- Email: [email protected]
Microemulsification of clove essential oil improves its in vitro and in vivo control of Penicillium digitatum
Accepted Manuscript Microemulsification of clove essential oil improves its in vitro and in vivo control of Penicillium digitatum Shoukui He, Xiaoyun Ren, Yangfan Lu, Yunbin Zhang, Yifei Wang, Linjun Sun PII:
S0956-7135(16)30021-4
DOI:
10.1016/j.foodcont.2016.01.020
Reference:
JFCO 4830
To appear in:
Food Control
Received Date: 7 September 2015 Revised Date:
12 January 2016
Accepted Date: 13 January 2016
Please cite this article as: He S., Ren X., Lu Y., Zhang Y., Wang Y. & Sun L., Microemulsification of clove essential oil improves its in vitro and in vivo control of Penicillium digitatum, Food Control (2016), doi: 10.1016/j.foodcont.2016.01.020. 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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Microemulsification of clove essential oil improves its in vitro and in vivo control
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of Penicillium digitatum
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Running title: Microemulsion improves clove oil antifungal activity
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4 Shoukui He, Xiaoyun Ren, Yangfan Lu, Yunbin Zhang, Yifei Wang*, Linjun Sun
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School of Perfume and Aroma Technology, Shanghai Institute of Technology, Haiquan
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Road 100, Shanghai 201418, People’s Republic of China
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9 *Correspondence
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Yifei Wang, School of Perfume and Aroma Technology, Shanghai Institute of
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Technology, Shanghai 201418, People’s Republic of China
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Email: [email protected]
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Tel.: +86 021 60873150
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Fax: +86 021 60873248
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Abstract: Clove essential oil (CEO) is a promising alternative to chemical fungicides for
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postharvest decay control. However, relatively high concentrations required for in
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vivo microbial growth inhibition limit its application. Hence, this study aimed to
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strengthen the antifungal activity of CEO by loading it in microemulsion system. Two
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CEO
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CEO/ethanol/Tween 80 = 1:3:6) were evaluated their antifungal activity against
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Penicillium digitatum in vitro and in navel oranges. Microemulsification of CEO
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caused a reduction of MIC (minimum inhibitory concentration) from 0.50 µl/ml to
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0.25 µl/ml, while the MFC (minimum fungicidal concentration) remained unchanged
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at 0.50 µl/ml, indicating enhancement of only fungistatic activity. The decay
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incidence of navel oranges treated with ME-1 and ME-2 was significantly (p < 0.05)
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reduced by 34.51% and 20.93%, respectively, in comparison to that of pure oil
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treatment after 5 days’ storage at 25 oC. In the vapor phase, CEO microemulsions had
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the best control of lesion diameter and decay development. Additionally, the
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improved antifungal activity of CEO microemulsions may be related to their stronger
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ability to suppress spore germination and germ tube elongation of P. digitatum. The
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enhanced control of postharvest green mold of navel oranges by CEO after
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microemulsification can broaden its application in food industry.
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Key words: clove essential oil; microemulsion; antifungal activity; navel oranges;
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Penicillium digitatum
(ME-1,
CEO/ethanol/Tween
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ME-2,
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1. Introduction Navel oranges (Citrus sinensis L., Osbeck) are popular due to their highly
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nutritional value. Unluckily, they are readily affected by pathogens (i.e., Penicillium
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digitatum and Penicillium italicum) (Zeng, Zhang, Chen, & Fu, 2012). For citrus fruit,
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green mold caused by P. digitatum accounts for 90% of postharvest losses (Kellerman,
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Erasmus, Cronjé, & Fourie, 2014). Traditionally, chemical fungicides are primarily
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utilized to control this disease. However, in consideration of antifungal resistance,
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environmental problems and human toxicity (Xu, Yan, Ni, Chen, Zhang, & Zheng,
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2014), natural antifungal products should be developed as alternatives.
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In recent years, environmentally friendly essential oils (EOs) have been
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extensively studied as natural food preservatives due to their broad antimicrobial
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activities (Davidson, Critzer, & Taylor, 2013). Since EOs can be derived from organic
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plants, application of EOs is an appealing approach to control postharvest diseases. A
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predominant defense mechanism of plants against pests and pathogens is believed to
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be the production of EOs (Feng, & Zheng, 2007). Indeed, EOs of citronella (Chen et
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al., 2014), Laurus nobilis (Xu, Yan, Ni, Chen, Zhang, & Zheng, 2014), lemongrass
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and cinnamon (Maqbool, Ali, Alderson, Mohamed, Siddiqui, & Zahid, 2011) have
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exhibited inhibitory activity against postharvest fungal pathogens in vitro and in vivo.
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Despite the high efficiency of EOs in in vitro tests, the same effect in food is achieved
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only with higher concentrations (Burt, 2004; Hulin, Mathot, Mafart, & Dufosse, 1998).
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This fact may imply a challenging issue of EOs utilization in food industry, namely,
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the balance between sensory acceptability and antifungal activity.
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applications. An ethyl oleate-based microemulsion system was active against five
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foodborne pathogens (Teixeira, Leite, Domingues, Silva, Gibbs, & Ferreira, 2007).
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Similarly, another research group found that a food-grade glycerol monolaurate
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microemulsion was effective in inhibiting Bacillus subtilis, Escherichia coli and
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Staphylococcus aureus (Zhang, Shen, Bao, He, Feng, & Zheng, 2008a; Zhang, Shen,
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Weng, Zhao, Feng, & Zheng, 2009). In this sense, microemulsion could be an
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approach to attain a balance between antimicrobial efficiency and odor acceptability
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of EOs. However, up to now very little research has been carried out to formulate EO
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microemulsions for antifungal purposes.
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In systematic preliminary investigations conducted in our laboratory, we tested
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the antimicrobial activity of various EOs. From those, cinnamon and clove EOs were
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screened as being effective. Moreover, a cinnamon EO microemulsion previously
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established in our group controlled postharvest gray mold of pears without adverse
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influence on fruit qualities (Wang, Zhao, Yu, Zhang, He, & Yao, 2014), indicating that
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such technologies are very promising to satisfy consumer demand for commercial
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formulations of EOs. Consequently, the present work aimed to (i) develop clove oil
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microemulsions; (ii) determine their in vitro inhibitory activity against P. digitatum;
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(iii) evaluate their capacity to control green mold in navel oranges by direct contact
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and vapor phase.
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2. Materials and methods
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2.1 Materials
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No.1 Factory (Shanghai, China). Analytical grade Tween 80 was supplied by
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Shanghai Shenyu Pharmaceutical & Chemical Co., Ltd. (Shanghai, China). These
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chemicals were utilized without further purification. Commercially available clove
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essential oil (CEO) from Syzygium aromaticum was purchased from Dongshi Essence
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Perfume Co., Ltd. (Shanghai, China) and was stored at 4 °C in dark bottles prior to
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use. According to the information given by the supplier, this oil was produced by
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steam distillation method. Its major components were found to be eugenol (76.190%)
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and β-caryophyllene (15.088%) (He et al., 2014).
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2.2 Preparation of pathogen
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The navel orange fruit pathogen (P. digitatum), obtained from Institute of
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Microbiology, Chinese Academy of Sciences (Beijing, China), was maintained on
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potato dextrose agar (PDA) at 4 oC. P. digitatum was incubated for one week on PDA
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at 28 oC prior to treatment. Spore suspensions were prepared by flooding and
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suspending 1-week-old cultures of the pathogen in sterile distilled water (SDW).
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Spore concentrations were determined by a haemocytometer and adjusted to
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approximately 1.0×105 and 1.0×107 spores/ml with SDW.
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2.3 Formulation of CEO microemulsions CEO microemulsions were prepared as previously described (Wang, Zhao, Yu,
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Zhang, He, & Yao, 2014). Two kinds of microemulsions were prepared: (1) ME-1: 1.0
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g CEO, 2.0 g ethanol, 7.0 g Tween 80; and (2) ME-2: 1.0 g CEO, 3.0 g ethanol, 6.0 g
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Tween 80. Briefly, CEO and ethanol were vigorously mixed at predetermined weight
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ACCEPTED MANUSCRIPT ratios in glass test tubes sealed with removable caps at 25 oC. Tween 80 was then
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added to the mixture under constant stirring using a magnetic stirrer (78-1B, Shanghai,
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China) for 30 min. CEO microemulsions were stored at 4 oC and allowed to
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equilibrate for at least 24 h at 25 oC before treatment to guarantee a steady-state
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condition. In the two formulated microemulsions, evaporation loss was negligible.
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After equilibration for a period of 24 h, the resulting microemulsions were diluted to
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the instrument sensitivity range with deionized water to measure the mean particle
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diameter using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a
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He-Ne laser (633.0 nm). The measurements were performed at a 90o scattering angle.
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ME-1 and ME-2 showed a droplet size of 241.1 nm and 150.0 nm, respectively. The
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stability of the developed microemulsions was then examined according to Zhang, Li,
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Zhu, Feng, and Zheng (2011) with modifications. ME-1 and ME-2 were subjected to
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centrifugation at 4000 g for 15 min and 1-month storage at 20 oC. It was observed that
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no phase separation or creaming occurred.
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2.4. In vitro antifungal effects of CEO microemulsions
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MIC (Minimum inhibitory concentration) and MFC (minimum fungicidal
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concentration) of CEO microemulsions were determined using 2-fold serial dilution
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method (Jordán, Lax, Rota, Lorán, & Sotomayor, 2013) in order to assess their in
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vitro antifungal activity. Aliquots (50 µl) of P. digitatum suspensions (approximately
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1.0×107 spores/ml) were inoculated into 5 ml of potato dextrose broth (PDB)
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containing suitable amounts of CEO microemulsions (0.0625-2 µl/ml). Both negative
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(sterile PDB) and positive (5 ml of PDB + 50 µl of inoculum) controls were included.
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The samples were then incubated at 28 oC with shaking (200 rpm) for 48 h. The MIC
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was recorded as the lowest concentration of microemulsions that completely inhibited
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visible growth of P. digitatum. In order to determine MFC, 100 µl of each case without visible microbial growth
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was plated on PDA. Plates were incubated at 28 oC for 48 h. The lowest concentration
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at which P. digitatum failed to grow on PDA plates was defined as MFC. The MIC
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and MFC of pure oil was also determined in a similar way. The assay was performed
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in triplicate.
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The fungistatic and fungicidal effects of CEO microemulsions were considered
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to be significantly (p < 0.05) enhanced when their MIC and MFC values were equal
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to or higher than a twofold decrease compared with those of pure oil, respectively
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(Monte et al., 2014).
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2.5 The capacity of CEO microemulsions to control green mold in navel oranges
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2.5.1 Direct addition assay
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Navel oranges (Citrus sinensis L., Osbeck), harvested at commercial maturity,
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were obtained from Gannan, China. Samples of uniform ripeness, size and free of
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injuries were selected. The test fruits were surface-sterilized in sodium hypochlorite
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(0.1%) for 60 s, scoured under running water and air dried at 25 oC. Fruits were gently
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wounded (3 mm deep, 5 mm diameter) with a sterile puncher. A 30 µl aliquot of spore
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suspension of P. digitatum (approximately 1.0×105 spores/ml) was pipetted into each
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wound. Samples were then treated with 30 µl CEO microemulsions or pure oil at the
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level of 0.25 µl/ml. SDW served as the control. After air drying for 1.5 h, the oranges
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(approximately 90%) at 25 oC. Disease incidence (infected wound number/total
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wound number) and corresponding lesion diameter of oranges were examined after 5
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days. Each treatment consisted of three replicates of 30 wounds each, and the
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experiment was performed twice.
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2.5.2 Vapor contact assay
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Vapor contact test was used to determine the antifungal effects of vapors of CEO
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microemulsions as previously stated (Wang, Zhao, Yu, Zhang, He, & Yao, 2014).
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Oranges were placed in 8-L plastic boxes after wounded and inoculated with the
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pathogen as described above. For each box, a culture dish (3.5 cm diameter)
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containing 30 µl SDW (control), pure oil (0.025 µl/ml) or CEO microemulsions
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(0.025 µl/ml) was placed in the center. These boxes were then sealed using
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polyethylene bags. Disease incidence (infected wound number/total wound number)
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and corresponding lesion diameter of oranges were examined after 5 days’ storage at
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25 oC. Each treatment consisted of three replicates of 30 wounds each, and the
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experiment was performed twice.
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2.6 Spore germination assay
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Pure oil and CEO microemulsions were added to 15-ml tubes containing 5 ml
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PDB to achieve a final level of 0.25 µl/ml, respectively. SDW was added as the
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control. Then a spore suspension (approximately 1×107 spores/ml) of 100µl was
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inoculated into each tube. Spore germination (%) and germ tube length (µm) were
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assessed following 18 h incubation at 28 oC/200 rpm. A spore was regarded as
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150 spores per replicate were microscopically examined for the presence of germ
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tubes. The percentage of germinated spores was then estimated from the evaluated
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spores. Furthermore, germ tube length (µm) was also determined. Each treatment
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consisted of three replicates, and the experiment was performed twice.
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2.7 Data analysis
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Statistical analyses were conducted with SAS version 8.2, via one-way ANOVA.
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Duncan's test was then used to detect statistical significance. Differences in mean
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values were considered significant when p < 0.05.
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3. Results
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3.1 In vitro antifungal activity of CEO microemulsions
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The MIC and MFC values of CEO microemulsions in comparison to pure oil,
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reported in Fig. 1, give a measurement of the antifungal activity. Microemulsification
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of CEO caused a reduction of MIC from 0.50 µl/ml to 0.25 µl/ml, while the MFC
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remained unchanged at 0.50 µl/ml. The lower MIC of CEO microemulsions suggested
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their enhancement in antifungal activity relative to pure oil.
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3.2 The capacity of CEO microemulsions to control green mold in navel oranges
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3.2.1 Direct addition assay
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Direct addition assay was used to evaluate inhibitory effect of CEO
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microemulsions on navel orange green mold. As shown in Fig. 2, pure oil and
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microemulsions greatly delayed decay development. Moreover, the disease incidence
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of navel oranges treated with microemulsion ME-1 and ME-2 was significantly (p <
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0.05) reduced by 34.51% and 20.93%, respectively, in comparison to the treatment
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with pure oil.
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3.2.2 Vapor contact assay Data shown in Fig. 3 indicated that pure oil and microemulsions in vapor phase
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could also significantly (p < 0.05) inhibit P. digitatum on navel oranges after 5 days’
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storage at 25 oC. CEO microemulsions exhibited the best control of lesion diameter
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and decay development (57.34%-59.52%, 8.28-8.59 mm), followed by pure oil
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(72.84%, 9.54 mm).
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3.3 Spore germination assay
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Determination of spore germination and germ tube elongation could help to
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explain the antifungal activity of CEO microemulsions. In the control group, all
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spores germed and the germ tubes were too long and enwind each other (Fig. 4).
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However, pure oil and microemulsions significantly (p < 0.05) inhibited fungal spore
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germination rate and reduced germ tube length. The average suppressive efficacy was
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in the following order: ME-1 ≈ ME-2 > pure oil > SDW control.
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4. Discussion
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Although microemulsions have been utilized to dissolve hydrophobic
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compounds for food, cosmetic, pharmaceutical and oil recovery applications (Ma &
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Zhong, 2015), only a limited number of reports relevant to their use for antimicrobial
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purposes are available. The utilization of microemulsions as antimicrobials is quite a
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new and promising application. In the current work, we explored the possibility of
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reinforcing CEO antimicrobial activity by loading it in microemulsion system.
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Interestingly, CEO microemulsions showed better control efficiency against P.
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digitatum than pure oil in vitro and in vivo. In in vitro tests, CEO microemulsions exhibited enhanced fungistatic activity as
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compared to pure oil. This phenomenon may result from the small droplet sizes of
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CEO microemulsions (ME-1, 241.1 nm; ME-2, 150.0 nm), which could improve the
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transport mechanisms through P. digitatum cell membrane (Donsìa, Annunziatab,
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Sessaa, & Ferraria, 2011). Similarly, microemulsion was found to improve the
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antimicrobial activity of glycerol monolaurate (the glycerol monoester of lauric acid)
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(Fu, Feng, & Huang, 2006; Zhang, Shen, Bao, He, Feng, & Zheng, 2008a; Zhang, Lu,
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Wang, Shen, Feng, & Zheng, 2008b). In the food additive industry, there are
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increasing applications of using combinations of antimicrobial substances at
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sub-lethal levels instead of putting reliance on individual food preservatives at high
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concentrations (Roller, 1999). In this regard, enhanced fungistatic activity may imply
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a promising application of CEO microemulsions.
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The functionality of antimicrobials needs to be checked in real food systems.
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Hence, the in vivo antifungal activity of CEO microemulsions in the liquid and vapor
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phases was determined subsequently. In agreement with a previous study, pure CEO
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had some control effects on citrus green mold caused by P. digitatum (Shao, Cao, Xu,
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Xie, Yu, & Wang, 2015). Additionally, better antifungal activity was found in CEO
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microemulsions in the liquid phase. Similarly, cinnamon oil microemulsions
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previously formulated in our laboratory exhibited higher in vivo antifungal activity
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than pure ones (Wang, Zhao, Yu, Zhang, He, & Yao, 2014).
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effect (Tyagi, Malik, Gottardi, & Guerzoni, 2012). The antimicrobial activity
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generated by CEO vapor has been reported in previous literature. Tullio et al. (2007)
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found that CEO vapor had a wide antifungal spectrum. Vapors of EO combinations of
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clove and cinnamon exerted a synergism or antagonistic effect for the inhibition of
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bacteria, depending on EO concentration applied (Goni, Lopez, Sanchez, Gomez-Lus,
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Becerril, & Nerin, 2009). In the present paper, we evaluated the effect of vapors of
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CEO microemulsions in controlling a major postharvest pathogen P. digitatum. Better
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control activity was observed in microemulsified CEO. Moreover, CEO
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microemulsions in the liquid and vapor phases exhibited quite similar antifungal
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efficacy, evidencing that microemulsification technology may be a promising
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approach to meet consumer demands for commercialization formulations of EOs.
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Exploring the mechanism of action could help to elucidate the remarkable
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performance of CEO microemulsions. In this study, spore germination and germ tube
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elongation of P. digitatum was significantly (p < 0.05) suppressed by pure oil and
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microemulsions, which was indirect evidence of disruption and dysfunction of cell
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walls and membranes. Furthermore, the greatest inhibition was observed in P.
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digitatum treated with CEO microemulsions. Thus, it can be inferred that the
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enhanced antifungal activity of CEO microemulsions might be due to the increase in
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cell membrane permeabilization caused by the small droplet sizes of these
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formulations.
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In conclusion, improved control activity of CEO against P. digitatum in vitro and
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CEO microemulsions exhibited stronger inhibitory activity on spore germination and
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germ tube elongation than pure oil. Due to the enhanced antifungal activity of
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microemulsified CEO in the vapor and liquid phases, lower antimicrobial
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concentrations are required for utilization in food. This fact is of paramount
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importance from a safety and organoleptic point of view. The sensory effect of CEO
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might thus be reduced. These results suggest that CEO microemulsions established in
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this work may be potential commercial formulations to control postharvest green
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mold of navel oranges. Further investigations should be conducted to explore the
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effects of these formulations on fruit quality traits, such as sensory acceptability,
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weight loss, firmness, total soluble solids, ascorbic acid and color.
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Acknowledgment
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This research was financially supported by Shanghai Municipal Education
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Commission Science and Technology Innovation Foundation (12YZ165) and Science
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and Technology Commission of Shanghai Municipality (13120503300).
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cinnamon essential oil microemulsion and its vapor phase for controlling postharvest
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gray mold of pears (pyrus pyrifolia). Journal of the Science of Food and Agriculture,
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94(5), 1000–1004.
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Xu, S., Yan, F., Ni, Z., Chen, Q., Zhang, H., & Zheng, X. (2014). In vitro and in vivo
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control of Alternaria alternata in cherry tomato by essential oil from Laurus nobilis
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of Chinese origin. Journal of the Science of Food and Agriculture, 94(7), 1403–1408.
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Zeng, R., Zhang, A., Chen, J., & Fu, Y. (2012). Postharvest quality and physiological
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138, 253–258.
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Zhang, H., Shen, Y., Bao, Y., He, Y., Feng, F., & Zheng, X. (2008a). Characterization
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and synergistic antimicrobial activities of food-grade dilution-stable microemulsions
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against Bacillus subtilis. Food Research International, 41(5), 495–499.
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Zhang, H., Lu, Z., Wang, S., Shen, Y., Feng, F., & Zheng, X. (2008b). Development
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and antifungal evaluation of a food-grade U-type microemulsion. Journal of Applied
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Microbiology, 105(4), 993–1001.
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Zhang, H., Shen, Y., Weng, P., Zhao, G., Feng, F., & Zheng, X. (2009). Antimicrobial
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activity of a food-grade fully dilutable microemulsion against Escherichia coli and
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Staphylococcus aureus. International Journal of Food Microbiology, 135(3),
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211–215.
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Zhang, H., Li, D., Zhu, S., Feng, F., & Zheng, X. (2011). Antibacterial activities of a
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food-grade dilution-stable microemulsion. Journal of Food Safety, 31(2), 232–237.
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Figure 1 Minimum inhibitory concentration (MIC) and minimum fungicidal
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concentration (MFC) values of clove essential oil microemulsions against P.
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digitatum.
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Figure 2 The inhibitory effects of clove essential oil microemulsions on P.digitatum
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decay development in artificially wounded and inoculated navel oranges. The disease
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incidence (A) and lesion diameter (B) were measured after 5 days’ storage at 25 oC.
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Vertical bars represent standard deviation. Different letters signify significant
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differences (p < 0.05).
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Figure 3 The inhibitory effects of vapors of clove essential oil microemulsions on
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P.digitatum decay development in artificially wounded and inoculated navel oranges.
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The disease incidence (A) and lesion diameter (B) were measured after 5 days’
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storage at 25 oC. Vertical bars represent standard deviation. Different letters signify
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significant differences (p < 0.05).
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Figure 4 Effects of clove essential oil microemulsions on spore germination (A),
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germ tube elongation (B) and microscope images (C) of P. digitatum. Data represent
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mean ± standard deviation. Different lowercase letters within a column indicate
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significant differences (p < 0.05). ND (not determined), the germ tube length could
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not be determined because the germ tubes were too long and enwind each other.
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Germination and germ tube length were determined microscopically (C) after 18 h
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incubation at 28 oC in potato dextrose broth (for control, magnification=960×; for
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other treatments, magnification=2560×).
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ACCEPTED MANUSCRIPT Highlights Two clove essential oil (CEO) microemulsions were formulated.
Microemulsions showed enhanced fungistatic activity against P. digitatum.
Microemulsion and its vapor controlled postharvest green mould of navel
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oranges.
Microemulsions decreased spore germination and germ tube elongation of P.
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DOI:
10.1016/j.foodcont.2016.01.020
Reference:
JFCO 4830
To appear in:
Food Control
Received Date: 7 September 2015 Revised Date:
12 January 2016
Accepted Date: 13 January 2016
Please cite this article as: He S., Ren X., Lu Y., Zhang Y., Wang Y. & Sun L., Microemulsification of clove essential oil improves its in vitro and in vivo control of Penicillium digitatum, Food Control (2016), doi: 10.1016/j.foodcont.2016.01.020. 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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Microemulsification of clove essential oil improves its in vitro and in vivo control
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of Penicillium digitatum
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Running title: Microemulsion improves clove oil antifungal activity
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4 Shoukui He, Xiaoyun Ren, Yangfan Lu, Yunbin Zhang, Yifei Wang*, Linjun Sun
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School of Perfume and Aroma Technology, Shanghai Institute of Technology, Haiquan
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Road 100, Shanghai 201418, People’s Republic of China
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9 *Correspondence
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Yifei Wang, School of Perfume and Aroma Technology, Shanghai Institute of
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Technology, Shanghai 201418, People’s Republic of China
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Email: [email protected]
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Tel.: +86 021 60873150
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Fax: +86 021 60873248
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Abstract: Clove essential oil (CEO) is a promising alternative to chemical fungicides for
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postharvest decay control. However, relatively high concentrations required for in
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vivo microbial growth inhibition limit its application. Hence, this study aimed to
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strengthen the antifungal activity of CEO by loading it in microemulsion system. Two
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CEO
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CEO/ethanol/Tween 80 = 1:3:6) were evaluated their antifungal activity against
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Penicillium digitatum in vitro and in navel oranges. Microemulsification of CEO
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caused a reduction of MIC (minimum inhibitory concentration) from 0.50 µl/ml to
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0.25 µl/ml, while the MFC (minimum fungicidal concentration) remained unchanged
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at 0.50 µl/ml, indicating enhancement of only fungistatic activity. The decay
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incidence of navel oranges treated with ME-1 and ME-2 was significantly (p < 0.05)
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reduced by 34.51% and 20.93%, respectively, in comparison to that of pure oil
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treatment after 5 days’ storage at 25 oC. In the vapor phase, CEO microemulsions had
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the best control of lesion diameter and decay development. Additionally, the
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improved antifungal activity of CEO microemulsions may be related to their stronger
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ability to suppress spore germination and germ tube elongation of P. digitatum. The
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enhanced control of postharvest green mold of navel oranges by CEO after
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microemulsification can broaden its application in food industry.
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Key words: clove essential oil; microemulsion; antifungal activity; navel oranges;
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Penicillium digitatum
(ME-1,
CEO/ethanol/Tween
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=
1:2:7;
ME-2,
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1. Introduction Navel oranges (Citrus sinensis L., Osbeck) are popular due to their highly
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nutritional value. Unluckily, they are readily affected by pathogens (i.e., Penicillium
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digitatum and Penicillium italicum) (Zeng, Zhang, Chen, & Fu, 2012). For citrus fruit,
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green mold caused by P. digitatum accounts for 90% of postharvest losses (Kellerman,
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Erasmus, Cronjé, & Fourie, 2014). Traditionally, chemical fungicides are primarily
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utilized to control this disease. However, in consideration of antifungal resistance,
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environmental problems and human toxicity (Xu, Yan, Ni, Chen, Zhang, & Zheng,
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2014), natural antifungal products should be developed as alternatives.
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In recent years, environmentally friendly essential oils (EOs) have been
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extensively studied as natural food preservatives due to their broad antimicrobial
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activities (Davidson, Critzer, & Taylor, 2013). Since EOs can be derived from organic
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plants, application of EOs is an appealing approach to control postharvest diseases. A
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predominant defense mechanism of plants against pests and pathogens is believed to
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be the production of EOs (Feng, & Zheng, 2007). Indeed, EOs of citronella (Chen et
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al., 2014), Laurus nobilis (Xu, Yan, Ni, Chen, Zhang, & Zheng, 2014), lemongrass
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and cinnamon (Maqbool, Ali, Alderson, Mohamed, Siddiqui, & Zahid, 2011) have
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exhibited inhibitory activity against postharvest fungal pathogens in vitro and in vivo.
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Despite the high efficiency of EOs in in vitro tests, the same effect in food is achieved
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only with higher concentrations (Burt, 2004; Hulin, Mathot, Mafart, & Dufosse, 1998).
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This fact may imply a challenging issue of EOs utilization in food industry, namely,
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the balance between sensory acceptability and antifungal activity.
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applications. An ethyl oleate-based microemulsion system was active against five
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foodborne pathogens (Teixeira, Leite, Domingues, Silva, Gibbs, & Ferreira, 2007).
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Similarly, another research group found that a food-grade glycerol monolaurate
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microemulsion was effective in inhibiting Bacillus subtilis, Escherichia coli and
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Staphylococcus aureus (Zhang, Shen, Bao, He, Feng, & Zheng, 2008a; Zhang, Shen,
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Weng, Zhao, Feng, & Zheng, 2009). In this sense, microemulsion could be an
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approach to attain a balance between antimicrobial efficiency and odor acceptability
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of EOs. However, up to now very little research has been carried out to formulate EO
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microemulsions for antifungal purposes.
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In systematic preliminary investigations conducted in our laboratory, we tested
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the antimicrobial activity of various EOs. From those, cinnamon and clove EOs were
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screened as being effective. Moreover, a cinnamon EO microemulsion previously
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established in our group controlled postharvest gray mold of pears without adverse
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influence on fruit qualities (Wang, Zhao, Yu, Zhang, He, & Yao, 2014), indicating that
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such technologies are very promising to satisfy consumer demand for commercial
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formulations of EOs. Consequently, the present work aimed to (i) develop clove oil
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microemulsions; (ii) determine their in vitro inhibitory activity against P. digitatum;
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(iii) evaluate their capacity to control green mold in navel oranges by direct contact
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and vapor phase.
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2. Materials and methods
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2.1 Materials
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No.1 Factory (Shanghai, China). Analytical grade Tween 80 was supplied by
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Shanghai Shenyu Pharmaceutical & Chemical Co., Ltd. (Shanghai, China). These
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chemicals were utilized without further purification. Commercially available clove
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essential oil (CEO) from Syzygium aromaticum was purchased from Dongshi Essence
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Perfume Co., Ltd. (Shanghai, China) and was stored at 4 °C in dark bottles prior to
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use. According to the information given by the supplier, this oil was produced by
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steam distillation method. Its major components were found to be eugenol (76.190%)
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and β-caryophyllene (15.088%) (He et al., 2014).
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2.2 Preparation of pathogen
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The navel orange fruit pathogen (P. digitatum), obtained from Institute of
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Microbiology, Chinese Academy of Sciences (Beijing, China), was maintained on
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potato dextrose agar (PDA) at 4 oC. P. digitatum was incubated for one week on PDA
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at 28 oC prior to treatment. Spore suspensions were prepared by flooding and
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suspending 1-week-old cultures of the pathogen in sterile distilled water (SDW).
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Spore concentrations were determined by a haemocytometer and adjusted to
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approximately 1.0×105 and 1.0×107 spores/ml with SDW.
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2.3 Formulation of CEO microemulsions CEO microemulsions were prepared as previously described (Wang, Zhao, Yu,
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Zhang, He, & Yao, 2014). Two kinds of microemulsions were prepared: (1) ME-1: 1.0
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g CEO, 2.0 g ethanol, 7.0 g Tween 80; and (2) ME-2: 1.0 g CEO, 3.0 g ethanol, 6.0 g
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Tween 80. Briefly, CEO and ethanol were vigorously mixed at predetermined weight
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ACCEPTED MANUSCRIPT ratios in glass test tubes sealed with removable caps at 25 oC. Tween 80 was then
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added to the mixture under constant stirring using a magnetic stirrer (78-1B, Shanghai,
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China) for 30 min. CEO microemulsions were stored at 4 oC and allowed to
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equilibrate for at least 24 h at 25 oC before treatment to guarantee a steady-state
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condition. In the two formulated microemulsions, evaporation loss was negligible.
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After equilibration for a period of 24 h, the resulting microemulsions were diluted to
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the instrument sensitivity range with deionized water to measure the mean particle
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diameter using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a
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He-Ne laser (633.0 nm). The measurements were performed at a 90o scattering angle.
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ME-1 and ME-2 showed a droplet size of 241.1 nm and 150.0 nm, respectively. The
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stability of the developed microemulsions was then examined according to Zhang, Li,
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Zhu, Feng, and Zheng (2011) with modifications. ME-1 and ME-2 were subjected to
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centrifugation at 4000 g for 15 min and 1-month storage at 20 oC. It was observed that
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no phase separation or creaming occurred.
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2.4. In vitro antifungal effects of CEO microemulsions
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MIC (Minimum inhibitory concentration) and MFC (minimum fungicidal
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concentration) of CEO microemulsions were determined using 2-fold serial dilution
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method (Jordán, Lax, Rota, Lorán, & Sotomayor, 2013) in order to assess their in
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vitro antifungal activity. Aliquots (50 µl) of P. digitatum suspensions (approximately
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1.0×107 spores/ml) were inoculated into 5 ml of potato dextrose broth (PDB)
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containing suitable amounts of CEO microemulsions (0.0625-2 µl/ml). Both negative
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(sterile PDB) and positive (5 ml of PDB + 50 µl of inoculum) controls were included.
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The samples were then incubated at 28 oC with shaking (200 rpm) for 48 h. The MIC
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was recorded as the lowest concentration of microemulsions that completely inhibited
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visible growth of P. digitatum. In order to determine MFC, 100 µl of each case without visible microbial growth
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was plated on PDA. Plates were incubated at 28 oC for 48 h. The lowest concentration
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at which P. digitatum failed to grow on PDA plates was defined as MFC. The MIC
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and MFC of pure oil was also determined in a similar way. The assay was performed
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in triplicate.
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The fungistatic and fungicidal effects of CEO microemulsions were considered
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to be significantly (p < 0.05) enhanced when their MIC and MFC values were equal
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to or higher than a twofold decrease compared with those of pure oil, respectively
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(Monte et al., 2014).
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2.5 The capacity of CEO microemulsions to control green mold in navel oranges
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2.5.1 Direct addition assay
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Navel oranges (Citrus sinensis L., Osbeck), harvested at commercial maturity,
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were obtained from Gannan, China. Samples of uniform ripeness, size and free of
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injuries were selected. The test fruits were surface-sterilized in sodium hypochlorite
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(0.1%) for 60 s, scoured under running water and air dried at 25 oC. Fruits were gently
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wounded (3 mm deep, 5 mm diameter) with a sterile puncher. A 30 µl aliquot of spore
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suspension of P. digitatum (approximately 1.0×105 spores/ml) was pipetted into each
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wound. Samples were then treated with 30 µl CEO microemulsions or pure oil at the
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level of 0.25 µl/ml. SDW served as the control. After air drying for 1.5 h, the oranges
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(approximately 90%) at 25 oC. Disease incidence (infected wound number/total
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wound number) and corresponding lesion diameter of oranges were examined after 5
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days. Each treatment consisted of three replicates of 30 wounds each, and the
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experiment was performed twice.
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2.5.2 Vapor contact assay
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Vapor contact test was used to determine the antifungal effects of vapors of CEO
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microemulsions as previously stated (Wang, Zhao, Yu, Zhang, He, & Yao, 2014).
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Oranges were placed in 8-L plastic boxes after wounded and inoculated with the
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pathogen as described above. For each box, a culture dish (3.5 cm diameter)
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containing 30 µl SDW (control), pure oil (0.025 µl/ml) or CEO microemulsions
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(0.025 µl/ml) was placed in the center. These boxes were then sealed using
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polyethylene bags. Disease incidence (infected wound number/total wound number)
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and corresponding lesion diameter of oranges were examined after 5 days’ storage at
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25 oC. Each treatment consisted of three replicates of 30 wounds each, and the
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experiment was performed twice.
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2.6 Spore germination assay
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Pure oil and CEO microemulsions were added to 15-ml tubes containing 5 ml
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PDB to achieve a final level of 0.25 µl/ml, respectively. SDW was added as the
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control. Then a spore suspension (approximately 1×107 spores/ml) of 100µl was
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inoculated into each tube. Spore germination (%) and germ tube length (µm) were
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assessed following 18 h incubation at 28 oC/200 rpm. A spore was regarded as
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150 spores per replicate were microscopically examined for the presence of germ
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tubes. The percentage of germinated spores was then estimated from the evaluated
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spores. Furthermore, germ tube length (µm) was also determined. Each treatment
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consisted of three replicates, and the experiment was performed twice.
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2.7 Data analysis
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Statistical analyses were conducted with SAS version 8.2, via one-way ANOVA.
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Duncan's test was then used to detect statistical significance. Differences in mean
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values were considered significant when p < 0.05.
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3. Results
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3.1 In vitro antifungal activity of CEO microemulsions
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The MIC and MFC values of CEO microemulsions in comparison to pure oil,
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reported in Fig. 1, give a measurement of the antifungal activity. Microemulsification
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of CEO caused a reduction of MIC from 0.50 µl/ml to 0.25 µl/ml, while the MFC
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remained unchanged at 0.50 µl/ml. The lower MIC of CEO microemulsions suggested
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their enhancement in antifungal activity relative to pure oil.
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3.2 The capacity of CEO microemulsions to control green mold in navel oranges
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3.2.1 Direct addition assay
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Direct addition assay was used to evaluate inhibitory effect of CEO
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microemulsions on navel orange green mold. As shown in Fig. 2, pure oil and
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microemulsions greatly delayed decay development. Moreover, the disease incidence
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of navel oranges treated with microemulsion ME-1 and ME-2 was significantly (p <
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0.05) reduced by 34.51% and 20.93%, respectively, in comparison to the treatment
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with pure oil.
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3.2.2 Vapor contact assay Data shown in Fig. 3 indicated that pure oil and microemulsions in vapor phase
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could also significantly (p < 0.05) inhibit P. digitatum on navel oranges after 5 days’
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storage at 25 oC. CEO microemulsions exhibited the best control of lesion diameter
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and decay development (57.34%-59.52%, 8.28-8.59 mm), followed by pure oil
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(72.84%, 9.54 mm).
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3.3 Spore germination assay
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Determination of spore germination and germ tube elongation could help to
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explain the antifungal activity of CEO microemulsions. In the control group, all
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spores germed and the germ tubes were too long and enwind each other (Fig. 4).
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However, pure oil and microemulsions significantly (p < 0.05) inhibited fungal spore
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germination rate and reduced germ tube length. The average suppressive efficacy was
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in the following order: ME-1 ≈ ME-2 > pure oil > SDW control.
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4. Discussion
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compounds for food, cosmetic, pharmaceutical and oil recovery applications (Ma &
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Zhong, 2015), only a limited number of reports relevant to their use for antimicrobial
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purposes are available. The utilization of microemulsions as antimicrobials is quite a
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new and promising application. In the current work, we explored the possibility of
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reinforcing CEO antimicrobial activity by loading it in microemulsion system.
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Interestingly, CEO microemulsions showed better control efficiency against P.
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digitatum than pure oil in vitro and in vivo. In in vitro tests, CEO microemulsions exhibited enhanced fungistatic activity as
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compared to pure oil. This phenomenon may result from the small droplet sizes of
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CEO microemulsions (ME-1, 241.1 nm; ME-2, 150.0 nm), which could improve the
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transport mechanisms through P. digitatum cell membrane (Donsìa, Annunziatab,
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Sessaa, & Ferraria, 2011). Similarly, microemulsion was found to improve the
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antimicrobial activity of glycerol monolaurate (the glycerol monoester of lauric acid)
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(Fu, Feng, & Huang, 2006; Zhang, Shen, Bao, He, Feng, & Zheng, 2008a; Zhang, Lu,
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Wang, Shen, Feng, & Zheng, 2008b). In the food additive industry, there are
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increasing applications of using combinations of antimicrobial substances at
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sub-lethal levels instead of putting reliance on individual food preservatives at high
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concentrations (Roller, 1999). In this regard, enhanced fungistatic activity may imply
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a promising application of CEO microemulsions.
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The functionality of antimicrobials needs to be checked in real food systems.
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Hence, the in vivo antifungal activity of CEO microemulsions in the liquid and vapor
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phases was determined subsequently. In agreement with a previous study, pure CEO
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had some control effects on citrus green mold caused by P. digitatum (Shao, Cao, Xu,
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Xie, Yu, & Wang, 2015). Additionally, better antifungal activity was found in CEO
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microemulsions in the liquid phase. Similarly, cinnamon oil microemulsions
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previously formulated in our laboratory exhibited higher in vivo antifungal activity
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than pure ones (Wang, Zhao, Yu, Zhang, He, & Yao, 2014).
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effect (Tyagi, Malik, Gottardi, & Guerzoni, 2012). The antimicrobial activity
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generated by CEO vapor has been reported in previous literature. Tullio et al. (2007)
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found that CEO vapor had a wide antifungal spectrum. Vapors of EO combinations of
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clove and cinnamon exerted a synergism or antagonistic effect for the inhibition of
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bacteria, depending on EO concentration applied (Goni, Lopez, Sanchez, Gomez-Lus,
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Becerril, & Nerin, 2009). In the present paper, we evaluated the effect of vapors of
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CEO microemulsions in controlling a major postharvest pathogen P. digitatum. Better
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control activity was observed in microemulsified CEO. Moreover, CEO
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microemulsions in the liquid and vapor phases exhibited quite similar antifungal
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efficacy, evidencing that microemulsification technology may be a promising
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approach to meet consumer demands for commercialization formulations of EOs.
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Exploring the mechanism of action could help to elucidate the remarkable
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performance of CEO microemulsions. In this study, spore germination and germ tube
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elongation of P. digitatum was significantly (p < 0.05) suppressed by pure oil and
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microemulsions, which was indirect evidence of disruption and dysfunction of cell
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walls and membranes. Furthermore, the greatest inhibition was observed in P.
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digitatum treated with CEO microemulsions. Thus, it can be inferred that the
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enhanced antifungal activity of CEO microemulsions might be due to the increase in
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cell membrane permeabilization caused by the small droplet sizes of these
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formulations.
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In conclusion, improved control activity of CEO against P. digitatum in vitro and
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CEO microemulsions exhibited stronger inhibitory activity on spore germination and
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germ tube elongation than pure oil. Due to the enhanced antifungal activity of
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microemulsified CEO in the vapor and liquid phases, lower antimicrobial
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concentrations are required for utilization in food. This fact is of paramount
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importance from a safety and organoleptic point of view. The sensory effect of CEO
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might thus be reduced. These results suggest that CEO microemulsions established in
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this work may be potential commercial formulations to control postharvest green
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mold of navel oranges. Further investigations should be conducted to explore the
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effects of these formulations on fruit quality traits, such as sensory acceptability,
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weight loss, firmness, total soluble solids, ascorbic acid and color.
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Acknowledgment
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This research was financially supported by Shanghai Municipal Education
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Commission Science and Technology Innovation Foundation (12YZ165) and Science
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and Technology Commission of Shanghai Municipality (13120503300).
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Figure 1 Minimum inhibitory concentration (MIC) and minimum fungicidal
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concentration (MFC) values of clove essential oil microemulsions against P.
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digitatum.
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Figure 2 The inhibitory effects of clove essential oil microemulsions on P.digitatum
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decay development in artificially wounded and inoculated navel oranges. The disease
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incidence (A) and lesion diameter (B) were measured after 5 days’ storage at 25 oC.
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Vertical bars represent standard deviation. Different letters signify significant
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differences (p < 0.05).
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Figure 3 The inhibitory effects of vapors of clove essential oil microemulsions on
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P.digitatum decay development in artificially wounded and inoculated navel oranges.
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The disease incidence (A) and lesion diameter (B) were measured after 5 days’
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storage at 25 oC. Vertical bars represent standard deviation. Different letters signify
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significant differences (p < 0.05).
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Figure 4 Effects of clove essential oil microemulsions on spore germination (A),
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germ tube elongation (B) and microscope images (C) of P. digitatum. Data represent
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mean ± standard deviation. Different lowercase letters within a column indicate
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significant differences (p < 0.05). ND (not determined), the germ tube length could
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not be determined because the germ tubes were too long and enwind each other.
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Germination and germ tube length were determined microscopically (C) after 18 h
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incubation at 28 oC in potato dextrose broth (for control, magnification=960×; for
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other treatments, magnification=2560×).
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Figure 1 (A)
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ACCEPTED MANUSCRIPT Highlights Two clove essential oil (CEO) microemulsions were formulated.
Microemulsions showed enhanced fungistatic activity against P. digitatum.
Microemulsion and its vapor controlled postharvest green mould of navel
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oranges.
Microemulsions decreased spore germination and germ tube elongation of P.
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digitatum.
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