Accepted Manuscript

Cinnamon oil nanoemulsions by spontaneous emulsification: Formulation,

characterization and antimicrobial activity

Simge Tutku Yildirim, Mecit Halil Oztop, Yesim Soyer

PII: S0023-6438(17)30356-0
DOI: 10.1016/j.lwt.2017.05.041
Reference: YFSTL 6257

To appear in: LWT - Food Science and Technology

Received Date: 5 August 2016

Revised Date: 23 April 2017
Accepted Date: 21 May 2017

Please cite this article as: Yildirim, S.T., Oztop, M.H., Soyer, Y., Cinnamon oil nanoemulsions by
spontaneous emulsification: Formulation, characterization and antimicrobial activity, LWT - Food
Science and Technology (2017), doi: 10.1016/j.lwt.2017.05.041.

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 ACCEPTED MANUSCRIPT
1 Cinnamon Oil Nanoemulsions by Spontaneous Emulsification: Formulation,

2 Characterization and Antimicrobial Activity

3 Simge Tutku Yildirim1, Mecit Halil Oztop1*, Yesim Soyer1

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Department of Food Engineering, Middle East Technical University, Ankara, Turkey

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11 Corresponding Author: Dr. Mecit Halil Oztop

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13 Phone: +90 312 210 5632

14 Fax: +90 312 210 2767

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15 E-mail: mecit@metu.edu.tr

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23 Running title: Cinnamon Oil Nanoemulsions by Spontaneous Emulsification (SE)…

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25 Abstract

26 The goal of this study was to formulate stable cinnamon oil nanoemulsions (NEs) exhibiting

27 high antimicrobial activity by using the low-energy approach: spontaneous emulsification

28 (SE) and compare it with two high-energy methods. To prepare the nanoemulsions by SE, oil

29 phase containing cinnamon oil (CO) and carrier oil (coconut oil (CNO)) at different ratios

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30 (2:8-10:0) and surfactant (Tween 80) at 10% (w/w) was titrated into an aqueous phase

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31 (distilled water). For antimicrobial activity, agar disc diffusion method with E.coli as the

32 model microorganism was used. NEs were characterized by Dynamic Light Scattering (DLS)

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33 and Transmission Electron Microscopy (TEM). Both DLS and TEM gave parallel results and

34 mean particle size were found as ~100 nm for 6:4 (CO: CNO) oil phase composition. These

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NEs also showed high physical stability during one-month storage. NEs were also prepared
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36 by using two high-energy homogenization methods: microfluidization and ultrasonication.
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37 Ultrasonication and SE showed similar trends for mean particle size and microbial activity.

38 Microfluidization resulted in the smallest mean particle size (p<0.05) and antimicrobial
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39 activity was not effected from cinnamon oil concentration (p>0.05).

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48 Key Words: Coconut Oil; Spontaneous Emulsification; Microfludization; Ultrasonication;

49 Antimicrobial Activity

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50 1. INTRODUCTION

51 Essential oils have strong antimicrobial, antioxidant and antiradical activity due to terpenoid,

52 aldehyde and phenolic constituents (Chang, Mclandsborough, & McClements, 2013). These

53 bioactive compounds present in essential oils show strong antimicrobial activity against

54 important food pathogens such as Escherichia coli, Listeria monocytogenes, Pseudomonas

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55 aeruginosa, Staphylococcus aureus, Helicobacter pylori and Salmonella Typhi (Sugumar et

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56 al., 2013; Ghosh, Mukherjee, & Chandrasekaran, 2014). To prevent the microorganism

57 proliferation and to substitute the synthetic antimicrobial compounds used in foods through

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58 natural ways, essential oils are considered as alternative antimicrobial additives (Chang et al.,

59 2013; Bassolé & Juliani, 2012). For the dispersion of these lipid particles, colloidal delivery

60
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systems such as oil-in- water (O/W) emulsions or nanoemulsions are used to entrap the
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61 functional components into the aqueous based foods, beverages and packaging materials
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62 (Chang, McLandsborough, & McClements, 2012).

63 Nanoemulsions have droplet sizes ranging between 20 nm to 300 nm and to distinguish them
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64 from conventional emulsion, there is not an identified clear size range (Anton & Vandamme,
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65 2009; Chang et al., 2013; Komaiko & McClements, 2015; Mahdi Jafari, He, & Bhandari,

66 2006). From a physiochemical point of view, nanoemulsions (NEs) are thermodynamically

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67 unstable but kinetically stable systems. High kinetic stability can be obtained when

preparation method, composition and component of the system is appropriately selected

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69 (Solans & Solé, 2012).

70 Fabrication of nanoemulsions are basically achieved by high and low energy approaches

71 (Acosta, 2009; Tadros, Izquierdo, Esquena, & Solans, 2004). Mechanical devices are used for

72 high energy methods such as high- pressure homogenizers (Quintanilla-Carvajal et al., 2010),

73 ultrasound generators (Maa & Hsu, 1999). By using mechanical devices, generation of

74 intensive disruptive forces lead to formation of oil droplets while breaking up the water and

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75 oil phases (McClements, 2012). On the other hand, ultrafine droplet formation with low

76 energy methods relies on the internal chemical energy of the system. Membrane

77 emulsification (Sanguansri & Augustin, 2006), spontaneous emulsification (Bouchemal,

78 Briançon, Perrier, & Fessi, 2004), solvent displacement (Yin, Chu, Kobayashi, & Nakajima,

79 2009), emulsion inversion point (Sadtler, Rondon-Gonzalez, Acrement, Choplin, & Marie,

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80 2010) and phase inversion point (Shinoda & Saito, 1969) are some of the used low energy

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81 methods.

82 Spontaneous emulsification (SE) is a widely used technique to form nano-scaled particles.

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83 Without causing a change in the curvature of the surfactant, its movement from the dispersed

84 phase to the continuous phase leads to the formation of a nanoemulsion (Solans & Solé,

85
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2012). Surfactant to oil ratio (SOR), surfactant type and oil type influence the size of the
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86 droplets produced by this method. High amount of surfactant is required to stabilize the
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87 droplets formed. If the concentration is insufficient, a protective coating does not form and

88 consequently particles collide with each other and droplet aggregation is observed (Komaiko
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89 & McClements, 2015). Requirement of the proper surfactant concentration to obtain small
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90 particle size strongly depends on the phase behavior of the surfactant- oil-water (SOW)

91 system that is used to form the nanoemulsion since formation of ultrafine droplets by using
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92 SE occurs only at certain SOW compositions (Rao & McClements, 2011b).

In this study, Tween 80 (Polysorbate 80) was used as the main surfactant. Tween 80 is a
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94 nonionic, single tail surfactant and is very commonly used as an emulsifier in foods and

95 pharmaceutical products (Athas et al., 2014).

96 Due to high eugenol (4-allyl-2-methoxyphenol) and cinnamaldehyde (3-Phenyl-2-propenal)

97 content, cinnamon leaf oil exhibits high antimicrobial and antifungal activities (Tzortzakis,

98 2009). There is recent study where cinnamon oil was encapsulated through spontaneous

99 emulsification by using Tween 80 and medium chain TGs (Tian, Lei, Zhang, & Li, 2016).

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101 In the current study, cinnamon leaf oil (C. zeylanicum) was used as the antimicrobial agent

102 and rather than a pure medium chain triglyceride mixture, coconut oil (Cocos nucifera) was

103 used as the carrier oil to formulate a stable nanoemulsion. In addition, the physical stability

104 and antimicrobial activity of the nanoemulsions were investigated and compared with two

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105 high-energy methods: microfluidization and ultrasonication techniques. In this study, it was

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106 hypothesized that stable nanoemulsions that maintain high antimicrobial activity could be

107 obtained by using coconut oil as a ripening inhibitor due to its high content of Medium Chain

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108 Fatty Acids (MCFAs) (> 50 wt. % of fatty acids) (Marten, Pfeuffer, & Schrezenmeir, 2006).

109 Antimicrobial activity of cinnamon oil nanoemulsions was tested against a model bacteria

110 strain: E. coli. ATCC 25922.

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111 2. MATERIALS AND METHODS
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112 Cinnamon oil, ethanol and barium chloride (for McFarland standard preparation) were

113 purchased from Sigma-Aldrich (St. Louis, MO, USA). Coconut oil (KRK Gıda, Turkey) was
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114 obtained from a local grocery store.

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115 Whatman (Maidstone, UK) filter papers were used for antimicrobial activity experiments. The

nonionic surfactant: Tween 80 and sulfuric acid were obtained from Merck chemicals
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117 (Darmstadt, UK). For antimicrobial tests, ATCC25922 E. coli strain was kindly provided by
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118 Food Safety Laboratory in Food Engineering Department at Middle East Technical
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119 University. To prepare brain heart infusion (BHI) agar, agar bacteriological (Agar No.1) and

120 BHI broth (Bury, Lancashire, UK) were purchased from OXOID. Mueller Hinton Agar

121 (MHA) and Mueller Hinton Broth (MHB) was purchased from OXOID (Basingstoke,

122 Hampshire, England). Antimicrobial susceptibility test disc, tetracycline (TE) was purchased

123 from OXOID (Basingstoke, Hampshire, England). Distilled water was used in all

124 experiments.

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125

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127 Compositional Analysis of Coconut Oil

128 Fatty acid composition analysis was conducted at the facilities of TUBITAK Marmara

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129 Research Center (Gebze, Kocaeli, Turkey). ISO 12966-2:2011 method was followed for the

130 analysis and Perkin Elmer gas chromatography system (Auto system GLX, Shelton, U.S.A.)

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131 that included a flame ionization detector (FID) was used. Fused silica capillary column SP™

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132 −2380 (100 m length × 0.25 mm with a 0.25 µm film thickness) that was obtained from

133 Supelco (Bellefonte, U.S.A.) was used for the chromatographic separation of fatty acid

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methyl esters (FAMEs). Results are given in Table S1.
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135 2.1 Preparation of Emulsions
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136 2.1.1 Low Energy Method

137 Spontaneous emulsification was carried out by the titration of an organic phase containing
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138 Tween 80 and different amount of cinnamon oil and coconut oil into an aqueous phase while
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139 the system was continuously being stirred at 750 rpm with a magnetic stirrer at a temperature

140 of 25 °C ±1. Experiments were performed using standardized conditions: 10 % (w/w) oil
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141 (cinnamon + coconut), 10 % surfactant (Tween 80) (w/w) and 80 wt % aqueous phase (w/w)
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142 (distilled water). Emulsions with varying cinnamon oil concentrations (2%, 4%, 6%, 8% and
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143 10%) were prepared for particle size and antimicrobial activity experiments. Coconut oil and

144 cinnamon oil were stirred for 30 minutes and then Tween 80 was added to the mixture and

145 stirred for 30 minutes. The resulting mixture was titrated into an aqueous phase at a rate of 1

146 ml /min using a 5 mL syringe. For some experiments, nanoemulsions were also prepared at

147 different surfactant to oil ratios (SOR: 5, 10, 20)

148 2.1.2 High Energy Methods

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149 2.1.2.1 Ultra- Turrax

150 Pre-homogenization of O/W nanoemulsions were achieved by mixing oil phase (cinnamon oil

151 + coconut oil + Tween 80) and aqueous phase (distillate water) at a total volume of 100 mL

152 with Ultra-Turrax (WiseTis Homogenizer, Witeg Labortechnik GmbH, Germany) at 10,000

153 rpm for 2 min. If excess heating of the solution is observed, beaker was soaked into an ice

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154 bath.

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155 2.1.2.2 Microfluidization

156 To investigate the effect of microfluidization on particle size and antimicrobial activity, pre-

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157 homogenized nanoemulsions containing 10 wt % oil, (2:8, 4:6, 5:5, 6:4, 8:2, 10:0, cinnamon

158 oil: coconut oil), 10 wt % surfactant (Tween 80) and 80 wt % aqueous phase (distillate water)

159
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were subjected to high pressure microfluidization (Nano Disperser - NLM 100, South Korea).
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160 The samples were treated at 900 bar for 3 passes. After passing through the microfluidizer, the
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161 sample was collected to conduct particle size measurements and antimicrobial tests.

162 2.2.2.3 Ultrasonication

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163 To examine the effect of ultrasonication on particle size and antimicrobial activity of
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164 nanoemulsions, oil mixtures (2:8, 4:6, 5:5, 6:4, 8:2, 10:0, cinnamon oil: coconut oil) were

165 obtained following 30 minutes mixing using the magnetic stirrer. Following stirring,
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166 surfactant was added to the mixtures and the obtained organic phase was mixed for another 30

minutes. Nanoemulsion was formed when organic phase (10 wt% (cinnamon oil + coconut
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168 oil) + 10 wt% Tween 80) and aqueous phase (distillate water) mixture was sonicated using an

169 ultrasonicator (Bandelin Sonoplus HD 3100, Bandelin electronic GmbH & Co. KG, Berlin

170 Germany) (sonotrode: TT13) for 10 min at 75 W.

171 2.2 Characterization of Nanoemulsions

172 2.2. 1 Mean Particle Size Measurement

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173 Experiments were conducted at METU Central Lab Facilities. Dynamic light scattering

174 (MALVERN Nano ZS90, Worcestershire, UK) was used to determine the particle size

175 distribution and to measure the mean particle diameter (Z-averages). By using intensity time

176 fluctuations of laser beam (633 nm) that is scattered from the sample with 173o, the mean

177 particle size of samples was determined. On an average, 15 run was done for each individual

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178 measurement. Samples were diluted with distilled water before the measurement to avoid

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179 multiple scattering effect.

180 Poly Dispersity Index (PDI), which indicates the homogeneity of the distribution, was also

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181 recorded during measurements.

182 2.2.2 Transmission Electron Microscopy (TEM)

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TEM experiments were conducted at METU Central Lab Facilities. Transmission Electron
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184 Microscopy (FEI Tecnai G2 Spirit BioTwin CTEM, Oregon USA) was used to examine the
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185 morphology and size of nanoparticles. Samples were prepared while transferring diluted

186 solution to a freshly glow discharged TEM copper grid (300 mesh copper Formvar / Carbon)
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187 Afterwards samples were allowed to dry at room temperature. TEM images were obtained for
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188 the most stable emulsions only.

189 2.3 Antimicrobial Activity

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190 2.3.1 Agar Disc Diffusion Method

Before the disc diffusion experiments, E. coli was inoculated on a BHI agar. It was incubated
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at 37 oC for 20-24 hours (ET 120 Oven, Şimşek Laborteknik, Turkey). Following incubation,
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193 several colonies were selected and suspended in MHB. After incubating at 37oC for 2 h,

194 turbidity of the suspensions was controlled with 0.5 McFarland standard. One colony was

195 chosen and inoculated on MHA for the disc diffusion tests. Inoculation was performed using a

196 cotton swab. Entire plate was covered by streaking back and forth from edge to edge.

197 Swabbing was repeated 3 times while rotating the plate 60o. Standard 6 mm paper discs that

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198 were obtained from filter paper were used for disc diffusion tests. 20 µl of the active

199 compound containing nanoemulsion was put on a disc. After 30 minutes the discs were placed

200 on the inoculated plate while pressing each disc down firmly. When the incubation was

201 completed, zone diameters around the discs were measured.

202

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203 2.4 Statistical Analysis

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204 Using freshly prepared samples, all experiments were carried out at least three times and

205 results were reported as the mean values. Analysis of Variance (ANOVA) was performed by

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206 using Minitab (ver.16.2.0.0, Minitab Inc., United Kingdom) with Tukey’s test and results

207 were considered as statistically significant when p < 0.05. Significant differences between

208
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209 constant variance requirements were checked for ANOVA to be meaningful. Particularly for
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210 the comparison of mean particle sizes wrt to homogenization type, these assumptions were

211 not satisfied for the raw data. Therefore, log 10 transformation on mean particle size was
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212 applied while making the comparison and ANOVA. Using Minitab, Anderson Darling and
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213 Bartlett’s test were used for normality and testing the equality of variances respectively.

214 3. RESULTS AND DISCUSSION

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215 3.1 Effect of Oil Phase Composition on Nanoemulsion Formation and Mean Particle Size

The effect of oil phase composition on the characteristics of the emulsions that were prepared
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217 by spontaneous emulsification was investigated. Effect of oil composition on polydispersity

218 index (PdI) and mean particle size of nanoemulsions were shown in Figure 1a. While

219 cinnamon oil concentration in organic phase increased, initial mean particle diameter

220 decreased until a minimum value. The mean particle diameter was d ≈ 343 nm at a CO: CNO

221 ratio of 2:8 and a milky white cream layer was observed on the top of the emulsion in a short

222 time. Nanoemulsions were highly unstable and phase separation was observed at cinnamon oil

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223 concentrations around 10%. Smaller droplets were obtained, 101 nm and 81 nm respectively

224 for 6% and 8% CO concentrations in the organic phase. However, PdIs at these

225 concentrations were quite different (Figure 1a). Despite the small particle size at 8:2 (CO:

226 CNO) ratio, phase separation was observed for undiluted samples in a short period. However,

227 for 6:4 CO: CNO nanoemulsions, narrower size distribution was observed. It was observed

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228 from the particle size distribution plot that at 8:2 CO to CNO ratio, two different peaks were

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229 present (Figure 1b). This was an indicator of the inhomogeneity on the samples. Likewise, for

230 10:0, 4:6 and 2:8 CO to CNO ratios a wide range of particle sizes was observed. Only for 6:4

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231 ratio, one peak existed. For this reason, physical stability test was conducted at 6:4 (CO:

232 CNO) ratio. These results indicated that by using spontaneous emulsification method, there

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was an optimal oil phase composition to obtain more stable, smaller droplets containing
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234 nanoemulsions. It is known that although to form nanoemulsions of small droplets is possible
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235 by using essential oils, Ostwald ripening (OR) could be a problem (Davidov-Pardo &

236 McClements, 2015). OR can be inhibited by the addition of the appropriate amount of highly
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237 hydrophobic materials (corn oil, MCT) into the oil phase. Those ripening inhibitors affects the
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238 initial size of the droplets and afterwards affects the stability of these droplets against growth

239 (Chang et al., 2013). Addition of a second oil into the organic phase decreased the droplet size
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240 to a certain extent (Davidov-Pardo & McClements, 2015). In this study, coconut oil was used

as the carrier oil and it was obvious that it had a ripening inhibitor effect since stable
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242 nanoemulsions could not be formed with cinnamon oil alone.

243 To confirm the particle shape and measured particle size in accordance with DLS

244 measurements, Transmission electron microscopy (TEM) analysis was also carried out

245 (Bouchemal et al., 2004). Transmission electron micrographs of cinnamon oil nanoemulsions

246 with 6:4 CO: CNO ratio, are given in Figure 2. Results indicated that emulsion droplets were

247 spherical in shape and the droplet size was in nanometric range. Moreover, size of particles in

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248 images (≈100 nm) were almost same with the results of dynamic light scattering (Figure 2).

249 Photos of the nanoemulsions prepared at various cinnamon oil concentrations are also given

250 in Fig. S2a.

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252

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253 3.2 Effect of Surfactant Concentration on Mean Particle Size

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254 For food-grade nanoemulsions, determination of the minimum surfactant concentration

255 resulting in the most stable nanoemulsions with small droplet size is very significant due to

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256 the safety (toxicity), legislative requirements, taste and economic reasons. In this study, to

257 determine the minimum surfactant concentration for a stable nanoemulsion, colloidal

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dispersions were prepared at three different SOR. Figure 3 showed that the mean particle
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259 diameter as measured by DLS decreased with increasing surfactant concentration. The mean
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260 particle diameters were about 171nm, 101 nm and 29 nm for 5 %, 10 % and 20% surfactant

261 concentrations, respectively. With increasing SOR, emulsions tend to be less turbid, since
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262 small droplets are formed and light scattering decreased (Ostertag, Weiss, & McClements,
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263 2012). Decrease in mean particle size depends on several physicochemical mechanisms.

264 Firstly, the interfacial tension decrease at oil-water boundary with increasing amount of
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265 surfactant and thus ultrafine droplets are generated spontaneously by formation of interfacial

turbulence. Secondly, smaller droplets can be formed with higher surfactant concentrations by
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267 stabilized larger oil-water interface. Third, smaller particle size can be associated with the

268 phase behavior of the surfactant-oil-water (SOW) system, since some of the structural

269 organizations between surfactant, oil, and water molecules favor the formation of ultrafine

270 droplets (Komaiko & McClements, 2015;Chang & McClements, 2014).

271 At the end of the study, usage of SOR- 1 was considered appropriate for other experiments

272 since nanoemulsions with ≈100 nm mean particle size could be obtained with SOR- 1. As

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273 known, the most important drawback of spontaneous emulsification is the need for high

274 surfactant concentrations. Thus if a stable nanoemulsion was obtained at a lower surfactant

275 concentration, this was considered as a reasonable choice. Rather than obtaining smaller

276 particle size with very high surfactant concentrations, for the remaining part of the study,

277 nanoemulsions were formulated using 10% Tween 80. Considering the surfactant

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278 concentrations examined ( 5%, 10%, 20%), 10% (w/w) was found to be the most reasonable

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279 one in terms of cost and it would have minimal effect in terms sensorial and legislative

280 aspects for commercial applications (Chang et al., 2013). Photos of the nanoemulsions

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281 prepared at various SORs are also given in Fig. S2b.

282 3.3 Storage Stability of Cinnamon Oil Nanoemulsion Formed by Spontaneous

283 Emulsification
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284 For practical applications, stability of emulsions is very important. For this study, kinetic
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285 stability of the nanoemulsions was investigated by measuring droplet size in a four-week

286 period. Nanoemulsion system with SOR- 1 and consisted of 10 % oil (CO: CNO 6:4 (w/w))
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287 phase was used for the storage experiments. Mean particle diameter slightly increased from
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288 101 nm to 103 nm during storage for 30 days at ∼25 °C (Figure 4). These results confirmed

289 the stability of this formulation. PdI gives information about the width of the droplet size
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290 distribution. In this study, PdI remained below 0.15 during a month storage time which

reflected the relative homogeneity of the emulsions. When the PdI is smaller than 0.2, it
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292 shows generally a monodispersed size distribution (Figure S1) (McClements, 2012).

293 3.4 Effect of Emulsification Method on Mean Particle Size

294 Change in particle size between nanoemulsions that were prepared with spontaneous

295 emulsification, microfluidization and ultrasonication are given in Figure 5.

296 When three emulsification method were compared, ANOVA results showed that

297 ultrasonication and spontaneous emulsification did not differ significantly in terms of mean

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298 particle size (p>0.05), and the minimum particle size was achieved through the

299 microfluidization (p<0.05). After microfluidization, all of the samples were classified as

300 nanoemulsions since they had relatively small mean droplet diameter (d < 200 nm). The

301 smallest particle size of 35.9 nm was obtained at 8:2 (CO: CNO) ratio with microfluidization

302 (p<0.05). However, a regular trend similar to U shaped behavior as observed in SE on particle

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303 size vs. concentration plots was not observed in microfluidized samples (Chang et al.,

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304 2013;Chang & McClements, 2014).

305 On the other hand, samples prepared by ultrasonication had similar decreasing/increasing

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306 trend with samples prepared by SE in terms of mean particle size. Minimum particle size was

307 obtained at 6% and 8% concentrations for both SE and ultrasonication (p<0.05). At that point,

308 it is important to mention

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309 significant impact on the mean particle size whereas in high energy methods ; processing
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310 conditions such as ultrasonication time , power and in the case of microfluidization, pressure

311 and number of cycles could all have effect on the mean particle size of the final emulsions
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312 (Mahdi Jafari et al., 2006).

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313 3.5 Effect of SOR on Antimicrobial Activity of the Nanoemulsions Prepared by SE

314 Although SOR of 1 was selected as the best combination for nanoemulsion formulation, to see
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315 the effect of particle size on antimicrobial activity, SOR of 2 was also tested. Average

inhibition zones were found to be 7.2±0.1 and 10.4±0.5 mm for SOR of 1 and 2 respectively.
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317 The concentration of surfactant had a pronounced effect on the mean particle diameter of the

318 colloidal dispersions and accordingly affected the antimicrobial activity of samples. With

319 increasing surfactant amount from 10% to 20%, antimicrobial activity increased by 44.4%.

320 The mean particle diameters were about 101 nm and 29 nm for SOR- 1 and SOR- 2,

321 respectively. It was obvious that, the increase in antimicrobial activity was due to droplet size

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322 decrease. Photos of the inhibition zones of nanoemulsions at SOR-1 and SOR-2 are given in

323 Fig S3.

324 3.6 Antimicrobial Efficacy of Cinnamon Oil Nanoemulsions obtained by low energy and

325 high energy methods

326 Antimicrobial activity of the nanoemulsions that were prepared with spontaneous

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327 emulsification, microfluidization and ultrasonication were given in Figure 6. Antimicrobial

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328 activity against E. coli was determined by measuring the inhibition zones using agar disc

329 diffusion method. Samples prepared with 2% CO concentration did not exhibit any

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330 antimicrobial activity against E. coli for all emulsification types.

331 Overall ANOVA results showed that , three methods were not significantly different from

332
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each other (p>0.05) but cinnamon oil concentration had a pronounced effect on the
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333 antimicrobial activity ; 4% having the lowest and 8 and 10 % having the highest and 6 % in
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334 between (p<0.05). Interaction between emulsification type and cinnamon oil concentration

335 was also found to be significant (p<0.05).

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336 When individual nanoemulsions were explored in terms of their antimicrobial activity against
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337 E. coli , it was observed that average inhibition zones obtained through SE were recorded as

338 6.3, 7.2, 8.6 and 9.3 mm and through ultrasonication, 6.4, 7.7, 8.3, 8.7 mm for 4%, 6%, 8%
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339 and 10% CO concentrations, respectively. Similar results were also obtained in previous

studies (Chang et al., 2013). For ultrasonication and spontaneous emulsification, at 6 and 8%
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341 CO concentrations, similar particle sizes were obtained (Figure 5) and antimicrobial activity

342 was not significant either (p>0.05). On the other hand, at 4 % CO concentration, antimicrobial

343 activity was similar (p>0.05) but SE resulted in significantly smaller particle size emulsions

344 (~283 nm) compared to ultrasonicated ones (~433 nm) (p<0.05). In contrast, at 10%

345 concentration, despite the reverse case on mean particle sizes, same affect was observed on

346 antimicrobial activity. Although the mean particle size of SE emulsions at 10% CO

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347 concentration was higher (~283 nm) compared to ultrasonicated ones (~172 nm),

348 antimicrobial activity did not differ significantly for these methods (p>0.05). It is also

349 important to mention that the at 10% cinnamon oil, nanoemulsions by SE were unstable

350 which could also have an effect on the mean particle size and thus on antimicrobial activity.

351 As explained before, mean particle size was an important parameter for antimicrobial activity

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352 in SE nanoemulsions where composition was the major player. However, in high-energy

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353 homogenization methods, the processing conditions could also affect the antimicrobial

354 activity of the emulsions. Concentration of the active agent, surfactant to active agent ratio

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355 (SOR) and most importantly processing parameters could all have a synergistic effect and

356 change the antimicrobial activity. Due to the localized heating during ultrasonication, active

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and volatile compounds of CO could have degraded and this loss could have resulted on a
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358 decrease on antimicrobial activity even at smaller particle sizes (Capelo-Martínez, 2009).
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359 Effect of processing was also observed on nanoemulsions prepared by microfludization,

360 which was another high-energy technique. Results showed that, antimicrobial effect was not
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361 enhanced after microfluidization. Antimicrobial activity was not significantly different
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362 between five different CO concentrations (p>0.05). Average inhibition zones were 7.6, 7.7,

363 7.5, 7.7 mm for 4%, 6%, 8%, 10% CO concentrations, respectively. In another study where
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364 trans-cinnamaldehyde nanoemulsions were characterized researchers found that on the

emulsion prepared by high-pressure homogenization, antimicrobial activity against E. coli

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366 was not affected from the active agent concentration (Jo et al., 2015). Since in other

367 emulsification techniques, concentration was found to have a signicant effect, the reason

368 could not be associated with a specific interaction between E. coli and cinnamon oil.

369 Therefore, it is hypothesized that the temperature or high shear during microfluidization had

370 effected the active components that were responsible for the antimicrobial effect. Subjecting

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371 high mechanical stress could have also caused volatile antimicrobial agent loss resulting in no

372 change in antimicrobial activity with increase in the concentration of active compounds.

373 4. Conclusion

374 In the study, it was shown that composition of oil phase (cinnamon oil / coconut oil ratio) and

375 surfactant concentration had a significant impact on the particle size and stability of the

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376 nanoemulsions. Smaller mean particle sizes were obtained for 6:4 and 8:2 CO- CNO ratios

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377 with spontaneous emulsification. However, when the size distribution graph was investigated

378 and tendency to phase separation was observed, the ratio of 6:4 was believed to be more

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379 appropriate for stability of the emulsions. Highly stable nanoemulsions were obtained with

380 spontaneous emulsification at 6:4 CO-CNO ratio with the aid of coconut oil. No phase

381
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separation was observed at the end of 4-week period and kinetic stability of this nanoemulsion
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382 was confirmed. Without using coconut oil, stable nanoemulsions were not formed. Both DLS
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383 and TEM gave parallel results and mean particle size of the stable NEs are found ~100 nm for

384 6:4 (cinnamon oil: coconut oil) combination. Moreover, effect of surfactant amount on
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385 particle size was investigated and with increasing SOR, particle size decreased.
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386 Nanoemulsions were also prepared by microfluidization and ultrasonication. When three

387 methods were compared at same CO % for antimicrobial activity, with increasing amount of
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388 cinnamon oil in organic phase, antimicrobial activity increased by spontaneous emulsification

and ultrasonication. Moreover, antimicrobial activity could not be enhanced by

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389
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390 microfluidization towards E. coli. The study showed that at intermediate amount of surfactant

391 concentrations (10%), stable cinnamon oil nanoemulsions was obtained by using spontaneous

392 emulsification method. Furthermore, coconut oil was utilized as a second oil (carrier oil) in

393 the system rather than using a pure MCT mixture. Considering the cost of the process and the

394 test volumes studied, spontaneous emulsification was found to be more appropriate for this

395 nanoemulsion system. These nanoemulsions did not inhibit the E. coli completely with agar

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396 disc diffusion method however they showed antimicrobial effect to some extent. The use of

397 essential oil loaded nanoemulsions in food applications is promising as they show critical

398 effects on final antimicrobial activity of the products even though complete inhibition is not

399 ensured.

400 5. ACKNOWLEDGEMENT

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401
402 The authors gratefully acknowledge the financial support of The Scientific Technological

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403 Council of Turkey (TUBITAK) with proposal number 113O442. The microfluidizer used in

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404 the study was funded through this grant.

405

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497 Food Hydrocolloids, 23, 1617–1622. http://doi.org/10.1016/j.foodhyd.2008.12.005

498

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499

500 LIST OF FIGURES

501 Figure 1a Effect of cinnamon oil concentration on mean particle size: (∎) and polydispersity

502 index (PdI): ( )

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503 Figure 1b Particle size distribution of nanoemulsions at various CO: CNO ratios

504 Figure 2 Transmission electron microscopy (TEM) images of 6:4 (CO: CNO) nanoemulsions

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505 at different magnification levels. (Surfactant to Oil Ratio = SOR=1)

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506 Figure 3 Effect of SOR on mean particle size of nanoemulsions

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508 storage

509 Figure 5 Effect of spontaneous emulsification (∎), microfluidization (∎) and ultrasonication
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510 (∎) on the mean particle size of nanoemulsions at various cinnamon oil concentrations
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511 (SOR=1)
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512 Figure 6 Effect of spontaneous emulsification (∎), microfluidization (∎) and ultrasonication

513 (∎) on the antimicrobial activity of nanoemulsions at various cinnamon oil concentrations
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514 (SOR=1)
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515 Supplementary Tables and Figures

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516 Table 1 Fatty Acid Composition of the Coconut Oil Used in the Study**

517 Figure S1 Mean particle size distribution of 6:4 (CNO: CO) nanoemulsions at the 1st day and

518 after 4 weeks of storage

519 Figure S2a Nanoemulsions at various CNO: CO ratios. (Left to right: 2:8,4:6,6:4;8:2,10:0)

520 Figure S2b Nanoemulsions at various SOR ratios. (Left to right: 0.5,1, 2)

521 Figure S3 Inhibition zone of 6 % CO nanoemulsions at SOR-1 (right) and SOR-2 (left)

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653 Supplemantary Tables and Figures

654 Table 1 Fatty Acid Composition of the Coconut Oil Used in the Study**

Compounds Percentage (%)

Oleic acid (C18:1) 5.32±0.12

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Arachidic acid (C20:0) 0.08±0.01

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Behenic acid (C22:0) 0.01±0.00

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Pentadecanoic acid (C15:0) 0.01±0.00
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Tricosanoic acid (C23:0) 0.01±0.00
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Capric acid (C10:0)* 6.44±0.47
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Lauric acid (C12:0)* 48.20±1.23

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Highlights

1 Coconut oil was used as the carrier oil to formulate cinnamon oil nanoemulsions.

2 Cinnamon oil: Coconut oil at ratio of 6:4 provided the most stable nanoemulsions.

3 Antimicrobial activity of nanoemulsions were enhanced by spontaneous emulsification.

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