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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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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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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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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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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
suggested
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after
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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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Anton, N., Benoit, J. P., & Saulnier, P. (2008). Design and production of nanoparticles formulated
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Hoar, T. P., Schulman, J. H. (1943). Transparent water-in-oil dispersions: the oleopathic hydro-micelle. Nature, 24 (152), 102-103.
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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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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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Roger, K., Cabane, B., & Olsson, U. (2010). Emulsification through surfactant hydration: the PIC process revisited. Langmuir, 27(2), 604-611.
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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
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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).
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Phase transition pathways for the production of 100 nm oil-in-water emulsions. Physical
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Sznitowska, M., Zurowska-Pryczkowska, K., Dabrowska, E., & Janicki, S. (2000). Increased
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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.
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Wang, Y., Lu, Z., Wu, H., & Lv, F. (2009). Study on the antibiotic activity of microcapsule
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curcumin against foodborne pathogens. International Journal of Food Microbiology, 136(1),
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71-74.
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Wu, S. Q., Li, S. M., Lang, Y. Y., Liu, H., & Liu, H. Z. (2005). Investigation on formation factors
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of oil-in-water microemulsion. Journal of Shenyang Pharmaceutical University, 22 (2), 97-99.
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Shan, C. Y., Ma, S. H., & Zhang, W. M. (2012). Application of spice oils in food preservation.
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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
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antimicrobial properties of starch films incorporated with ε-poly-l-lysine. Food Chemistry,
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166, 107-114.
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mix -su
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mix -su
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417 418 419 420
0.0
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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.
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Fig.2 The particle size distribution of the blended cloves/cinnamon nanoenulsion
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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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Different letters in the same column indicate significantly different (p ≤0.05).
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Table 2 Antimicrobial activity of the blended cloves/cinnamon essential oil
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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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E. coli
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Blank nanoemulsion b Cinnamon oil Cloves oil Blended nanoemulsion
Diameter of the inhibition zone/mm
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Table 3 Flavor substances of mushroom sauces with (sample) and without (control)
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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-
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4H-Pyran-4-one, 2-ethyl-3-hydroxyPhenol, 2-methoxyPhenol, 2-methyl-
Control
Sample
2.34
0.60
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1,2,4-Trithiolane Trisulfide, dimethyl Carbon disulfide 2-Acetylthiazole Disulfide, dimethyl Disulfide, di-2-propenyl Thiophene, 3,4-dimethyl-
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Nitrogen-containing heterocyclics 11.819 13.318 14.894 13.7 14.516 26.183 22.378 7.585
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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
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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
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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
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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
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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
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2-Heptenal, (E)Heptanal Pentanal Nonanal 2,4-Heptadienal, (E,E)Benzeneacetaldehyde Octanal 2-Pentenal, 2-methyl-
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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
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0.26
0.14 0.10 0.50 2.64
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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-
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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:
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A nanoemulsion composed of the blended clove/cinnamon essential oil was prepared The nanoemulsion preparation process was optimized
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The nanoemulsion showed good structural, thermal and storage stability
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The nanoemulsion showed higher antimicrobial activity against the common
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microorganisms
The substances in nanoemulsion could help food feature durable fragrance
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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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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
136
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
139
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
154
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
165
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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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
170
of the nanoemulsion). The flavor compounds of the mushroom sauces with and
171
without the nanoemulsion were analyzed on a gas chromatography-mass spectrometer
172
(GC-MS, SCION SQ 456-GC, Bruker Daltonics Inc., Billerica, MA, USA). The
173
detection parameters were as follows:
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Chromatographic conditions: Column: TRACE TR-50 MS (30 m × 0.25 mm, 0.25 µm,
175
Thermo Scientific, Pittsburgh, PA, USA); heating up programs: the temperature of
176
40 °C was kept for 3 mins, raised to 90 °C in 5 °C/min, then raised to 230 °C in 10 °C
177
/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
184
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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is at the optimal mass ratio, the cosurfactant is completely embedded into the
191
surfactant, so the solubilization space for the oil phase is formed to the maximum
192
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
194
< 1), the emulsifying ability is weak and only a small nanoemulsion area (the particle
195
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
197
(Wu, Li, Lang, Liu, & Liu, 2005). Different ratios of surfactant to cosurfactant (2:1,
198
3:1 and 4:1) were tested in this study. As shown in Fig. 1, the maximum nanoemulsion
199
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
206
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
208
the emulsions. With the increase of surfactant concentration, the decrease in particle
209
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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prepared at such an oil-surfactant ratio are able to maintain good stability and particle
213
size distribution, even being largely diluted (Men & Zhang, 2010). However, if the
214
surfactant amount is increased further, the oil phase load will become less, which
215
would not be a good property as the active agent would be too low to achieve the
216
target functionality (e.g. antimicrobial activity). Therefore, the oil-surfactant ratio of
217
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
219
blended nano cloves/cinnamon nanoemulsion were at Km of 3:1 and oil to surfactant
220
ratio of 1:9 (Table 1). This nanoemulsion was composed of cloves/cinnamon essential
221
oil 4%, tween 80 27%, ethanol 9% and water 60%. With this formulation, a clear and
222
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
225
very important indicators that describe the quality, stability, uniformity and
226
dispersibility of the nanoemulsions (Mason, Wilking, Meleson, Chang, & Graves,
227
2006). After DLS measurement, the average particle size of the blended
228
cloves/cinnamon nanoemulsion was about 8.69 nm. The peak width of the distribution
229
diagram was very narrow which comprised more than 97% of the droplets, indicating
230
that the size of the nanoemulsion particles were substantially uniform (Fig. 2).
231
Meanwhile, PDI gave information on the deviation from the average size, and the
232
lower value of 0.22 was desirable which suggested that the nanoemulsion droplets
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(Kelmann, Kuminek, Teixeira, & Koester, 2007).
235
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
238
yellowish, clear and transparent liquid, without observed stratification, turbidity, and
239
precipitate. After centrifugation at 10,000 rpm for 20 min, the system was maintained
240
the same clear and transparent appearance. The particle size distribution of the
241
nanoemulsion before and after the centrifugation was also in the same range,
242
indicating good stability. The nanoemulsion was stored at 25 ± 1 °C, 37 ± 1 °C and 60
243
± 1 °C for one month respectively. The results showed that the nanoemulsions did not
244
change during the storage below 37 °C and remained the clear and transparent state.
245
The particle size was even smaller when the emulsion was stored at higher
246
temperature of 60 °C. The small PDI range of 0.22 ~ 0.29 also showed the good
247
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
252
maintained the clear and transparent state. However, when the temperature was 90 °C
253
or above, the nanoemulsion became turbid. Interestingly, when the temperature was
254
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
257
and PDI about 0.19 ~ 0.33, similar to the samples before heat treatment (Table 1).
258
This
259
cloves/cinnamon essential oil nanoemulsion structure could be recovered to its
260
original state. This is probably because the solubility of the surface-active agent
261
(Tween 80 in this study) increases with the temperature increasing, but when the
262
temperature exceeds its cloud points, its solubility declines and jeopardizes the
263
stability of the nanoemulsions with the appearance of cloudiness. However, when the
264
temperature is cooled below the cloud point, the solubility of the surfactant resumes
265
and the nanoemulsions return to the transparent appearance (Quina, & Hinze, 1999).
266
When the temperature is close to or above the cloud point, the possibility of breaking
267
the nanoemulsion system is very high. However, the high temperature treatment in
268
this study did not cause an irreversible change on the stability of the cloves/cinnamon
269
essential oil emulsions. When the temperature was reduced below the surfactant’s
270
cloud point, the nanoemulsion restored its original properties. After one month's
271
storage at room temperature, the appearance, particle size distribution, mean particle
272
size, and PDI remained stable, indicating very good thermal stability of the
273
nanoemulsion.
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3.4 Antimicrobial activity
suggested
that
after
the
heat
treatment,
the
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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
281
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
283
nanoemulsion exhibited the highest antibacterial activity on the four microbial strains.
284
According to Sections 2.3.2 and 3.1.2, the nanoemulsion contained only 4% blended
285
cloves/cinnamon essential oil, whereas original cloves essential oil and cinnamon
286
essential oil extracts were used in the cloves and cinnamon treatment samples. The
287
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
301
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.
304
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
307
of cloves and cinnamon (Shan, Ma, & Zhang, 2012). In addition, the control group
308
contained 21.01% of sorbic acid (Table 3), which was mainly because of the
309
preservative of potassium sorbate in the mushroom sauce. Therefore, compared with
310
the preservative of potassium sorbate, the cloves/cinnamon nanoemulsion contained
311
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
315
cloves/cinnamon essential oil was optimized. At the Km = 3:1 and oil/surfactant ratio
316
1:9, the average particle size of the nanoemulsion was 8.69 nm and PDI 0.22. The
317
nanoemulsion maintained good stability and dispersion after centrifuging at 10,000
318
rpm for 20 min, stored at 60 °C for 1 month, or heated at 80 °C for 30 min,
319
respectively. Furthermore, even with far lower concentrations, the blended
320
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
323
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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417 418 419 420
0.0
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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.
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421 422
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Fig.2 The particle size distribution of the blended cloves/cinnamon nanoenulsion
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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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Different letters in the same column indicate significantly different (p ≤0.05).
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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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E. coli
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Blank nanoemulsion b Cinnamon oil Cloves oil Blended nanoemulsion
Diameter of the inhibition zone/mm
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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
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1,2,4-Trithiolane Trisulfide, dimethyl Carbon disulfide 2-Acetylthiazole Disulfide, dimethyl Disulfide, di-2-propenyl Thiophene, 3,4-dimethyl-
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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
M AN U
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
TE D
instead of harming flavor
M AN U
•