cinnamon essential oil nanoemulsions

Accepted Manuscript Preparation and characterization of blended cloves/cinnamon essential oil nanoemulsions Shengjiang Zhang, Min Zhang, Zhongxiang Fang, Yaping Liu PII:

S0023-6438(16)30532-1

DOI:

10.1016/j.lwt.2016.08.046

Reference:

YFSTL 5695

To appear in:

LWT - Food Science and Technology

Received Date: 5 August 2015 Revised Date:

11 February 2016

Accepted Date: 20 August 2016

Please cite this article as: Zhang, S., Zhang, M., Fang, Z., Liu, Y., Preparation and characterization of blended cloves/cinnamon essential oil nanoemulsions, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.08.046. 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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Preparation and characterization of blended cloves/cinnamon essential oil

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nanoemulsions

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Shengjiang Zhanga, Min Zhanga*, Zhongxiang Fangb,*, Yaping Liuc

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a State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

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China

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b Faculty of Veterinary and Agricultural Sciences, The University of Melbourne,

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Parkville, Victoria 3010, Australia.

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c Guangdong Galore Food Co. Ltd, Zhongshan 528447, China

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* Corresponding authors: Tel.: +86 510 85877225; Fax: +86 510 85877225; E-mail:

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[email protected] (M. Zhang) & Tel.: +61 3 90356663; E-mail:

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[email protected] (Z.X. Fang).

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Abstract Blended cloves/cinnamon essential oil nanoemulsions were prepared using

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Tween 80 and ethanol as surfactant and cosurfactant respectively. The preparation

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process was optimized via a pseudo-ternary phase diagram. The nanoemulsion

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showed a steady state with an average particle size of 8.69 nm under the surfactant to

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cosurfactant ratio (Km) of 3:1 and oil to the mixed surfactant/cosurfactant ratio of 1:9.

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The nanoemulsion was stable after centrifuging at 10,000 rpm for 20 min, stored at

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60 °C for 1 month, or even heated at 80 °C for 30 min, respectively. Compared with

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the non-nanoemulsion counterparts, the nanoemulsion showed higher antimicrobial

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activity against four tested microorganisms of Escherichia coli，Bacillus subtilis，

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Salmonella typhimurium，and Staphylococcus aureus, even at far lower concentrations.

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Furthermore, the addition of the cloves/cinnamon nanoemulsion in a model food of

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mushroom sauce did not alter its major flavor, except offered some attractive fragrant

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flavors. This research suggested that the blended cloves/cinnamon essential oil

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nanoemulsions have the potential to be developed as a natural antimicrobial agent in

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food industry.

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Keywords: blended cloves/cinnamon essential oil; nanoemulsion; physicochemical

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properties: antimicrobial activity

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1 Introduction Both cloves (Syringa oblata) and cinnamon (Cinnamomum cassia Presl) are rich

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in essential oils, which have inhibitory effect on the proliferation and growth of a

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wide range of microorganisms (Li, Ji, Zhou, & Li, 2006). Each of the essential oils

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could be used as natural preservative in food and other industries to extend the shelf

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life of the products. However, the blended cloves/cinnamon essential oils could have

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stronger antimicrobial effects because of the potential synergic effects of the essential

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oils from two different sources (Li et al., 2006; Hoar et al., 1943).

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Nanoemulsions are a sub-group of emulsions which have the droplet diameter in

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the range of 1~ 100 nm (Hoar & Schulman, 1943). There is an increasing interest in

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the formulation, preparation and utilization of nanoemulsions due to their novel

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physicochemical properties and higher bioavailability of the encapsulated active

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ingredients (Sznitowska, Zurowska-Pryczkowska, Dabrowska, & Janicki, 2000;

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Mason, Wilking, Meleson, Chang, & Graves, 2006; McClements, 2011).

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Nanoemulsions with their unique subcellular size can effectively increase the

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distribution of antimicrobial agents in food matrices where microorganisms are

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preferably located, and therefore greater antimicrobial activity could be achieved. In

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addition, the physical stability of the encapsulated active substances is highly

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improved in the nano-sized status (Weiss, Gaysinsky, Davidson, & McClements,

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2009). Nanoemulsions using essential oils as the active agents have shown excellent

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antimicrobial properties against a range of different microorganisms (Donsì,

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Annunziata, Sessa, & Ferrari, 2011). Thus, it is expected that the blended

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nanoemulsion of cloves/cinnamon essential oils could exhibit higher stability and

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antimicrobial activity than that of the non-nano state counterparts. Emulsion phase inversion (EPI) method is a commonly used low-energy

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approach (Anton et al, 2008; Mayer et al, 2013) where an emulsion is formed when

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water is added to an oil-surfactant mixture (Solè, Pey, Maestro, González, Porras,

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Solans, & Gutiérrez, 2010). The EPI method uses the chemical energy released from

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the emulsification process as a consequence of change in the spontaneous curvature of

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surfactant molecules from negative to positive (obtaining oil-in-water nanoemulsions,

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O/W) or from positive to negative (obtaining water-in-oil nanoemulsions, W/O).

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Studies on nanoemulsion formation by EPI method have shown the presence of

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lamellar liquid crystalline phases or bicontinuous microemulsions (i.e. aggregates in

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which the mean curvature of the surfactant molecules is zero) to achieve minimum

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droplet sizes (Sonneville-Aubrun et al, 2009; Roger et al, 2010; Morales et al, 2006;

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Heunemann et al, 2011; Izquierdo et al, 2004).

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The O/W emulsions are exploited in a wide variety of industries to encapsulate,

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protect, and deliver lipophilic components, such as pharmaceuticals, cosmetics, foods,

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and agrochemicals. Emulsions are formed when one of two immiscible liquids is

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dispersed in the other liquid as small spherical droplets (Anton, Benoit, & Saulnier,

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2008). The O/W nanoemulsions can be well dissolved in the aqueous medium and is

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able to hold the active agents to exert long time steady functionalities like

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antimicrobial activity.

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Up to date, limited information is available on the physicochemical properties

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oils as active agents. Therefore, the objectives of this study were to prepare O/W

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nanoemulsions of cloves/cinnamon essential oils, and investigate its stability and

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antimicrobial activity. This study could have provided practical information in

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utilization of cloves/cinnamon essential oil nanoemulsions as natural preservatives.

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2 Materials and methods

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2.1. Materials

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Cloves and cinnamon powders were purchased from Shengtelun Food Ingredient

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Store (Wuxi city, Jiangsu province, China). Tween 80 was ordered from Sinopharm

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Chemical Reagent Co. Ltd, Shanghai, China. Deionized water was used in this work.

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The microbial test strains, Escherichia coli (E. coli, strain number ATCC 25922),

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Bacillus subtilis (B. subtilis, strain number ATCC 6633), Staphylococcus Aureus (S.

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aureus, strain number ATCC 12600) and Salmonella Typhimurium (S. typhimurium,

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strain number CMCC50093) were obtained from the Laboratory of Microbiology of

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Jiangnan University, China.

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2.2 Preparation of the combined extract

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Dried cloves and cinnamon powders (passed through an 80 mesh sieve) were

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extracted in beakers using 95% ethanol (V/V) with a solid to liquid ratio of 1:4 (g/ml).

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After being extracted for 3 h at the temperature of 40 °C using a magnetic stirrer, the

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extract solution was vacuum filtered. The residue was re-extracted for another three

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times until the extraction solution became clear. The filtrate was combined and

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concentrated to 1g/ml soluble solid under vacuum at 40 °C with a rotary evaporator.

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ACCEPTED MANUSCRIPT The cloves and cinnamon extracts were combined in a 1:1 ratio, and kept in a

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refrigerator at 4 °C until further use (Hessien et al, 2011; Xia et al, 2011).

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2.3 Preparation of nanoemulsions

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2.3.1 Selection of surfactant and cosurfactant

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As Tween 80 has a high hydrophilic and lipophilic balance (HLB) value of 15, it

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has been widely used as a surfactant in O/W emulsion preparation (Nakabayashi et al,

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2011; Ghosh et al, 2013). Among the hydrophilic surfactants, Tween 80 has a very

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good solubility for essential oils and also miscible with water, and therefore was used

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in this study. In addition, cosurfactant can regulate the HLB of a surfactant, reduce the

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interfacial tension between oil and water, and increase the mobility of interfacial film

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(Nakabayashi, Amemiya, Fuchigami, Machida, Takeda, Tamamitsu, & Atobe, 2011),

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which can improve the stability of the O/W emulsion system. As the cloves and

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cinnamon essential oils were extracted by ethanol, suggesting they are mainly ethanol

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soluble compounds, absolute ethanol was selected as the cosurfactant.

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2.3.2 The mass ratio (Km) of surfactant to cosurfactant

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At room temperature, the surfactant and cosurfactant were mixed at different Km

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(v/v) 2:1, 3:1 and 4:1, followed by mixing essential oil and surfactant/cosurfactant

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with varying mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 to 9:1. Then, water was

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added dropwise. The flow rate of water was kept constantly at approximately 1.0

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ml/min. The clarity of the system was visually observed, and the water was added

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until it turned from turbid to clear. The ternary phase diagram was drawn using the oil

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phase, the surfactant/co-surfactant and the water phase as three vertices. The optimum

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value of Km is obtained according to the size of the emulsion area (Liu, Ouyang, Song,

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Wu, & Rui, 2011).

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2.4 Characterization of the nanoemulsion The particle size distribution, mean particle diameter (Z-averages), and

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polydispersity index (PDI) of the samples were determined using dynamic light

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scattering (DLS) at 25 °C on a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern,

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Worcestershire, UK). The analysis was performed at a scattering angle of 90 °, and

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each recorded measurement was an average of 3 scans.

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2.5 Stability of the nanoemulsion

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2.5.1 Centrifugation and storage stability

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The blended cloves/cinnamon nanoemulsions were centrifuged at 10,000 g for

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20 min to evaluate the stability. Intrinsic stability was investigated by storing 50 ml of

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the nanoemulsions at temperature of 25 ± 1 °C, 37 ± 1 °C and 60 ± 1 °C for one

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month (Ghosh, Mukherjee, & Chandrasekaran, 2013). The samples were observed for

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instability phase separation, creaming or flocculation. Meanwhile the mean particle

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diameter and PDI（polydispersity index）of the samples were determined after the

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storage.

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2.5.2 Thermal stability

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An equal amount (50 ml) of the nanoemulsions were heated to 80 °C, 90 °C, and

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100°C for 30 min to mimic the pasteurization during food processing. The visual

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changes of the nanoemulsions were observed. Then the particle size distribution,

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mean particle diameter, and PDI of the samples were determined after the

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nanoemulsions were cooled down to room temperature.

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2.6 Antimicrobial activity The inhibition zone assay was used to determine the antimicrobial activity of the

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nanoemulsions against E. coli, B. subtilis, S. typhimurium and S. aureus (Zhang, Li,

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Dong, Tian, Li, & Dai, 2015), using the method of Oxford cup (Wang, Lu, Wu, & Lv,

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2009). The strains were activated and obtained at a 5×105 CFU/ml concentration of

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the cell suspension by tenfold serial dilution (Li, Ji, Zhou, & Li, 2006). Four Oxford

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cups were separated on a prepared agar plate covered with 1 ml bacteria solution.

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Then 100 µl of blank nanoemulsion, cloves essential oil extract, cinnamon essential

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oil extract and blended cloves/cinnamon essential oil nanoemulsion were dropped into

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the 4 respective Oxford cups. The strains were incubated under their required

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temperatures for 24 or 48 h. After the incubation, the plates were examined to

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determine the inhibition zone of different samples, and the diameter of the inhibition

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zone was measured with a sliding caliper in triplicate (Zhang, Li, Dong, Tian, Li, &

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Dai, 2015).

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2.7 Effect of the nanoemulsion addition on the flavor of food

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A commercial mushroom sauce was selected as a model food to evaluate the

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effect of addition of the nanoemulsion on the food flavor. The sauce was

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manufactured by our collaborative food company of Guangdong Galore Food Co.

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Ltd., using the mushroom variety of Lentinus edodes, which is very popular in China

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market. Referring to the National Standard of Preservatives on Foods (GB 2760-2011,

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2011), the nanoemulsion was added at 0.5g/kg to the mushroom sauce. Then the sauce

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ACCEPTED MANUSCRIPT was sterilized using the same process (100 °C for 30 min) of the commercial

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mushroom sauce, and cooled to room temperature. The commercial mushroom sauce

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(preservative of potassium sorbate) was used as a control of the sample (preservative

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of the nanoemulsion). The flavor compounds of the mushroom sauces with and

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without the nanoemulsion were analyzed on a gas chromatography-mass spectrometer

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(GC-MS, SCION SQ 456-GC, Bruker Daltonics Inc., Billerica, MA, USA). The

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detection parameters were as follows:

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Chromatographic conditions: Column: TRACE TR-50 MS (30 m × 0.25 mm, 0.25 µm,

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Thermo Scientific, Pittsburgh, PA, USA); heating up programs: the temperature of

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40 °C was kept for 3 mins, raised to 90 °C in 5 °C/min, then raised to 230 °C in 10 °C

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/min, and maintained for 7 min; the flow rate of carrier gas (He) was 0.8 mL/min.

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Mass Spectrometer Conditions: Electron impact (EI) ion source; electron energy was

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70 eV; transfer line temperature was 250 °C; ion source temperature was 200 °C.

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2.8 Statistical analyses

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Each determination was repeated three times and two replications of one treatment

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were performed. All the data were statistically analyzed by the software of SPSS 17.0

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(SPSS Inc., Chicago, IL, USA), using one-way analysis of variance. The significant

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differences were determined at the 95 % level.

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3 Results and discussion

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3.1 Preparation of the nanoemulsions

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3.1.1 The mass ratio (Km) of surfactant to cosurfactant

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The parameter of Km affects the formation and stability of a nanoemulsion

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ACCEPTED MANUSCRIPT system (Liu, Ouyang, Song, Wu, & Rui, 2011). When the surfactant and cosurfactant

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is at the optimal mass ratio, the cosurfactant is completely embedded into the

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surfactant, so the solubilization space for the oil phase is formed to the maximum

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level. This suggests that an optimal Km value results in the highest stability of the

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O/W emulsion system. Some studies have shown that when the Km is less than 1 (Km

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< 1), the emulsifying ability is weak and only a small nanoemulsion area (the particle

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size of the emulsion in this area is in the nano-size range) is formed. When Km > 1,

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the nanoemulsion area is first increased but then reduced as the Km value is too great

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(Wu, Li, Lang, Liu, & Liu, 2005). Different ratios of surfactant to cosurfactant (2:1,

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3:1 and 4:1) were tested in this study. As shown in Fig. 1, the maximum nanoemulsion

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area was formed when the Km was 3:1, and then this parameter was selected in the

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following experiments.

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3.1.2 Effect of oil-surfactant ratio

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The major role of the surfactants is to stabilize the emulsion by reducing surface

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tension at the oil/water interface (Ghosh, Mukherjee, & Chandrasekaran, 2013).

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Although emulsions could be formed in a range of oil-surfactant ratio, the particle size

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and stability varies significantly in some circumstances. Only sufficient surfactants

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are able to dissolve oils and adjust the interfacial tension of the emulsions. Thus the

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oil-surfactant ratio plays a crucial role in determining the particle size and stability of

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the emulsions. With the increase of surfactant concentration, the decrease in particle

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size was observed. When the oil-surfactant ratio was reached to 1:9, the nanoemulsion

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showed the highest stability and dispersion stability, with the smallest particle size

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ACCEPTED MANUSCRIPT and lower PDI in this experiment (Table 1). It has been reported that nanoemulsions

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prepared at such an oil-surfactant ratio are able to maintain good stability and particle

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size distribution, even being largely diluted (Men & Zhang, 2010). However, if the

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surfactant amount is increased further, the oil phase load will become less, which

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would not be a good property as the active agent would be too low to achieve the

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target functionality (e.g. antimicrobial activity). Therefore, the oil-surfactant ratio of

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1:9 was the most appropriate parameter in the present work.

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The above results indicated that the optimal parameters for the preparation of the

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blended nano cloves/cinnamon nanoemulsion were at Km of 3:1 and oil to surfactant

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ratio of 1:9 (Table 1). This nanoemulsion was composed of cloves/cinnamon essential

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oil 4%, tween 80 27%, ethanol 9% and water 60%. With this formulation, a clear and

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transparent blended cloves/cinnamon essential oil nanoemulsion was obtained.

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3.2 Characterization of the nanoemulsion

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The particle size distribution, mean particle diameter (Z-averages), and PDI are

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very important indicators that describe the quality, stability, uniformity and

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dispersibility of the nanoemulsions (Mason, Wilking, Meleson, Chang, & Graves,

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2006). After DLS measurement, the average particle size of the blended

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cloves/cinnamon nanoemulsion was about 8.69 nm. The peak width of the distribution

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diagram was very narrow which comprised more than 97% of the droplets, indicating

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that the size of the nanoemulsion particles were substantially uniform (Fig. 2).

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Meanwhile, PDI gave information on the deviation from the average size, and the

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lower value of 0.22 was desirable which suggested that the nanoemulsion droplets

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(Kelmann, Kuminek, Teixeira, & Koester, 2007).

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3.3 Stability of the nanoemulsions

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3.3.1 Centrifugation and storage stability

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The appearance of the blended cloves/cinnamon essential oil nanoemulsion is a

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yellowish, clear and transparent liquid, without observed stratification, turbidity, and

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precipitate. After centrifugation at 10,000 rpm for 20 min, the system was maintained

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the same clear and transparent appearance. The particle size distribution of the

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nanoemulsion before and after the centrifugation was also in the same range,

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indicating good stability. The nanoemulsion was stored at 25 ± 1 °C, 37 ± 1 °C and 60

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± 1 °C for one month respectively. The results showed that the nanoemulsions did not

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change during the storage below 37 °C and remained the clear and transparent state.

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The particle size was even smaller when the emulsion was stored at higher

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temperature of 60 °C. The small PDI range of 0.22 ~ 0.29 also showed the good

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stability of the nanoemulsion under 60 °C storage.

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3.3.2 Thermal stability

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Most nanoemulsions have good thermal stability, but high temperature

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treatments (e.g. pasteurization and sterilization) could destabilize the system (Wu, Li,

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Lang, Liu, & Liu, 2005). When the nanoemulsion was heated at 80 °C for 30 min, it

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maintained the clear and transparent state. However, when the temperature was 90 °C

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or above, the nanoemulsion became turbid. Interestingly, when the temperature was

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cooled down below 90 °C, the nanoemulsion returned to the transparent and clear

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state again. Furthermore, the particle size distribution were consistent with the

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non-heat-treated sample, with the average particle diameter at about 8.68 ~ 9.8 nm

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and PDI about 0.19 ~ 0.33, similar to the samples before heat treatment (Table 1).

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This

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cloves/cinnamon essential oil nanoemulsion structure could be recovered to its

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original state. This is probably because the solubility of the surface-active agent

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(Tween 80 in this study) increases with the temperature increasing, but when the

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temperature exceeds its cloud points, its solubility declines and jeopardizes the

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stability of the nanoemulsions with the appearance of cloudiness. However, when the

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temperature is cooled below the cloud point, the solubility of the surfactant resumes

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and the nanoemulsions return to the transparent appearance (Quina, & Hinze, 1999).

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When the temperature is close to or above the cloud point, the possibility of breaking

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the nanoemulsion system is very high. However, the high temperature treatment in

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this study did not cause an irreversible change on the stability of the cloves/cinnamon

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essential oil emulsions. When the temperature was reduced below the surfactant’s

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cloud point, the nanoemulsion restored its original properties. After one month's

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storage at room temperature, the appearance, particle size distribution, mean particle

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size, and PDI remained stable, indicating very good thermal stability of the

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nanoemulsion.

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3.4 Antimicrobial activity

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phenomenon

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In this study, four different microorganism strains including Gram positive and

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negative bacteria, e.g. E. coli, B. subtilis, S. typhimurium and S. aureus, were used to

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and the results are presented in Table 2. Obviously, a larger diameter of the inhibition

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zone shows a higher antibacterial activity of the substance. All the cloves, cinnamon,

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and cloves/cinnamon essential oil samples showed good antibacterial activity for the

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four tested strains, but no antibacterial activity was observed for the blank

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nanoemulsion (Table 2). However, the blended cloves/cinnamon essential oil

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nanoemulsion exhibited the highest antibacterial activity on the four microbial strains.

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According to Sections 2.3.2 and 3.1.2, the nanoemulsion contained only 4% blended

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cloves/cinnamon essential oil, whereas original cloves essential oil and cinnamon

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essential oil extracts were used in the cloves and cinnamon treatment samples. The

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results suggested that the blended cloves/cinnamon essential oil nanoemulsion

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showed higher antimicrobial activity than their individual non-nanoemulsion

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counterparts, even at far lower concentrations.

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3.5 Effect of addition of the nanoemulsion on the flavor of food

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It is reported that some natural food preservatives have limits in practical

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applications, such as they may alter the original food flavor which is unacceptable to

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some consumers (Feng, & Chen, 2011). Therefore, investigating the influence of

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nanoemulsion on the food flavor could be an important step to

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commercial natural preservative. After analysis the GC-MS spectra and referring to

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CAS (Chinese Academy of Sciences) databases, the flavor compounds and relative

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contents in the nanoemulsion added sauce (sample) and control sauce were obtained

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(Table 3). The main

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develop it as a

flavor substances of the mushroom sauces were

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heterocycles, phenols, aldehydes, alcohols, acids, esters, and hydrocarbons. Compared

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with the control, the flavor substances in the nanoemulsion added mushroom sauce

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sample were similar, and there were only small differences in the relative contents.

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However, flavor compounds of eugenol (13.02%), 2-propenal, 3-phenyl- (i.e.

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cinnamic aldehyde, 23.49%), methyl salicylate (14.37%), ethanol (7.34%), and benzyl

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alcohol (7.13%) were significantly increased after the addition of nanoemulsion.

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These increased flavor substances are the well-known attractive fragrance substances

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of cloves and cinnamon (Shan, Ma, & Zhang, 2012). In addition, the control group

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contained 21.01% of sorbic acid (Table 3), which was mainly because of the

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preservative of potassium sorbate in the mushroom sauce. Therefore, compared with

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the preservative of potassium sorbate, the cloves/cinnamon nanoemulsion contained

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some attractive flavor substances by itself, and the major flavors of the mushroom

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sauce were not changed.

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4 Conclusion

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In this study, the preparation process of a nanoemulsion composed of blended

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cloves/cinnamon essential oil was optimized. At the Km = 3:1 and oil/surfactant ratio

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1:9, the average particle size of the nanoemulsion was 8.69 nm and PDI 0.22. The

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nanoemulsion maintained good stability and dispersion after centrifuging at 10,000

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rpm for 20 min, stored at 60 °C for 1 month, or heated at 80 °C for 30 min,

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respectively. Furthermore, even with far lower concentrations, the blended

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cloves/cinnamon essential oil nanoemulsion showed higher antimicrobial activity than

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the non-nanoemulsion counterparts. This research indicated that the blended

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cloves/cinnamon essential oil nanoemulsion may have the potential to be developed

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as a natural antimicrobial agent for food preservation.

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Acknowledgment

We acknowledge the financial support by Jiangsu Province(China) “Collaborative

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Innovation Center for Food Safety and Quality Control”Industry Development

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Program, Jiangsu Province (China) Infrastructure Project (Contract No. BM2014051),

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and Guangdong Province(China) R & D Project (No.2012B091000125).which have

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enabled us to carry out this study.

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References

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Anton, N., Benoit, J. P., & Saulnier, P. (2008). Design and production of nanoparticles formulated

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from nano-emulsion templates. Journal of Controlled Release, 128(3), 185-199. Donsì, F., Annunziata, M., Sessa, M., & Ferrari, G. (2011). Nanoencapsulation of essential oils to

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enhance their antimicrobial activity in foods. LWT-Food Science and Technology, 44(9),

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Feng, W. W., & Chen, Z. W. (2011). Advances in natural food preservatives. Journal of Anhui Agriculture, 39(18), 11015-11017. GB 2760-2011, (2011). National food safety standards for uses of food additives. Beijing: China Standard Press. Ghosh, V., Mukherjee, A., & Chandrasekaran, N. (2013). Ultrasonic emulsification of food-grade

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nanoemulsion formulation and evaluation of its bactericidal activity. Ultrasonics sonochemistry,

344

20(1), 338-344.

346

Hoar, T. P., Schulman, J. H. (1943). Transparent water-in-oil dispersions: the oleopathic hydro-micelle. Nature, 24 (152), 102-103.

RI PT

345

Heunemann, P., Prévost, S., Grillo, I., Marino, C. M., Meyer, J., & Gradzielski, M. (2011).

348

Formation and structure of slightly anionically charged nanoemulsions obtained by the phase

349

inversion concentration (PIC) method. Journal of Soft Matter, 7(12), 5697-5710.

351

Hessien, M., Singh, N., Kim, C., & Prouzet, E. (2011). Stability and tunability of O/W

M AN U

350

SC

347

nanoemulsions prepared by phase inversion composition. Langmuir, 27(6), 2299-2307. Izquierdo, P., Esquena, J., Tadros, T. F., Dederen, J. C., Feng, J., Garcia-Celma, M. J., & Solans, C.

353

(2004). Phase behavior and nano-emulsion formation by the phase inversion temperature

354

method. Langmuir, 20(16), 6594-6598.

TE D

352

Kelmann, R. G., Kuminek, G., Teixeira, H., & Koester, L. S. (2007). Carbamazepine parenteral

356

nanoemulsions prepared by spontaneous emulsification process. International Journal of

357

Pharmaceutics, 342, 231-239.

EP

355

Li, J. J., Ji, B. P., Zhou, F., & Li, B. (2006). Study on the extraction, main component and

359

antimicrobial activity of clove and cinnamon essential oil. Journal of Food Science, 27(8),

360

64-68.

361 362 363 364

AC C

358

Liu, M. X., Ouyang, W. Q., Song, B., Wu, J. C., & Rui, X. (2011). Preparation and stability evaluation of thymol nanoemulsion. Journal of Shanghai Jiaotong University. 29(6), 29-34. McClements, D. J. (2011). Edible nanoemulsions: fabrication, properties, and functional performance. Journal of Soft Matter, 7(6), 2297-2316.

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Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B., & Graves, S. M. (2006). Nanoemulsions:

366

formation, structure, and physical properties. Journal of Physics: Condensed Matter, 18(41),

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R635. Mayer, S., Weiss, J., & McClements, D. J. (2013). Vitamin E-enriched nanoemulsions formed by

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emulsion phase inversion: Factors influencing droplet size and stability. Journal of colloid and

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interface science, 402, 122-130.

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Morales, D., Solans, C., Gutiérrez, J. M., Garcia-Celma, M. J., & Olsson, U. (2006). Oil/water

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droplet formation by temperature change in the water/C16E6/mineral oil system. Langmuir,

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22(7), 3014-3020.

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Men, Z. Z., & Zhang, L. F. (2010). Preparation and application of microemulsified sorbic acid nano-dispersion system. Cereals and Oils Processing, (12), 138-141. Nakabayashi, K., Amemiya, F., Fuchigami, T., Machida, K., Takeda, S., Tamamitsu, K., & Atobe,

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M. (2011). Highly clear and transparent nanoemulsion preparation under surfactant-free

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conditions using tandem acoustic emulsification. Chemical Communications, 47(20),

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5765-5767.

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Quina, F. H., & Hinze, W. L. (1999). Surfactant-mediated cloud point extractions: an

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environmentally benign alternative separation approach. Industrial & Engineering Chemistry

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Research, 38(11), 4150-4168.

383 384

AC C

380

Roger, K., Cabane, B., & Olsson, U. (2010). Emulsification through surfactant hydration: the PIC process revisited. Langmuir, 27(2), 604-611.

385

Saberi, A. H., Fang, Y., & McClements, D. J. (2013). Fabrication of vitamin E-enriched

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nanoemulsions: factors affecting particle size using spontaneous emulsification. Journal of

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colloid and interface science, 391, 95-102. Solè, I., Pey, C. M., Maestro, A., González, C., Porras, M., Solans, C., & Gutiérrez, J. M. (2010).

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Nano-emulsions prepared by the phase inversion composition method: Preparation variables

390

and scale up. Journal of colloid and interface science, 344(2), 417-423.

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Sonneville-Aubrun, O., Babayan, D., Bordeaux, D., Lindner, P., Rata, G., & Cabane, B. (2009).

392

Phase transition pathways for the production of 100 nm oil-in-water emulsions. Physical

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Chemistry Chemical Physics, 11(1), 101-110.

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391

Sznitowska, M., Zurowska-Pryczkowska, K., Dabrowska, E., & Janicki, S. (2000). Increased

395

partitioning of pilocarpine to the oily phase of submicron emulsion does not result in improved

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ocular bioavailability. International journal of pharmaceutics, 202(1), 161-164.

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Weiss, J., Gaysinsky, S., Davidson, M., & McClements, J. (2009). Chapter 24, Nanostructured

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encapsulation systems: food antimicrobials. In Barbosa-Cánovas G., Mortimer, A., Lineback, D.,

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Spiess, W., Buckle K., & Colonna, P. (Eds). Global issues in food science and technology,

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Academic Press, Oxford, UK, P425-479.

TE D

397

Wang, Y., Lu, Z., Wu, H., & Lv, F. (2009). Study on the antibiotic activity of microcapsule

402

curcumin against foodborne pathogens. International Journal of Food Microbiology, 136(1),

403

71-74.

AC C

EP

401

404

Wu, S. Q., Li, S. M., Lang, Y. Y., Liu, H., & Liu, H. Z. (2005). Investigation on formation factors

405

of oil-in-water microemulsion. Journal of Shenyang Pharmaceutical University, 22 (2), 97-99.

406

Shan, C. Y., Ma, S. H., & Zhang, W. M. (2012). Application of spice oils in food preservation.

407 408

Journal of China Condiment, 37(3), 26-31. Xia, X. Q. (2011). Effects of natural spices ethanol extracts adding method on beef the shelf life of

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cold spiced. Journal of Meat Industry, 3, 15-18. Zhang, L., Li, R., Dong, F., Tian, A., Li, Z., & Dai, Y. (2015). Physical, mechanical and

411

antimicrobial properties of starch films incorporated with ε-poly-l-lysine. Food Chemistry,

412

166, 107-114.

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410

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413

20

ACCEPTED MANUSCRIPT 0.0

1.0

0.8

0.4

0.6

mix -su

te wa

fac

tan

t

0.2

0.6

r

0.4

0.8

1.0

0.0

0.0

0.2

0.4

0.6

0.8

1.0

oil

a.

0.0

1.0

0.8

0.4

0.6

mix -su

te wa

fac

tan

t

0.2

0.6

M AN U

r

0.8

0.4

SC

414

RI PT

0.2

0.2

1.0

0.0

0.0

0.2

0.4

0.6

0.8

1.0

oil

415

b.

0.0

1.0

0.2

0.8

0.6

mix -su

te wa

fac

tan

t

TE D

0.4

0.6

r

0.4

0.8

0.2

1.0

417 418 419 420

0.0

0.4

0.6

0.8

1.0

oil

c.

Fig.1 Pseudo-ternary phase diagrams for Km determination. (a), (b), and (c) represent Km range of 2:1, 3:1, and 4:1 respectively. The black area indicates the nano-sized area of the emulsion particles.

AC C

416

0.2

EP

0.0

21

421 422

RI PT

ACCEPTED MANUSCRIPT

Fig.2 The particle size distribution of the blended cloves/cinnamon nanoenulsion

AC C

EP

TE D

M AN U

SC

423

22

ACCEPTED MANUSCRIPT Table 1 Effect of different oil-surfactants ratio on the particle size of the nanoemulsions

426

a

oil-surfactants ratio

particle size (nm)

PDI

3:7

28.50 ± 1.21c

0.35 ± 0.03b

2:8

13.56 ± 0.54b

0.45 ± 0.01c

1:9

8.69 ± 0.15a

0.22 ± 0.02a

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424 425

Different letters in the same column indicate significantly different (p ≤0.05).

SC

427

AC C

EP

TE D

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428

23

ACCEPTED MANUSCRIPT 429

Table 2 Antimicrobial activity of the blended cloves/cinnamon essential oil

430

nanoemulsions B. subtilis

S. typhimurium

S. aureus

0.00a

0.00a

0.00a

0.00a

16.87 ± 0.42b

15.43 ± 0.40b

15.00 ± 1.00b

18.63 ± 0.47b

27.27 ± 0.64d

16.50 ± 1.32c

24.57 ± 0.40c

30.00 ± 1.00c

22.63 ± 0.71c

14.17 ± 0.76d

21.87 ± 0.81d

25.50 ±1.32d

a

a

Different letters in the same column indicate significantly different (p ≤0.05). Cinnamon oil and cloves oil were used as pure extracts but their concentration in the blended nanoemulsion was about 4%. b

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431 432 433

E. coli

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Blank nanoemulsion b Cinnamon oil Cloves oil Blended nanoemulsion

Diameter of the inhibition zone/mm

SC

Samples

AC C

EP

TE D

434

24

ACCEPTED MANUSCRIPT 435

Table 3 Flavor substances of mushroom sauces with (sample) and without (control)

436

nanoemulsion addition

Pyrazine, methylPyrazine, 2,6-dimethylPyrazine, trimethylPyrazine, 2,3-dimethylPyrazine, 2-ethyl-6-methyl3-benzyl-1,4-diaza-2,5-dioxobicyclo[4.3. Ethanone, 1-(1H-pyrrol-2-yl)Undecane, 4,7-dimethyl-

AC C Aldehydes 3.89 16.85

4H-Pyran-4-one, 2-ethyl-3-hydroxyPhenol, 2-methoxyPhenol, 2-methyl-

Control

Sample

2.34

0.60

RI PT

1,2,4-Trithiolane Trisulfide, dimethyl Carbon disulfide 2-Acetylthiazole Disulfide, dimethyl Disulfide, di-2-propenyl Thiophene, 3,4-dimethyl-

EP

Nitrogen-containing heterocyclics 11.819 13.318 14.894 13.7 14.516 26.183 22.378 7.585

M AN U

Sulfur-containing compounds 20.044 14.357 2.176 18.637 7.026 16.188 11.438

2-Furancarboxaldehyde Furan, 2-pentylEthanone, 1-(2-furanyl)Butyrolactone 2-Furancarboxaldehyde, 5-methyl2(3H)-Furanone, dihydro-5-methyl2(5H)-furanone Isocineole

TE D

Oxygen-containing heterocyclics 15.849 10.947 16.543 18.36 17.575 18.114 19.985 9.598

phenols 22.89 21.17 22.66 24.33 25.96

Relative content/%

Compounds

0.97 0.57 0.26 0.20 0.17 0.10 0.08

SC

R.T/min

1.00

2.55 0.93 0.26 0.18 0.16 0.15 0.15

0.73 0.20

1.59 0.53 0.23 0.14 0.04 0.02 0.41 0.22 29.02 28.86 0.09 0.07

0.03 0.04

0.17

0.03

0.08 0.06 23.28 10.18

13.02 0.08

Phenol, 2-methoxy-4-(1-propenyl)5.65 1.47 0.92 25

0.087

4.37

eugenol

Butanal, 3-methylBenzaldehyde

0.23 0.14 0.06 0.04 0.06

27.10 0.26 1.77

ACCEPTED MANUSCRIPT

2-Octenal

2-Nonenal, (Z)2,4-Decadienal, (E,E)2-Propenal, 3-phenyl2-Propenal, 3-phenylNonenal

Esters 3.47 3.24 20.31 19.13

2-Furanmethanol 1-Octen-3-ol

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Acetic acid, ethyl ester 2-Ethylhexyl acrylate Methyl salicylate Formic acid, phenylmethyl ester

1.73 1.15 0.58

Acids 24.07 15.63 18.91 21.01 23.57 19.76 22.19

Hydrocarbons 10.05 7.30

0.21 0.24 0.13 0.10

4.05 1.29 1.20 0.50 0.40 0.20 0.13 0.12 0.11 0.10

l-Limonene Butane, 2-iodo-2-methyl26

0.16 23.49 0.44 0.11

15.09

15.48 0.26 0.28 0.10 0.13

7.34 7.13 0.12 0.11

Fenchyl alcohol trans-Geraniol

Sorbic Acid Acetic acid Butanoic acid, 3-methylHexanoic acid 2,4-Hexadienoic acid, (E,E)Pentanoic acid Heptanoic acid

0.20

14.37 0.72

EP

TE D

Linalool Phenylethyl Alcohol 2-Propen-1-ol 1,6-Octadien-3-ol, 3,7-dimethyl2-Octen-1-ol, (E)3-Heptanol, 5-methyl1-Octanol, 2-butylEthanol Benzyl alcohol

AC C

Alcohols 18.74 15.68 7.64 21.77 8.31 17.20 18.19 14.68 7.79 4.29 21.37 19.29 7.64

0.76 0.43 0.37 0.34 0.25 0.21 0.20 0.19 0.19 0.17 0.16

RI PT

2-Heptenal, (E)Heptanal Pentanal Nonanal 2,4-Heptadienal, (E,E)Benzeneacetaldehyde Octanal 2-Pentenal, 2-methyl-

SC

13.16 9.80 5.01 14.64 16.38 18.54 12.35 9.14 15.30 17.06 20.65 23.19 21.63 17.05

24.60 21.01 1.64 0.46 0.41 0.39 0.11 0.59 16.78 6.14 2.11

0.82 0.35 0.09 0.25 0.05 0.08 6.30 0.78 0.57

ACCEPTED MANUSCRIPT

AC C

EP

TE D

437

27

0.26

0.14 0.10 0.50 2.64

RI PT

1.98 1.00 0.97 0.90 0.72 0.54 0.51 0.39 0.39 0.32 0.30 0.29 0.25

SC

Tetradecane Styrene Tetradecane Heptane, 2,4-dimethylHeptadecane Dodecane .alpha.-pinene, (-)Tridecane trans-Caryophyllene Eicosane Decane, 5-methyl1-Octene .beta.-Myrcene .alpha.-Humulene Benzene, 1-methyl-2-(1-methylethyl)Benzene, hexyl-

M AN U

6.29 11.53 6.39 2.67 11.31 7.41 8.72 12.59 18.04 6.18 7.52 3.14 9.20 19.02 11.83 16.80

0.13 0.52 0.40 0.26

ACCEPTED MANUSCRIPT Highlights:

•

A nanoemulsion composed of the blended clove/cinnamon essential oil was prepared The nanoemulsion preparation process was optimized

•

The nanoemulsion showed good structural, thermal and storage stability

•

The nanoemulsion showed higher antimicrobial activity against the common

SC

RI PT

•

microorganisms

The substances in nanoemulsion could help food feature durable fragrance

AC C

EP

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instead of harming flavor

M AN U

•