvel, Six, & Durand, 2013).The
54
second approach, also known as utilization of surface ripening inhibitor, is preferable since the
55
concentration of bioactive component in the system can be increased (Chang et al., 2012;
56
Ghaderi, Moghimi, Aliahmadi, McClements, & Rafati, 2017).
57
Nanoemulsions can be fabricated by a number of different processing methods, which are
58
usually categorized as either high- or low-energy methods (McClements & Rao, 2011). High
59
energy approaches utilize different devices to generate intense disruptive forces leading to the
60
formation of the tiny oil droplets e.g., high pressure homogenizers, high speed homogenizers,
61
microfluidizers and ultrasonication methods (Ryu, McClements, Corradini, & McLandsborough,
62
2018). High speed homogenizers apply shear force for producing tiny droplets in emulsion
63
systems (Karthik
64
nanoemulsion by cavitation phenomenon where formation and collapse of vapor cavities in the
65
liquid medium occurred due to high intensity ultrasound (Mahdi Jafari, He, & Bhandari, 2006).
66
A literature survey revealed that no study has been conducted on the fabrication of Satureja
67
khuzestanica EO (SKEO) nanoemulsion. However, in recent years several reports have been
68
published concerning nanoemulsification of carvacrol as an antimicrobial agent (Chang,
69
McLandsborough, & McClements, 2013; Donsi, Annunziata, Vincensi, & Ferrari, 2012; Landry,
70
Chang, McClements, & McLandsborough, 2014; Landry et al., 2016; Nash & Erk, 2017; Tastan,
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Ferrari, Baysal, & Donsì, 2016). Ostwald ripening is known as the most important mechanism
72
affecting the stability of carvacrol nanoemulsions (Chang et al., 2013; McClements & Rao,
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2011). To the best of our knowledge, all methods reported for fabricating of carvacrol
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2016). Ultrasonication
process
produces
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nanoemulsions, utilize carrier oils e.g. MCT oil or sunflower oil in their formulations that
75
simultaneously decrease the rate of droplet growth due to Ostwald ripening (Chang et al., 2013;
76
Donsi et al., 2012; Landry et al., 2014; Landry et al., 2016; Nash & Erk, 2017). However, in
77
most cases, when the percentage of carvacrol in the nanoemulsions exceeds 2.5%, a significant
78
increase in particle size is observed after one month. Therefore, Ostwald ripening is still
79
recognized as the most important challenge for carvacrol containing nanoemulsions.
80
In the present study, we investigated the possibility of fabricating a stable nanoemulsion from
81
carvacrol-rich SKEO without the incorporation of carrier oil. A series of surfactants mixture
82
were utilized and the efficiency of two high energy methods was evaluated to determine the best
83
preparation method that led to the production of stable nanoemulsions with fine droplets. The
84
optimized nanoemulsion properties were applied for fabrication of carvacrol nanoemulsion that
85
showed appreciable stability over long time storage. Finally, the antimicrobial effects of pure
86
carvacrol and SKEO as well as their nanoemulsion formulations were evaluated against some
87
selected food-borne bacteria including Escherichia coli, Staphylococcus aureus and Salmonella
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enterica.
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1. Materials and Methods
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2.1. Chemicals
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Tween 80, Span 80, Tween 20 and Span 20 were purchased from Merck Millipore (Darmstadt,
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Germany). Carvacrol (98%) was purchased from Sigma-Aldrich (Germany). Satureja
94
khuzestanica essential oil was kindly provided by Dr. J. Hadian from Khorraman Pharmaceutical
95
Co.
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2.2. GC chromatography/Mass spectrometry (GC-FID, GC-MS)
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To analyze the EO components, GC analysis was carried out using a TRACE mass spectrometer
98
(Thermoquest, Manchester, UK), equipped with a flame ionization detector (FID) and a DB-5
99
column (30 m × 0.25 mm × 0.25 µm film thickness). Nitrogen was used as a carrier gas with a
100
flow rate of 1.1 mL/min. The oven temperature was gradually increased from 60 to 250°C at the
101
rate of 5℃/min, and then maintained at 250℃ for 10 min. The temperatures of the injection
102
chamber and detector were maintained at 250 and 300℃, respectively.
103
The constituents of the EO were identified using a GC coupled to a mass spectrometer
104
(Thermoquest-Finnigan, Manchester, UK) equipped with a DB-5 column (60 m × 0.25 mm ×
105
0.25µm film thickness). Helium gas with a flow rate of 1.1 mL/min was used as the mobile phase
106
and ionization energy was set to 70 eV. The temperature settings were similar to those given
107
above for the GC analysis.
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2.3. Nanoemulsion Preparation
109
Essential oil nanoemulsions were prepared from a mixture of SKEO (2 or 3% w/w), surfactants
110
(including Tween 20 or 80 and Span 20 or 80) with surfactant to oil ratio (SOR) of 2, 3 (4, 6 or
111
6, 9% w/w) and deionized water. The aqueous phase (solution of Tween in deionized water) was
112
poured into the oil phase consists of SKEO and Span. Nanoemulsions were then fabricated either
113
by utilizing a high speed homogenizer (SilentCrusher M, Heidolph, Germany) or a probe
114
sonicator (MPI, Dattwil, Switzerland). Homogenization process was performed 5 min at 15000
115
rpm applying a 12F dispersion tool which immersed in 5 mL sample. For fabricating of
116
nanoemulsions by ultrasonication method, a probe sonicator was used with 20.5 KHz frequency
117
and 30% maximum of power output (120 W). The energy input was provided using an ultrasonic
118
probe containing a piezoelectric crystal with a maximum probe diameter of 8 mm. The sonicator
119
was dipped into 3 mL of the mixture placed in ice bath and ultrasonic waves were applied for 10
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min. The hydrophilic lipophilic balance (HLB) value of SKEO was optimized according to the
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surfactant HLB values ranging from 8 to 15 using different combinations of Span 80 (HLB 4.3)
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and Tween 80 (HLB 15.0) (Nirmal, Mereddy, Li, & Sultanbawa, 2018).
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2.4. Particle Size Measurements
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Measurement of droplet size and particle size distribution of the nanoemulsions was performed
125
using a Dynamic Light Scattering (DLS) instrument (Nanophox Sympatec GmbH, Claushtal,
126
Germany). Nanoemulsions were diluted with deionized water to have a specified particle count
127
range, between 200 and 1000 kCPS.
128
2.5. Nanoemulsion Stability
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Long-term stability of nanoemulsions was evaluated by determination of mean particle diameter
130
for 60-90 days storage at room temperature and refrigerator respectively.
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2.6. Transmission Electron Microscopy
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Morphology and structure of optimum nanoemulsion were evaluated using transmission electron
133
microscopy (TEM). A 20 L drop of the sample was placed on a Carbon film coated on 300 mesh
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copper grid (Agar) for 2 min. Excess liquid was absorbed with filter paper then negatively
135
stained with a 20 L drop of 2% w/v uranyl acetate for 1-2 min and the grid was allowed to air
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dry. Grid was examined on a Zeiss EM900 transmission electron microscope operating at an
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accelerating voltage of 80 kV.
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2.7. Antimicrobial Activity
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Determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations
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The antimicrobial activity of pure SKEO, pure carvacrol and their related nanoemulsions were
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evaluated against Escherichia coli PTCC1339, Staphylococcus aureus ATCC25923, or
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Salmonella enterica PTCC1639 using a serial dilution method. Aliquots of samples were serially
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diluted in Mueller Hinton Broth (MHB) medium in 96 well plates, to produce a concentration
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range of 0.03 to 64 mg/mL. To prevent possible solubility problem in MIC determination of pure
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SKEO or carvacrol, a 0.5% v/v Tween 80 solution was added to the medium. The final
146
concentration of microorganisms was adjusted to 5 × 106 CFU/mL. Plates were incubated at
147
37ºC for 24 h and the MIC values were determined as the lowest concentration of samples that
148
demonstrated no visible growth of microorganisms. For MBC determination, 100 µL of the
149
samples from wells without visible growth were transferred onto Mueller Hinton Agar (MHA)
150
and were incubated for 24 h at 37ºC. The lowest concentration of the sample that could kill all of
151
the initial bacterial population is reported as the MBCs.
152
2.8. Statistical analysis:
153
Each experiment was performed in triplicates and all values are reported as mean ± standard
154
deviation (SD) by Microsoft Excel. The data was statistically analyzed by one-way analysis of
155
variance (ANOVA) and p values less than 0.05 were considered statistically significant.
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3. Results and Discussions
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3.1. Essential Oil Analysis
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The SKEO was analyzed by GC-FID and GC-MS methods. Twenty-one components were
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identified representing 99.12% of the total oil. The qualitative and quantitative EO compositions
161
are presented in Table 1, where compounds are listed according to the elution time on the DB-5
162
column. The major constituents of the EO were carvacrol (87.16%) and p-cymene (6.39%). High
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level of carvacrol content (between 87.7 and 93.9%) in the SKEO were reported in previous
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studies which proposes this endemic medicinal plant as a natural source of carvacrol for
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application in food industries (Farsam et al., 2004; Hashemi et al., 2012).
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3.2. Effect of Surfactant Type on Particle Size
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Initially, a SKEO nanoemulsion was prepared by homogenization of 3% w/w EO with 6%w/w
168
surfactants (Tween 80 and Span 80) with a required HLB 10 (determined based on the
169
preliminary studies), and 91%w/w water. To assess whether the defined HLB value could be
170
suitable for fabrication of stable SKEO nanoemulsions, different combinations of surfactants
171
including Tween 20, Tween 80, Span 20 and Span 80 were utilized for determination of the best
172
surfactant mixture. The mean particle diameter of the resulting nanoemulsions, just after
173
preparation and a week are represented in Figure 1A. The results implied that the initial diameter
174
of nanoemulsion droplets was strongly depended on the type of the oil soluble surfactant. Using
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Span 80 as the oil soluble surfactant in the presence of either Tween 20 or Tween 80 resulted in
176
the production of nanodroplets with almost the same particle size (p>0.05). The use of Span 20
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resulted in the production of much smaller nanoemulsion droplets at the preparation time
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compared to Span 80. Since usually emulsifiers having different HLB value are being used in
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nanoemulsion preparation, hydrophobic emulsifiers having low HLB value are located inside
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the oil droplets and the hydrophilic emulsifiers are outside, which makes hydrocarbon chains in
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tail in close contact (Cho, Kim, Bae, Mok, & Park, 2008). Span 20 contains a saturated 12
182
carbon atom chain of lauric acid with a fairly linear structure compared to the more kinked
183
structure of unsaturated oleic acid moiety presents in Span 80. This spatial difference affects the
184
arrangement of the surfactant molecules in the oil-water interface that can cause more compact
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structure and facilitate the production of smaller droplets when Span 20 is used as the oil soluble
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surfactant. In the case of Span 20, the utilization of Tween 80 as the water soluble surfactant
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results to production of smaller droplets compared to Tween 20 (p<0.05). Tween 20 contains a
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fairly linear structure of saturated lauric acid compared to the more kinked structure of
189
unsaturated oleic acid moiety presents in Tween 80 similar to those of Spans. It has been
190
reported that the presence of double bond in the hydrophobic tail of nonionic surfactants is
191
preferable for producing of smaller nanoemulsion droplets (Wang, Dong, Chen, Eastoe, & Li,
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2009).
193
It is noteworthy that there is not a strong correlation between the primary particle size of
194
nanoemulsions and stability over a period of time. Nanoemulsions that contain Span 20 as the oil
195
soluble surfactant produced smaller droplets at the preparation time, however, the significant
196
enhancement of droplet size was observed after a week. Using Span 80 as the oil soluble
197
surfactant resulted in the production of nanoemulsions which were more resistant against droplet
198
size enhancement due to Ostwald ripening. It has been reported that packing form of the
199
surfactant molecules at the oil-water interface that conducted from their molecular geometry, has
200
a significant impact on the formation of nanoemulsions (Israelachvili, 2011). The packing
201
parameter (p) of a surfactant defined as the cross-sectional area of the tail group relative to that
202
of the head group (p=aT/aH) (Komaiko & McClements, 2016). Span 20 has a saturated linear tail
203
group whereas Span 80 has an unsaturated kinked one. Thus, Span 80 would be expected to have
204
a higher packing parameter (due to a larger aT) that correlates to production of more stable
205
droplets compared to those produced by Span 20. Therefore, it can be concluded that, in addition
206
of the hydrophilic-lipophilic balance (HLB) value of surfactants mixture, the geometry of
207
surfactants plays a crucial role in the stability of nanoemulsions droplets (Schmidts, Dobler,
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Nissing, & Runkel, 2009).
209
3.3. Effect of Nanoemulsion Preparation Method on Particle Size
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To evaluate the impact of preparation method on the primary droplet size and stability of
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nanoemulsions, the above mentioned nanoemulsions were fabricated using a probe sonicator.
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The mean particle diameter of the resulting nanoemulsions that measured at preparation time and
213
after one week represented in Figure 1B. Comparison of the mean particle diameter of prepared
214
nanoemulsions with those fabricated by high speed homogenizer, revealed that the
215
homogenization method is more efficient in the producing of smaller droplets. Although higher
216
energy level is used in the ultrasonication method and consequently, smaller droplets are
217
expected to be produced, but the cavitation phenomenon does not seem to provide the sufficient
218
time required for the proper arrangement of surfactant molecules around the oil. This could be
219
correlated to the high surfactant concentration used to prepare nanoemulsions that led to the
220
aggregation of surfactant molecules around the oil droplets. Both homogenization and
221
ultrasoniaction method showed similar pattern in the primary particle size and stability of
222
nanoemulsions over a week. Some probable consideration was implied in the previous section.
223
3.4. Effect of Surfactant and EO Concentration on Particle Size and Long-Term Stability
224
To evaluate the effect of surfactant and EO concentration on the primary droplet size and long
225
term stability, fabrication of SKEO nanoemulsion was performed by homogenization of 2 or 3
226
wt% EO and surfactant to oil ratio (SOR) of 2 and 3 (Tween 80 and Span 80 in HLB 10).
227
Preliminary experiments indicated that the equal concentration of surfactant and EO (SOR=1)
228
could not result in the formation of nanoemulsion. Figure 2 represents the changes in mean
229
particle diameter of nanoemulsions over a period of two months. As expected, increasing the
230
percentage of EO led to the formation of larger droplets when the percentage of surfactants
231
remained constant. It is also clear that when the percentage of the EO is constant, the particle
232
size decreases with increasing surfactant percentage. Increasing the surfactant concentration
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leads to the accumulation of more surfactant molecules at the interface and from the organic
234
phase into the aqueous phase that facilitates the formation of smaller droplets at the oil-water
235
interface. In addition, concentrated surfactant solutions provide different structural arrangements
236
of the surfactant, oil and water molecules in the system, and some of these arrangements are
237
favorable for nanoemulsion formation (Lamaallam, Bataller, Dicharry, & Lachaise, 2005). Long-
238
term stability study of nanoemulsions based on the mean particle diameter measurement revealed
239
that some fluctuations were observed for nanoemulsion contain 2 % EO with SOR=3. Three
240
other nanoemulsions showed acceptable stability over two months storage at room temperature.
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3.5. Effect of Different HLB Values of SKEO on Particle Size and Long-Term Stability
242
For determination of the best HLB system, SKEO nanoemulsions were prepared by
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homogenization (5 min, 15000 rpm) of 3 wt% EO and 9 wt% surfactant mixture (Tween 80 and
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Span 80) with HLB values ranging from 8 to 15. The first nanoemulsion with HLB value of 8,
245
resulted in an apparent creaming process and was eliminated from further investigations. A
246
portion of each of the nanoemulsions was stored in the refrigerator and the other part was kept at
247
ambient temperature. Figure 3 represents the changes in mean particle diameter of
248
nanoemulsions over a period of two and three month storage at room and refrigerator
249
temperatures. The primary particle diameter of prepared nanoemulsion with HLB=9 was 148 nm
250
and nanoemulsions with HLB values of 10 to 15 formed smaller droplets ranging from 70 to 95
251
nm. The long-term stability studies indicated that nanoemulsions with HLB values more than 10
252
showed significant enhancement in the mean particle diameter so that phase separation was
253
occurred for the nanoemulsion with HLB=15 after six weeks. This remarkable difference in the
254
long-term stability of nanoemulsions indicates that the HLB value of surfactants mixture could
255
be considered as an important factor that controls the rate of droplets growth due to Ostwald
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ripening mechanism. In addition, as shown in Figure 3B, the storage of the nanoemulsions at a
257
refrigerated temperature showed that the rate of droplet growth decreases significantly. This
258
observation is in accordance with the theory proposed by Lifshitz-Slezov and Wagner that
259
implied the rate of droplet growth due to Ostwald ripening is inversely correlated to the absolute
260
temperature (Delmas et al., 2011; Ee, Duan, Liew, & Nguyen, 2008).
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Since the optimum nanoemulsion with HLB value of 10 represented appreciable stability over
262
long time storage, the pure carvacrol nanoemulsion was fabricated based on the defined
263
properties with the same method. No significant differences were observed between SKEO
264
nanoemulsion and pure carvacrol nanoemulsion based on the mean particle diameter and long-
265
term stability (data not shown). Previous reports concerning nanoemulsification of carvacrol
266
utilized carrier oils in their formulation that couldn’t inhibit the Ostwald ripening completely
267
when carvacrol was more than 2.5% in formulation (Chang et al., 2013; Donsi et al., 2012;
268
Landry et al., 2014; Landry et al., 2016). Therefore, the present study is the only report that has
269
been able to form a nanoemulsion containing 3% carvacrol without the incorporation of carrier
270
oil, and showed considerable long term stability. In addition, as shown in Figure 4, the small
271
polydispersity index (PDI) value of optimized formulation (<0.1) indicated that the particles
272
were of a narrow size range.
273
3.6. Antimicrobial Efficacies of Carvacrol and SKEO Nanoemulsions
274
The antimicrobial effects of SKEO, carvacrol and their optimized nanoemulsions against two
275
strains of foodborne Gram negative (Escherichia coli PTCC1339 and Salmonella enterica
276
PTCC1639) and Gram positive (Staphylococcus aureus ATCC25923) pathogenic microorganism
277
are shown in Table 2. The results indicated that the optimized nanoemulsions showed better
278
antimicrobial activity than the bulk oil against S. enterica and S. aureus for carvacrol and SKEO,
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respectively. An improved antimicrobial activity of the nanoemulsions compared to the bulk oil
280
was also observed in our previous study (Moghimi, Ghaderi, Rafati, Aliahmadi, & McClements,
281
2016). In other cases, the antimicrobial activity of optimized nanoemulsions was similar to that
282
of the bulk oils for all three bacterial strains. These results are in agreement with some previous
283
reports that indicated nanoemulsions have similar antimicrobial activity to bulk oils (Ghaderi et
284
al., 2017). In addition, no significant difference was observed in the antimicrobial activity of
285
carvacrol, SKEO and their optimized nanoemulsions against both gram positive and gram
286
negative bacteria.
287
It has been reported that hydrophobicity of natural EO components such as carvacrol could be
288
an advantage for inducing antibacterial properties. It is well known that lipophilic compounds
289
possess a high affinity for cell membranes and their insertions induce changes in membrane
290
physicochemical properties. The interactions of antimicrobial compounds and cell membranes
291
are considered to affect both the lipid ordering and the bilayer stability, resulting in a membrane
292
integrity decrease and potential depolarization (Ben Arfa, Combes, Preziosi-Belloy, Gontard, &
293
Chalier, 2006; Xu, Zhou, Ji, Pei, & Xu, 2008). The bacterial membrane perturbations caused by
294
carvacrol, lead to the leakage of intracellular ATP, proton and potassium ions and ultimately cell
295
death (Liolios, Gortzi, Lalas, Tsaknis, & Chinou, 2009). Microencapsulation prevents reactivity
296
of bioactive component with the environment (water, oxygen, light) and decreases the
297
evaporation or the transfer rate to the outside region (Liolios et al., 2009).
298
nanoemulsions in order to deliver EOs bioactive components to the biological membranes, can
299
also provide the necessary protection against their oxidation and evaporation (Flores et al.,
300
2011), while the incorporation of food antimicrobials could aid in the protection of food products
301
against growth of spoilage and pathogenic microorganisms.
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4. Conclusion
304
In the present work, carvacrol and SKEO nanoemulsions were fabricated without the
305
incorporation of any other carrier oil. Hydrophilic Lipophilic Balance (HLB) value and
306
molecular geometry of surfactants showed significant impact on initial droplet size and
307
nanoemulsions stability. Comparison of two high energy preparation methods implied that high
308
speed homogenization was more efficacious than ultrasonication in the production of smaller
309
droplets. In addition, the impact of surfactant to oil ratio and HLB value of surfactants mixture
310
were evaluated on the nanoemulsions stability during long-term storage. Optimized
311
nanoemulsions containing 3 % EO and 9 % surfactant mixture (Tween 80+Span 80) with HLB
312
value of 10 showed appreciable stability against droplet growth due to Ostwald ripening. The
313
antimicrobial activity of optimized carvacrol and SKEO nanoemulsions improved compared to
314
that of the bulk oils. Therefore, the optimized formulation could be considered as a stable
315
carvacrol delivering system that preserved the antibacterial activity for utilization in food
316
industries.
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Acknowledgment
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This work has been supported by Shahid Beheshti University Research Council and the authors
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gratefully acknowledge the support provided by MPDRI. The kind assistance of Ms. Lida
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Ghaderi in determination of antibacterial effects of nanoemulsions is gratefully appreciated. The
322
authors appreciate help provided by Khorraman Pharmaceutical Co.
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Figure captions:
433
Figure 1. Effect of surfactant type on mean particle diameter of SKEO nanoemulsions produced
434
by (A) homogenization method and (B) ultrasonication method.
435
T80: Tween 80, T20: Tween 20, S80: Span 80, S20: Span 20. (HLB value of surfactants mixture
436
=10). (
437
Figure 2. Effect of surfactant to oil ratio (SOR) on mean particle diameter and stability of SKEO
438
nanoemulsions. (
439
SOR 3) SKEO: Satureja khuzistanica essential oil
440
Figure 3. Effect of HLB value of surfactant mixture (Tween 80 and Span 80) on mean particle
441
diameter of SKEO nanoemulsions and storage stability at different temperatures (A) 25°C and
442
(B) 4°C. (
443
Satureja khuzistanica essential oil
444
Figure 4. (A) Particle size distribution diagram of optimized SKEO nanoemulsion measured by
445
DLS (B) Transmission electron microscopy (TEM) image of optimized SKEO nanoemulsion.
446
SKEO: Satureja khuzistanica essential oil
RI PT
432
After one week) SKEO: Satureja khuzistanica essential oil
10,
11,
12,
AC C
EP
TE D
9,
2% EO, SOR 3.
3% EO, SOR 2.
M AN U
2% EO, SOR 2.
SC
Preparation time,
21
13,
14,
3% EO,
15) SKEO:
ACCEPTED MANUSCRIPT
Compounds
RI
%
1
α-pinene
935
0.63
2
camphene
951
0.05
3
β-myrcene
992
4
α-phellandrene
1007
5
3-carene
1013
6
α-terpinene
1018
7
p-cymene
8
γ-terpinene
9
p-cymenene
1091
0.17
10
linalool
1100
0.51
11
borneol
1167
0.7
12
terpinen-4-ol
1178
0.08
13
L-α-terpineol
1191
0.23
14
carvacrol methyl ether
1242
0.11
15
thymol
1294
0.25
carvacrol
1325
87.16
carvacrol acetate
1372
0.53
trans-β-caryophyllene
1418
0.09
trans-α-bergamotene
1432
0.09
β-bisabolene
1506
0.67
α-bisabolene
1538
0.05
17 18 19
21
SC
0.89
0.06
0.06
0.34
1027
6.39
1059
0.06
M AN U
AC C
20
EP
16
RI PT
No.
TE D
Table 1. Chemical composition of the essential oil of Satureja khuzistanica Jamzad
RI: retention indices relative to C9-C22 n-alkanes on the DB-5 column
ACCEPTED MANUSCRIPT
Table 2. Antibacterial activity of carvacrol, SKEO and their optimized nanoemulsions against selected bacteria
SKEOa SKEO nanoemulsion
ATCC25923
PTCC1639
MIC
MBC
MIC
MBC
MIC
MBC
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
0.25
0.25
0.125
2
0.5
0.5
0.25
0.25
0.125
1
0.125
0.25
0.25
0.25
0.25
0.25
0.25
0.125
EP
TE D
SKEO: Satureja khuzestanica essential oil
AC C
a
PTCC1339
RI PT
nanoemulsion
Salmonella enterica
SC
Carvacrol
Staphylococcus aureus
2
0.25
0.25
1
0.25
0.25
M AN U
Carvacrol
Escherichia coli
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
•
SKEO : Satureja khuzestanica essential oil, HLB : hydrophilic lipophilic balance
2
•
Stable nanoemulsions were fabricated from carvacrol and SKEO
3
•
The best surfactants mixture defined as Tween 80: Span 80 with HLB value of 10
4
•
Ostwald ripening was inhibited by optimization of HLB value of surfactants
5
•
The MIC value of nanoemulsions improved 2-4 times compared to the bulk oils in two
EP
TE D
M AN U
SC
strains
AC C
6
RI PT
1
1